Enzymatic modification of saponins

Abstract

The present invention relates to a method for enzymatically modifying saponin produced in plant cell culture, a product produced thereby, uses of the product, and other related aspects. The saponin can be obtained from a plant cell culture extract of Quillaja species, such as an extract of Quillaja Saponaria Molina.

Description

[Technical Field]

[0001] This application relates generally to methods for the enzymatic modification of saponins, particularly saponins produced in in vitro plant cell culture, the saponin products produced thereby, uses of the saponin products, and other related aspects. This application further relates to the use of glucosidases and rhamnosidases, particularly mutant glucosidases and rhamnosidases, to increase cell culture production of a saponin of interest. Saponins may be obtainable from plant cells capable of synthesizing saponins, particularly plant cells from Quillaja species, including plant cell culture extracts thereof. [Background technology]

[0002] Saponins are steroid or terpenoid glycosides with a wide range of uses, from firefighting foams to food additives and immune stimulants (Reichert, 2019).

[0003] Saponins have been of interest as immunostimulating agents for several decades (see, e.g., Hyslop, 1969). Traditionally, saponins have been purified from plants, such as the tree Quillaja saponaria Molina. Quil A is a saponin fraction derived from an aqueous extract of Quillaja saponaria bark (Dalsgaard, 1974). Quil A itself contains multiple components, with four most predominant Quil A fractions purified by reversed-phase chromatography: QS-7, QS-17, QS-18, and QS-21. All of these fractions possess immunostimulating activity but differ in hemolytic activity and toxicity (Kensil, 1991; Kensil, 1995). The main saponin fraction, QS-18, was found to be highly toxic in mice, whereas saponin fractions QS-7 and QS-21 were much less toxic. QS-21, which is more abundant than QS-7, is the most widely studied saponin adjuvant (Ragupathi, 2011).

[0004] Liquid chromatography / mass spectrometry of Quillaja saponaria bark water / methanol extracts has revealed over 100 saponins, many of which have been assigned structures (Nyberg, 2000; Nyberg, 2003; Kite, 2004).

[0005] Quillaja brasiliensis (A St.-Hil&Tul) Mart. extracts have been described, along with the identities of various components therein, as determined by mass spectrometry. Numerous saponin components in Quillaja brasiliensis extracts correspond to saponins found in Quillaja saponaria extracts (Wallace, 2017; Wallace, 2019), and Quillaja brasiliensis extracts have also been shown to have immunostimulatory properties (Cibulski, 2018; Yendo, 2017).

[0006] Adjuvant System 01 (AS01) is a liposome-based adjuvant containing two immunostimulants, 3-O-desacyl-4'-monophosphoryl lipid A (3D-MPL) and QS-21 (Garcon, 2011; Didierlaurent, 2017). 3D-MPL is a non-toxic derivative of lipopolysaccharide derived from Salmonella minnesota. AS01 is a liposome-based adjuvant containing 3D-MPL and QS-21, both of which are immunostimulants. ... TM ) and shingles (HZ / su-Shingrix TM ) and is included in several candidate vaccines. AS01 injection results in rapid and transient activation of innate immunity in animal models. Neutrophils and monocytes are rapidly recruited to the draining lymph nodes (dLN) upon immunization. Furthermore, AS01 inhibits MHCII, which is required for T cell activation. highIt induces the recruitment and activation of dendritic cells (DCs) (Didierlaurent, 2014). Some data are also available on the mechanism of action of the components of AS01. 3D-MPL signals through TLR4 to stimulate NK-κB transcriptional activity and cytokine production, and directly activates antigen-presenting cells (APCs) in both humans and mice (De Becker, 2000; Ismaili, 2002; Martin, 2003; Mata-Haro, 2007). QS-21 induces high antigen-specific antibody responses and CD8 + It promotes T cell responses (Kensil, 1998; Newman, 1992; Soltysik, 1995) and antigen-specific antibody responses in humans (Livingston, 1994). Due to its physical properties, QS-21 may act as a danger signal in vivo (Lambrecht, 2009; Li, 2008). Although QS-21 has been shown to activate the ASC-NLRP3 inflammasome and subsequent IL-1β / IL-18 release (Marty-Roix, 2016), the actual molecular pathways involved in the adjuvant action of saponins remain to be clearly defined.

[0007] Extracts of Quillaja saponaria are commercially available, including its fractions with various degrees of purity, such as Quil A, Fraction A, Fraction B, Fraction C, QS-7, QS-17, QS-18, and QS-21.

[0008] Enzymatic hydrolysis of Quil-A by Rapidase® Revelation Aroma has been described during the development of a quality control method to provide a degradation reference sample ( Lecas, 2021 ).

[0009] Current sources of saponins rely largely on natural sources, which can be limited. As an alternative to Quillaja saponaria bark, cell culture-derived approaches have been developed and are expected to represent a more sustainable source of saponins. For example, International Publication No. 94 / 10291 discloses cultured Quillaja saponaria cells and methods for preparing saponins for use as active substances useful as adjuvants. However, the amount of target saponins so produced may be small. Furthermore, separation of specific saponins from other components, particularly other saponin components that may have similar structures, can be laborious. As a result, there remains a need for novel methods that can improve the yield of target saponins produced in plant cell cultures and / or facilitate the removal of undesired saponin components.

[0010] Modestobacter marinus glucosidase (Uniparc reference UPI000260A2FA, Uniprot reference I4EYD5) is a naturally occurring glucosidase. There remains a need for additional glucosidases that may have improved properties.

[0011] Kribbella flavida rhamnosidase (Uniparc reference UPI00019BDB13, Uniprot reference D2PMT) is a naturally occurring rhamnosidase. There remains a need for additional rhamnosidases that may have improved properties. Summary of the Invention

[0012] The present invention comprises the following steps: (i) providing a plant cell culture extract containing saponins; and (ii) enzymatically converting the starting saponin from the plant cell culture extract to a product saponin. The present invention provides a method for producing the product saponin, comprising:

[0013] Steps below: (i) culturing plant cells capable of synthesizing saponins under conditions conducive to the synthesis of saponins; (ii) recovering the saponin from the plant cell culture; and (iii) enzymatically converting the starting saponin from the recovered saponin into a product saponin. Further provided is a method for producing the product saponin, comprising:

[0014] Steps below: (i) culturing plant cells capable of synthesizing saponins under conditions conducive to the synthesis of saponins; (ii) enzymatically converting the starting saponin derived from the synthesized saponin into the product saponin; and (iii) recovering the saponin from the plant cell culture A method for producing the product saponin is provided, comprising:

[0015] Steps below: (i) culturing plant cells capable of synthesizing saponins under conditions conducive to the synthesis of saponins; (ii) recovering the saponin from the plant cell culture; and (iii) enzymatically converting the starting saponin from the recovered saponin into a product saponin.

[0013] A method for increasing the amount of product saponin obtainable from a plant cell culture is provided, comprising:

[0016] Steps below: (i) culturing plant cells capable of synthesizing saponins under conditions conducive to the synthesis of saponins; (ii) enzymatically converting the starting saponin derived from the synthesized saponin into the product saponin; and (iii) recovering the saponin from the plant cell culture

[0013] A method for increasing the amount of product saponin obtainable from a plant cell culture is provided, comprising:

[0017] Steps below: (i) culturing plant cells capable of synthesizing saponins under conditions conducive to the synthesis of saponins; (ii) recovering the saponin from the plant cell culture; and (iii) enzymatically converting the starting saponin from the recovered saponin into a product saponin. In accordance with the present invention, there is provided a method for reducing the amount of starting saponin obtainable from a plant cell culture, comprising:

[0018] Steps below: (i) culturing plant cells capable of synthesizing saponins under conditions conducive to the synthesis of saponins; (ii) enzymatically converting the starting saponin derived from the synthesized saponin into the product saponin; and (iii) recovering the saponin from the plant cell culture In accordance with the present invention, there is provided a method for reducing the amount of starting saponin obtainable from a plant cell culture, comprising:

[0019] Additionally, the following steps: (i) culturing plant cells capable of synthesizing saponins under conditions conducive to the synthesis of saponins; and (ii) recovering the saponin from the plant cell culture 1. A method for producing saponin by plant cell culture, comprising: The yield of product saponin is increased by enzymatically converting the starting saponin from the recovered saponin to the product saponin. Methods for producing saponins by plant cell culture are provided.

[0020] The use of a glycosidase and / or rhamnosidase to enzymatically convert a starting saponin obtained from a plant cell culture according to the methods of the invention to a product saponin is also provided by the invention.

[0021] Preferably, the glucosidase is (i) or (ii) an amino acid sequence according to SEQ ID NO: 262, 208, 63, 229, 250, 5, 101, 207, 169, 247, 302, 324, 319, 9, 240, 325, 338, 850, 879, 868, 826, 804, 888, 881, 891, 816, 827, 857, 853, 842, 814, 886, 885, 838, 829, 808, 828, 870, 873, 844, 882, 874, 825, 824, 823, 810, 894, 849, 803, 890, 841, 832, 830, 845, 871, 837, 883, or 809, or a functional variant thereof; or The enzyme may comprise, for example consist of, an amino acid sequence according to SEQ ID NO: 262, 208, 63, 229, 250, 5, 101, 207, 169, 247, 302, 324, 319, 9, 240, 325, 338, 850, 879, 868, 826, 804, 888, 881, 891, 816, 827, 857, 853, 842, 814, 886, 885, 838, 829, 808, 828, 870, 873, 844, 882, 874, 825, 824, 823, 810, 894, 849, 803, 890, 841, 832, 830, 845, 871, 837, 883 or 809 or a functional variant thereof.

[0022] Suitably the rhamnosidase is an enzyme comprising, e.g. consisting of, an amino acid sequence according to SEQ ID NO: 992, 1003, 1052, 1073, 1017, 1055, 1075, 1001, 1007, 1061, 1079, 1027, 1039, 1041, 989, 1053, 1018, 1066, 1082, 1076, 993, 1077, 1046, 1015, 1063, 1054, 1074, 1067 or 1033 or a functional variant thereof.

[0023] Also provided by the present invention is the use of an engineered glycosidase and / or an engineered rhamnosidase polypeptide to enzymatically convert a starting saponin obtained from a plant cell culture according to the methods of the present invention to a product saponin.

[0024] Preferably, the engineered glucosidase polypeptide comprises, e.g., consists of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 262, or a functional fragment thereof, wherein the engineered glucosidase polypeptide is F44Y; V60L; G117A; F170N; V263G or V263L; N351H or N351Q; A355H, A355I, A355L, A355M, A355R, A355T or A355W; A356P; R357A, R357C, R357K, R357M or R357Q; G362C; T365A, T365N or T365S; L367C; V394R; V395Y; Q396E, Q396G, Q396N, Q396P, Q396R, Q396S or Q396Y; F430W; R435F; V438T; V440F; F442M or F442Q; G444T; A473F or A473R; L474C, L474I or L474V; I475F; L492C, L492G, L492H, L492I, L492N, L492Q, L492V, L492W or L492Y; Q493F or Q493H; P494H or P494I; S495I, S495K or S495Q; G496P or G496W; D498A, D498E, D498F, D498I, D498K, D498L, D498N, D498P, D498R, D498S, D498T or D498V; A502R; M504G or M504R; L507A or L507R; T508M; L529M; F535P; A536D or A536E; A537R; F541A, F541I, F541L, F541M or F541V; L542I; Q543G or Q543L; E547L; and Y585W It contains at least one residue substitution from

[0025] Suitably, the engineered rhamnosidase polypeptide comprises, e.g. consists of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1017, or a functional fragment thereof, wherein the engineered rhamnosidase polypeptide is (i) A56C (ii) A143P (iii) Q181H, Q181R, or Q181S (iv) L214M (v) G215S (vi) F216M (vii) G218D or G218N (viii) K219G (ix) A238M (x) T252Y (xi) T311W (xii) V326C (xiii) G357C (xiv) S369C, S369I, S369K, or S369M (xv) I487M, I487Q, or I487V (xvi) K492N (xvii) V499T (xviii) G508S (xix) R543C (xx) L557Y (xxi) G634A (xxii) S635N (xxiii) A690C and (xxiv) Q921H It contains at least one residue substitution from

[0026] The present invention also provides saponins prepared by the methods herein, saponin-containing compositions comprising the product saponins prepared by the methods herein, adjuvant compositions comprising the saponins or saponin-containing compositions, and adjuvant compositions prepared using the saponins or saponin-containing compositions.

[0027] Also provided is a method for preparing an adjuvant comprising preparing a saponin or saponin-containing composition from a plant cell culture extract according to the methods of the invention, and formulating the saponin or saponin-containing composition into an adjuvant.

[0028] There is also provided the use of a saponin or saponin-containing composition obtained from a plant cell culture extract of the invention in the manufacture of an adjuvant composition.

[0029] Further provided is an immunogenic composition comprising a saponin or saponin-containing composition according to the invention and an antigen or a polynucleotide encoding the antigen.

[0030] Also provided is a method for preparing an immunogenic composition comprising preparing an adjuvant according to the methods of the invention and formulating it into a composition comprising an antigen or a polynucleotide encoding the antigen.

[0031] Also provided is a kit comprising (i) a saponin or saponin-containing composition according to the invention and (ii) an antigen or a polynucleotide encoding the antigen. [Brief explanation of the drawings]

[0032] [Figure 1] FIG. 1 shows an HPLC chromatogram of a crude aqueous Quillaja saponaria bark extract. [Figure 2] FIG. 1 shows the HPLC-UV chromatogram of a crude aqueous Quillaja saponaria bark extract. [Figure 3] Figure 1 shows the UPLC-UV chromatogram of crude aqueous Quillaja saponaria bark extract. [Figure 4] Figure 1 shows the UPLC-UV chromatogram of polystyrene purified Quillaja saponaria QS-21 saponin extract with low content of 2018 components. [Figure 5] Figure 1 shows the UPLC-UV / MS chromatogram of Quillaja saponaria QS-21 purified saponin extract with low content of 2018 components. [Figure 6] Figure 1 shows the detailed UPLC-UV / MS chromatogram of Quillaja saponaria QS-21 purified saponin extract with low content of 2018 components. [Figure 7] Figure 7A shows extracted mass chromatograms for the 1988 (Figure 7A) and 2002 (Figure 7B) molecular weight ions of Quillaja saponaria QS-21 purified saponin extract with low content of the 2018 component. [Figure 8] Figure 1 shows the combined centroid spectrum of Quillaja saponaria QS-21 purified saponin extract with low content of 2018 components. [Figure 9]Figure 2 shows the UPLC-UV chromatogram of Quillaja saponaria QS-21 purified saponin extract with low 2018 components. [Figure 10] FIG. 1 shows LCMS / MS chromatograms for the QS-18 2150 (panel A) and QS-21 1988 (panel B) component contents in the QS-21 standard in Example 4-2. [Figure 11] FIG. 1 shows LCMS / MS chromatograms for the component contents of QS-18 2150 (panel A) and QS-21 1988 (panel B) after negative control treatment in Example 4-2. [Figure 12] FIG. 1 shows LCMS / MS chromatograms of the component contents of QS-18 2150 (panel A) and QS-21 1988 (panel B) after treatment with glucosidase SEQ ID NO: 262 in Example 4-2. [Figure 13] FIG. 1 shows UV HPLC chromatograms of crude bark extract (CBE) after glucosidase SEQ ID NO: 262 treatment (upper trace) and negative control treatment (lower trace) in Example 4-4. [Figure 14] FIG. 1 shows LCMS / MS chromatograms for the component contents of QS-17 2296 (panel A) and QS-18 2150 (panel B) after negative control treatment in Example 6-1. [Figure 15] FIG. 1 shows LCMS / MS chromatograms for the desglucosyl QS-17 2134 (panel A) and QS-21 1988 (panel B) component contents after negative control treatment in Example 6-1. [Figure 16] FIG. 1 shows LCMS / MS chromatograms for the content of components QS-17 2310 (panel A) and QS-18 2164 (panel B) after negative control treatment in Example 6-1. [Figure 17] FIG. 1 shows LCMS / MS chromatograms for the content of QS-17 2296 (panel A) and QS-18 2150 (panel B) components after treatment with rhamnosidase SEQ ID NO: 1017 in Example 6-1. [Figure 18]FIG. 1 shows LCMS / MS chromatograms for the desglucosyl QS-17 2134 (panel A) and QS-21 1988 (panel B) component contents after treatment with rhamnosidase SEQ ID NO: 1017 in Example 6-1. [Figure 19] FIG. 1 shows LCMS / MS chromatograms for the content of QS-17 2310 (panel A) and QS-18 2164 (panel B) components after treatment with rhamnosidase SEQ ID NO: 1017 in Example 6-1. [Figure 20] FIG. 1 shows UV HPLC chromatograms of treated bark extract (TBE) from Example 6-2 after rhamnosidase SEQ ID NO: 1017 treatment (upper trace) and negative control treatment (lower trace). [Figure 21] FIG. 1 shows UV HPLC chromatograms of CBE after rhamnosidase SEQ ID NO: 1017 treatment (upper trace) and negative control treatment (lower trace) in Example 6-3. [Figure 22] FIG. 1 shows LCMS / MS chromatograms of QS-21 1988 component content at TO (Panel A) and 24 hours (Panel B) after dual enzyme treatment of CBE in Example 7. [Figure 23a] FIG. 1 shows an exemplary UV HPLC chromatogram of CBE after glucosidase enzyme treatment in Example 8. [Figure 23b] FIG. 10 shows an exemplary UV HPLC chromatogram after negative control treatment of CBE in Example 8. [Figure 24] FIG. 1 shows exemplary UV HPLC chromatograms following rhamnosidase enzyme treatment of CBE in Example 9 (top trace) and negative control treatment of CBE (bottom trace). [Figure 25] FIG. 1 shows HPLC-UV chromatograms of untreated and enzyme-treated CBE at 1 L scale from Example 11. [Figure 26] FIG. 10 shows UPLC-UV chromatograms (total acquisition) after purification of untreated and enzyme-treated CBE at 1 L scale from Example 11. [Figure 27]FIG. 10 shows UPLC-UV chromatograms (zoom) after purification of untreated and enzyme-treated CBE at 1 L scale from Example 11. [Figure 28] Figure 1 shows the saponin production potential of untreated and dual enzyme-treated plant cell cultures from Example 12. The content of the indicated saponin components was analyzed and measured by LCMS / MS in crude cell extracts (CCE) obtained from the indicated plant cell cultures. The corresponding saponin component concentrations (expressed in μg / ml of culture) were calculated retrospectively relative to the indicated plant cell cultures. To facilitate visualization, the concentration of each analyzed saponin component has been regrouped into a single bar in the bar graph. The identity of the analyzed saponin component is indicated by the color code provided in the figure. [Figure 29] Figure 1 shows saponin profiles and ratios in plant cell culture extracts. The indicated saponin components were analyzed and measured by LCMS / MS at the indicated steps of the process described in Example 13: (i) pre-enzyme treatment (pre-enzyme treatment); (ii) post-enzyme treatment (post-enzyme treatment); (iii) after UF / DF concentration; and (iv) after phenyl chromatography. Results are expressed as the percentage of each given saponin component compared to the sum of the percentages of all saponin components analyzed (which totals 100%). The identity of the saponin components analyzed is indicated by the color code provided in the figure. DETAILED DESCRIPTION OF THE INVENTION

[0033] Brief description of sequence identifiers SEQ ID NO: 1: Amino acid sequence for Cyberlindnera fabianii glucosidase, Uniparc reference UPI00049B1A8C, Uniprot reference A0A061B3J2. SEQ ID NO: 2: Amino acid sequence for Flavobacterium gilvum glucosidase, Uniparc reference UPI0004E3EF7B, Uniprot reference A0A085EII0. SEQ ID NO: 3: Amino acid sequence for Algibacter lectus glucosidase, Uniparc reference UPI00050EE490, Uniprot reference A0A090X649. SEQ ID NO: 4: Amino acid sequence for Microbacterium azadirachtae glucosidase, Uniparc reference UPI0005ECB51E, Uniprot reference A0A0F0LB94. SEQ ID NO: 5: Amino acid sequence for Actinobacteria bacterial glucosidase, Uniparc reference UPI0006588DAD, Uniprot reference A0A0J0UT37. SEQ ID NO: 6: Amino acid sequence for Chloroflexi bacterial glucosidase, Uniparc reference UPI0007968552, Uniprot reference A0A136KWB3. SEQ ID NO: 7: Amino acid sequence for Komagataeibacter rhaeticus glucosidase, Uniparc reference UPI0002080410, Uniprot reference A0A181C809. SEQ ID NO: 8: Amino acid sequence for Bacteroides sp. glucosidase, Uniparc reference UPI0008211BFC, Uniprot reference A0A1C5WEL8. SEQ ID NO: 9: Amino acid sequence for Streptomyces rubrolavendulae glucosidase, Uniparc reference UPI00085A2BD0, Uniprot reference A0A1D8FZW3. SEQ ID NO: 10: Amino acid sequence for Clostridium roseum glucosidase, Uniparc reference UPI00098C60F6, Uniprot reference A0A1S8KYM5.

[0034] SEQ ID NO: 11: Amino acid sequence for uncultured bacterial glucosidase, Uniparc reference UPI0009CE0D4C, Uniprot reference A0A1V5M6V6. SEQ ID NO: 12: Amino acid sequence for a Firmicutes bacterial glucosidase, Uniparc reference UPI0009D4127D, Uniprot reference A0A1V6AN95. SEQ ID NO: 13: Amino acid sequence for Anthracocystis flocculosa glucosidase, Uniparc reference UPI00045601AB, Uniprot reference A0A061H1Z3. SEQ ID NO: 14: Amino acid sequence for Bifidobacterium boum glucosidase, Uniparc reference UPI0004FF77C7, Uniprot reference A0A086ZKU2. SEQ ID NO: 15: Amino acid sequence for Jejuia pallidilutea glucosidase, Uniparc reference UPI00051EDBDE, Uniprot reference A0A098LTR2. SEQ ID NO: 16: Amino acid sequence for Ceratocystis fimbriata glucosidase, Uniparc reference UPI00062105AB, Uniprot reference A0A0F8B2B0. SEQ ID NO: 17: Amino acid sequence for Actinobacteria bacterial glucosidase, Uniparc reference UPI0006583AB1, Uniprot reference A0A0J0UVW7. SEQ ID NO: 18: Amino acid sequence for Rhodococcus sp. glucosidase, Uniparc reference UPI0007AABFAD, Uniprot reference A0A143QAX3. SEQ ID NO: 19: Amino acid sequence for Valsa mali glucosidase, Uniparc reference UPI0007F2D02D, Uniprot reference A0A194VF47. SEQ ID NO: 20: Amino acid sequence for uncultured Bacteroides sp. glucosidase, Uniparc reference UPI000821004D, Uniprot reference A0A1C5WSI4.

[0035] SEQ ID NO: 21: Amino acid sequence for Eisenbergiella tayi glucosidase, Uniparc reference UPI00084089B2, Uniprot reference A0A1E3ALT2. SEQ ID NO: 22: Amino acid sequence for Streptomyces sp. glucosidase, Uniparc reference UPI000978E914, Uniprot reference A0A1V2MYI4. SEQ ID NO: 23: Amino acid sequence for a Firmicutes bacterial glucosidase, Uniparc reference UPI0009CBF21C, Uniprot reference A0A1V5MH90. SEQ ID NO: 24: Amino acid sequence for a Tenericutes bacterial glucosidase, Uniparc reference UPI0009D5B1F0, Uniprot reference A0A1V6BAK3. SEQ ID NO: 25: Amino acid sequence for Gluconobacter oxydans glucosidase, Uniparc reference UPI0004A87350, Uniprot reference A0A067Z479. SEQ ID NO: 26: Amino acid sequence for Bifidobacterium catenulatum glucosidase, Uniparc reference UPI00050787A2, Uniprot reference A0A087B8Q8. SEQ ID NO: 27: Amino acid sequence for Bionectria ochroleuca glucosidase, Uniparc reference UPI0005965863, Uniprot reference A0A0B7K538. SEQ ID NO: 28: Amino acid sequence for Parcubacteria sp. glucosidase, Uniparc reference UPI0006377CA4, Uniprot reference A0A0G0GD78. SEQ ID NO: 29: Amino acid sequence for Microbacterium ketosireducens glucosidase, Uniparc reference UPI0006228575, Uniprot reference A0A0M2H276. SEQ ID NO: 30: Amino acid sequence for Roseburia faecis glucosidase, Uniparc reference UPI0006C454B4, Uniprot reference A0A173R3W4.

[0036] SEQ ID NO: 31: Amino acid sequence for Kwoniella dejecticola glucosidase, Uniparc reference UPI0007F1D695, Uniprot reference A0A1A6A050. SEQ ID NO: 32: Amino acid sequence for uncultured Clostridium sp. glucosidase, Uniparc reference UPI0008232A70, Uniprot reference A0A1C6C862. SEQ ID NO: 33: Amino acid sequence for Cyberlindnera jadinii glucosidase, Uniparc reference UPI000866CB30, Uniprot reference A0A1E4S2F8. SEQ ID NO: 34: Amino acid sequence for a Bacteroidetes bacterial glucosidase, Uniparc reference UPI0009D3483D, Uniprot reference A0A1V5G4W6. SEQ ID NO: 35: Amino acid sequence for a Verrucomicrobia bacterial glucosidase, Uniparc reference UPI0009CC9AEA, Uniprot reference A0A1V5Q4R4. SEQ ID NO: 36: Amino acid sequence for a Bacteroidetes bacterial glucosidase, Uniparc reference UPI0009C6DE73, Uniprot reference A0A1V6BV25. SEQ ID NO: 37: Amino acid sequence for Lichtheimia ramosa glucosidase, Uniparc reference UPI0004E051A9, Uniprot reference A0A077WUK7. SEQ ID NO: 38: Amino acid sequence for Bifidobacterium mongoliense glucosidase, Uniparc reference UPI0005060F52, Uniprot reference A0A087BWT7. SEQ ID NO: 39: Amino acid sequence for Vibrio ishigakensis glucosidase, Uniparc reference UPI000591CED9, Uniprot reference A0A0B8NZY1. SEQ ID NO: 40: Amino acid sequence for Phaeomoniella chlamydospora glucosidase, Uniparc reference UPI00063B706F, Uniprot reference A0A0G2HEV5.

[0037] SEQ ID NO: 41: Amino acid sequence for Ardenticatena maritima glucosidase, Uniparc reference UPI0006C04F59, Uniprot reference A0A0M8K5H7. SEQ ID NO: 42: Amino acid sequence for Coprococcus comes glucosidase, Uniparc reference UPI000197E031, Uniprot reference A0A173WPC4. SEQ ID NO: 43: Amino acid sequence for Nocardioides dokdonensis glucosidase, Uniparc reference UPI0007DDBAB3, Uniprot reference A0A1A9GNJ0. SEQ ID NO: 44: Amino acid sequence for uncultured Clostridium sp. glucosidase, Uniparc reference UPI000822F7EB, Uniprot reference A0A1C6EBC1. SEQ ID NO: 45: Amino acid sequence for Acetobacterium wieringae glucosidase, Uniparc reference UPI000878EB40, Uniprot reference A0A1F2PFB4. SEQ ID NO: 46: Amino acid sequence for a Tenericutes bacterial glucosidase, Uniparc reference UPI0009D0A2C1, Uniprot reference A0A1V5HNJ3. SEQ ID NO: 47: Amino acid sequence for a Tenericutes bacterial glucosidase, Uniparc reference UPI0009CB8A73, Uniprot reference A0A1V5UFI1. SEQ ID NO: 48: Amino acid sequence for uncultured bacterial glucosidase, Uniparc reference UPI0009CC90AF, Uniprot reference A0A1V6CDT2. SEQ ID NO: 49: Amino acid sequence for Parabacteroides distasonis glucosidase, Uniparc reference UPI0004D8E473, Uniprot reference A0A078SYD0. SEQ ID NO: 50: Amino acid sequence for Bifidobacterium psychraerophilum glucosidase, Uniparc reference UPI0005006C6A, Uniprot reference A0A087CJB0.

[0038] SEQ ID NO: 51: Amino acid sequence for Hebeloma cylindrosporum glucosidase, Uniparc reference UPI00059A3BA8, Uniprot reference A0A0C2YJF2. SEQ ID NO: 52: Amino acid sequence for Brenneria goodwinii glucosidase, Uniparc reference UPI0006579FEA, Uniprot reference A0A0G4JRR8. SEQ ID NO: 53: Amino acid sequence for Aspergillus calidoustus glucosidase, Uniparc reference UPI00073C9E60, Uniprot reference A0A0U5GN20. SEQ ID NO: 54: Amino acid sequence for Bacteroides finegoldii glucosidase, Uniparc reference UPI0006C6E0C3, Uniprot reference A0A174BEZ2. SEQ ID NO: 55: Amino acid sequence for Altererythrobacter dongtanensis glucosidase, Uniparc reference UPI0008153D6D, Uniprot reference A0A1B2A943. SEQ ID NO: 56: Amino acid sequence for uncultured Anaerotruncus sp. glucosidase, Uniparc reference UPI0008206E7B, Uniprot reference A0A1C6FWD9. SEQ ID NO: 57: Amino acid sequence for Candidatus firestone glucosidase, Uniparc reference UPI0008AC0E17, Uniprot reference A0A1F5UJR6. SEQ ID NO: 58: Amino acid sequence for a Tenericutes bacterial glucosidase, Uniparc reference UPI0009C6C8E4, Uniprot reference A0A1V5HS22. SEQ ID NO: 59: Amino acid sequence for a Lentisphaerae bacterial glucosidase, Uniparc reference UPI0009CEE1AB, Uniprot reference A0A1V5VBL2. SEQ ID NO: 60: Amino acid sequence for a Planctomycetes bacterial glucosidase, Uniparc reference UPI0009CAAB0A, Uniprot reference A0A1V6FZ47.

[0039] SEQ ID NO: 61: Amino acid sequence for Pseudallescheria apiosperma glucosidase, Uniparc reference UPI0004DD62AC, Uniprot reference A0A084G332. SEQ ID NO: 62: Amino acid sequence for Nonlabens sediminis glucosidase, Uniparc reference UPI000507F00A, Uniprot reference A0A090Q4N8. SEQ ID NO: 63: Amino acid sequence for Gynuella sunshinyii glucosidase, Uniparc reference UPI0005CC42CA, Uniprot reference A0A0C5VDU3. SEQ ID NO: 64: Amino acid sequence for Verticillium longisporum glucosidase, Uniparc reference UPI00063E4005, Uniprot reference A0A0G4N9Q7. SEQ ID NO: 65: Amino acid sequence for Cellulomonas sp. glucosidase, Uniparc reference UPI00073C6CD3, Uniprot reference A0A0V8TAB5. SEQ ID NO: 66: Amino acid sequence for Hungatella hathewayi glucosidase, Uniparc reference UPI0006C024BB, Uniprot reference A0A174FBX7. SEQ ID NO: 67: Amino acid sequence for Mesorhizobium sp. glucosidase, Uniparc reference UPI000688E74D, Uniprot reference A0A1C2DG64. SEQ ID NO: 68: Amino acid sequence for Clostridium sp. glucosidase, Uniparc reference UPI000822FAB7, Uniprot reference A0A1C6GRT5. SEQ ID NO: 69: Amino acid sequence for Chlamydiales bacterial glucosidase, Uniparc reference UPI0009284E74, Uniprot reference A0A1M3CSY6. SEQ ID NO: 70: Amino acid sequence for a Spirochaetes bacterial glucosidase, Uniparc reference UPI0009CB461D, Uniprot reference A0A1V5HUX3.

[0040] SEQ ID NO: 71: Amino acid sequence for bacterial glucosidase, Uniparc reference UPI0009C92D6A, Uniprot reference A0A1V5VHB9. SEQ ID NO: 72: Amino acid sequence for a Thermotogae bacterial glucosidase, Uniparc reference UPI0009D12B86, Uniprot reference A0A1V6H2W5. SEQ ID NO: 73: Amino acid sequence for Pseudallescheria apiosperma glucosidase, Uniparc reference UPI0004DCBED3, Uniprot reference A0A084GGE2. SEQ ID NO: 74: Amino acid sequence for Algibacter lectus glucosidase, Uniparc reference UPI00050E0BE3, Uniprot reference A0A090VF17. SEQ ID NO: 75: Amino acid sequence for Paxillus involutus glucosidase, Uniparc reference UPI0005B075C8, Uniprot reference A0A0C9TWP5. SEQ ID NO: 76: Amino acid sequence for Verticillium longisporum glucosidase, Uniparc reference UPI00063DF006, Uniprot reference A0A0G4NA55. SEQ ID NO: 77: Amino acid sequence for Mucilaginibacter gotjawali glucosidase, Uniparc reference UPI00076F8EA4, Uniprot reference A0A110B1H1. SEQ ID NO: 78: Amino acid sequence for Bacteroides uniformis glucosidase, Uniparc reference UPI0006C0FFEF, Uniprot reference A0A174IWW4. SEQ ID NO: 79: Amino acid sequence for Coprococcus sp. glucosidase, Uniparc reference UPI0008222B77, Uniprot reference A0A1C5W0N6. SEQ ID NO: 80: Amino acid sequence for Blautia sp. glucosidase, Uniparc reference UPI0006C36823, Uniprot reference A0A1C6K2X5.

[0041] SEQ ID NO: 81: Amino acid sequence for Cellulomonas sp. glucosidase, Uniparc reference UPI00092B3CD4, Uniprot reference A0A1M3ELH4. SEQ ID NO: 82: Amino acid sequence for bacterial glucosidase, Uniparc reference UPI0009C85428, Uniprot reference A0A1V5J984. SEQ ID NO: 83: Amino acid sequence for a Spirochaetes bacterial glucosidase, Uniparc reference UPI0009CA1D5C, Uniprot reference A0A1V5WHQ7. SEQ ID NO: 84: Amino acid sequence for a Tenericutes bacterial glucosidase, Uniparc reference UPI0009C7041F, Uniprot reference A0A1V6IJ57. SEQ ID NO: 85: Amino acid sequence for Flavobacterium gilvum glucosidase, Uniparc reference UPI0004E2A41F, Uniprot reference A0A085EG29. SEQ ID NO: 86: Amino acid sequence for Algibacter lectus glucosidase, Uniparc reference UPI0005102470, Uniprot reference A0A090WWZ1. SEQ ID NO: 87: Amino acid sequence for Hydnomerulius pinastri glucosidase, Uniparc reference UPI0005B0F0AE, Uniprot reference A0A0C9WDY0. SEQ ID NO: 88: Amino acid sequence for Nocardia farcinica glucosidase, Uniparc reference UPI00065C2666, Uniprot reference A0A0H5NWN2. SEQ ID NO: 89: Amino acid sequence for Bacteroides cellulosilyticus glucosidase, Uniparc reference UPI000760375B, Uniprot reference A0A125MG18. SEQ ID NO: 90: Amino acid sequence for Fonsecaea erecta glucosidase, Uniparc reference UPI0007DF4250, Uniprot reference A0A178ZBN2.

[0042] SEQ ID NO: 91: Amino acid sequence for Bacteroides sp. glucosidase, Uniparc reference UPI0008209852, Uniprot reference A0A1C5W9N9. SEQ ID NO: 92: Amino acid sequence for Tannerella forsythia glucosidase, Uniparc reference UPI00086C34C1, Uniprot reference A0A1D3UGH8. SEQ ID NO: 93: Amino acid sequence for Microbacterium esteraromaticum glucosidase, Uniparc reference UPI00097E83BB, Uniprot reference A0A1R4KI92. SEQ ID NO: 94: Amino acid sequence for bacterial glucosidase, Uniparc reference UPI0009D255E5, Uniprot reference A0A1V5LJK9. SEQ ID NO: 95: Amino acid sequence for Candidatus hydrogenedentes glucosidase, Uniparc reference UPI0009C5A3CF, Uniprot reference A0A1V5Z2L2. SEQ ID NO: 96: Amino acid sequence for a Bacteroidetes bacterial glucosidase, Uniparc reference UPI0009C55799, Uniprot reference A0A1V6J4J8. SEQ ID NO: 97: Amino acid sequence for Penicillium solitum glucosidase, Uniparc reference UPI0009D4067F, Uniprot reference A0A1V6RQ41. SEQ ID NO: 98: Amino acid sequence for Weissella soli glucosidase, Uniparc reference UPI0008737AA2, Uniprot reference A0A288Q8I2. SEQ ID NO: 99: Amino acid sequence for Acetatifactor muris glucosidase, Uniparc reference UPI000CAC57D4, Uniprot reference A0A2K4ZN91. SEQ ID NO: 100: Amino acid sequence for Corynespora cassiicola glucosidase, Uniparc reference UPI000D237A4A, Uniprot reference A0A2T2NYD4.

[0043] SEQ ID NO: 101: Amino acid sequence for Meira miltonrushii glucosidase, Uniparc reference UPI000D77C91D, Uniprot reference A0A316V6M3. SEQ ID NO: 102: Amino acid sequence for Bacteroides fragilis glucosidase, Uniparc reference UPI00004E1F76, Uniprot reference A0A380YVC7. SEQ ID NO: 103: Amino acid sequence for Malassezia restricta glucosidase, Uniparc reference UPI000F0C30E8, Uniprot reference A0A3G2SB79. SEQ ID NO: 104: Amino acid sequence for Fusarium euwallaceae glucosidase, Uniparc reference UPI000FFFEFB2, Uniprot reference A0A430LYA2. SEQ ID NO: 105: Amino acid sequence for Psathyrella aberdarensis glucosidase, Uniparc reference UPI0010251887, Uniprot reference A0A4Q2E070. SEQ ID NO: 106: Amino acid sequence for Aeromonas hydrophila glucosidase, Uniparc reference UPI0000E69509, Uniprot reference A0KLP6. SEQ ID NO: 107: Amino acid sequence for Saccharopolyspora erythraea glucosidase, Uniparc reference UPI00000B86CB, Uniprot reference A4F7P9. SEQ ID NO: 108: Amino acid sequence for Streptomyces sviceus glucosidase, Uniparc reference UPI000180240E, Uniprot reference B5I181. SEQ ID NO: 109: Amino acid sequence for Naematelia encephala glucosidase, Uniparc reference UPI000A250F78, Uniprot reference A0A1Y2AWB7. SEQ ID NO: 110: Amino acid sequence for Hartmannibacter diazotrophicus glucosidase, Uniparc reference UPI000C0221F1, Uniprot reference A0A2C9D6I2.

[0044] SEQ ID NO: 111: Amino acid sequence for Pontimonas salivibrio glucosidase, Uniparc reference UPI000CEB5AB1, Uniprot reference A0A2L2BPE2. SEQ ID NO: 112: Amino acid sequence for Cadophora sp. glucosidase, Uniparc reference UPI000D5B9C38, Uniprot reference A0A2V1CH24. SEQ ID NO: 113: Amino acid sequence for Meira miltonrushii glucosidase, Uniparc reference UPI000D779558, Uniprot reference A0A316V8S0. SEQ ID NO: 114: Amino acid sequence for Monilinia fructigena glucosidase, Uniparc reference UPI000DC42E3D, Uniprot reference A0A395IJW4. SEQ ID NO: 115: Amino acid sequence for Hortaea werneckii glucosidase, Uniparc reference UPI000F3E476C, Uniprot reference A0A3M6XGS0. SEQ ID NO: 116: Amino acid sequence for Streptomyces netropsis glucosidase, Uniparc reference UPI00101460D7, Uniprot reference A0A445N7U0. SEQ ID NO: 117: Amino acid sequence for Aureobasidium pullulans glucosidase, Uniparc reference UPI001139C6A8, Uniprot reference A0A4S9IFI0. SEQ ID NO: 118: Amino acid sequence for Aspergillus clavatus glucosidase, Uniparc reference UPI0000EA5CFF, Uniprot reference A1CTN9. SEQ ID NO: 119: Amino acid sequence for Clavibacter michiganensis glucosidase, Uniparc reference UPI0001523037, Uniprot reference A5CT94. SEQ ID NO: 120: Amino acid sequence for Penicillium rubens glucosidase, Uniparc reference UPI0001831CF5, Uniprot reference B6H7R5.

[0045] SEQ ID NO: 121: Amino acid sequence for Lachnoclostridium sp. glucosidase, Uniparc reference UPI000B365547, Uniprot reference A0A1Y4NTL9. SEQ ID NO: 122: Amino acid sequence for a Rhodobacteraceae bacterial glucosidase, Uniparc reference UPI000C09BF88, Uniprot reference A0A2D5IXB9. SEQ ID NO: 123: Amino acid sequence for Bacteroides fragilis glucosidase, Uniparc reference UPI0004B5EEF2, Uniprot reference A0A2M9UUC4. SEQ ID NO: 124: Amino acid sequence for Aspergillus indologenus glucosidase, Uniparc reference UPI000D7FE1ED, Uniprot reference A0A2V5IY78. SEQ ID NO: 125: Amino acid sequence for Acaromyces ingoldii glucosidase, Uniparc reference UPI000D802B25, Uniprot reference A0A316YR39. SEQ ID NO: 126: Amino acid sequence for Monilinia fructigena glucosidase, Uniparc reference UPI000DC60823, Uniprot reference A0A395J1U5. SEQ ID NO: 127: Amino acid sequence for Paenibacillus xylanexedens glucosidase, Uniparc reference UPI000F52D773, Uniprot reference A0A3N6CA02. SEQ ID NO: 128: Amino acid sequence for Actinomyces howellii glucosidase, Uniparc reference UPI000F6DAAAE, Uniprot reference A0A448HIG0. SEQ ID NO: 129: Amino acid sequence for Friedmanniomyces endolithicus glucosidase, Uniparc reference UPI00113D19DF, Uniprot reference A0A4V5N9I4. SEQ ID NO: 130: Amino acid sequence for Neosartorya fischeri glucosidase, Uniparc reference UPI0000EA8672, Uniprot reference A1DNS0.

[0046] SEQ ID NO: 131: Amino acid sequence for Pseudomonas aeruginosa glucosidase, Uniparc reference UPI0000D7314B, Uniprot reference A6V4K6. SEQ ID NO: 132: Amino acid sequence for Talaromyces stipitatus glucosidase, Uniparc reference UPI00018E7266, Uniprot reference B8MF24. SEQ ID NO: 133: Amino acid sequence for Aquimixticola soesokkakensis glucosidase, Uniparc reference UPI000A1A5FD7, Uniprot reference A0A1Y5RVF8. SEQ ID NO: 134: Amino acid sequence for a Rhodobacterales bacterial glucosidase, Uniparc reference UPI000C98D37C, Uniprot reference A0A2D9YGV1. SEQ ID NO: 135: Amino acid sequence for Methylorubrum extorquens glucosidase, Uniparc reference UPI0006F9793E, Uniprot reference A0A2N9AS40. SEQ ID NO: 136: Amino acid sequence for Clostridium perfringens glucosidase, Uniparc reference UPI00000CF7C8, Uniprot reference A0A2X2YBP0. SEQ ID NO: 137: Amino acid sequence for Acholeplasmatales bacterial glucosidase, Uniparc reference UPI0008AEBEA3, Uniprot reference A0A348NID6. SEQ ID NO: 138: Amino acid sequence for a Bacteroidetes bacterial glucosidase, Uniparc reference UPI000EC3C979, Uniprot reference A0A3B8VHE8. SEQ ID NO: 139: Amino acid sequence for Clostridium carnis glucosidase, Uniparc reference UPI000F637E38, Uniprot reference A0A3P6K8E8. SEQ ID NO: 140: Amino acid sequence for Mycolicibacterium flavescens glucosidase, Uniparc reference UPI000B93B5C9, Uniprot reference A0A448HNB5.

[0047] SEQ ID NO: 141: Amino acid sequence for Streptococcus gallolyticus glucosidase, Uniparc reference UPI000F6EFA71, Uniprot reference A0A4V6LJ94. SEQ ID NO: 142: Amino acid sequence for Yersinia enterocolitica glucosidase, Uniparc reference UPI0000EB54CC, Uniprot reference A1JNB7. SEQ ID NO: 143: Amino acid sequence for Anaeromyxobacter sp. glucosidase, Uniparc reference UPI0000ED8A80, Uniprot reference A7HFG4. SEQ ID NO: 144: Amino acid sequence for Talaromyces stipitatus glucosidase, Uniparc reference UPI00018E7D70, Uniprot reference B8MK55. SEQ ID NO: 145: Amino acid sequence for Hortaea werneckii glucosidase, Uniparc reference UPI000A2E3FAA, Uniprot reference A0A1Z5SL14. SEQ ID NO: 146: Amino acid sequence for Micavibrio sp. glucosidase, Uniparc reference UPI000C529025, Uniprot reference A0A2E2Q8X2. SEQ ID NO: 147: Amino acid sequence for an Acidobacteriia bacterial glucosidase, Uniparc reference UPI000CE6B996, Uniprot reference A0A2N9MBS0. SEQ ID NO: 148: Amino acid sequence for Corynebacterium jeikeium glucosidase, Uniparc reference UPI000DA3A972, Uniprot reference A0A2X4T570. SEQ ID NO: 149: Amino acid sequence for a Clostridiaceae bacterial glucosidase, Uniparc reference UPI000E8D37A1, Uniprot reference A0A353PZH8. SEQ ID NO: 150: Amino acid sequence for Anaerolineaceae bacterial glucosidase, Uniparc reference UPI000748C096, Uniprot reference A0A3B9PA35.

[0048] SEQ ID NO: 151: Amino acid sequence for Gymnopilus dilepis glucosidase, Uniparc reference UPI000FF41956, Uniprot reference A0A409WSY0. SEQ ID NO: 152: Amino acid sequence for Kocuria rosea glucosidase, Uniparc reference UPI000F7105D4, Uniprot reference A0A448R8N0. SEQ ID NO: 153: Amino acid sequence for Teredinibacter sp. glucosidase, Uniparc reference UPI0011696FAB, Uniprot reference A0A509DWZ3. SEQ ID NO: 154: Amino acid sequence for Aspergillus niger glucosidase, Uniparc reference UPI0000EFB564, Uniprot reference A2QS42. SEQ ID NO: 155: Amino acid sequence for Laccaria bicolor glucosidase, Uniparc reference UPI000164423D, Uniprot reference B0D734. SEQ ID NO: 156: Amino acid sequence for Pedosphaera parvula glucosidase, Uniparc reference UPI00017357F6, Uniprot reference B9XH33. SEQ ID NO: 157: Amino acid sequence for Megamonas hypermegale glucosidase, Uniparc reference UPI00042469F6, Uniprot reference A0A239TGH2. SEQ ID NO: 158: Amino acid sequence for Armillaria gallica glucosidase, Uniparc reference UPI000BC209C3, Uniprot reference A0A2H3E300. SEQ ID NO: 159: Amino acid sequence for Micromonospora sp. glucosidase, Uniparc reference UPI000D2EAE87, Uniprot reference A0A2P8AV03. SEQ ID NO: 160: Amino acid sequence for Klebsiella oxytoca glucosidase, Uniparc reference UPI000DA286EE, Uniprot reference A0A2X5CJC5.

[0049] SEQ ID NO: 161: Amino acid sequence for Candidatus ozemobacter glucosidase, Uniparc reference UPI000DFAEF6C, Uniprot reference A0A367ZIH8. SEQ ID NO: 162: Amino acid sequence for Coleophoma crateriformis glucosidase, Uniparc reference UPI000E399EE8, Uniprot reference A0A3D8R2C2. SEQ ID NO: 163: Amino acid sequence for Apiotrichum porosum glucosidase, Uniparc reference UPI000FBC01E3, Uniprot reference A0A427XHS2. SEQ ID NO: 164: Amino acid sequence for Acholeplasma hippikon glucosidase, Uniparc reference UPI00068E4E50, Uniprot reference A0A449BJ27. SEQ ID NO: 165: Amino acid sequence for Streptomyces spectabilis glucosidase, Uniparc reference UPI001185F074, Uniprot reference A0A516RGT1. SEQ ID NO: 166: Amino acid sequence for Aspergillus niger glucosidase, Uniparc reference UPI0000EFCED2, Uniprot reference A2R8G2. SEQ ID NO: 167: Amino acid sequence for Xanthomonas campestris glucosidase, Uniparc reference UPI00000D8BFA, Uniprot reference B0RYA0. SEQ ID NO: 168: Amino acid sequence for Lactobacillus paracasei glucosidase, Uniparc reference UPI00019C9CD7, Uniprot reference C2FDL2. SEQ ID NO: 169: Amino acid sequence for Bifiguratus adelaidae glucosidase, Uniparc reference UPI000BC490A3, Uniprot reference A0A261XUH4. SEQ ID NO: 170: Amino acid sequence for bacterial glucosidase, Uniparc reference UPI000CC31AE2, Uniprot reference A0A2H5YYA1.

[0050] SEQ ID NO: 171: Amino acid sequence for Actinomadura parvosata glucosidase, Uniparc reference UPI000D26C9C6, Uniprot reference A0A2P9IY35. SEQ ID NO: 172: Amino acid sequence for Melissococcus plutonius glucosidase, Uniparc reference UPI00024F22B8, Uniprot reference A0A2Z5Y4P3. SEQ ID NO: 173: Amino acid sequence for Enterococcus durans glucosidase, Uniparc reference UPI000E020871, Uniprot reference A0A377KJS3. SEQ ID NO: 174: Amino acid sequence for Malassezia restricta glucosidase, Uniparc reference UPI000DD17A7D, Uniprot reference A0A3G2S2J6. SEQ ID NO: 175: Amino acid sequence for Apiotrichum porosum glucosidase, Uniparc reference UPI000FA2DE87, Uniprot reference A0A427XZQ0. SEQ ID NO: 176: Amino acid sequence for Streptomonospora sp. glucosidase, Uniparc reference UPI0010355193, Uniprot reference A0A4P6PWP5. SEQ ID NO: 177: Amino acid sequence for Lactobacillus gasseri glucosidase, Uniparc reference UPI00119640A1, Uniprot reference A0A558LH47. SEQ ID NO: 178: Amino acid sequence for Aspergillus niger glucosidase, Uniparc reference UPI0000EFD0AA, Uniprot reference A2RAJ1. SEQ ID NO: 179: Amino acid sequence for Neosartorya fumigata glucosidase, Uniparc reference UPI000170BF91, Uniprot reference B0XXG1. SEQ ID NO: 180: Amino acid sequence for Eubacterium eligens glucosidase, Uniparc reference UPI0001A5B234, Uniprot reference C4Z6T5.

[0051] SEQ ID NO: 181: Amino acid sequence for Bifiguratus adelaidae glucosidase, Uniparc reference UPI000BC64A75, Uniprot reference A0A261XVM8. SEQ ID NO: 182: Amino acid sequence for bacterial glucosidase, Uniparc reference UPI000CAA854E, Uniprot reference A0A2H6EX57. SEQ ID NO: 183: Amino acid sequence for Corynespora cassiicola glucosidase, Uniparc reference UPI000D24EC2C, Uniprot reference A0A2T2N5H9. SEQ ID NO: 184: Amino acid sequence for Pseudomicrostroma glucosiphilum glucosidase, Uniparc reference UPI000D77A0B7, Uniprot reference A0A316UK83. SEQ ID NO: 185: Amino acid sequence for Staphylococcus saprophyticus glucosidase, Uniparc reference UPI0002DAAFCC, Uniprot reference A0A380HD31. SEQ ID NO: 186: Amino acid sequence for Malassezia restricta glucosidase, Uniparc reference UPI000F0C6B8E, Uniprot reference A0A3G2S932. SEQ ID NO: 187: Amino acid sequence for Saitozyma podzolica glucosidase, Uniparc reference UPI000FBA70E2, Uniprot reference A0A427YCL1. SEQ ID NO: 188: Amino acid sequence for Tremella mesenterica glucosidase, Uniparc reference UPI00102822B4, Uniprot reference A0A4Q1BRC6. SEQ ID NO: 189: Amino acid sequence for Arthrobacter sp. glucosidase, Uniparc reference UPI0000527506, Uniprot reference A0JZ86. SEQ ID NO: 190: Amino acid sequence for Scheffersomyces stipitis glucosidase, Uniparc reference UPI000157388C, Uniprot reference A3LRB0.

[0052] SEQ ID NO: 191: Amino acid sequence for Leptothrix cholodnii glucosidase, Uniparc reference UPI0001712E02, Uniprot reference B1XZK8. SEQ ID NO: 192: Amino acid sequence for Thauera sp. glucosidase, Uniparc reference UPI000166883C, Uniprot reference C4ZLL7. SEQ ID NO: 193: Amino acid sequence for Kosmotoga olearia glucosidase, Uniparc reference UPI00018494AB, Uniprot reference C5CDW5. SEQ ID NO: 194: Amino acid sequence for Roseburia intestinalis glucosidase, Uniparc reference UPI0001CD671F, Uniprot reference D4L3Y2. SEQ ID NO: 195: Amino acid sequence for Streptococcus equinus glucosidase, Uniparc reference UPI0001E0DC00, Uniprot reference E0PDF8. SEQ ID NO: 196: Amino acid sequence for Streptococcus cristatus glucosidase, Uniparc reference UPI0001F80C4B, Uniprot reference E8JUK5. SEQ ID NO: 197: Amino acid sequence for Cellulosilyticum lentocellum glucosidase, Uniparc reference UPI0001D2DBBC, Uniprot reference F2JLH3. SEQ ID NO: 198: Amino acid sequence for Streptococcus gallolyticus glucosidase, Uniparc reference UPI0001C48657, Uniprot reference F5WYI5. SEQ ID NO: 199: Amino acid sequence for Ketogulonicigenium vulgare glucosidase, Uniparc reference UPI00021D443A, Uniprot reference F9Y8Z7. SEQ ID NO: 200: Amino acid sequence for Spathaspora passalidarum glucosidase, Uniparc reference UPI000228255D, Uniprot reference G3AGX1.

[0053] SEQ ID NO: 201: Amino acid sequence for Niastella koreensis glucosidase, Uniparc reference UPI00023F6F5F, Uniprot reference G8T9J3. SEQ ID NO: 202: Amino acid sequence for Cellvibrio sp. glucosidase, Uniparc reference UPI000260108C, Uniprot reference I3IDC0. SEQ ID NO: 203: Amino acid sequence for Flavobacterium sp. glucosidase, Uniparc reference UPI000272D1E0, Uniprot reference J1ACA0. SEQ ID NO: 204: Amino acid sequence for Macrophomina phaseolina glucosidase, Uniparc reference UPI00028E7FE1, Uniprot reference K2S5D3. SEQ ID NO: 205: Amino acid sequence for Kosmotoga olearia glucosidase, Uniparc reference UPI00018483A2, Uniprot reference C5CHI5. SEQ ID NO: 206: Amino acid sequence for Blautia obeum glucosidase, Uniparc reference UPI0001CD5918, Uniprot reference D4LRF6. SEQ ID NO: 207: Amino acid sequence for Bifidobacterium dentium glucosidase, Uniparc reference UPI0001E18CDA, Uniprot reference E0Q541. SEQ ID NO: 208: Amino acid sequence for Anaerolinea thermophila glucosidase, Uniparc reference UPI0001F55F8F, Uniprot reference E8N5R8. SEQ ID NO: 209: Amino acid sequence for Coriobacterium glomerans glucosidase, Uniparc reference UPI0002050DA2, Uniprot reference F2N7E4. SEQ ID NO: 210: Amino acid sequence for Microlunatus phosphovorus glucosidase, Uniparc reference UPI000210C886, Uniprot reference F5XJQ3.

[0054] SEQ ID NO: 211: Amino acid sequence for Streptomyces sp. glucosidase, Uniparc reference UPI0001C18877, Uniprot reference G0Q1U8. SEQ ID NO: 212: Amino acid sequence for Spathaspora passalidarum glucosidase, Uniparc reference UPI0002282B61, Uniprot reference G3AIV6. SEQ ID NO: 213: Amino acid sequence for Glarea lozoyensis glucosidase, Uniparc reference UPI0002402F4A, Uniprot reference H0ET34. SEQ ID NO: 214: Amino acid sequence for Glaciozyma antarctica glucosidase, Uniparc reference UPI0002633B36, Uniprot reference I3UJK0. SEQ ID NO: 215: Amino acid sequence for Arthrobacter sp. glucosidase, Uniparc reference UPI00027DFD35, Uniprot reference J7LN00. SEQ ID NO: 216: Amino acid sequence for Fusarium pseudograminearum glucosidase, Uniparc reference UPI00028D698C, Uniprot reference K3VMA9. SEQ ID NO: 217: Amino acid sequence for Hypocrea rufa glucosidase, Uniparc reference UPI000006AA61, Uniprot reference C6GGC9. SEQ ID NO: 218: Amino acid sequence for Ruminococcus torques glucosidase, Uniparc reference UPI0001CDA964, Uniprot reference D4M6W6. SEQ ID NO: 219: Amino acid sequence for Bifidobacterium dentium glucosidase, Uniparc reference UPI0001E17370, Uniprot reference E0Q9Z7. SEQ ID NO: 220: Amino acid sequence for Bacteroides salanitronis glucosidase, Uniparc reference UPI0001FC71F6, Uniprot reference F0R2D7.

[0055] SEQ ID NO: 221: Amino acid sequence for Bacteroides coprosuis glucosidase, Uniparc reference UPI00020E6D19, Uniprot reference F3ZQ40. SEQ ID NO: 222: Amino acid sequence for Marinomonas posidonica glucosidase, Uniparc reference UPI00020D4CC6, Uniprot reference F6CWF6. SEQ ID NO: 223: Amino acid sequence for Chaetomium thermophilum glucosidase, Uniparc reference UPI000227E8ED, Uniprot reference G0SE64. SEQ ID NO: 224: Amino acid sequence for Tetragenococcus halophilus glucosidase, Uniparc reference UPI00022B9A15, Uniprot reference G4L5K1. SEQ ID NO: 225: Amino acid sequence for Paenibacillus sp. glucosidase, Uniparc reference UPI00024F0867, Uniprot reference H6CCZ8. SEQ ID NO: 226: Amino acid sequence for Turneriella parva glucosidase, Uniparc reference UPI000265AA4B, Uniprot reference I4B8U7. SEQ ID NO: 227: Amino acid sequence for Arthrobacter sp. glucosidase, Uniparc reference UPI00027DFDB2, Uniprot reference J7LQK9. SEQ ID NO: 228: Amino acid sequence for Agaricus bisporus glucosidase, Uniparc reference UPI00029074B1, Uniprot reference K5W7V1. SEQ ID NO: 229: Amino acid sequence for Nectria haematococca glucosidase, Uniparc reference UPI0001B67634, Uniprot reference C7YIP3. SEQ ID NO: 230: Amino acid sequence for Bacteroides xylanisolvens glucosidase, Uniparc reference UPI0001A25287, Uniprot reference D4VSZ0.

[0056] SEQ ID NO: 231: Amino acid sequence for Sediminispirochaeta smaragdinae glucosidase, Uniparc reference UPI0001DD9790, Uniprot reference E1R331. SEQ ID NO: 232: Amino acid sequence for Deinococcus proteolyticus glucosidase, Uniparc reference UPI0001FC42E9, Uniprot reference F0RPV2. SEQ ID NO: 233: Amino acid sequence for Sphingobacterium sp. glucosidase, Uniparc reference UPI0002033A0A, Uniprot reference F4C226. SEQ ID NO: 234: Amino acid sequence for Sphingobium chlorophenolicum glucosidase, Uniparc reference UPI0001E54133, Uniprot reference F6ET40. SEQ ID NO: 235: Amino acid sequence for Caloramator australicus glucosidase, Uniparc reference UPI00021CACC4, Uniprot reference G0V3V5. SEQ ID NO: 236: Amino acid sequence for Commensalibacter intestini glucosidase, Uniparc reference UPI000230E3BF, Uniprot reference G6F370. SEQ ID NO: 237: Amino acid sequence for Paenibacillus sp. glucosidase, Uniparc reference UPI00024F07AC, Uniprot reference H6CIT2. SEQ ID NO: 238: Amino acid sequence for Nitrolancea hollandica glucosidase, Uniparc reference UPI0002638AF3, Uniprot reference I4EIA9. SEQ ID NO: 239: Amino acid sequence for Cryptococcus neoformans glucosidase, Uniparc reference UPI000392C3ED, Uniprot reference J9VVK7. SEQ ID NO: 240: Amino acid sequence for Acidipropionibacterium acidipropionici glucosidase, Uniparc reference UPI0002988588, Uniprot reference K7S596.

[0057] SEQ ID NO: 241: Amino acid sequence for Prevotella sp. glucosidase, Uniparc reference UPI0001B93465, Uniprot reference C9PT75. SEQ ID NO: 242: Amino acid sequence for Rhodobacter capsulatus glucosidase, Uniparc reference UPI0001D08095, Uniprot reference D5ALU0. SEQ ID NO: 243: Amino acid sequence for Stigmatella aurantiaca glucosidase, Uniparc reference UPI0001E74370, Uniprot reference E3FJ05. SEQ ID NO: 244: Amino acid sequence for Sphaerochaeta globosa glucosidase, Uniparc reference UPI0002010060, Uniprot reference F0RVK3. SEQ ID NO: 245: Amino acid sequence for Sphaerochaeta coccoides glucosidase, Uniparc reference UPI000207D78C, Uniprot reference F4GH96. SEQ ID NO: 246: Amino acid sequence for Novosphingobium sp. glucosidase, Uniparc reference UPI00020EFBCB, Uniprot reference F6ICQ5. SEQ ID NO: 247: Amino acid sequence for Arthrobotrys oligospora glucosidase, Uniparc reference UPI000225331F, Uniprot reference G1XH86. SEQ ID NO: 248: Amino acid sequence for Lactococcus lactis glucosidase, Uniparc reference UPI00000C6907, Uniprot reference G6FFS4. SEQ ID NO: 249: Amino acid sequence for Phaeospirillum molischianum glucosidase, Uniparc reference UPI000255313A, Uniprot reference H8FXH7. SEQ ID NO: 250: Amino acid sequence for Modestobacter marinus glucosidase, Uniparc reference UPI0002609C68, Uniprot reference I4EW72.

[0058] SEQ ID NO: 251: Amino acid sequence for Saccharothrix espanaensis glucosidase, Uniparc reference UPI00028AC337, Uniprot reference K0K125. SEQ ID NO: 252: Amino acid sequence for Cronobacter sakazakii glucosidase, Uniparc reference UPI00029BA293, Uniprot reference K8DAK0. SEQ ID NO: 253: Amino acid sequence for Verticillium alfalfae glucosidase, Uniparc reference UPI0001BBDF1E, Uniprot reference C9SVX1. SEQ ID NO: 254: Amino acid sequence for Bacteroides xylanisolvens glucosidase, Uniparc reference UPI0001CCFD36, Uniprot reference D6CY10. SEQ ID NO: 255: Amino acid sequence for Leadbetterella byssophila glucosidase, Uniparc reference UPI0001EBD98A, Uniprot reference E4RUR0. SEQ ID NO: 256: Amino acid sequence for Sphaerochaeta globosa glucosidase, Uniparc reference UPI00020102A5, Uniprot reference F0RYB6. SEQ ID NO: 257: Amino acid sequence for Sphaerochaeta coccoides glucosidase, Uniparc reference UPI000207D6A0, Uniprot reference F4GLH6. SEQ ID NO: 258: Amino acid sequence for Haloplasma contractile glucosidase, Uniparc reference UPI000212252C, Uniprot reference F7Q0Y2. SEQ ID NO: 259: Amino acid sequence for Nitrospirillum amazonense glucosidase, Uniparc reference UPI0002265447, Uniprot reference G1Y4C7. SEQ ID NO: 260: Amino acid sequence for Azospirillum brasilense glucosidase, Uniparc reference UPI00023420B8, Uniprot reference G8AWD9.

[0059] SEQ ID NO: 261: Amino acid sequence for Phaeospirillum molischianum glucosidase, Uniparc reference UPI000255314F, Uniprot reference H8FXJ8. SEQ ID NO: 262: Amino acid sequence for Modestobacter marinus glucosidase, Uniparc reference UPI000260A2FA, Uniprot reference I4EYD5. SEQ ID NO: 263: Amino acid sequence for Wickerhamomyces ciferrii glucosidase, Uniparc reference UPI000283EB8D, Uniprot reference K0KVJ2. SEQ ID NO: 264: Amino acid sequence for Gloeocapsa sp. glucosidase, Uniparc reference UPI0002A5D085, Uniprot reference K9XKL8. SEQ ID NO: 265: Amino acid sequence for Sphaerobacter thermophilus glucosidase, Uniparc reference UPI0001A3BCB6, Uniprot reference D1C7U8. SEQ ID NO: 266: Amino acid sequence for Bacteroides xylanisolvens glucosidase, Uniparc reference UPI0001CCEF1F, Uniprot reference D6D4V2. SEQ ID NO: 267: Amino acid sequence for Prevotella buccae glucosidase, Uniparc reference UPI0001F149E8, Uniprot reference E6K4W5. SEQ ID NO: 268: Amino acid sequence for Grosmannia clavigera glucosidase, Uniparc reference UPI0001FF1101, Uniprot reference F0XBR0. SEQ ID NO: 269: Amino acid sequence for Melampsora larici-populina glucosidase, Uniparc reference UPI00020F9774, Uniprot reference F4R4W2. SEQ ID NO: 270: Amino acid sequence for Prevotella multisaccharivorax glucosidase, Uniparc reference UPI0002138E9C, Uniprot reference F8N7G1.

[0060] SEQ ID NO: 271: Amino acid sequence for Streptomyces zinciresistens glucosidase, Uniparc reference UPI0002255A63, Uniprot reference G2G8K4. SEQ ID NO: 272: Amino acid sequence for Granulicella mallensis glucosidase, Uniparc reference UPI0001D9FC40, Uniprot reference G8NY42. SEQ ID NO: 273: Amino acid sequence for Gibberella zeae glucosidase, Uniparc reference UPI00021F1FEE, Uniprot reference I1RH94. SEQ ID NO: 274: Amino acid sequence for Modestobacter marinus glucosidase, Uniparc reference UPI00026090A2, Uniprot reference I4EYK6. SEQ ID NO: 275: Amino acid sequence for Lactobacillus equicursoris glucosidase, Uniparc reference UPI0002869F55, Uniprot reference K0NRS8. SEQ ID NO: 276: Amino acid sequence for Colletotrichum fructicola glucosidase, Uniparc reference UPI0002A93280, Uniprot reference L2F9W0. SEQ ID NO: 277: Amino acid sequence for Streptosporangium roseum glucosidase, Uniparc reference UPI0001BF8AF6, Uniprot reference D2B261. SEQ ID NO: 278: Amino acid sequence for Listeria grayi glucosidase, Uniparc reference UPI00019F252A, Uniprot reference D7UX19. SEQ ID NO: 279: Amino acid sequence for Enterococcus italicus glucosidase, Uniparc reference UPI0001F11EFF, Uniprot reference E6LF07. SEQ ID NO: 280: Amino acid sequence for Fluviicola taffensis glucosidase, Uniparc reference UPI000203D9EA, Uniprot reference F2IIT6.

[0061] SEQ ID NO: 281: Amino acid sequence for Shigella flexneri glucosidase, Uniparc reference UPI00020CA641, Uniprot reference F5N4W9. SEQ ID NO: 282: Amino acid sequence for Actinomyces sp. glucosidase, Uniparc reference UPI0002189080, Uniprot reference F9EFW5. SEQ ID NO: 283: Amino acid sequence for Verticillium dahliae glucosidase, Uniparc reference UPI00022EBE5A, Uniprot reference G2X5V6. SEQ ID NO: 284: Amino acid sequence for Actinoplanes sp. glucosidase, Uniparc reference UPI00023ED5A8, Uniprot reference G8S0M0. SEQ ID NO: 285: Amino acid sequence for Gibberella zeae glucosidase, Uniparc reference UPI000023EA29, Uniprot reference I1S320. SEQ ID NO: 286: Amino acid sequence for Auricularia subglabra glucosidase, Uniparc reference UPI00027CE685, Uniprot reference J0WV06. SEQ ID NO: 287: Amino acid sequence for Nitratireductor indicus glucosidase, Uniparc reference UPI00028E86D6, Uniprot reference K2N268. SEQ ID NO: 288: Amino acid sequence for Thermoclostridium stercorarium glucosidase, Uniparc reference UPI0002AD999E, Uniprot reference L7VH66. SEQ ID NO: 289: Amino acid sequence for Geobacillus sp. glucosidase, Uniparc reference UPI0002AF2DE3, Uniprot reference L8A1S2. SEQ ID NO: 290: Amino acid sequence for uncultured bacterial glucosidase, Uniparc reference UPI000327C3CC, Uniprot reference M9ZC55.

[0062] SEQ ID NO: 291: Amino acid sequence for Burkholderia ambifaria glucosidase, Uniparc reference UPI000059ACE1, Uniprot reference Q0BAK2. SEQ ID NO: 292: Amino acid sequence for Aspergillus oryzae glucosidase, Uniparc reference UPI0000676B8D, Uniprot reference Q2UIR4. SEQ ID NO: 293: Amino acid sequence for Yersinia pseudotuberculosis glucosidase, Uniparc reference UPI00004269B5, Uniprot reference Q66DJ0. SEQ ID NO: 294: Amino acid sequence for Wallemia ichthyophaga glucosidase, Uniparc reference UPI000331CC98, Uniprot reference R9AF64. SEQ ID NO: 295: Amino acid sequence for Glarea lozoyensis glucosidase, Uniparc reference UPI0003522404, Uniprot reference S3DIM6. SEQ ID NO: 296: Amino acid sequence for Moniliophthora roreri glucosidase, Uniparc reference UPI0003BF753A, Uniprot reference V2YU31. SEQ ID NO: 297: Amino acid sequence for Zhouia amylolytica glucosidase, Uniparc reference UPI0003DBCB4F, Uniprot reference W2ULL3. SEQ ID NO: 298: Amino acid sequence for Fusarium oxysporum glucosidase, Uniparc reference UPI0003F362FF, Uniprot reference W9NQQ3. SEQ ID NO: 299: Amino acid sequence for Flavobacterium johnsoniae glucosidase, Uniparc reference UPI00006EA168, Uniprot reference A5FAA5. SEQ ID NO: 300: Amino acid sequence for rice (Oryza sativa) glucosidase, Uniparc reference UPI00002394F5, Uniprot reference Q75I93.

[0063] SEQ ID NO: 301: Amino acid sequence for Clostridium saccharoperbutylacetonicum glucosidase, Uniparc reference UPI0002B65681, Uniprot reference M1MCD1. SEQ ID NO: 302: Amino acid sequence for Rhodococcus sp. glucosidase, Uniparc reference UPI0002D21DB1, Uniprot reference N1MBN6. SEQ ID NO: 303: Amino acid sequence for Burkholderia ambifaria glucosidase, Uniparc reference UPI000059C0CD, Uniprot reference Q0BCV8. SEQ ID NO: 304: Amino acid sequence for Xanthomonas campestris glucosidase, Uniparc reference UPI00005CE9E7, Uniprot reference Q3BVH7. SEQ ID NO: 305: Amino acid sequence for Caulobacter vibrioides glucosidase, Uniparc reference UPI00000C7604, Uniprot reference Q9A6F8. SEQ ID NO: 306: Amino acid sequence for Arcticibacter svalbardensis glucosidase, Uniparc reference UPI000338772A, Uniprot reference R9GRA7. SEQ ID NO: 307: Amino acid sequence for Winogradskyella psychrotolerans glucosidase, Uniparc reference UPI00035A4604, Uniprot reference S7VQ28. SEQ ID NO: 308: Amino acid sequence for Methyloglobulus morosus glucosidase, Uniparc reference UPI0003C4ED6A, Uniprot reference V5DXT8. SEQ ID NO: 309: Amino acid sequence for Pestalotiopsis fici glucosidase, Uniparc reference UPI0003E05C04, Uniprot reference W3WV37. SEQ ID NO: 310: Amino acid sequence for Capronia coronata glucosidase, Uniparc reference UPI000434E698, Uniprot reference W9YNR7.

[0064] SEQ ID NO: 311: Amino acid sequence for Aspergillus aculeatus glucosidase, Uniparc reference UPI00001268FD, Uniprot reference P48825. SEQ ID NO: 312: Amino acid sequence for Thermotoga neapolitana glucosidase, Uniparc reference UPI0000DD5996, Uniprot reference Q0GC07. SEQ ID NO: 313: Amino acid sequence for Clostridium saccharoperbutylacetonicum glucosidase, Uniparc reference UPI0002B666E8, Uniprot reference M1MJF4. SEQ ID NO: 314: Amino acid sequence for Schizosaccharomyces pombe glucosidase, Uniparc reference UPI000006B001, Uniprot reference O74799. SEQ ID NO: 315: Amino acid sequence for Phaeosphaeria nodorum glucosidase, Uniparc reference UPI000161BD2B, Uniprot reference Q0TXF6. SEQ ID NO: 316: Amino acid sequence for Xylella fastidiosa Dixon glucosidase, Uniparc reference UPI00003806C8, Uniprot reference Q3RGJ3. SEQ ID NO: 317: Amino acid sequence for Schizosaccharomyces pombe glucosidase, Uniparc reference UPI000006A330, Uniprot reference Q9P6J6. SEQ ID NO: 318: Amino acid sequence for Arcticibacter svalbardensis glucosidase, Uniparc reference UPI000337E887, Uniprot reference R9GWD6. SEQ ID NO: 319: Amino acid sequence for Colletotrichum gloeosporioides glucosidase, Uniparc reference UPI0003885717, Uniprot reference T0KJI7. SEQ ID NO: 320: Amino acid sequence for uncultured bacterial glucosidase, Uniparc reference UPI0003C9E340, Uniprot reference V5R1E8.

[0065] SEQ ID NO: 321: Amino acid sequence for Xanthomonas arboricola glucosidase, Uniparc reference UPI0003E06A61, Uniprot reference W4S7I5. SEQ ID NO: 322: Amino acid sequence for Fusarium oxysporum glucosidase, Uniparc reference UPI00021EC697, Uniprot reference X0A8X8. SEQ ID NO: 323: Amino acid sequence for Paenibacillus polymyxa glucosidase, Uniparc reference UPI00001108D0, Uniprot reference P22073. SEQ ID NO: 324: Amino acid sequence for Kluyveromyces marxianus glucosidase, Uniparc reference UPI0001BE5ADA, Uniprot reference D1GCC6. SEQ ID NO: 325: Amino acid sequence for Ilumatobacter coccineus glucosidase, Uniparc reference UPI0002C04A25, Uniprot reference M5A594. SEQ ID NO: 326: Amino acid sequence for Agrobacterium sp. glucosidase, Uniparc reference UPI0000126912, Uniprot reference P12614. SEQ ID NO: 327: Amino acid sequence for Cytophaga hutchinsonii glucosidase, Uniparc reference UPI000038ECF7, Uniprot reference Q11P53. SEQ ID NO: 328: Amino acid sequence for Thermobifida fusca glucosidase, Uniparc reference UPI00003C5CA4, Uniprot reference Q47PF5. SEQ ID NO: 329: Amino acid sequence for Botryotinia fuckeliana glucosidase, Uniparc reference UPI0000069E5E, Uniprot reference Q9UVJ6. SEQ ID NO: 330: Amino acid sequence for Agarivorans albus glucosidase, Uniparc reference UPI00033994D2, Uniprot reference R9PTK2.

[0066] SEQ ID NO: 331: Amino acid sequence for Enterococcus sp. glucosidase, Uniparc reference UPI00038B7639, Uniprot reference T0UDU2. SEQ ID NO: 332: Amino acid sequence for Salinispira pacifica glucosidase, Uniparc reference UPI0003D8A7DC, Uniprot reference V5WKT4. SEQ ID NO: 333: Amino acid sequence for Bacteroides xylanisolvens glucosidase, Uniparc reference UPI0003ECF15E, Uniprot reference W6P696. SEQ ID NO: 334: Amino acid sequence for Fusarium oxysporum glucosidase, Uniparc reference UPI000430029A, Uniprot reference X0AB46. SEQ ID NO: 335: Amino acid sequence for Thermotoga maritima glucosidase, Uniparc reference UPI0000126906, Uniprot reference Q08638B. SEQ ID NO: 336: Amino acid sequence for Neotermes koshunensis glucosidase, Uniparc reference UPI0000083EA8, Uniprot reference Q8T0W7. SEQ ID NO: 337: Amino acid sequence for Thanatephorus cucumeris glucosidase, Uniparc reference UPI0002BF34B3, Uniprot reference M5CHG9. SEQ ID NO: 338: Amino acid sequence for Hungateiclostridium thermocellum glucosidase, Uniparc reference UPI000053581D, Uniprot reference P14002. SEQ ID NO: 339: Amino acid sequence for Koribacter versatilis glucosidase, Uniparc reference UPI0000D76A70, Uniprot reference Q1IJ89. SEQ ID NO: 340: Amino acid sequence for Neosartorya fumigata glucosidase, Uniparc reference UPI00005203D8, Uniprot reference Q4WLX5.

[0067] SEQ ID NO: 341: Amino acid sequence for Amycolatopsis vancoresmycina glucosidase, Uniparc reference UPI00032DB8C1, Uniprot reference R1FKF0. SEQ ID NO: 342: Amino acid sequence for Gibberella fujikuroi glucosidase, Uniparc reference UPI0003519C76, Uniprot reference S0EKU1. SEQ ID NO: 343: Amino acid sequence for Bifidobacterium longum glucosidase, Uniparc reference UPI000390F839, Uniprot reference T2I2H5. SEQ ID NO: 344: Amino acid sequence for uncultured bacterial glucosidase, Uniparc reference UPI0003DFF0DA, Uniprot reference W0FLD1. SEQ ID NO: 345: Amino acid sequence for Bacteroides xylanisolvens glucosidase, Uniparc reference UPI0002D3D994, Uniprot reference W6P9J9. SEQ ID NO: 346: Amino acid sequence for Fusarium oxysporum glucosidase, Uniparc reference UPI00042F8016, Uniprot reference X0HA35. SEQ ID NO: 347: Amino acid sequence for Sorghum bicolor glucosidase, Uniparc reference UPI00000A7F40, Uniprot reference Q41290. SEQ ID NO: 348: Amino acid sequence for Clostridium cellulovorans glucosidase, Uniparc reference UPI000050B701, Uniprot reference Q53EH2. SEQ ID NO: 349: Amino acid sequence for Dacryopinax primogenitus glucosidase, Uniparc reference UPI0002C29EAA, Uniprot reference M5G1U5. SEQ ID NO: 350: Amino acid sequence for Rhizobium radiobacter glucosidase, Uniparc reference UPI0000126913, Uniprot reference P27034.

[0068] SEQ ID NO: 351: Amino acid sequence for Phanerochaete chrysosporium glucosidase, Uniparc reference UPI00006E0CC7, Uniprot reference Q25BW5A. SEQ ID NO: 352: Amino acid sequence for Enterobacter agglomerans glucosidase, Uniparc reference UPI0000126905, Uniprot reference Q59437. SEQ ID NO: 353: Amino acid sequence for Candidatus microthrix glucosidase, Uniparc reference UPI00033056C4, Uniprot reference R4YWU4. SEQ ID NO: 354: Amino acid sequence for Chthonomonas calidirosea glucosidase, Uniparc reference UPI0003427F29, Uniprot reference S0ET37. SEQ ID NO: 355: Amino acid sequence for Acholeplasma brassicae glucosidase, Uniparc reference UPI0003B04208, Uniprot reference U4KMR7. SEQ ID NO: 356: Amino acid sequence for Chania multitudinisentens glucosidase, Uniparc reference UPI0003E13BD3, Uniprot reference W0LJ28. SEQ ID NO: 357: Amino acid sequence for Gibberella moniliformis glucosidase, Uniparc reference UPI0003ECCF80, Uniprot reference W7MW98. SEQ ID NO: 358: Amino acid sequence for Fusarium oxysporum glucosidase, Uniparc reference UPI0004305856, Uniprot reference X0I2U8. SEQ ID NO: 359: Amino acid sequence for Phanerochaete chrysosporium glucosidase, Uniparc reference UPI00006E0CC7, Uniprot reference Q25BW5B. SEQ ID NO: 360: Amino acid sequence for rye (Secale cereale) glucosidase, Uniparc reference UPI00000A7EFC, Uniprot reference Q9FYS3.

[0069] SEQ ID NO: 361: Amino acid sequence for Anoxybacillus gonensis glucosidase, Uniparc reference UPI0002C0273E, Uniprot reference M5QUM2. SEQ ID NO: 362: Amino acid sequence for Thermotoga maritima glucosidase, Uniparc reference UPI0000126906, Uniprot reference Q08638A. SEQ ID NO: 363: Amino acid sequence for Rhodospirillum rubrum glucosidase, Uniparc reference UPI00003C2ACC, Uniprot reference Q2RP51. SEQ ID NO: 364: Amino acid sequence for Thermotoga neapolitana glucosidase, Uniparc reference UPI00000B9013, Uniprot reference Q60038. SEQ ID NO: 365: Amino acid sequence for Candidatus microthrix glucosidase, Uniparc reference UPI00032F466E, Uniprot reference R4Z6M6. SEQ ID NO: 366: Amino acid sequence for Ruminiclostridium cellobioparum glucosidase, Uniparc reference UPI00032874EC, Uniprot reference S0FPI8. SEQ ID NO: 367: Amino acid sequence for Acholeplasma brassicae glucosidase, Uniparc reference UPI0003B0490C, Uniprot reference U4KMV4. SEQ ID NO: 368: Amino acid sequence for Klebsiella pneumoniae glucosidase, Uniparc reference UPI0003DB6EFD, Uniprot reference W1BBP5. SEQ ID NO: 369: Amino acid sequence for Enterobacter sp. glucosidase, Uniparc reference UPI0003ED0A8E, Uniprot reference W7P6Y1. SEQ ID NO: 370: Amino acid sequence for Rhizoctonia solani glucosidase, Uniparc reference UPI00045BB507, Uniprot reference X8JI88.

[0070] SEQ ID NO: 371: Amino acid sequence for human (Homo sapiens) glucosidase, Uniparc reference UPI0000072C73, Uniprot reference Q9H227. SEQ ID NO: 372: Amino acid sequence for uncultured bacterial glucosidase, Uniparc reference UPI0000DD5304, Uniprot reference Q0GMU3. SEQ ID NO: 373: Amino acid sequence for Thermoanaerobacter thermohydrosulfuricus glucosidase, Uniparc reference UPI0002CA9E4F, Uniprot reference M8CQD9. SEQ ID NO: 374: Amino acid sequence for Stigmatella aurantiaca glucosidase, Uniparc reference UPI0000E297D8, Uniprot reference Q091M8. SEQ ID NO: 375: Amino acid sequence for Burkholderia thailandensis glucosidase, Uniparc reference UPI00006676B1, Uniprot reference Q2T7C9. SEQ ID NO: 376: Amino acid sequence for Yersinia pseudotuberculosis glucosidase, Uniparc reference UPI0000426E8F, Uniprot reference Q665S2. SEQ ID NO: 377: Amino acid sequence for Togninia minima glucosidase, Uniparc reference UPI00032BF71F, Uniprot reference R8BQM8. SEQ ID NO: 378: Amino acid sequence for Ophiostoma piceae glucosidase, Uniparc reference UPI00035213C2, Uniprot reference S3BVJ8. SEQ ID NO: 379: Amino acid sequence for Pyronema omphalodes glucosidase, Uniparc reference UPI0003B117B7, Uniprot reference U4LIL3. SEQ ID NO: 380: Amino acid sequence for Ogataea parapolymorpha glucosidase, Uniparc reference UPI0001F76D1F, Uniprot reference W1QIW0.

[0071] SEQ ID NO: 381: Amino acid sequence for Hymenobacter swuensis glucosidase, Uniparc reference UPI0003F2059F, Uniprot reference W8F6U0. SEQ ID NO: 382: Amino acid sequence for Flavobacterium johnsoniae glucosidase, Uniparc reference UPI00006E8E1B, Uniprot reference A5FEF5. SEQ ID NO: 383: Amino acid sequence for Paenibacillus polymyxa glucosidase, Uniparc reference UPI000012690B, Uniprot reference P22505. SEQ ID NO: 384: Amino acid sequence for rice (Oryza sativa) glucosidase, Uniparc reference UPI000009D014, Uniprot reference Q8L7J2. SEQ ID NO: 385: Amino acid sequence for rice (Oryza sativa) glucosidase, Uniparc reference UPI0000E580F2, Uniprot reference B8AVF0. SEQ ID NO: 386: Amino acid sequence for Nannochloris glucosidase, Uniparc reference UPI000EA7F16F, Uniprot reference A0A452CSM4. SEQ ID NO: 387: Amino acid sequence for Halothermothrix orenii glucosidase, Uniparc reference UPI00006AE508, Uniprot reference B8CYA8. SEQ ID NO: 388: Amino acid sequence for Neurospora crassa glucosidase, Uniparc reference UPI000018B2B4, Uniprot reference Q7RWP2. SEQ ID NO: 389: Amino acid sequence for Micrococcus antarcticus glucosidase, Uniparc reference UPI000192BB5F, Uniprot reference B9V8P5. SEQ ID NO: 390: Amino acid sequence for Exiguobacterium antarcticum glucosidase, Uniparc reference UPI000285E79E, Uniprot reference K0A8J9.

[0072] SEQ ID NO: 391: Amino acid sequence for Thermus thermophilus glucosidase, Uniparc reference UPI00000BEB61, Uniprot reference Q9RA61. SEQ ID NO: 392: Amino acid sequence for Trichoderma harzianum glucosidase, Uniparc reference UPI00078BF747, Uniprot reference A0A2T4AR08. SEQ ID NO: 393: Amino acid sequence for Hypocrea jecorina glucosidase, Uniparc reference UPI000006AA61, Uniprot reference Q12715. SEQ ID NO: 394: Amino acid sequence for Streptomyces sp. glucosidase, Uniparc reference UPI00000B411B, Uniprot reference Q59976. SEQ ID NO: 395: Amino acid sequence for Streptococcus pyogenes glucosidase, Uniparc reference UPI00000C7E56, Uniprot reference Q99YP9. SEQ ID NO: 396: Amino acid sequence for white clover (Trifolium repens) glucosidase, Uniparc reference UPI000012691B, Uniprot reference P26205. SEQ ID NO: 397: Amino acid sequence for Talaromyces emersonii glucosidase, Uniparc reference UPI000006C8FF, Uniprot reference Q8TGI8. SEQ ID NO: 398: Amino acid sequence for Hungateiclostridium thermocellum glucosidase, Uniparc reference UPI0000126903, Uniprot reference P26208. SEQ ID NO: 399: Amino acid sequence for Lactobacillus plantarum glucosidase, Uniparc reference UPI000219FE3E, Uniprot reference F9ULH8. SEQ ID NO: 400: Amino acid sequence for Agrobacterium tumefaciens glucosidase, Uniparc reference UPI0003F2033A, Uniprot reference A0A2I4PGZ0.

[0073] SEQ ID NO: 401: Codon-optimized DNA encoding 6xHis Cyberlindnera fabianii glucosidase, Uniparc reference UPI00049B1A8C, Uniprot reference A0A061B3J2. SEQ ID NO: 402: Codon-optimized DNA encoding 6xHis Flavobacterium gilvum glucosidase, Uniparc reference UPI0004E3EF7B, Uniprot reference A0A085EII0. SEQ ID NO: 403: Codon-optimized DNA encoding 6xHis Algibacter lectus glucosidase, Uniparc reference UPI00050EE490, Uniprot reference A0A090X649. SEQ ID NO: 404: Codon-optimized DNA encoding 6xHis Microbacterium azadirachtae glucosidase, Uniparc reference UPI0005ECB51E, Uniprot reference A0A0F0LB94. SEQ ID NO: 405: Codon-optimized DNA encoding a 6xHis Actinobacteria bacterial glucosidase, Uniparc reference UPI0006588DAD, Uniprot reference A0A0J0UT37. SEQ ID NO: 406: Codon-optimized DNA encoding a 6xHis Chloroflexi bacterial glucosidase, Uniparc reference UPI0007968552, Uniprot reference A0A136KWB3. SEQ ID NO: 407: 6xHis codon-optimized DNA encoding Komagataeibacter rhaeticus glucosidase, Uniparc reference UPI0002080410, Uniprot reference A0A181C809. SEQ ID NO: 408: Codon-optimized DNA encoding 6xHis Bacteroides sp. glucosidase, Uniparc reference UPI0008211BFC, Uniprot reference A0A1C5WEL8. SEQ ID NO: 409: Codon-optimized DNA encoding 6xHis Streptomyces rubrolavendulae glucosidase, Uniparc reference UPI00085A2BD0, Uniprot reference A0A1D8FZW3. SEQ ID NO: 410: Codon-optimized DNA encoding 6xHis Clostridium roseum glucosidase, Uniparc reference UPI00098C60F6, Uniprot reference A0A1S8KYM5.

[0074] SEQ ID NO: 411: Codon-optimized DNA encoding 6xHis uncultured bacterial glucosidase, Uniparc reference UPI0009CE0D4C, Uniprot reference A0A1V5M6V6. SEQ ID NO: 412: Codon-optimized DNA encoding a 6xHis Firmicutes bacterial glucosidase, Uniparc reference UPI0009D4127D, Uniprot reference A0A1V6AN95. SEQ ID NO: 413: Codon-optimized DNA encoding 6xHis Anthracocystis flocculosa glucosidase, Uniparc reference UPI00045601AB, Uniprot reference A0A061H1Z3. SEQ ID NO: 414: Codon-optimized DNA encoding 6xHis Bifidobacterium boum glucosidase, Uniparc reference UPI0004FF77C7, Uniprot reference A0A086ZKU2. SEQ ID NO: 415: Codon-optimized DNA encoding 6xHis Jejuia pallidilutea glucosidase, Uniparc reference UPI00051EDBDE, Uniprot reference A0A098LTR2. SEQ ID NO: 416: Codon-optimized DNA encoding 6xHis Ceratocystis fimbriata glucosidase, Uniparc reference UPI00062105AB, Uniprot reference A0A0F8B2B0. SEQ ID NO: 417: Codon-optimized DNA encoding a 6xHis Actinobacteria bacterial glucosidase, Uniparc reference UPI0006583AB1, Uniprot reference A0A0J0UVW7. SEQ ID NO: 418: Codon-optimized DNA encoding 6xHis Rhodococcus sp. glucosidase, Uniparc reference UPI0007AABFAD, Uniprot reference A0A143QAX3. SEQ ID NO: 419: Codon-optimized DNA encoding 6xHis Valsa mali glucosidase, Uniparc reference UPI0007F2D02D, Uniprot reference A0A194VF47. SEQ ID NO: 420: Codon-optimized DNA encoding 6xHis uncultured Bacteroides sp. glucosidase, Uniparc reference UPI000821004D, Uniprot reference A0A1C5WSI4.

[0075] SEQ ID NO: 421: Codon-optimized DNA encoding 6xHis Eisenbergiella tayi glucosidase, Uniparc reference UPI00084089B2, Uniprot reference A0A1E3ALT2. SEQ ID NO: 422: Codon-optimized DNA encoding 6xHis Streptomyces sp. glucosidase, Uniparc reference UPI000978E914, Uniprot reference A0A1V2MYI4. SEQ ID NO: 423: Codon-optimized DNA encoding a 6xHis Firmicutes bacterial glucosidase, Uniparc reference UPI0009CBF21C, Uniprot reference A0A1V5MH90. SEQ ID NO: 424: Codon-optimized DNA encoding a 6xHis Tenericutes bacterial glucosidase, Uniparc reference UPI0009D5B1F0, Uniprot reference A0A1V6BAK3. SEQ ID NO: 425: Codon-optimized DNA encoding 6xHis Gluconobacter oxydans glucosidase, Uniparc reference UPI0004A87350, Uniprot reference A0A067Z479. SEQ ID NO: 426: Codon-optimized DNA encoding 6xHis Bifidobacterium catenulatum glucosidase, Uniparc reference UPI00050787A2, Uniprot reference A0A087B8Q8. SEQ ID NO: 427: Codon-optimized DNA encoding 6xHis Bionectria ochroleuca glucosidase, Uniparc reference UPI0005965863, Uniprot reference A0A0B7K538. SEQ ID NO: 428: Codon-optimized DNA encoding 6xHis Parcubacteria sp. glucosidase, Uniparc reference UPI0006377CA4, Uniprot reference A0A0G0GD78. SEQ ID NO: 429: Codon-optimized DNA encoding 6xHis Microbacterium ketosireducens glucosidase, Uniparc reference UPI0006228575, Uniprot reference A0A0M2H276. SEQ ID NO: 430: Codon-optimized DNA encoding 6xHis Roseburia faecis glucosidase, Uniparc reference UPI0006C454B4, Uniprot reference A0A173R3W4.

[0076] SEQ ID NO: 431: Codon-optimized DNA encoding 6xHis Kwoniella dejecticola glucosidase, Uniparc reference UPI0007F1D695, Uniprot reference A0A1A6A050. SEQ ID NO: 432: Codon-optimized DNA encoding 6xHis uncultured Clostridium sp. glucosidase, Uniparc reference UPI0008232A70, Uniprot reference A0A1C6C862. SEQ ID NO: 433: Codon-optimized DNA encoding 6xHis Cyberlindnera jadinii glucosidase, Uniparc reference UPI000866CB30, Uniprot reference A0A1E4S2F8. SEQ ID NO: 434: Codon-optimized DNA encoding a 6xHis Bacteroidetes bacterial glucosidase, Uniparc reference UPI0009D3483D, Uniprot reference A0A1V5G4W6. SEQ ID NO: 435: Codon-optimized DNA encoding 6xHis Verrucomicrobia bacterial glucosidase, Uniparc reference UPI0009CC9AEA, Uniprot reference A0A1V5Q4R4. SEQ ID NO: 436: Codon-optimized DNA encoding a 6xHis Bacteroidetes bacterial glucosidase, Uniparc reference UPI0009C6DE73, Uniprot reference A0A1V6BV25. SEQ ID NO: 437: Codon-optimized DNA encoding 6xHis Lichtheimia ramosa glucosidase, Uniparc reference UPI0004E051A9, Uniprot reference A0A077WUK7. SEQ ID NO: 438: Codon-optimized DNA encoding 6xHis Bifidobacterium mongoliense glucosidase, Uniparc reference UPI0005060F52, Uniprot reference A0A087BWT7. SEQ ID NO: 439: Codon-optimized DNA encoding 6xHis Vibrio ishigakensis glucosidase, Uniparc reference UPI000591CED9, Uniprot reference A0A0B8NZY1. SEQ ID NO: 440: Codon-optimized DNA encoding 6xHis Phaeomoniella chlamydospora glucosidase, Uniparc reference UPI00063B706F, Uniprot reference A0A0G2HEV5.

[0077] SEQ ID NO: 441: Codon-optimized DNA encoding 6xHis Ardenticatena maritima glucosidase, Uniparc reference UPI0006C04F59, Uniprot reference A0A0M8K5H7. SEQ ID NO: 442: Codon-optimized DNA encoding 6xHis Coprococcus cometh glucosidase, Uniparc reference UPI000197E031, Uniprot reference A0A173WPC4. SEQ ID NO: 443: Codon-optimized DNA encoding 6xHis Nocardioides dokdonensis glucosidase, Uniparc reference UPI0007DDBAB3, Uniprot reference A0A1A9GNJ0. SEQ ID NO: 444: Codon-optimized DNA encoding 6xHis uncultured Clostridium sp. glucosidase, Uniparc reference UPI000822F7EB, Uniprot reference A0A1C6EBC1. SEQ ID NO: 445: Codon-optimized DNA encoding 6xHis Acetobacterium wieringae glucosidase, Uniparc reference UPI000878EB40, Uniprot reference A0A1F2PFB4. SEQ ID NO: 446: Codon-optimized DNA encoding a 6xHis Tenericutes bacterial glucosidase, Uniparc reference UPI0009D0A2C1, Uniprot reference A0A1V5HNJ3. SEQ ID NO: 447: Codon-optimized DNA encoding a 6xHis Tenericutes bacterial glucosidase, Uniparc reference UPI0009CB8A73, Uniprot reference A0A1V5UFI1. SEQ ID NO: 448: Codon-optimized DNA encoding 6xHis uncultured bacterial glucosidase, Uniparc reference UPI0009CC90AF, Uniprot reference A0A1V6CDT2. SEQ ID NO: 449: Codon-optimized DNA encoding 6xHis Parabacteroides distasonis glucosidase, Uniparc reference UPI0004D8E473, Uniprot reference A0A078SYD0. SEQ ID NO: 450: Codon-optimized DNA encoding 6xHis Bifidobacterium psychraerophilum glucosidase, Uniparc reference UPI0005006C6A, Uniprot reference A0A087CJB0.

[0078] SEQ ID NO: 451: Codon-optimized DNA encoding 6xHis Hebeloma cylindrosporum glucosidase, Uniparc reference UPI00059A3BA8, Uniprot reference A0A0C2YJF2. SEQ ID NO: 452: Codon-optimized DNA encoding 6xHis Brenneria goodwinii glucosidase, Uniparc reference UPI0006579FEA, Uniprot reference A0A0G4JRR8. SEQ ID NO: 453: Codon-optimized DNA encoding 6xHis Aspergillus calidoustus glucosidase, Uniparc reference UPI00073C9E60, Uniprot reference A0A0U5GN20. SEQ ID NO: 454: Codon-optimized DNA encoding 6xHis Bacteroides finegoldii glucosidase, Uniparc reference UPI0006C6E0C3, Uniprot reference A0A174BEZ2. SEQ ID NO: 455: Codon-optimized DNA encoding 6xHis Altererythrobacter dongtanensis glucosidase, Uniparc reference UPI0008153D6D, Uniprot reference A0A1B2A943. SEQ ID NO: 456: Codon-optimized DNA encoding 6xHis uncultured Anaerotruncus sp. glucosidase, Uniparc reference UPI0008206E7B, Uniprot reference A0A1C6FWD9. SEQ ID NO: 457: Codon-optimized DNA encoding 6xHis Candidatus firestone glucosidase, Uniparc reference UPI0008AC0E17, Uniprot reference A0A1F5UJR6. SEQ ID NO: 458: Codon-optimized DNA encoding a 6xHis Tenericutes bacterial glucosidase, Uniparc reference UPI0009C6C8E4, Uniprot reference A0A1V5HS22. SEQ ID NO: 459: Codon-optimized DNA encoding 6xHis Lentisphaerae bacterial glucosidase, Uniparc reference UPI0009CEE1AB, Uniprot reference A0A1V5VBL2. SEQ ID NO: 460: Codon-optimized DNA encoding a 6xHis Planctomycetes bacterial glucosidase, Uniparc reference UPI0009CAAB0A, Uniprot reference A0A1V6FZ47.

[0079] SEQ ID NO: 461: Codon-optimized DNA encoding 6xHis Pseudallescheria apiosperma glucosidase, Uniparc reference UPI0004DD62AC, Uniprot reference A0A084G332. SEQ ID NO: 462: Codon-optimized DNA encoding 6xHis Nonlabens sediminis glucosidase, Uniparc reference UPI000507F00A, Uniprot reference A0A090Q4N8. SEQ ID NO: 463: Codon-optimized DNA encoding 6xHis Gynuella sunshinyii glucosidase, Uniparc reference UPI0005CC42CA, Uniprot reference A0A0C5VDU3. SEQ ID NO: 464: Codon-optimized DNA encoding 6xHis Verticillium longisporum glucosidase, Uniparc reference UPI00063E4005, Uniprot reference A0A0G4N9Q7. SEQ ID NO: 465: Codon-optimized DNA encoding 6xHis Cellulomonas sp. glucosidase, Uniparc reference UPI00073C6CD3, Uniprot reference A0A0V8TAB5. SEQ ID NO: 466: Codon-optimized DNA encoding 6xHis Hungatella hathewayi glucosidase, Uniparc reference UPI0006C024BB, Uniprot reference A0A174FBX7. SEQ ID NO: 467: Codon-optimized DNA encoding 6xHis Mesorhizobium sp. glucosidase, Uniparc reference UPI000688E74D, Uniprot reference A0A1C2DG64. SEQ ID NO: 468: Codon-optimized DNA encoding 6xHis Clostridium sp. glucosidase, Uniparc reference UPI000822FAB7, Uniprot reference A0A1C6GRT5. SEQ ID NO: 469: Codon-optimized DNA encoding 6xHis Chlamydiales bacterial glucosidase, Uniparc reference UPI0009284E74, Uniprot reference A0A1M3CSY6. SEQ ID NO: 470: Codon-optimized DNA encoding a 6xHis Spirochaetes bacterial glucosidase, Uniparc reference UPI0009CB461D, Uniprot reference A0A1V5HUX3.

[0080] SEQ ID NO: 471: Codon-optimized DNA encoding 6xHis bacterial glucosidase, Uniparc reference UPI0009C92D6A, Uniprot reference A0A1V5VHB9. SEQ ID NO: 472: Codon-optimized DNA encoding a 6xHis Thermotogae bacterial glucosidase, Uniparc reference UPI0009D12B86, Uniprot reference A0A1V6H2W5. SEQ ID NO: 473: Codon-optimized DNA encoding 6xHis Pseudallescheria apiosperma glucosidase, Uniparc reference UPI0004DCBED3, Uniprot reference A0A084GGE2. SEQ ID NO: 474: Codon-optimized DNA encoding 6xHis Algibacter lectus glucosidase, Uniparc reference UPI00050E0BE3, Uniprot reference A0A090VF17. SEQ ID NO: 475: Codon-optimized DNA encoding 6xHis Paxillus involutus glucosidase, Uniparc reference UPI0005B075C8, Uniprot reference A0A0C9TWP5. SEQ ID NO: 476: Codon-optimized DNA encoding 6xHis Verticillium longisporum glucosidase, Uniparc reference UPI00063DF006, Uniprot reference A0A0G4NA55. SEQ ID NO: 477: Codon-optimized DNA encoding 6xHis Mucilaginibacter gotjawali glucosidase, Uniparc reference UPI00076F8EA4, Uniprot reference A0A110B1H1. SEQ ID NO: 478: Codon-optimized DNA encoding 6xHis Bacteroides uniformis glucosidase, Uniparc reference UPI0006C0FFEF, Uniprot reference A0A174IWW4. SEQ ID NO: 479: Codon-optimized DNA encoding 6xHis Coprococcus sp. glucosidase, Uniparc reference UPI0008222B77, Uniprot reference A0A1C5W0N6. SEQ ID NO: 480: Codon-optimized DNA encoding 6xHis Blautia sp. glucosidase, Uniparc reference UPI0006C36823, Uniprot reference A0A1C6K2X5.

[0081] SEQ ID NO: 481: Codon-optimized DNA encoding 6xHis Cellulomonas sp. glucosidase, Uniparc reference UPI00092B3CD4, Uniprot reference A0A1M3ELH4. SEQ ID NO: 482: Codon-optimized DNA encoding 6xHis bacterial glucosidase, Uniparc reference UPI0009C85428, Uniprot reference A0A1V5J984. SEQ ID NO: 483: Codon-optimized DNA encoding a 6xHis Spirochaetes bacterial glucosidase, Uniparc reference UPI0009CA1D5C, Uniprot reference A0A1V5WHQ7. SEQ ID NO: 484: Codon-optimized DNA encoding a 6xHis Tenericutes bacterial glucosidase, Uniparc reference UPI0009C7041F, Uniprot reference A0A1V6IJ57. SEQ ID NO: 485: Codon-optimized DNA encoding 6xHis Flavobacterium gilvum glucosidase, Uniparc reference UPI0004E2A41F, Uniprot reference A0A085EG29. SEQ ID NO: 486: Codon-optimized DNA encoding 6xHis Algibacter lectus glucosidase, Uniparc reference UPI0005102470, Uniprot reference A0A090WWZ1. SEQ ID NO: 487: Codon-optimized DNA encoding 6xHis Hydnomerulius pinastri glucosidase, Uniparc reference UPI0005B0F0AE, Uniprot reference A0A0C9WDY0. SEQ ID NO: 488: Codon-optimized DNA encoding 6xHis Nocardia farcinica glucosidase, Uniparc reference UPI00065C2666, Uniprot reference A0A0H5NWN2. SEQ ID NO: 489: Codon-optimized DNA encoding 6xHis Bacteroides cellulosilyticus glucosidase, Uniparc reference UPI000760375B, Uniprot reference A0A125MG18. SEQ ID NO: 490: Codon-optimized DNA encoding 6xHis Fonsecaea erecta glucosidase, Uniparc reference UPI0007DF4250, Uniprot reference A0A178ZBN2.

[0082] SEQ ID NO: 491: Codon-optimized DNA encoding 6xHis Bacteroides sp. glucosidase, Uniparc reference UPI0008209852, Uniprot reference A0A1C5W9N9. SEQ ID NO: 492: Codon-optimized DNA encoding 6xHis Tannerella forsythia glucosidase, Uniparc reference UPI00086C34C1, Uniprot reference A0A1D3UGH8. SEQ ID NO: 493: Codon-optimized DNA encoding 6xHis Microbacterium esteraromaticum glucosidase, Uniparc reference UPI00097E83BB, Uniprot reference A0A1R4KI92. SEQ ID NO: 494: Codon-optimized DNA encoding 6xHis bacterial glucosidase, Uniparc reference UPI0009D255E5, Uniprot reference A0A1V5LJK9. SEQ ID NO: 495: Codon-optimized DNA encoding 6xHis Candidatus hydrogenedentes glucosidase, Uniparc reference UPI0009C5A3CF, Uniprot reference A0A1V5Z2L2. SEQ ID NO: 496: Codon-optimized DNA encoding a 6xHis Bacteroidetes bacterial glucosidase, Uniparc reference UPI0009C55799, Uniprot reference A0A1V6J4J8. SEQ ID NO: 497: Codon-optimized DNA encoding 6xHis Penicillium solitum glucosidase, Uniparc reference UPI0009D4067F, Uniprot reference A0A1V6RQ41. SEQ ID NO: 498: Codon-optimized DNA encoding 6xHis Weissella soli glucosidase, Uniparc reference UPI0008737AA2, Uniprot reference A0A288Q8I2. SEQ ID NO: 499: Codon-optimized DNA encoding 6xHis Acetatifactor muris glucosidase, Uniparc reference UPI000CAC57D4, Uniprot reference A0A2K4ZN91. SEQ ID NO: 500: Codon-optimized DNA encoding 6xHis Corynespora cassiicola glucosidase, Uniparc reference UPI000D237A4A, Uniprot reference A0A2T2NYD4.

[0083] SEQ ID NO: 501: Codon-optimized DNA encoding 6xHis Meira miltonrushii glucosidase, Uniparc reference UPI000D77C91D, Uniprot reference A0A316V6M3. SEQ ID NO: 502: Codon-optimized DNA encoding 6xHis Bacteroides fragilis glucosidase, Uniparc reference UPI00004E1F76, Uniprot reference A0A380YVC7. SEQ ID NO: 503: Codon-optimized DNA encoding 6xHis Malassezia restricta glucosidase, Uniparc reference UPI000F0C30E8, Uniprot reference A0A3G2SB79. SEQ ID NO: 504: 6×His codon-optimized DNA encoding Fusarium euwallaceae glucosidase, Uniparc reference UPI000FFFEFB2, Uniprot reference A0A430LYA2. SEQ ID NO: 505: Codon-optimized DNA encoding 6xHis Psathyrella aberdarensis glucosidase, Uniparc reference UPI0010251887, Uniprot reference A0A4Q2E070. SEQ ID NO: 506: Codon-optimized DNA encoding 6×His Aeromonas hydrophila glucosidase, Uniparc reference UPI0000E69509, Uniprot reference A0KLP6. SEQ ID NO: 507: Codon-optimized DNA encoding 6xHis Saccharopolyspora erythraea glucosidase, Uniparc reference UPI00000B86CB, Uniprot reference A4F7P9. SEQ ID NO: 508: Codon-optimized DNA encoding 6xHis Streptomyces sviceus glucosidase, Uniparc reference UPI000180240E, Uniprot reference B5I181. SEQ ID NO: 509: Codon-optimized DNA encoding 6xHis Naematelia encephala glucosidase, Uniparc reference UPI000A250F78, Uniprot reference A0A1Y2AWB7. SEQ ID NO: 510: Codon-optimized DNA encoding 6xHis Hartmannibacter diazotrophicus glucosidase, Uniparc reference UPI000C0221F1, Uniprot reference A0A2C9D6I2.

[0084] SEQ ID NO: 511: Codon-optimized DNA encoding 6xHis Pontimonas salivibrio glucosidase, Uniparc reference UPI000CEB5AB1, Uniprot reference A0A2L2BPE2. SEQ ID NO: 512: Codon-optimized DNA encoding 6xHis Cadophora sp. glucosidase, Uniparc reference UPI000D5B9C38, Uniprot reference A0A2V1CH24. SEQ ID NO: 513: Codon-optimized DNA encoding 6xHis Meira miltonrushii glucosidase, Uniparc reference UPI000D779558, Uniprot reference A0A316V8S0. SEQ ID NO: 514: Codon-optimized DNA encoding 6xHis Monilinia fructigena glucosidase, Uniparc reference UPI000DC42E3D, Uniprot reference A0A395IJW4. SEQ ID NO: 515: Codon-optimized DNA encoding 6xHis Hortaea werneckii glucosidase, Uniparc reference UPI000F3E476C, Uniprot reference A0A3M6XGS0. SEQ ID NO: 516: Codon-optimized DNA encoding 6xHis Streptomyces netropsis glucosidase, Uniparc reference UPI00101460D7, Uniprot reference A0A445N7U0. SEQ ID NO: 517: Codon-optimized DNA encoding 6xHis Aureobasidium pullulans glucosidase, Uniparc reference UPI001139C6A8, Uniprot reference A0A4S9IFI0. SEQ ID NO: 518: Codon-optimized DNA encoding 6xHis Aspergillus clavatus glucosidase, Uniparc reference UPI0000EA5CFF, Uniprot reference A1CTN9. SEQ ID NO: 519: Codon-optimized DNA encoding 6xHis Clavibacter michiganensis glucosidase, Uniparc reference UPI0001523037, Uniprot reference A5CT94. SEQ ID NO: 520: Codon-optimized DNA encoding 6xHis Penicillium rubens glucosidase, Uniparc reference UPI0001831CF5, Uniprot reference B6H7R5.

[0085] SEQ ID NO: 521: Codon-optimized DNA encoding 6xHis Lachnoclostridium sp. glucosidase, Uniparc reference UPI000B365547, Uniprot reference A0A1Y4NTL9. SEQ ID NO: 522: Codon-optimized DNA encoding 6xHis Rhodobacteraceae bacterial glucosidase, Uniparc reference UPI000C09BF88, Uniprot reference A0A2D5IXB9. SEQ ID NO: 523: Codon-optimized DNA encoding 6xHis Bacteroides fragilis glucosidase, Uniparc reference UPI0004B5EEF2, Uniprot reference A0A2M9UUC4. SEQ ID NO: 524: Codon-optimized DNA encoding 6xHis Aspergillus indologenus glucosidase, Uniparc reference UPI000D7FE1ED, Uniprot reference A0A2V5IY78. SEQ ID NO: 525: Codon-optimized DNA encoding 6xHis Acaromyces ingoldii glucosidase, Uniparc reference UPI000D802B25, Uniprot reference A0A316YR39. SEQ ID NO: 526: Codon-optimized DNA encoding 6xHis Monilinia fructigena glucosidase, Uniparc reference UPI000DC60823, Uniprot reference A0A395J1U5. SEQ ID NO: 527: Codon-optimized DNA encoding 6xHis Paenibacillus xylanexedens glucosidase, Uniparc reference UPI000F52D773, Uniprot reference A0A3N6CA02. SEQ ID NO: 528: Codon-optimized DNA encoding 6xHis Actinomyces howellii glucosidase, Uniparc reference UPI000F6DAAAE, Uniprot reference A0A448HIG0. SEQ ID NO: 529: Codon-optimized DNA encoding 6xHis Friedmanniomyces endolithicus glucosidase, Uniparc reference UPI00113D19DF, Uniprot reference A0A4V5N9I4. SEQ ID NO: 530: Codon-optimized DNA encoding 6xHis Neosartorya fischeri glucosidase, Uniparc reference UPI0000EA8672, Uniprot reference A1DNS0.

[0086] SEQ ID NO: 531: Codon-optimized DNA encoding 6xHis Pseudomonas aeruginosa glucosidase, Uniparc reference UPI0000D7314B, Uniprot reference A6V4K6. SEQ ID NO: 532: Codon-optimized DNA encoding 6xHis Talaromyces stipitatus glucosidase, Uniparc reference UPI00018E7266, Uniprot reference B8MF24. SEQ ID NO: 533: Codon-optimized DNA encoding 6xHis Aquimixticola soesokkakensis glucosidase, Uniparc reference UPI000A1A5FD7, Uniprot reference A0A1Y5RVF8. SEQ ID NO: 534: Codon-optimized DNA encoding 6xHis Rhodobacterales bacterial glucosidase, Uniparc reference UPI000C98D37C, Uniprot reference A0A2D9YGV1. SEQ ID NO: 535: Codon-optimized DNA encoding 6xHis Methylorubrum extorquens glucosidase, Uniparc reference UPI0006F9793E, Uniprot reference A0A2N9AS40. SEQ ID NO: 536: Codon-optimized DNA encoding 6xHis Clostridium perfringens glucosidase, Uniparc reference UPI00000CF7C8, Uniprot reference A0A2X2YBP0. SEQ ID NO: 537: Codon-optimized DNA encoding 6xHis Acholeplasmatales bacterial glucosidase, Uniparc reference UPI0008AEBEA3, Uniprot reference A0A348NID6. SEQ ID NO: 538: Codon-optimized DNA encoding a 6xHis Bacteroidetes bacterial glucosidase, Uniparc reference UPI000EC3C979, Uniprot reference A0A3B8VHE8. SEQ ID NO: 539: Codon-optimized DNA encoding 6xHis Clostridium carnis glucosidase, Uniparc reference UPI000F637E38, Uniprot reference A0A3P6K8E8. SEQ ID NO: 540: Codon-optimized DNA encoding 6xHis Mycolicibacterium flavescens glucosidase, Uniparc reference UPI000B93B5C9, Uniprot reference A0A448HNB5.

[0087] SEQ ID NO: 541: Codon-optimized DNA encoding 6xHis Streptococcus gallolyticus glucosidase, Uniparc reference UPI000F6EFA71, Uniprot reference A0A4V6LJ94. SEQ ID NO: 542: Codon-optimized DNA encoding 6xHis Yersinia enterocolitica glucosidase, Uniparc reference UPI0000EB54CC, Uniprot reference A1JNB7. SEQ ID NO: 543: Codon-optimized DNA encoding 6xHis Anaeromyxobacter sp. glucosidase, Uniparc reference UPI0000ED8A80, Uniprot reference A7HFG4. SEQ ID NO: 544: Codon-optimized DNA encoding 6xHis Talaromyces stipitatus glucosidase, Uniparc reference UPI00018E7D70, Uniprot reference B8MK55. SEQ ID NO: 545: Codon-optimized DNA encoding 6xHis Hortaea werneckii glucosidase, Uniparc reference UPI000A2E3FAA, Uniprot reference A0A1Z5SL14. SEQ ID NO: 546: Codon-optimized DNA encoding 6xHis Micavibrio sp. glucosidase, Uniparc reference UPI000C529025, Uniprot reference A0A2E2Q8X2. SEQ ID NO: 547: Codon-optimized DNA encoding a 6xHis Acidobacteriia bacterial glucosidase, Uniparc reference UPI000CE6B996, Uniprot reference A0A2N9MBS0. SEQ ID NO: 548: Codon-optimized DNA encoding 6xHis Corynebacterium jeikeium glucosidase, Uniparc reference UPI000DA3A972, Uniprot reference A0A2X4T570. SEQ ID NO: 549: Codon-optimized DNA encoding 6xHis Clostridiaceae bacterial glucosidase, Uniparc reference UPI000E8D37A1, Uniprot reference A0A353PZH8. SEQ ID NO: 550: Codon-optimized DNA encoding 6xHis Anaerolineaceae bacterial glucosidase, Uniparc reference UPI000748C096, Uniprot reference A0A3B9PA35.

[0088] SEQ ID NO: 551: Codon-optimized DNA encoding 6xHis Gymnopilus dilepis glucosidase, Uniparc reference UPI000FF41956, Uniprot reference A0A409WSY0. SEQ ID NO: 552: Codon-optimized DNA encoding 6xHis Kocuria rosea glucosidase, Uniparc reference UPI000F7105D4, Uniprot reference A0A448R8N0. SEQ ID NO: 553: Codon-optimized DNA encoding 6xHis Teredinibacter sp. glucosidase, Uniparc reference UPI0011696FAB, Uniprot reference A0A509DWZ3. SEQ ID NO: 554: Codon-optimized DNA encoding 6xHis Aspergillus niger glucosidase, Uniparc reference UPI0000EFB564, Uniprot reference A2QS42. SEQ ID NO: 555: Codon-optimized DNA encoding 6xHis Laccaria bicolor glucosidase, Uniparc reference UPI000164423D, Uniprot reference B0D734. SEQ ID NO: 556: Codon-optimized DNA encoding 6xHis Pedosphaera parvula glucosidase, Uniparc reference UPI00017357F6, Uniprot reference B9XH33. SEQ ID NO: 557: Codon-optimized DNA encoding 6xHis Megamonas hypermegale glucosidase, Uniparc reference UPI00042469F6, Uniprot reference A0A239TGH2. SEQ ID NO: 558: Codon-optimized DNA encoding 6xHis Armillaria gallica glucosidase, Uniparc reference UPI000BC209C3, Uniprot reference A0A2H3E300. SEQ ID NO: 559: Codon-optimized DNA encoding 6xHis Micromonospora sp. glucosidase, Uniparc reference UPI000D2EAE87, Uniprot reference A0A2P8AV03. SEQ ID NO: 560: Codon-optimized DNA encoding 6xHis Klebsiella oxytoca glucosidase, Uniparc reference UPI000DA286EE, Uniprot reference A0A2X5CJC5.

[0089] SEQ ID NO: 561: Codon-optimized DNA encoding 6xHis Candidatus ozemobacter glucosidase, Uniparc reference UPI000DFAEF6C, Uniprot reference A0A367ZIH8. SEQ ID NO: 562: Codon-optimized DNA encoding 6xHis Coleophoma crateriformis glucosidase, Uniparc reference UPI000E399EE8, Uniprot reference A0A3D8R2C2. SEQ ID NO: 563: Codon-optimized DNA encoding 6xHis Apiotrichum porosum glucosidase, Uniparc reference UPI000FBC01E3, Uniprot reference A0A427XHS2. SEQ ID NO: 564: Codon-optimized DNA encoding 6xHis Acholeplasma hippikon glucosidase, Uniparc reference UPI00068E4E50, Uniprot reference A0A449BJ27. SEQ ID NO: 565: Codon-optimized DNA encoding 6xHis Streptomyces spectabilis glucosidase, Uniparc reference UPI001185F074, Uniprot reference A0A516RGT1. SEQ ID NO: 566: Codon-optimized DNA encoding 6xHis Aspergillus niger glucosidase, Uniparc reference UPI0000EFCED2, Uniprot reference A2R8G2. SEQ ID NO: 567: Codon-optimized DNA encoding 6xHis Xanthomonas campestris glucosidase, Uniparc reference UPI00000D8BFA, Uniprot reference B0RYA0. SEQ ID NO: 568: Codon-optimized DNA encoding 6xHis Lactobacillus paracasei glucosidase, Uniparc reference UPI00019C9CD7, Uniprot reference C2FDL2. SEQ ID NO: 569: Codon-optimized DNA encoding 6xHis Bifiguratus adelaidae glucosidase, Uniparc reference UPI000BC490A3, Uniprot reference A0A261XUH4. SEQ ID NO: 570: Codon-optimized DNA encoding 6xHis bacterial glucosidase, Uniparc reference UPI000CC31AE2, Uniprot reference A0A2H5YYA1.

[0090] SEQ ID NO: 571: Codon-optimized DNA encoding 6xHis Actinomadura parvosata glucosidase, Uniparc reference UPI000D26C9C6, Uniprot reference A0A2P9IY35. SEQ ID NO: 572: Codon-optimized DNA encoding 6xHis Melissococcus plutonius glucosidase, Uniparc reference UPI00024F22B8, Uniprot reference A0A2Z5Y4P3. SEQ ID NO: 573: Codon-optimized DNA encoding 6xHis Enterococcus durans glucosidase, Uniparc reference UPI000E020871, Uniprot reference A0A377KJS3. SEQ ID NO: 574: Codon-optimized DNA encoding 6xHis Malassezia restricta glucosidase, Uniparc reference UPI000DD17A7D, Uniprot reference A0A3G2S2J6. SEQ ID NO: 575: Codon-optimized DNA encoding 6xHis Apiotrichum porosum glucosidase, Uniparc reference UPI000FA2DE87, Uniprot reference A0A427XZQ0. SEQ ID NO: 576: Codon-optimized DNA encoding 6xHis Streptomonospora sp. glucosidase, Uniparc reference UPI0010355193, Uniprot reference A0A4P6PWP5. SEQ ID NO: 577: Codon-optimized DNA encoding 6xHis Lactobacillus gasseri glucosidase, Uniparc reference UPI00119640A1, Uniprot reference A0A558LH47. SEQ ID NO: 578: Codon-optimized DNA encoding 6xHis Aspergillus niger glucosidase, Uniparc reference UPI0000EFD0AA, Uniprot reference A2RAJ1. SEQ ID NO: 579: Codon-optimized DNA encoding 6xHis Neosartorya fumigata glucosidase, Uniparc reference UPI000170BF91, Uniprot reference B0XXG1. SEQ ID NO: 580: Codon-optimized DNA encoding 6xHis Eubacterium eligens glucosidase, Uniparc reference UPI0001A5B234, Uniprot reference C4Z6T5.

[0091] SEQ ID NO: 581: Codon-optimized DNA encoding 6xHis Bifiguratus adelaidae glucosidase, Uniparc reference UPI000BC64A75, Uniprot reference A0A261XVM8. SEQ ID NO: 582: Codon-optimized DNA encoding 6xHis bacterial glucosidase, Uniparc reference UPI000CAA854E, Uniprot reference A0A2H6EX57. SEQ ID NO: 583: Codon-optimized DNA encoding 6xHis Corynespora cassiicola glucosidase, Uniparc reference UPI000D24EC2C, Uniprot reference A0A2T2N5H9. SEQ ID NO: 584: Codon-optimized DNA encoding 6xHis Pseudomicrostroma glucosiphilum glucosidase, Uniparc reference UPI000D77A0B7, Uniprot reference A0A316UK83. SEQ ID NO: 585: Codon-optimized DNA encoding 6xHis Staphylococcus saprophyticus glucosidase, Uniparc reference UPI0002DAAFCC, Uniprot reference A0A380HD31. SEQ ID NO: 586: Codon-optimized DNA encoding 6xHis Malassezia restricta glucosidase, Uniparc reference UPI000F0C6B8E, Uniprot reference A0A3G2S932. SEQ ID NO: 587: Codon-optimized DNA encoding 6xHis Saitozyma podzolica glucosidase, Uniparc reference UPI000FBA70E2, Uniprot reference A0A427YCL1. SEQ ID NO: 588: Codon-optimized DNA encoding 6xHis Tremella mesenterica glucosidase, Uniparc reference UPI00102822B4, Uniprot reference A0A4Q1BRC6. SEQ ID NO: 589: Codon-optimized DNA encoding 6xHis Arthrobacter sp. glucosidase, Uniparc reference UPI0000527506, Uniprot reference A0JZ86. SEQ ID NO: 590: Codon-optimized DNA encoding 6xHis Scheffersomyces stipitis glucosidase, Uniparc reference UPI000157388C, Uniprot reference A3LRB0.

[0092] SEQ ID NO: 591: Codon-optimized DNA encoding 6xHis Leptothrix cholodnii glucosidase, Uniparc reference UPI0001712E02, Uniprot reference B1XZK8. SEQ ID NO: 592: Codon-optimized DNA encoding 6xHis Thauera sp. glucosidase, Uniparc reference UPI000166883C, Uniprot reference C4ZLL7. SEQ ID NO: 593: Codon-optimized DNA encoding 6xHis Kosmotoga olearia glucosidase, Uniparc reference UPI00018494AB, Uniprot reference C5CDW5. SEQ ID NO: 594: Codon-optimized DNA encoding 6xHis Roseburia intestinalis glucosidase, Uniparc reference UPI0001CD671F, Uniprot reference D4L3Y2. SEQ ID NO: 595: Codon-optimized DNA encoding 6xHis Streptococcus equinus glucosidase, Uniparc reference UPI0001E0DC00, Uniprot reference E0PDF8. SEQ ID NO: 596: Codon-optimized DNA encoding 6xHis Streptococcus cristatus glucosidase, Uniparc reference UPI0001F80C4B, Uniprot reference E8JUK5. SEQ ID NO: 597: Codon-optimized DNA encoding 6xHis Cellulosilyticum lentocellum glucosidase, Uniparc reference UPI0001D2DBBC, Uniprot reference F2JLH3. SEQ ID NO: 598: Codon-optimized DNA encoding 6xHis Streptococcus gallolyticus glucosidase, Uniparc reference UPI0001C48657, Uniprot reference F5WYI5. SEQ ID NO: 599: Codon-optimized DNA encoding 6xHis Ketogulonicigenium vulgare glucosidase, Uniparc reference UPI00021D443A, Uniprot reference F9Y8Z7. SEQ ID NO: 600: Codon-optimized DNA encoding 6xHis Spathaspora passalidarum glucosidase, Uniparc reference UPI000228255D, Uniprot reference G3AGX1.

[0093] SEQ ID NO: 601: 6×His codon-optimized DNA encoding Niastella koreensis glucosidase, Uniparc reference UPI00023F6F5F, Uniprot reference G8T9J3. SEQ ID NO: 602: Codon-optimized DNA encoding 6xHis Cellvibrio sp. glucosidase, Uniparc reference UPI000260108C, Uniprot reference I3IDC0. SEQ ID NO: 603: Codon-optimized DNA encoding 6xHis Flavobacterium sp. glucosidase, Uniparc reference UPI000272D1E0, Uniprot reference J1ACA0. SEQ ID NO: 604: Codon-optimized DNA encoding 6xHis Macrophomina phaseolina glucosidase, Uniparc reference UPI00028E7FE1, Uniprot reference K2S5D3. SEQ ID NO: 605: Codon-optimized DNA encoding 6xHis Kosmotoga olearia glucosidase, Uniparc reference UPI00018483A2, Uniprot reference C5CHI5. SEQ ID NO: 606: Codon-optimized DNA encoding 6xHis Blautia obeum glucosidase, Uniparc reference UPI0001CD5918, Uniprot reference D4LRF6. SEQ ID NO: 607: Codon-optimized DNA encoding 6xHis Bifidobacterium dentium glucosidase, Uniparc reference UPI0001E18CDA, Uniprot reference E0Q541. SEQ ID NO: 608: Codon-optimized DNA encoding 6xHis Anaerolinea thermophila glucosidase, Uniparc reference UPI0001F55F8F, Uniprot reference E8N5R8. SEQ ID NO: 609: Codon-optimized DNA encoding 6xHis Coriobacterium glomerans glucosidase, Uniparc reference UPI0002050DA2, Uniprot reference F2N7E4. SEQ ID NO: 610: Codon-optimized DNA encoding 6xHis Microlunatus phosphovorus glucosidase, Uniparc reference UPI000210C886, Uniprot reference F5XJQ3.

[0094] SEQ ID NO: 611: Codon-optimized DNA encoding 6xHis Streptomyces sp. glucosidase, Uniparc reference UPI0001C18877, Uniprot reference G0Q1U8. SEQ ID NO: 612: Codon-optimized DNA encoding 6xHis Spathaspora passalidarum glucosidase, Uniparc reference UPI0002282B61, Uniprot reference G3AIV6. SEQ ID NO: 613: Codon-optimized DNA encoding 6xHis Glarea lozoyensis glucosidase, Uniparc reference UPI0002402F4A, Uniprot reference H0ET34. SEQ ID NO: 614: Codon-optimized DNA encoding 6xHis Glaciozyma antarctica glucosidase, Uniparc reference UPI0002633B36, Uniprot reference I3UJK0. SEQ ID NO: 615: Codon-optimized DNA encoding 6xHis Arthrobacter sp. glucosidase, Uniparc reference UPI00027DFD35, Uniprot reference J7LN00. SEQ ID NO: 616: Codon-optimized DNA encoding 6xHis Fusarium pseudograminearum glucosidase, Uniparc reference UPI00028D698C, Uniprot reference K3VMA9. SEQ ID NO: 617: Codon-optimized DNA encoding 6xHis Hypocrea rufa glucosidase, Uniparc reference UPI000006AA61, Uniprot reference C6GGC9. SEQ ID NO: 618: Codon-optimized DNA encoding 6xHis Ruminococcus torques glucosidase, Uniparc reference UPI0001CDA964, Uniprot reference D4M6W6. SEQ ID NO: 619: Codon-optimized DNA encoding 6xHis Bifidobacterium dentium glucosidase, Uniparc reference UPI0001E17370, Uniprot reference E0Q9Z7. SEQ ID NO: 620: Codon-optimized DNA encoding 6xHis Bacteroides salanitronis glucosidase, Uniparc reference UPI0001FC71F6, Uniprot reference F0R2D7.

[0095] SEQ ID NO: 621: Codon-optimized DNA encoding 6xHis Bacteroides coprosuis glucosidase, Uniparc reference UPI00020E6D19, Uniprot reference F3ZQ40. SEQ ID NO: 622: Codon-optimized DNA encoding 6xHis Marinomonas posidonica glucosidase, Uniparc reference UPI00020D4CC6, Uniprot reference F6CWF6. SEQ ID NO: 623: Codon-optimized DNA encoding 6xHis Chaetomium thermophilum glucosidase, Uniparc reference UPI000227E8ED, Uniprot reference G0SE64. SEQ ID NO: 624: Codon-optimized DNA encoding 6xHis Tetragenococcus halophilus glucosidase, Uniparc reference UPI00022B9A15, Uniprot reference G4L5K1. SEQ ID NO: 625: Codon-optimized DNA encoding 6xHis Paenibacillus sp. glucosidase, Uniparc reference UPI00024F0867, Uniprot reference H6CCZ8. SEQ ID NO: 626: Codon-optimized DNA encoding 6xHis Turneriella parva glucosidase, Uniparc reference UPI000265AA4B, Uniprot reference I4B8U7. SEQ ID NO: 627: Codon-optimized DNA encoding 6xHis Arthrobacter sp. glucosidase, Uniparc reference UPI00027DFDB2, Uniprot reference J7LQK9. SEQ ID NO: 628: Codon-optimized DNA encoding 6xHis Agaricus bisporus glucosidase, Uniparc reference UPI00029074B1, Uniprot reference K5W7V1. SEQ ID NO: 629: Codon-optimized DNA encoding 6xHis Nectria haematococca glucosidase, Uniparc reference UPI0001B67634, Uniprot reference C7YIP3. SEQ ID NO: 630: Codon-optimized DNA encoding 6xHis Bacteroides xylanisolvens glucosidase, Uniparc reference UPI0001A25287, Uniprot reference D4VSZ0.

[0096] SEQ ID NO: 631: Codon-optimized DNA encoding 6xHis Sediminispirochaeta smaragdinae glucosidase, Uniparc reference UPI0001DD9790, Uniprot reference E1R331. SEQ ID NO: 632: Codon-optimized DNA encoding 6xHis Deinococcus proteolyticus glucosidase, Uniparc reference UPI0001FC42E9, Uniprot reference F0RPV2. SEQ ID NO: 633: Codon-optimized DNA encoding 6xHis Sphingobacterium sp. glucosidase, Uniparc reference UPI0002033A0A, Uniprot reference F4C226. SEQ ID NO: 634: Codon-optimized DNA encoding 6xHis Sphingobium chlorophenolicum glucosidase, Uniparc reference UPI0001E54133, Uniprot reference F6ET40. SEQ ID NO: 635: Codon-optimized DNA encoding 6xHis Caloramator australicus glucosidase, Uniparc reference UPI00021CACC4, Uniprot reference G0V3V5. SEQ ID NO: 636: Codon-optimized DNA encoding 6xHis Commensalibacter intestini glucosidase, Uniparc reference UPI000230E3BF, Uniprot reference G6F370. SEQ ID NO: 637: Codon-optimized DNA encoding 6xHis Paenibacillus sp. glucosidase, Uniparc reference UPI00024F07AC, Uniprot reference H6CIT2. SEQ ID NO: 638: Codon-optimized DNA encoding 6xHis Nitrolancea hollandica glucosidase, Uniparc reference UPI0002638AF3, Uniprot reference I4EIA9. SEQ ID NO: 639: Codon-optimized DNA encoding 6xHis Cryptococcus neoformans glucosidase, Uniparc reference UPI000392C3ED, Uniprot reference J9VVK7. SEQ ID NO: 640: Codon-optimized DNA encoding 6xHis Acidipropionibacterium acidipropionici glucosidase, Uniparc reference UPI0002988588, Uniprot reference K7S596.

[0097] SEQ ID NO: 641: Codon-optimized DNA encoding 6xHis Prevotella sp. glucosidase, Uniparc reference UPI0001B93465, Uniprot reference C9PT75. SEQ ID NO: 642: Codon-optimized DNA encoding 6xHis Rhodobacter capsulatus glucosidase, Uniparc reference UPI0001D08095, Uniprot reference D5ALU0. SEQ ID NO: 643: Codon-optimized DNA encoding 6xHis Stigmatella aurantiaca glucosidase, Uniparc reference UPI0001E74370, Uniprot reference E3FJ05. SEQ ID NO: 644: Codon-optimized DNA encoding 6xHis Sphaerochaeta globosa glucosidase, Uniparc reference UPI0002010060, Uniprot reference F0RVK3. SEQ ID NO: 645: Codon-optimized DNA encoding 6xHis Sphaerochaeta coccoides glucosidase, Uniparc reference UPI000207D78C, Uniprot reference F4GH96. SEQ ID NO: 646: Codon-optimized DNA encoding 6xHis Novosphingobium sp. glucosidase, Uniparc reference UPI00020EFBCB, Uniprot reference F6ICQ5. SEQ ID NO: 647: Codon-optimized DNA encoding 6xHis Arthrobotrys oligospora glucosidase, Uniparc reference UPI000225331F, Uniprot reference G1XH86. SEQ ID NO: 648: Codon-optimized DNA encoding 6xHis Lactococcus lactis glucosidase, Uniparc reference UPI00000C6907, Uniprot reference G6FFS4. SEQ ID NO: 649: Codon-optimized DNA encoding 6xHis Phaeospirillum molischianum glucosidase, Uniparc reference UPI000255313A, Uniprot reference H8FXH7. SEQ ID NO: 650: Codon-optimized DNA encoding 6xHis Modestobacter marinus glucosidase, Uniparc reference UPI0002609C68, Uniprot reference I4EW72.

[0098] SEQ ID NO: 651: Codon-optimized DNA encoding 6xHis Saccharothrix espanaensis glucosidase, Uniparc reference UPI00028AC337, Uniprot reference K0K125. SEQ ID NO: 652: Codon-optimized DNA encoding 6xHis Cronobacter sakazakii glucosidase, Uniparc reference UPI00029BA293, Uniprot reference K8DAK0. SEQ ID NO: 653: Codon-optimized DNA encoding 6xHis Verticillium alfalfae glucosidase, Uniparc reference UPI0001BBDF1E, Uniprot reference C9SVX1. SEQ ID NO: 654: Codon-optimized DNA encoding 6xHis Bacteroides xylanisolvens glucosidase, Uniparc reference UPI0001CCFD36, Uniprot reference D6CY10. SEQ ID NO: 655: Codon-optimized DNA encoding 6xHis Leadbetterella byssophila glucosidase, Uniparc reference UPI0001EBD98A, Uniprot reference E4RUR0. SEQ ID NO: 656: Codon-optimized DNA encoding 6xHis Sphaerochaeta globosa glucosidase, Uniparc reference UPI00020102A5, Uniprot reference F0RYB6. SEQ ID NO: 657: Codon-optimized DNA encoding 6xHis Sphaerochaeta coccoides glucosidase, Uniparc reference UPI000207D6A0, Uniprot reference F4GLH6. SEQ ID NO: 658: Codon-optimized DNA encoding 6xHis Haloplasma contractile glucosidase, Uniparc reference UPI000212252C, Uniprot reference F7Q0Y2. SEQ ID NO: 659: Codon-optimized DNA encoding 6xHis Nitrospirillum amazonense glucosidase, Uniparc reference UPI0002265447, Uniprot reference G1Y4C7. SEQ ID NO: 660: Codon-optimized DNA encoding 6xHis Azospirillum brasilense glucosidase, Uniparc reference UPI00023420B8, Uniprot reference G8AWD9.

[0099] SEQ ID NO: 661: Codon-optimized DNA encoding 6xHis Phaeospirillum molischianum glucosidase, Uniparc reference UPI000255314F, Uniprot reference H8FXJ8. SEQ ID NO: 662: Codon-optimized DNA encoding 6xHis Modestobacter marinus glucosidase, Uniparc reference UPI000260A2FA, Uniprot reference I4EYD5. SEQ ID NO: 663: Codon-optimized DNA encoding 6xHis Wickerhamomyces ciferrii glucosidase, Uniparc reference UPI000283EB8D, Uniprot reference K0KVJ2. SEQ ID NO: 664: Codon-optimized DNA encoding 6xHis Gloeocapsa sp. glucosidase, Uniparc reference UPI0002A5D085, Uniprot reference K9XKL8. SEQ ID NO: 665: Codon-optimized DNA encoding 6xHis Sphaerobacter thermophilus glucosidase, Uniparc reference UPI0001A3BCB6, Uniprot reference D1C7U8. SEQ ID NO: 666: Codon-optimized DNA encoding 6xHis Bacteroides xylanisolvens glucosidase, Uniparc reference UPI0001CCEF1F, Uniprot reference D6D4V2. SEQ ID NO: 667: Codon-optimized DNA encoding 6xHis Prevotella buccae glucosidase, Uniparc reference UPI0001F149E8, Uniprot reference E6K4W5. SEQ ID NO: 668: Codon-optimized DNA encoding 6xHis Grosmannia clavigera glucosidase, Uniparc reference UPI0001FF1101, Uniprot reference F0XBR0. SEQ ID NO: 669: Codon-optimized DNA encoding 6xHis Melampsora larici-populina glucosidase, Uniparc reference UPI00020F9774, Uniprot reference F4R4W2. SEQ ID NO: 670: Codon-optimized DNA encoding 6xHis Prevotella multisaccharivorax glucosidase, Uniparc reference UPI0002138E9C, Uniprot reference F8N7G1.

[0100] SEQ ID NO: 671: Codon-optimized DNA encoding 6xHis Streptomyces zinciresistens glucosidase, Uniparc reference UPI0002255A63, Uniprot reference G2G8K4. SEQ ID NO: 672: Codon-optimized DNA encoding 6xHis Granulicella mallensis glucosidase, Uniparc reference UPI0001D9FC40, Uniprot reference G8NY42. SEQ ID NO: 673: Codon-optimized DNA encoding 6xHis Gibberella zeae glucosidase, Uniparc reference UPI00021F1FEE, Uniprot reference I1RH94. SEQ ID NO: 674: Codon-optimized DNA encoding 6xHis Modestobacter marinus glucosidase, Uniparc reference UPI00026090A2, Uniprot reference I4EYK6. SEQ ID NO: 675: Codon-optimized DNA encoding 6xHis Lactobacillus equicursoris glucosidase, Uniparc reference UPI0002869F55, Uniprot reference K0NRS8. SEQ ID NO: 676: Codon-optimized DNA encoding 6xHis Colletotrichum fructicola glucosidase, Uniparc reference UPI0002A93280, Uniprot reference L2F9W0. SEQ ID NO: 677: Codon-optimized DNA encoding 6xHis Streptosporangium roseum glucosidase, Uniparc reference UPI0001BF8AF6, Uniprot reference D2B261. SEQ ID NO: 678: Codon-optimized DNA encoding 6xHis Listeria grayi glucosidase, Uniparc reference UPI00019F252A, Uniprot reference D7UX19. SEQ ID NO: 679: Codon-optimized DNA encoding 6xHis Enterococcus italicus glucosidase, Uniparc reference UPI0001F11EFF, Uniprot reference E6LF07. SEQ ID NO: 680: Codon-optimized DNA encoding 6xHis Fluviicola taffensis glucosidase, Uniparc reference UPI000203D9EA, Uniprot reference F2IIT6.

[0101] SEQ ID NO: 681: Codon-optimized DNA encoding 6xHis Shigella flexneri glucosidase, Uniparc reference UPI00020CA641, Uniprot reference F5N4W9. SEQ ID NO: 682: Codon-optimized DNA encoding 6xHis Actinomyces sp. glucosidase, Uniparc reference UPI0002189080, Uniprot reference F9EFW5. SEQ ID NO: 683: Codon-optimized DNA encoding 6xHis Verticillium dahliae glucosidase, Uniparc reference UPI00022EBE5A, Uniprot reference G2X5V6. SEQ ID NO: 684: Codon-optimized DNA encoding 6xHis Actinoplanes sp. glucosidase, Uniparc reference UPI00023ED5A8, Uniprot reference G8S0M0. SEQ ID NO: 685: Codon-optimized DNA encoding 6xHis Gibberella zeae glucosidase, Uniparc reference UPI000023EA29, Uniprot reference I1S320. SEQ ID NO: 686: Codon-optimized DNA encoding 6xHis Auricularia subglabra glucosidase, Uniparc reference UPI00027CE685, Uniprot reference J0WV06. SEQ ID NO: 687: Codon-optimized DNA encoding 6xHis Nitratireductor indicus glucosidase, Uniparc reference UPI00028E86D6, Uniprot reference K2N268. SEQ ID NO: 688: Codon-optimized DNA encoding 6xHis Thermoclostridium stercorarium glucosidase, Uniparc reference UPI0002AD999E, Uniprot reference L7VH66. SEQ ID NO: 689: Codon-optimized DNA encoding 6xHis Geobacillus sp. glucosidase, Uniparc reference UPI0002AF2DE3, Uniprot reference L8A1S2. SEQ ID NO: 690: Codon-optimized DNA encoding 6xHis uncultured bacterial glucosidase, Uniparc reference UPI000327C3CC, Uniprot reference M9ZC55.

[0102] SEQ ID NO: 691: Codon-optimized DNA encoding 6xHis Burkholderia ambifaria glucosidase, Uniparc reference UPI000059ACE1, Uniprot reference Q0BAK2. SEQ ID NO: 692: Codon-optimized DNA encoding 6xHis Aspergillus oryzae glucosidase, Uniparc reference UPI0000676B8D, Uniprot reference Q2UIR4. SEQ ID NO: 693: Codon-optimized DNA encoding 6xHis Yersinia pseudotuberculosis glucosidase, Uniparc reference UPI00004269B5, Uniprot reference Q66DJ0. SEQ ID NO: 694: Codon-optimized DNA encoding 6xHis Wallemia ichthyophaga glucosidase, Uniparc reference UPI000331CC98, Uniprot reference R9AF64. SEQ ID NO: 695: Codon-optimized DNA encoding 6xHis Glarea lozoyensis glucosidase, Uniparc reference UPI0003522404, Uniprot reference S3DIM6. SEQ ID NO: 696: Codon-optimized DNA encoding 6xHis Moniliophthora roreri glucosidase, Uniparc reference UPI0003BF753A, Uniprot reference V2YU31. SEQ ID NO: 697: Codon-optimized DNA encoding 6xHis Zhouia amylolytica glucosidase, Uniparc reference UPI0003DBCB4F, Uniprot reference W2ULL3. SEQ ID NO: 698: Codon-optimized DNA encoding 6xHis Fusarium oxysporum glucosidase, Uniparc reference UPI0003F362FF, Uniprot reference W9NQQ3. SEQ ID NO: 699: Codon-optimized DNA encoding 6xHis Flavobacterium johnsoniae glucosidase, Uniparc reference UPI00006EA168, Uniprot reference A5FAA5. SEQ ID NO: 700: Codon-optimized DNA encoding 6xHis rice (Oryza sativa) glucosidase, Uniparc reference UPI00002394F5, Uniprot reference Q75I93.

[0103] SEQ ID NO: 701: Codon-optimized DNA encoding 6xHis Clostridium saccharoperbutylacetonicum glucosidase, Uniparc reference UPI0002B65681, Uniprot reference M1MCD1. SEQ ID NO: 702: Codon-optimized DNA encoding 6xHis Rhodococcus sp. glucosidase, Uniparc reference UPI0002D21DB1, Uniprot reference N1MBN6. SEQ ID NO: 703: Codon-optimized DNA encoding 6xHis Burkholderia ambifaria glucosidase, Uniparc reference UPI000059C0CD, Uniprot reference Q0BCV8. SEQ ID NO: 704: Codon-optimized DNA encoding 6xHis Xanthomonas campestris glucosidase, Uniparc reference UPI00005CE9E7, Uniprot reference Q3BVH7. SEQ ID NO: 705: Codon-optimized DNA encoding 6xHis Caulobacter vibrioides glucosidase, Uniparc reference UPI00000C7604, Uniprot reference Q9A6F8. SEQ ID NO: 706: Codon-optimized DNA encoding 6xHis Arcticibacter svalbardensis glucosidase, Uniparc reference UPI000338772A, Uniprot reference R9GRA7. SEQ ID NO: 707: 6xHis codon-optimized DNA encoding Winogradskyella psychrotolerans glucosidase, Uniparc reference UPI00035A4604, Uniprot reference S7VQ28. SEQ ID NO: 708: Codon-optimized DNA encoding 6xHis Methyloglobulus morosus glucosidase, Uniparc reference UPI0003C4ED6A, Uniprot reference V5DXT8. SEQ ID NO: 709: Codon-optimized DNA encoding 6xHis Pestalotiopsis fici glucosidase, Uniparc reference UPI0003E05C04, Uniprot reference W3WV37. SEQ ID NO: 710: Codon-optimized DNA encoding 6xHis Capronia coronata glucosidase, Uniparc reference UPI000434E698, Uniprot reference W9YNR7.

[0104] SEQ ID NO: 711: Codon-optimized DNA encoding 6xHis Aspergillus aculeatus glucosidase, Uniparc reference UPI00001268FD, Uniprot reference P48825. SEQ ID NO: 712: Codon-optimized DNA encoding 6xHis Thermotoga neapolitana glucosidase, Uniparc reference UPI0000DD5996, Uniprot reference Q0GC07. SEQ ID NO: 713: Codon-optimized DNA encoding 6xHis Clostridium saccharoperbutylacetonicum glucosidase, Uniparc reference UPI0002B666E8, Uniprot reference M1MJF4. SEQ ID NO: 714: Codon-optimized DNA encoding 6xHis Schizosaccharomyces pombe glucosidase, Uniparc reference UPI000006B001, Uniprot reference O74799. SEQ ID NO: 715: Codon-optimized DNA encoding 6xHis Phaeosphaeria nodorum glucosidase, Uniparc reference UPI000161BD2B, Uniprot reference Q0TXF6. SEQ ID NO: 716: Codon-optimized DNA encoding 6xHis Xylella fastidiosa Dixon glucosidase, Uniparc reference UPI00003806C8, Uniprot reference Q3RGJ3. SEQ ID NO: 717: Codon-optimized DNA encoding 6xHis Schizosaccharomyces pombe glucosidase, Uniparc reference UPI000006A330, Uniprot reference Q9P6J6. SEQ ID NO: 718: Codon-optimized DNA encoding 6xHis Arcticibacter svalbardensis glucosidase, Uniparc reference UPI000337E887, Uniprot reference R9GWD6. SEQ ID NO: 719: Codon-optimized DNA encoding 6xHis Colletotrichum gloeosporioides glucosidase, Uniparc reference UPI0003885717, Uniprot reference T0KJI7. SEQ ID NO: 720: Codon-optimized DNA encoding 6xHis uncultured bacterial glucosidase, Uniparc reference UPI0003C9E340, Uniprot reference V5R1E8.

[0105] SEQ ID NO: 721: Codon-optimized DNA encoding 6xHis Xanthomonas arboricola glucosidase, Uniparc reference UPI0003E06A61, Uniprot reference W4S7I5. SEQ ID NO: 722: Codon-optimized DNA encoding 6xHis Fusarium oxysporum glucosidase, Uniparc reference UPI00021EC697, Uniprot reference X0A8X8. SEQ ID NO: 723: Codon-optimized DNA encoding 6xHis Paenibacillus polymyxa glucosidase, Uniparc reference UPI00001108D0, Uniprot reference P22073. SEQ ID NO: 724: Codon-optimized DNA encoding 6xHis Kluyveromyces marxianus glucosidase, Uniparc reference UPI0001BE5ADA, Uniprot reference D1GCC6. SEQ ID NO: 725: Codon-optimized DNA encoding 6xHis Ilumatobacter coccineus glucosidase, Uniparc reference UPI0002C04A25, Uniprot reference M5A594. SEQ ID NO: 726: Codon-optimized DNA encoding 6xHis Agrobacterium sp. glucosidase, Uniparc reference UPI0000126912, Uniprot reference P12614. SEQ ID NO: 727: Codon-optimized DNA encoding 6xHis Cytophaga hutchinsonii glucosidase, Uniparc reference UPI000038ECF7, Uniprot reference Q11P53. SEQ ID NO: 728: Codon-optimized DNA encoding 6xHis Thermobifida fusca glucosidase, Uniparc reference UPI00003C5CA4, Uniprot reference Q47PF5. SEQ ID NO: 729: Codon-optimized DNA encoding 6xHis Botryotinia fuckeliana glucosidase, Uniparc reference UPI0000069E5E, Uniprot reference Q9UVJ6. SEQ ID NO: 730: Codon-optimized DNA encoding 6xHis Agarivorans albus glucosidase, Uniparc reference UPI00033994D2, Uniprot reference R9PTK2.

[0106] SEQ ID NO: 731: Codon-optimized DNA encoding 6xHis Enterococcus sp. glucosidase, Uniparc reference UPI00038B7639, Uniprot reference T0UDU2. SEQ ID NO: 732: Codon-optimized DNA encoding 6xHis Salinispira pacifica glucosidase, Uniparc reference UPI0003D8A7DC, Uniprot reference V5WKT4. SEQ ID NO: 733: Codon-optimized DNA encoding 6xHis Bacteroides xylanisolvens glucosidase, Uniparc reference UPI0003ECF15E, Uniprot reference W6P696. SEQ ID NO: 734: Codon-optimized DNA encoding 6xHis Fusarium oxysporum glucosidase, Uniparc reference UPI000430029A, Uniprot reference X0AB46. SEQ ID NO: 735: Codon-optimized DNA encoding 6xHis Thermotoga maritima glucosidase, Uniparc reference UPI0000126906, Uniprot reference Q08638B. SEQ ID NO: 736: Codon-optimized DNA encoding 6xHis Neotermes koshunensis glucosidase, Uniparc reference UPI0000083EA8, Uniprot reference Q8T0W7. SEQ ID NO: 737: Codon-optimized DNA encoding 6xHis Thanatephorus cucumeris glucosidase, Uniparc reference UPI0002BF34B3, Uniprot reference M5CHG9. SEQ ID NO: 738: Codon-optimized DNA encoding 6xHis Hungateiclostridium thermocellum glucosidase, Uniparc reference UPI000053581D, Uniprot reference P14002. SEQ ID NO: 739: Codon-optimized DNA encoding 6xHis Koribacter versatilis glucosidase, Uniparc reference UPI0000D76A70, Uniprot reference Q1IJ89. SEQ ID NO: 740: Codon-optimized DNA encoding 6xHis Neosartorya fumigata glucosidase, Uniparc reference UPI00005203D8, Uniprot reference Q4WLX5.

[0107] SEQ ID NO: 741: Codon-optimized DNA encoding 6xHis Amycolatopsis vancoresmycina glucosidase, Uniparc reference UPI00032DB8C1, Uniprot reference R1FKF0. SEQ ID NO: 742: Codon-optimized DNA encoding 6xHis Gibberella fujikuroi glucosidase, Uniparc reference UPI0003519C76, Uniprot reference S0EKU1. SEQ ID NO: 743: Codon-optimized DNA encoding 6xHis Bifidobacterium longum glucosidase, Uniparc reference UPI000390F839, Uniprot reference T2I2H5. SEQ ID NO: 744: Codon-optimized DNA encoding 6xHis uncultured bacterial glucosidase, Uniparc reference UPI0003DFF0DA, Uniprot reference W0FLD1. SEQ ID NO: 745: Codon-optimized DNA encoding 6xHis Bacteroides xylanisolvens glucosidase, Uniparc reference UPI0002D3D994, Uniprot reference W6P9J9. SEQ ID NO: 746: Codon-optimized DNA encoding 6xHis Fusarium oxysporum glucosidase, Uniparc reference UPI00042F8016, Uniprot reference X0HA35. SEQ ID NO: 747: Codon-optimized DNA encoding 6xHis Sorghum bicolor glucosidase, Uniparc reference UPI00000A7F40, Uniprot reference Q41290. SEQ ID NO: 748: Codon-optimized DNA encoding 6xHis Clostridium cellulovorans glucosidase, Uniparc reference UPI000050B701, Uniprot reference Q53EH2. SEQ ID NO: 749: Codon-optimized DNA encoding 6xHis Dacryopinax primogenitus glucosidase, Uniparc reference UPI0002C29EAA, Uniprot reference M5G1U5. SEQ ID NO:750: Codon-optimized DNA encoding 6xHis Rhizobium radiobacter glucosidase, Uniparc reference UPI0000126913, Uniprot reference P27034.

[0108] SEQ ID NO: 751: Codon-optimized DNA encoding 6xHis Phanerochaete chrysosporium glucosidase, Uniparc reference UPI00006E0CC7, Uniprot reference Q25BW5A. SEQ ID NO: 752: Codon-optimized DNA encoding 6xHis Enterobacter agglomerans glucosidase, Uniparc reference UPI0000126905, Uniprot reference Q59437. SEQ ID NO: 753: Codon-optimized DNA encoding 6xHis Candidatus microthrix glucosidase, Uniparc reference UPI00033056C4, Uniprot reference R4YWU4. SEQ ID NO: 754: Codon-optimized DNA encoding 6xHis Chthonomonas calidirosea glucosidase, Uniparc reference UPI0003427F29, Uniprot reference S0ET37. SEQ ID NO: 755: Codon-optimized DNA encoding 6xHis Acholeplasma brassicae glucosidase, Uniparc reference UPI0003B04208, Uniprot reference U4KMR7. SEQ ID NO: 756: Codon-optimized DNA encoding 6xHis Chania multitudinisentens glucosidase, Uniparc reference UPI0003E13BD3, Uniprot reference W0LJ28. SEQ ID NO: 757: Codon-optimized DNA encoding 6xHis Gibberella moniliformis glucosidase, Uniparc reference UPI0003ECCF80, Uniprot reference W7MW98. SEQ ID NO: 758: Codon-optimized DNA encoding 6xHis Fusarium oxysporum glucosidase, Uniparc reference UPI0004305856, Uniprot reference X0I2U8. SEQ ID NO: 759: Codon-optimized DNA encoding 6xHis Phanerochaete chrysosporium glucosidase, Uniparc reference UPI00006E0CC7, Uniprot reference Q25BW5B. SEQ ID NO: 760: Codon-optimized DNA encoding 6xHis rye (Secale cereale) glucosidase, Uniparc reference UPI00000A7EFC, Uniprot reference Q9FYS3.

[0109] SEQ ID NO: 761: Codon-optimized DNA encoding 6xHis Anoxybacillus gonensis glucosidase, Uniparc reference UPI0002C0273E, Uniprot reference M5QUM2. SEQ ID NO: 762: Codon-optimized DNA encoding 6xHis Thermotoga maritima glucosidase, Uniparc reference UPI0000126906, Uniprot reference Q08638A. SEQ ID NO: 763: Codon-optimized DNA encoding 6xHis Rhodospirillum rubrum glucosidase, Uniparc reference UPI00003C2ACC, Uniprot reference Q2RP51. SEQ ID NO: 764: Codon-optimized DNA encoding 6xHis Thermotoga neapolitana glucosidase, Uniparc reference UPI00000B9013, Uniprot reference Q60038. SEQ ID NO: 765: Codon-optimized DNA encoding 6xHis Candidatus microthrix glucosidase, Uniparc reference UPI00032F466E, Uniprot reference R4Z6M6. SEQ ID NO: 766: Codon-optimized DNA encoding 6xHis Ruminiclostridium cellobioparum glucosidase, Uniparc reference UPI00032874EC, Uniprot reference S0FPI8. SEQ ID NO: 767: Codon-optimized DNA encoding 6xHis Acholeplasma brassicae glucosidase, Uniparc reference UPI0003B0490C, Uniprot reference U4KMV4. SEQ ID NO: 768: Codon-optimized DNA encoding 6xHis Klebsiella pneumoniae glucosidase, Uniparc reference UPI0003DB6EFD, Uniprot reference W1BBP5. SEQ ID NO: 769: Codon-optimized DNA encoding 6xHis Enterobacter sp. glucosidase, Uniparc reference UPI0003ED0A8E, Uniprot reference W7P6Y1. SEQ ID NO: 770: Codon-optimized DNA encoding 6xHis Rhizoctonia solani glucosidase, Uniparc reference UPI00045BB507, Uniprot reference X8JI88.

[0110] SEQ ID NO: 771: Codon-optimized DNA encoding 6xHis human (Homo sapiens) glucosidase, Uniparc reference UPI0000072C73, Uniprot reference Q9H227. SEQ ID NO: 772: Codon-optimized DNA encoding 6xHis uncultured bacterial glucosidase, Uniparc reference UPI0000DD5304, Uniprot reference Q0GMU3. SEQ ID NO: 773: 6xHis Codon-optimized DNA encoding Thermoanaerobacter thermohydrosulfuricus glucosidase, Uniparc reference UPI0002CA9E4F, Uniprot reference M8CQD9. SEQ ID NO: 774: Codon-optimized DNA encoding 6xHis Stigmatella aurantiaca glucosidase, Uniparc reference UPI0000E297D8, Uniprot reference Q091M8. SEQ ID NO: 775: Codon-optimized DNA encoding 6xHis Burkholderia thailandensis glucosidase, Uniparc reference UPI00006676B1, Uniprot reference Q2T7C9. SEQ ID NO: 776: Codon-optimized DNA encoding 6xHis Yersinia pseudotuberculosis glucosidase, Uniparc reference UPI0000426E8F, Uniprot reference Q665S2. SEQ ID NO: 777: Codon-optimized DNA encoding 6xHis Togninia minima glucosidase, Uniparc reference UPI00032BF71F, Uniprot reference R8BQM8. SEQ ID NO: 778: Codon-optimized DNA encoding 6xHis Ophiostoma piceae glucosidase, Uniparc reference UPI00035213C2, Uniprot reference S3BVJ8. SEQ ID NO: 779: Codon-optimized DNA encoding 6xHis Pyronema omphalodes glucosidase, Uniparc reference UPI0003B117B7, Uniprot reference U4LIL3. SEQ ID NO: 780: Codon-optimized DNA encoding 6xHis Ogataea parapolymorpha glucosidase, Uniparc reference UPI0001F76D1F, Uniprot reference W1QIW0.

[0111] SEQ ID NO: 781: Codon-optimized DNA encoding 6xHis Hymenobacter swuensis glucosidase, Uniparc reference UPI0003F2059F, Uniprot reference W8F6U0. SEQ ID NO: 782: Codon-optimized DNA encoding 6xHis Flavobacterium johnsoniae glucosidase, Uniparc reference UPI00006E8E1B, Uniprot reference A5FEF5. SEQ ID NO: 783: Codon-optimized DNA encoding 6xHis Paenibacillus polymyxa glucosidase, Uniparc reference UPI000012690B, Uniprot reference P22505. SEQ ID NO: 784: Codon-optimized DNA encoding 6xHis rice (Oryza sativa) glucosidase, Uniparc reference UPI000009D014, Uniprot reference Q8L7J2. SEQ ID NO: 785: Codon-optimized DNA encoding 6xHis rice (Oryza sativa) glucosidase, Uniparc reference UPI0000E580F2, Uniprot reference B8AVF0. SEQ ID NO: 786: Codon-optimized DNA encoding 6xHis Nannochloris glucosidase, Uniparc reference UPI000EA7F16F, Uniprot reference A0A452CSM4. SEQ ID NO: 787: Codon-optimized DNA encoding 6xHis Halothermothrix orenii glucosidase, Uniparc reference UPI00006AE508, Uniprot reference B8CYA8. SEQ ID NO: 788: Codon-optimized DNA encoding 6xHis Neurospora crassa glucosidase, Uniparc reference UPI000018B2B4, Uniprot reference Q7RWP2. SEQ ID NO: 789: Codon-optimized DNA encoding 6xHis Micrococcus antarcticus glucosidase, Uniparc reference UPI000192BB5F, Uniprot reference B9V8P5. SEQ ID NO: 790: Codon-optimized DNA encoding 6xHis Exiguobacterium antarcticum glucosidase, Uniparc reference UPI000285E79E, Uniprot reference K0A8J9.

[0112] SEQ ID NO: 791: Codon-optimized DNA encoding 6xHis Thermus thermophilus glucosidase, Uniparc reference UPI00000BEB61, Uniprot reference Q9RA61. SEQ ID NO: 792: Codon-optimized DNA encoding 6xHis Trichoderma harzianum glucosidase, Uniparc reference UPI00078BF747, Uniprot reference A0A2T4AR08. SEQ ID NO: 793: Codon-optimized DNA encoding 6xHis Hypocrea jecorina glucosidase, Uniparc reference UPI000006AA61, Uniprot reference Q12715. SEQ ID NO: 794: Codon-optimized DNA encoding 6xHis Streptomyces sp. glucosidase, Uniparc reference UPI00000B411B, Uniprot reference Q59976. SEQ ID NO: 795: Codon-optimized DNA encoding 6xHis Streptococcus pyogenes glucosidase, Uniparc reference UPI00000C7E56, Uniprot reference Q99YP9. SEQ ID NO: 796: Codon-optimized DNA encoding 6xHis white clover (Trifolium repens) glucosidase, Uniparc reference UPI000012691B, Uniprot reference P26205. SEQ ID NO: 797: Codon-optimized DNA encoding 6xHis Talaromyces emersonii glucosidase, Uniparc reference UPI000006C8FF, Uniprot reference Q8TGI8. SEQ ID NO: 798: Codon-optimized DNA encoding 6xHis Hungateiclostridium thermocellum glucosidase, Uniparc reference UPI0000126903, Uniprot reference P26208. SEQ ID NO: 799: Codon-optimized DNA encoding 6xHis Lactobacillus plantarum glucosidase, Uniparc reference UPI000219FE3E, Uniprot reference F9ULH8. SEQ ID NO: 800: Codon-optimized DNA encoding 6xHis Agrobacterium tumefaciens glucosidase, Uniparc reference UPI0003F2033A, Uniprot reference A0A2I4PGZ0.

[0113] SEQ ID NO: 801: Amino acid sequence for Bifidobacterium actinocoloniiforme glucosidase, Uniparc reference UPI000503B26C, Uniprot reference A0A086YYS8. SEQ ID NO: 802: Amino acid sequence for Bifidobacterium psychraerophilum glucosidase, Uniparc reference UPI000502B461, Uniprot reference A0A087CD28. SEQ ID NO: 803: Amino acid sequence for Penicillium italicum glucosidase, Uniparc reference UPI00052B8681, Uniprot reference A0A0A2K704. SEQ ID NO: 804: Amino acid sequence for Microbacterium trichothecenolyticum glucosidase, Uniparc reference UPI0005ED0AE6, Uniprot reference A0A0M2HDB3. SEQ ID NO: 805: Amino acid sequence for uncultured Clostridium sp. glucosidase, Uniparc reference UPI0008206F38, Uniprot reference A0A1C6I6A3. SEQ ID NO: 806: Amino acid sequence for Bifiguratus adelaidae glucosidase, Uniparc reference UPI000BC57319, Uniprot reference A0A261Y7Q8. SEQ ID NO: 807: Amino acid sequence for Paenibacillus thiaminolyticus glucosidase, Uniparc reference UPI000B3B73CD, Uniprot reference A0A378ZIK3. SEQ ID NO: 808: Amino acid sequence for Microbacterium lemovicicum glucosidase, Uniparc reference UPI000F8FB9BA, Uniprot reference A0A3S9WE68. SEQ ID NO: 809: Amino acid sequence for Rhodococcus erythropolis glucosidase, Uniparc reference UPI00019923DC, Uniprot reference C1A1N4. SEQ ID NO: 810: Amino acid sequence for Cutibacterium avidum glucosidase, Uniparc reference UPI00022C19B5, Uniprot reference G4CZU8.

[0114] SEQ ID NO: 811: Amino acid sequence for Clavibacter michiganensis glucosidase, Uniparc reference UPI0002C5A938, Uniprot reference M5BD10. SEQ ID NO: 812: Amino acid sequence for Microbacterium sp. glucosidase, Uniparc reference UPI0003DE3509, Uniprot reference W0ZC23. SEQ ID NO: 813: Amino acid sequence for Bifidobacterium actinocoloniiforme glucosidase, Uniparc reference UPI000507F38A, Uniprot reference A0A086YZL4. SEQ ID NO: 814: Amino acid sequence for Bifidobacterium reuteri glucosidase, Uniparc reference UPI000506119A, Uniprot reference A0A087CR26. SEQ ID NO: 815: Amino acid sequence for Propionibacterium freudenreichii glucosidase, Uniparc reference UPI0005A5CAC2, Uniprot reference A0A0A8RX48. SEQ ID NO: 816: Amino acid sequence for Microbacterium hydrocarbonoxydans glucosidase, Uniparc reference UPI0005EC18F9, Uniprot reference A0A0M2HRU0. SEQ ID NO: 817: Amino acid sequence for Pseudonocardia sp. glucosidase, Uniparc reference UPI00094B6D3B, Uniprot reference A0A1Q8KSV8. SEQ ID NO: 818: Amino acid sequence for Tuber aestivum glucosidase, Uniparc reference UPI000BC13DF0, Uniprot reference A0A292PKV5. SEQ ID NO: 819: Amino acid sequence for Propionibacterium australiense glucosidase, Uniparc reference UPI000E5B4EA5, Uniprot reference A0A383S7A9. SEQ ID NO: 820: Amino acid sequence for Fusarium sp. glucosidase, Uniparc reference UPI001004B2C9, Uniprot reference A0A428T6E2.

[0115] SEQ ID NO: 821: Amino acid sequence for Nectria haematococca glucosidase, Uniparc reference UPI0001B69B5C, Uniprot reference C7ZBV0. SEQ ID NO: 822: Amino acid sequence for Actinoplanes sp. glucosidase, Uniparc reference UPI00023EBB15, Uniprot reference G8S3E7. SEQ ID NO: 823: Amino acid sequence for Streptomyces fulvissimus glucosidase, Uniparc reference UPI0003289BC6, Uniprot reference N0CMW2. SEQ ID NO: 824: Amino acid sequence for Pestalotiopsis fici glucosidase, Uniparc reference UPI0003E02BF1, Uniprot reference W3WXF1. SEQ ID NO: 825: Amino acid sequence for Bifidobacterium bohemicum glucosidase, Uniparc reference UPI0005025F2E, Uniprot reference A0A086ZGP0. SEQ ID NO: 826: Amino acid sequence for Bifidobacterium saeculare glucosidase, Uniparc reference UPI0005084E52, Uniprot reference A0A087D0Q2. SEQ ID NO: 827: Amino acid sequence for Bionectria ochroleuca glucosidase, Uniparc reference UPI00059674D6, Uniprot reference A0A0B7JW24. SEQ ID NO: 828: Amino acid sequence for Bifidobacterium pseudocatenulatum glucosidase, Uniparc reference UPI0006C6D6F9, Uniprot reference A0A174AU04. SEQ ID NO: 829: Amino acid sequence for Pseudonocardia sp. glucosidase, Uniparc reference UPI00094ABAB3, Uniprot reference A0A1Q8LPB4. SEQ ID NO: 830: Amino acid sequence for bacterial glucosidase, Uniparc reference UPI000CB49A0B, Uniprot reference A0A2H5Z8Q4.

[0116] SEQ ID NO: 831: Amino acid sequence for Coleophoma crateriformis glucosidase, Uniparc reference UPI000E38A995, Uniprot reference A0A3D8Q771. SEQ ID NO: 832: Amino acid sequence for Arthrobotrys oligospora glucosidase, Uniparc reference UPI001102DFA3, Uniprot reference A0A4Z0Y5Y8. SEQ ID NO: 833: Amino acid sequence for Pyrenophora teres glucosidase, Uniparc reference UPI0001ECDCBD, Uniprot reference E3RFS2. SEQ ID NO: 834: Amino acid sequence for Gordonia polyisoprenivorans glucosidase, Uniparc reference UPI00024F2A26, Uniprot reference H6MTQ7. SEQ ID NO: 835: Amino acid sequence for Stigmatella aurantiaca glucosidase, Uniparc reference UPI0000E28E5D, Uniprot reference Q08S21. SEQ ID NO: 836: Amino acid sequence for Pestalotiopsis fici glucosidase, Uniparc reference UPI0003E03A52, Uniprot reference W3WZ03. SEQ ID NO: 837: Amino acid sequence for Bifidobacterium magnum glucosidase, Uniparc reference UPI0003B7B6EE, Uniprot reference A0A087BEN9. SEQ ID NO: 838: Amino acid sequence for Bifidobacterium stellenboschense glucosidase, Uniparc reference UPI000503F283, Uniprot reference A0A087DFL8. SEQ ID NO: 839: Amino acid sequence for Bionectria ochroleuca glucosidase, Uniparc reference UPI00059673EE, Uniprot reference A0A0B7K316. SEQ ID NO: 840: Amino acid sequence for Hungatella hathewayi glucosidase, Uniparc reference UPI0006C069F3, Uniprot reference A0A174LVE3.

[0117] SEQ ID NO: 841: Amino acid sequence for Mycetocola reblochoni glucosidase, Uniparc reference UPI00097EB800, Uniprot reference A0A1R4J2F9. SEQ ID NO: 842: Amino acid sequence for Nonomuraea sp. glucosidase, Uniparc reference UPI0009ABD7B3, Uniprot reference A0A2P9IX34. SEQ ID NO: 843: Amino acid sequence for Coleophoma crateriformis glucosidase, Uniparc reference UPI000E391DF6, Uniprot reference A0A3D8T9C2. SEQ ID NO: 844: Amino acid sequence for Paenarthrobacter aurescens glucosidase, Uniparc reference UPI0000EC83AE, Uniprot reference A1R2K1. SEQ ID NO: 845: Amino acid sequence for Kitasatospora setae glucosidase, Uniparc reference UPI0001F21F08, Uniprot reference E4N4F6. SEQ ID NO: 846: Amino acid sequence for Nocardiopsis alba glucosidase, Uniparc reference UPI00027E251E, Uniprot reference J7L3Z8. SEQ ID NO: 847: Amino acid sequence for Rhodococcus jostii glucosidase, Uniparc reference UPI0000DBA338, Uniprot reference Q0SCI4. SEQ ID NO: 848: Amino acid sequence for uncultured microbial glucosidase, Uniparc reference UPI0003EC942D, Uniprot reference W5X324. SEQ ID NO: 849: Amino acid sequence for Bifidobacterium merycicum glucosidase, Uniparc reference UPI0005084B63, Uniprot reference A0A087BJ88. SEQ ID NO: 850: Amino acid sequence for Bifidobacterium scardovii glucosidase, Uniparc reference UPI0005018B5B, Uniprot reference A0A087DGT3.

[0118] SEQ ID NO: 851: Amino acid sequence for Fusarium oxysporum glucosidase, Uniparc reference UPI00021EC81E, Uniprot reference A0A0C4DJL4. SEQ ID NO: 852: Amino acid sequence for Paraphaeosphaeria sporulosa glucosidase, Uniparc reference UPI0007CE199C, Uniprot reference A0A177BXH1. SEQ ID NO: 853: Amino acid sequence for Clostridium oryzae glucosidase, Uniparc reference UPI0009A50F8D, Uniprot reference A0A1V4IY77. SEQ ID NO: 854: Amino acid sequence for Corynespora cassiicola glucosidase, Uniparc reference UPI000D22F5EB, Uniprot reference A0A2T2N4T6. SEQ ID NO: 855: Amino acid sequence for Choiromyces venosus glucosidase, Uniparc reference UPI000F7332F8, Uniprot reference A0A3N4J9R6. SEQ ID NO: 856: Amino acid sequence for Saccharopolyspora erythraea glucosidase, Uniparc reference UPI0000F550EC, Uniprot reference A4F982. SEQ ID NO: 857: Amino acid sequence for Streptomyces venezuelae glucosidase, Uniparc reference UPI000204906E, Uniprot reference F2R0Y2. SEQ ID NO: 858: Amino acid sequence for Arthrobacter sp. glucosidase, Uniparc reference UPI00027DFDB0, Uniprot reference J7LVB1. SEQ ID NO: 859: Amino acid sequence for Thermobrachium celere glucosidase, Uniparc reference UPI00033420CB, Uniprot reference R7RMQ6. SEQ ID NO: 860: Amino acid sequence for Drechslerella stenobrocha glucosidase, Uniparc reference UPI0003EA65B9, Uniprot reference W7HT93.

[0119] SEQ ID NO: 861: Amino acid sequence for Bifidobacterium minimum glucosidase, Uniparc reference UPI0003B38542, Uniprot reference A0A087BMR5. SEQ ID NO: 862: Amino acid sequence for Bifidobacterium thermacidophilum glucosidase, Uniparc reference UPI000409825E, Uniprot reference A0A087E3K8. SEQ ID NO: 863: Amino acid sequence for Verruconis gallopava glucosidase, Uniparc reference UPI0005C0229F, Uniprot reference A0A0D1X9G0. SEQ ID NO: 864: Amino acid sequence for Stagonospora sp. glucosidase, Uniparc reference UPI0007CECAD5, Uniprot reference A0A178AER7. SEQ ID NO: 865: Amino acid sequence for a Firmicutes bacterial glucosidase, Uniparc reference UPI0009D58C49, Uniprot reference A0A1V6ALF9. SEQ ID NO: 866: Amino acid sequence for Tuber borchii glucosidase, Uniparc reference UPI000D50EED1, Uniprot reference A0A2T6ZNU7. SEQ ID NO: 867: Amino acid sequence for Morchella conica glucosidase, Uniparc reference UPI000F728167, Uniprot reference A0A3N4KUR3. SEQ ID NO: 868: Amino acid sequence for Dictyoglomus thermophilum glucosidase, Uniparc reference UPI00018152EB, Uniprot reference B5YC96. SEQ ID NO: 869: Amino acid sequence for Microlunatus phosphovorus glucosidase, Uniparc reference UPI000210C01C, Uniprot reference F5XL24. SEQ ID NO: 870: Amino acid sequence for Bifidobacterium asteroides glucosidase, Uniparc reference UPI00028BB1B6, Uniprot reference K4IPD2.

[0120] SEQ ID NO: 871: Amino acid sequence for Dactylellina haptotyla glucosidase, Uniparc reference UPI00035AE576, Uniprot reference S8BQ60. SEQ ID NO: 872: Amino acid sequence for Fusarium oxysporum glucosidase, Uniparc reference UPI0003F2D7DD, Uniprot reference W9JF54. SEQ ID NO: 873: Amino acid sequence for Bifidobacterium longum glucosidase, Uniparc reference UPI0005067FF9, Uniprot reference A0A087BRY2. SEQ ID NO: 874: Amino acid sequence for Bifidobacterium tsurumiense glucosidase, Uniparc reference UPI0004105550, Uniprot reference A0A087EK01. SEQ ID NO: 875: Amino acid sequence for Exophiala spinifera glucosidase, Uniparc reference UPI0005BF9DF9, Uniprot reference A0A0D1YG50. SEQ ID NO: 876: Amino acid sequence for Pyrenochaeta sp. glucosidase, Uniparc reference UPI0007CE7DBA, Uniprot reference A0A178E5I1. SEQ ID NO: 877: Amino acid sequence for a Firmicutes bacterial glucosidase, Uniparc reference UPI0009CDA3F1, Uniprot reference A0A1V6FLK2. SEQ ID NO: 878: Amino acid sequence for Cadophora sp. glucosidase, Uniparc reference UPI000D5A9E68, Uniprot reference A0A2V1B599. SEQ ID NO: 879: Amino acid sequence for Morchella conica glucosidase, Uniparc reference UPI000F7330CD, Uniprot reference A0A3N4L4M3. SEQ ID NO: 880: Amino acid sequence for Bifidobacterium animalis glucosidase, Uniparc reference UPI000189C68F, Uniprot reference B8DV42.

[0121] SEQ ID NO: 881: Amino acid sequence for Treponema azotonutricium glucosidase, Uniparc reference UPI00020ED2C9, Uniprot reference F5YGD5. SEQ ID NO: 882: Amino acid sequence for Acidipropionibacterium acidipropionici glucosidase, Uniparc reference UPI0002988429, Uniprot reference K7S0E5. SEQ ID NO: 883: Amino acid sequence for Salinispira pacifica glucosidase, Uniparc reference UPI0003D93613, Uniprot reference V5WNB6. SEQ ID NO: 884: Amino acid sequence for Bifidobacterium mongoliense glucosidase, Uniparc reference UPI000503588E, Uniprot reference A0A087C760. SEQ ID NO: 885: Amino acid sequence for Bifidobacterium indicum glucosidase, Uniparc reference UPI000499F5D1, Uniprot reference A0A087VUW4. SEQ ID NO: 886: Amino acid sequence for Brachyspira suanatina glucosidase, Uniparc reference UPI000659257F, Uniprot reference A0A0G4K5C2. SEQ ID NO: 887: Amino acid sequence for Phialocephala scopiformis glucosidase, Uniparc reference UPI0007F2FD44, Uniprot reference A0A194X8Q9. SEQ ID NO: 888: Amino acid sequence for uncultured Microbacterium sp. glucosidase, Uniparc reference UPI000A2BB4A5, Uniprot reference A0A1Y5P895. SEQ ID NO: 889: Amino acid sequence for Periconia macrospinosa glucosidase, Uniparc reference UPI000D5BC9BD, Uniprot reference A0A2V1E673. SEQ ID NO: 890: Amino acid sequence for Arthrobacter ulcerisalmonis glucosidase, Uniparc reference UPI000F3DC30B, Uniprot reference A0A3P5WTE2.

[0122] SEQ ID NO: 891: Amino acid sequence for Pseudarthrobacter chlorophenolicus glucosidase, Uniparc reference UPI0001664880, Uniprot reference B8H9A2. SEQ ID NO: 892: Amino acid sequence for Haloplasma contractile glucosidase, Uniparc reference UPI0002120C63, Uniprot reference F7PUF3. SEQ ID NO: 893: Amino acid sequence for Cochliobolus heterostrophus glucosidase, Uniparc reference UPI0002B73341, Uniprot reference M2ULB2. SEQ ID NO: 894: Amino acid sequence for Microbacterium sp. glucosidase, Uniparc reference UPI0003DE58EA, Uniprot reference W0Z818. SEQ ID NO: 895: Codon-optimized DNA encoding 6xHis Bifidobacterium actinocoloniiforme glucosidase, Uniparc reference UPI000503B26C, Uniprot reference A0A086YYS8. SEQ ID NO: 896: Codon-optimized DNA encoding 6xHis Bifidobacterium psychraerophilum glucosidase, Uniparc reference UPI000502B461, Uniprot reference A0A087CD28. SEQ ID NO: 897: Codon-optimized DNA encoding 6xHis Penicillium italicum glucosidase, Uniparc reference UPI00052B8681, Uniprot reference A0A0A2K704. SEQ ID NO: 898: Codon-optimized DNA encoding 6xHis Microbacterium trichothecenolyticum glucosidase, Uniparc reference UPI0005ED0AE6, Uniprot reference A0A0M2HDB3. SEQ ID NO: 899: Codon-optimized DNA encoding 6xHis uncultured Clostridium sp. glucosidase, Uniparc reference UPI0008206F38, Uniprot reference A0A1C6I6A3. SEQ ID NO: 900: Codon-optimized DNA encoding 6xHis Bifiguratus adelaidae glucosidase, Uniparc reference UPI000BC57319, Uniprot reference A0A261Y7Q8.

[0123] SEQ ID NO: 901: Codon-optimized DNA encoding 6xHis Paenibacillus thiaminolyticus glucosidase, Uniparc reference UPI000B3B73CD, Uniprot reference A0A378ZIK3. SEQ ID NO: 902: Codon-optimized DNA encoding 6xHis Microbacterium lemovicicum glucosidase, Uniparc reference UPI000F8FB9BA, Uniprot reference A0A3S9WE68. SEQ ID NO: 903: Codon-optimized DNA encoding 6xHis Rhodococcus erythropolis glucosidase, Uniparc reference UPI00019923DC, Uniprot reference C1A1N4. SEQ ID NO: 904: Codon-optimized DNA encoding 6xHis Cutibacterium avidum glucosidase, Uniparc reference UPI00022C19B5, Uniprot reference G4CZU8. SEQ ID NO: 905: Codon-optimized DNA encoding 6xHis Clavibacter michiganensis glucosidase, Uniparc reference UPI0002C5A938, Uniprot reference M5BD10. SEQ ID NO: 906: Codon-optimized DNA encoding 6xHis Microbacterium sp. glucosidase, Uniparc reference UPI0003DE3509, Uniprot reference W0ZC23. SEQ ID NO: 907: Codon-optimized DNA encoding 6xHis Bifidobacterium actinocoloniiforme glucosidase, Uniparc reference UPI000507F38A, Uniprot reference A0A086YZL4. SEQ ID NO: 908: Codon-optimized DNA encoding 6xHis Bifidobacterium reuteri glucosidase, Uniparc reference UPI000506119A, Uniprot reference A0A087CR26. SEQ ID NO: 909: Codon-optimized DNA encoding 6xHis Propionibacterium freudenreichii glucosidase, Uniparc reference UPI0005A5CAC2, Uniprot reference A0A0A8RX48. SEQ ID NO: 910: Codon-optimized DNA encoding 6xHis Microbacterium hydrocarbonoxydans glucosidase, Uniparc reference UPI0005EC18F9, Uniprot reference A0A0M2HRU0.

[0124] SEQ ID NO: 911: Codon-optimized DNA encoding 6xHis Pseudonocardia sp. glucosidase, Uniparc reference UPI00094B6D3B, Uniprot reference A0A1Q8KSV8. SEQ ID NO: 912: Codon-optimized DNA encoding 6xHis Tuber aestivum glucosidase, Uniparc reference UPI000BC13DF0, Uniprot reference A0A292PKV5. SEQ ID NO: 913: Codon-optimized DNA encoding 6xHis Propionibacterium australiense glucosidase, Uniparc reference UPI000E5B4EA5, Uniprot reference A0A383S7A9. SEQ ID NO: 914: Codon-optimized DNA encoding 6xHis Fusarium sp. glucosidase, Uniparc reference UPI001004B2C9, Uniprot reference A0A428T6E2. SEQ ID NO: 915: Codon-optimized DNA encoding 6xHis Nectria haematococca glucosidase, Uniparc reference UPI0001B69B5C, Uniprot reference C7ZBV0. SEQ ID NO: 916: Codon-optimized DNA encoding 6xHis Actinoplanes sp. glucosidase, Uniparc reference UPI00023EBB15, Uniprot reference G8S3E7. SEQ ID NO: 917: Codon-optimized DNA encoding 6xHis Streptomyces fulvissimus glucosidase, Uniparc reference UPI0003289BC6, Uniprot reference N0CMW2. SEQ ID NO: 918: Codon-optimized DNA encoding 6xHis Pestalotiopsis fici glucosidase, Uniparc reference UPI0003E02BF1, Uniprot reference W3WXF1. SEQ ID NO: 919: Codon-optimized DNA encoding 6xHis Bifidobacterium bohemicum glucosidase, Uniparc reference UPI0005025F2E, Uniprot reference A0A086ZGP0. SEQ ID NO: 920: Codon-optimized DNA encoding 6xHis Bifidobacterium saeculare glucosidase, Uniparc reference UPI0005084E52, Uniprot reference A0A087D0Q2.

[0125] SEQ ID NO: 921: Codon-optimized DNA encoding 6xHis Bionectria ochroleuca glucosidase, Uniparc reference UPI00059674D6, Uniprot reference A0A0B7JW24. SEQ ID NO: 922: Codon-optimized DNA encoding 6xHis Bifidobacterium pseudocatenulatum glucosidase, Uniparc reference UPI0006C6D6F9, Uniprot reference A0A174AU04. SEQ ID NO: 923: Codon-optimized DNA encoding 6xHis Pseudonocardia sp. glucosidase, Uniparc reference UPI00094ABAB3, Uniprot reference A0A1Q8LPB4. SEQ ID NO: 924: Codon-optimized DNA encoding 6xHis bacterial glucosidase, Uniparc reference UPI000CB49A0B, Uniprot reference A0A2H5Z8Q4. SEQ ID NO: 925: Codon-optimized DNA encoding 6xHis Coleophoma crateriformis glucosidase, Uniparc reference UPI000E38A995, Uniprot reference A0A3D8Q771. SEQ ID NO: 926: Codon-optimized DNA encoding 6xHis Arthrobotrys oligospora glucosidase, Uniparc reference UPI001102DFA3, Uniprot reference A0A4Z0Y5Y8. SEQ ID NO: 927: Codon-optimized DNA encoding 6xHis Pyrenophora teres glucosidase, Uniparc reference UPI0001ECDCBD, Uniprot reference E3RFS2. SEQ ID NO: 928: Codon-optimized DNA encoding 6xHis Gordonia polyisoprenivorans glucosidase, Uniparc reference UPI00024F2A26, Uniprot reference H6MTQ7. SEQ ID NO: 929: Codon-optimized DNA encoding 6xHis Stigmatella aurantiaca glucosidase, Uniparc reference UPI0000E28E5D, Uniprot reference Q08S21. SEQ ID NO: 930: Codon-optimized DNA encoding 6xHis Pestalotiopsis fici glucosidase, Uniparc reference UPI0003E03A52, Uniprot reference W3WZ03.

[0126] SEQ ID NO: 931: Codon-optimized DNA encoding 6xHis Bifidobacterium magnum glucosidase, Uniparc reference UPI0003B7B6EE, Uniprot reference A0A087BEN9. SEQ ID NO: 932: Codon-optimized DNA encoding 6xHis Bifidobacterium stellenboschense glucosidase, Uniparc reference UPI000503F283, Uniprot reference A0A087DFL8. SEQ ID NO: 933: Codon-optimized DNA encoding 6xHis Bionectria ochroleuca glucosidase, Uniparc reference UPI00059673EE, Uniprot reference A0A0B7K316. SEQ ID NO: 934: Codon-optimized DNA encoding 6xHis Hungatella hathewayi glucosidase, Uniparc reference UPI0006C069F3, Uniprot reference A0A174LVE3. SEQ ID NO: 935: Codon-optimized DNA encoding 6xHis Mycetocola reblochoni glucosidase, Uniparc reference UPI00097EB800, Uniprot reference A0A1R4J2F9. SEQ ID NO: 936: Codon-optimized DNA encoding 6xHis Nonomuraea sp. glucosidase, Uniparc reference UPI0009ABD7B3, Uniprot reference A0A2P9IX34. SEQ ID NO: 937: Codon-optimized DNA encoding 6xHis Coleophoma crateriformis glucosidase, Uniparc reference UPI000E391DF6, Uniprot reference A0A3D8T9C2. SEQ ID NO: 938: Codon-optimized DNA encoding 6xHis Paenarthrobacter aurescens glucosidase, Uniparc reference UPI0000EC83AE, Uniprot reference A1R2K1. SEQ ID NO: 939: Codon-optimized DNA encoding 6xHis Kitasatospora setae glucosidase, Uniparc reference UPI0001F21F08, Uniprot reference E4N4F6. SEQ ID NO: 940: Codon-optimized DNA encoding 6xHis Nocardiopsis alba glucosidase, Uniparc reference UPI00027E251E, Uniprot reference J7L3Z8.

[0127] SEQ ID NO: 941: Codon-optimized DNA encoding 6xHis Rhodococcus jostii glucosidase, Uniparc reference UPI0000DBA338, Uniprot reference Q0SCI4. SEQ ID NO: 942: Codon-optimized DNA encoding 6xHis uncultured microbial glucosidase, Uniparc reference UPI0003EC942D, Uniprot reference W5X324. SEQ ID NO: 943: Codon-optimized DNA encoding 6xHis Bifidobacterium merycicum glucosidase, Uniparc reference UPI0005084B63, Uniprot reference A0A087BJ88. SEQ ID NO: 944: Codon-optimized DNA encoding 6xHis Bifidobacterium scardovii glucosidase, Uniparc reference UPI0005018B5B, Uniprot reference A0A087DGT3. SEQ ID NO: 945: Codon-optimized DNA encoding 6xHis Fusarium oxysporum glucosidase, Uniparc reference UPI00021EC81E, Uniprot reference A0A0C4DJL4. SEQ ID NO: 946: Codon-optimized DNA encoding 6xHis Paraphaeosphaeria sporulosa glucosidase, Uniparc reference UPI0007CE199C, Uniprot reference A0A177BXH1. SEQ ID NO: 947: Codon-optimized DNA encoding 6xHis Clostridium oryzae glucosidase, Uniparc reference UPI0009A50F8D, Uniprot reference A0A1V4IY77. SEQ ID NO: 948: Codon-optimized DNA encoding 6xHis Corynespora cassiicola glucosidase, Uniparc reference UPI000D22F5EB, Uniprot reference A0A2T2N4T6. SEQ ID NO: 949: Codon-optimized DNA encoding 6xHis Choiromyces venosus glucosidase, Uniparc reference UPI000F7332F8, Uniprot reference A0A3N4J9R6. SEQ ID NO: 950: Codon-optimized DNA encoding 6xHis Saccharopolyspora erythraea glucosidase, Uniparc reference UPI0000F550EC, Uniprot reference A4F982.

[0128] SEQ ID NO: 951: Codon-optimized DNA encoding 6xHis Streptomyces venezuelae glucosidase, Uniparc reference UPI000204906E, Uniprot reference F2R0Y2. SEQ ID NO: 952: Codon-optimized DNA encoding 6xHis Arthrobacter sp. glucosidase, Uniparc reference UPI00027DFDB0, Uniprot reference J7LVB1. SEQ ID NO: 953: Codon-optimized DNA encoding 6xHis Thermobrachium celere glucosidase, Uniparc reference UPI00033420CB, Uniprot reference R7RMQ6. SEQ ID NO: 954: Codon-optimized DNA encoding 6xHis Drechslerella stenobrocha glucosidase, Uniparc reference UPI0003EA65B9, Uniprot reference W7HT93. SEQ ID NO: 955: Codon-optimized DNA encoding 6xHis Bifidobacterium minimum glucosidase, Uniparc reference UPI0003B38542, Uniprot reference A0A087BMR5. SEQ ID NO: 956: Codon-optimized DNA encoding 6xHis Bifidobacterium thermacidophilum glucosidase, Uniparc reference UPI000409825E, Uniprot reference A0A087E3K8. SEQ ID NO: 957: Codon-optimized DNA encoding 6xHis Verruconis gallopava glucosidase, Uniparc reference UPI0005C0229F, Uniprot reference A0A0D1X9G0. SEQ ID NO: 958: Codon-optimized DNA encoding 6xHis Stagonospora sp. glucosidase, Uniparc reference UPI0007CECAD5, Uniprot reference A0A178AER7. SEQ ID NO: 959: Codon-optimized DNA encoding a 6xHis Firmicutes bacterial glucosidase, Uniparc reference UPI0009D58C49, Uniprot reference A0A1V6ALF9. SEQ ID NO: 960: Codon-optimized DNA encoding 6xHis Tuber borchii glucosidase, Uniparc reference UPI000D50EED1, Uniprot reference A0A2T6ZNU7.

[0129] SEQ ID NO: 961: Codon-optimized DNA encoding 6xHis Morchella conica glucosidase, Uniparc reference UPI000F728167, Uniprot reference A0A3N4KUR3. SEQ ID NO: 962: Codon-optimized DNA encoding 6xHis Dictyoglomus thermophilum glucosidase, Uniparc reference UPI00018152EB, Uniprot reference B5YC96. SEQ ID NO: 963: Codon-optimized DNA encoding 6xHis Microlunatus phosphovorus glucosidase, Uniparc reference UPI000210C01C, Uniprot reference F5XL24. SEQ ID NO: 964: Codon-optimized DNA encoding 6xHis Bifidobacterium asteroides glucosidase, Uniparc reference UPI00028BB1B6, Uniprot reference K4IPD2. SEQ ID NO: 965: Codon-optimized DNA encoding 6xHis Dactylellina haptotyla glucosidase, Uniparc reference UPI00035AE576, Uniprot reference S8BQ60. SEQ ID NO: 966: Codon-optimized DNA encoding 6xHis Fusarium oxysporum glucosidase, Uniparc reference UPI0003F2D7DD, Uniprot reference W9JF54. SEQ ID NO: 967: Codon-optimized DNA encoding 6xHis Bifidobacterium longum glucosidase, Uniparc reference UPI0005067FF9, Uniprot reference A0A087BRY2. SEQ ID NO: 968: Codon-optimized DNA encoding 6xHis Bifidobacterium tsurumiense glucosidase, Uniparc reference UPI0004105550, Uniprot reference A0A087EK01. SEQ ID NO: 969: Codon-optimized DNA encoding 6xHis Exophiala spinifera glucosidase, Uniparc reference UPI0005BF9DF9, Uniprot reference A0A0D1YG50. SEQ ID NO: 970: Codon-optimized DNA encoding 6xHis Pyrenochaeta sp. glucosidase, Uniparc reference UPI0007CE7DBA, Uniprot reference A0A178E5I1.

[0130] SEQ ID NO: 971: Codon-optimized DNA encoding a 6xHis Firmicutes bacterial glucosidase, Uniparc reference UPI0009CDA3F1, Uniprot reference A0A1V6FLK2. SEQ ID NO: 972: Codon-optimized DNA encoding 6xHis Cadophora sp. glucosidase, Uniparc reference UPI000D5A9E68, Uniprot reference A0A2V1B599. SEQ ID NO: 973: Codon-optimized DNA encoding 6xHis Morchella conica glucosidase, Uniparc reference UPI000F7330CD, Uniprot reference A0A3N4L4M3. SEQ ID NO: 974: Codon-optimized DNA encoding 6xHis Bifidobacterium animalis glucosidase, Uniparc reference UPI000189C68F, Uniprot reference B8DV42. SEQ ID NO: 975: Codon-optimized DNA encoding 6xHis Treponema azotonutricium glucosidase, Uniparc reference UPI00020ED2C9, Uniprot reference F5YGD5. SEQ ID NO: 976: Codon-optimized DNA encoding 6xHis Acidipropionibacterium acidipropionici glucosidase, Uniparc reference UPI0002988429, Uniprot reference K7S0E5. SEQ ID NO: 977: Codon-optimized DNA encoding 6xHis Salinispira pacifica glucosidase, Uniparc reference UPI0003D93613, Uniprot reference V5WNB6. SEQ ID NO: 978: Codon-optimized DNA encoding 6xHis Bifidobacterium mongoliense glucosidase, Uniparc reference UPI000503588E, Uniprot reference A0A087C760. SEQ ID NO: 979: Codon-optimized DNA encoding 6xHis Bifidobacterium indicum glucosidase, Uniparc reference UPI000499F5D1, Uniprot reference A0A087VUW4. SEQ ID NO: 980: Codon-optimized DNA encoding 6xHis Brachyspira suanatina glucosidase, Uniparc reference UPI000659257F, Uniprot reference A0A0G4K5C2.

[0131] SEQ ID NO: 981: Codon-optimized DNA encoding 6xHis Phialocephala scopiformis glucosidase, Uniparc reference UPI0007F2FD44, Uniprot reference A0A194X8Q9. SEQ ID NO: 982: Codon-optimized DNA encoding 6xHis uncultured Microbacterium sp. glucosidase, Uniparc reference UPI000A2BB4A5, Uniprot reference A0A1Y5P895. SEQ ID NO: 983: Codon-optimized DNA encoding 6xHis Periconia macrospinosa glucosidase, Uniparc reference UPI000D5BC9BD, Uniprot reference A0A2V1E673. SEQ ID NO: 984: Codon-optimized DNA encoding 6xHis Arthrobacter ulcerisalmonis glucosidase, Uniparc reference UPI000F3DC30B, Uniprot reference A0A3P5WTE2. SEQ ID NO: 985: Codon-optimized DNA encoding 6xHis Pseudarthrobacter chlorophenolicus glucosidase, Uniparc reference UPI0001664880, Uniprot reference B8H9A2. SEQ ID NO: 986: Codon-optimized DNA encoding 6xHis Haloplasma contractile glucosidase, Uniparc reference UPI0002120C63, Uniprot reference F7PUF3. SEQ ID NO: 987: Codon-optimized DNA encoding 6xHis Cochliobolus heterostrophus glucosidase, Uniparc reference UPI0002B73341, Uniprot reference M2ULB2. SEQ ID NO: 988: Codon-optimized DNA encoding 6xHis Microbacterium sp. glucosidase, Uniparc reference UPI0003DE58EA, Uniprot reference W0Z818. SEQ ID NO: 989: Amino acid sequence for Rhodothermus marinus rhamnosidase, Uniparc reference UPI0001A31108, Uniprot reference D0MFR0. SEQ ID NO: 990: Amino acid sequence for Streptomyces bingchenggensis rhamnosidase, Uniparc reference UPI0001D90BFE, Uniprot reference D7C463.

[0132] SEQ ID NO: 991: Amino acid sequence for Spirosoma linguale rhamnosidase, Uniparc reference UPI0001A3AEAC, Uniprot reference D2QL60. SEQ ID NO: 992: Amino acid sequence for Roseburia intestinalis rhamnosidase, Uniparc reference UPI0001CD6D48, Uniprot reference D4L2K8. SEQ ID NO: 993: Amino acid sequence for Draconibacterium orientale rhamnosidase, Uniparc reference UPI000442EF22, Uniprot reference X5DG83. SEQ ID NO: 994: Amino acid sequence for Catenulispora acidiphila rhamnosidase, Uniparc reference UPI00019E052B, Uniprot reference C7QC24. SEQ ID NO: 995: Amino acid sequence for Bacteroides thetaiotaomicron rhamnosidase, Uniparc reference UPI000005ADE1, Uniprot reference Q8A916. SEQ ID NO: 996: Amino acid sequence for Opitutus terrae rhamnosidase, Uniparc reference UPI000172B2E3, Uniprot reference B1ZRE4. SEQ ID NO: 997: Amino acid sequence for Lachnoclostridium phytofermentans rhamnosidase, Uniparc reference UPI00015FE0BE, Uniprot reference A9KJP8. SEQ ID NO: 998: Amino acid sequence for Rhodanobacter denitrificans rhamnosidase, Uniparc reference UPI00022DA3ED, Uniprot reference M4NH01. SEQ ID NO: 999: Amino acid sequence for Prevotella ruminicola rhamnosidase, Uniparc reference UPI0001D07633, Uniprot reference D5ETD9. SEQ ID NO: 1000: Amino acid sequence for Aspergillus terreus rhamnosidase, Uniparc reference UPI000259E388, Uniprot reference I0AZ41.

[0133] SEQ ID NO: 1001: Amino acid sequence for Brachybacterium faecium rhamnosidase, Uniparc reference UPI0001A461B6, Uniprot reference C7MA58. SEQ ID NO: 1002: Amino acid sequence for Flavobacterium johnsoniae rhamnosidase, Uniparc reference UPI00006E4A0D, Uniprot reference A5FCH3. SEQ ID NO: 1003: Amino acid sequence for Rahnella aquatilis rhamnosidase, Uniparc reference UPI000245C507, Uniprot reference H2IYR2. SEQ ID NO: 1004: Amino acid sequence for Bifidobacterium moukalabense rhamnosidase, Uniparc reference UPI0003E4E711, Uniprot reference W4N6H0. SEQ ID NO: 1005: Amino acid sequence for Enterococcus casseliflavus rhamnosidase, Uniparc reference UPI000353BD9F, Uniprot reference S4BBS9. SEQ ID NO: 1006: Amino acid sequence for Geobacillus sp. rhamnosidase, Uniparc reference UPI0001789C0D, Uniprot reference D3EED1. SEQ ID NO: 1007: Amino acid sequence for Modestobacter marinus rhamnosidase, Uniparc reference UPI000260A2FE, Uniprot reference I4EYD9. SEQ ID NO: 1008: Amino acid sequence for Pedobacter heparinus rhamnosidase, Uniparc reference UPI00019EF0EE, Uniprot reference C6XYM6. SEQ ID NO: 1009: Amino acid sequence for Dyadobacter fermentans rhamnosidase, Uniparc reference UPI00019B5915, Uniprot reference C6VZL3. SEQ ID NO: 1010: Amino acid sequence for Paenibacillus mucilaginosus rhamnosidase, Uniparc reference UPI000212C156, Uniprot reference F8FQQ3.

[0134] SEQ ID NO: 1011: Amino acid sequence for Paenibacillus sp. rhamnosidase, Uniparc reference UPI0003E2544D, Uniprot reference W4D866. SEQ ID NO: 1012: Amino acid sequence for Bacteroides thetaiotaomicron rhamnosidase, Uniparc reference UPI000005ADD2, Uniprot reference A0A0P0FM19. SEQ ID NO: 1013: Amino acid sequence for Chloroflexus aurantiacus rhamnosidase, Uniparc reference UPI00005BA60B, Uniprot reference A9WDK5. SEQ ID NO: 1014: Amino acid sequence for Thermoclostridium stercorarium rhamnosidase, Uniparc reference UPI00000B098C, Uniprot reference Q9S3L0. SEQ ID NO: 1015: Amino acid sequence for Bifidobacterium moukalabense rhamnosidase, Uniparc reference UPI0003E5C314, Uniprot reference W4N6I2. SEQ ID NO: 1016: Amino acid sequence for Olsenella profusa rhamnosidase, Uniparc reference UPI0003AE032C, Uniprot reference U2USP4. SEQ ID NO: 1017: Amino acid sequence for Kribbella flavida rhamnosidase, Uniparc reference UPI00019BDB13, Uniprot reference D2PMT5. SEQ ID NO: 1018: Amino acid sequence for Caulobacter vibrioides rhamnosidase, Uniparc reference UPI00000C7226, Uniprot reference Q9A9K2. SEQ ID NO: 1019: Amino acid sequence for Bacteroides thetaiotaomicron rhamnosidase, Uniparc reference UPI000005BA09, Uniprot reference Q8A076. SEQ ID NO: 1020: Amino acid sequence for Rhodonellum psychrophilum rhamnosidase, Uniparc reference UPI0003745394, Uniprot reference U5BUY4.

[0135] SEQ ID NO: 1021: Amino acid sequence for Paenibacillus sp. rhamnosidase, Uniparc reference UPI0004F6D660, Uniprot reference A0A089M3T2. SEQ ID NO: 1022: Amino acid sequence for Catenovulum agarivorans rhamnosidase, Uniparc reference UPI0003ED860D, Uniprot reference W7QMH5. SEQ ID NO: 1023: Amino acid sequence for Zobellia galactanivorans rhamnosidase, Uniparc reference UPI000217D8B1, Uniprot reference G0L382. SEQ ID NO: 1024: Amino acid sequence for Bacteroides thetaiotaomicron rhamnosidase, Uniparc reference UPI000005B845, Uniprot reference Q8A1H5. SEQ ID NO: 1025: Amino acid sequence for Bacteroides xylanisolvens rhamnosidase, Uniparc reference UPI0001CD02E9, Uniprot reference D6CYE5. SEQ ID NO: 1026: Amino acid sequence for Pseudarthrobacter chlorophenolicus rhamnosidase, Uniparc reference UPI00018E07C6, Uniprot reference B8HAH3. SEQ ID NO: 1027: Amino acid sequence for Dictyoglomus thermophilum rhamnosidase, Uniparc reference UPI0001815896, Uniprot reference B5YC64. SEQ ID NO: 1028: Amino acid sequence for Formosa agariphila rhamnosidase, Uniparc reference UPI00039231C1, Uniprot reference T2KPL4. SEQ ID NO: 1029: Amino acid sequence for Rhodococcus jostii rhamnosidase, Uniparc reference UPI0000DBA6EB, Uniprot reference Q0S9T4. SEQ ID NO: 1030: Amino acid sequence for Lactobacillus crispatus rhamnosidase, Uniparc reference UPI0001D10896, Uniprot reference D5GZ45.

[0136] SEQ ID NO: 1031: Amino acid sequence for Pedobacter heparinus rhamnosidase, Uniparc reference UPI0001B17DE6, Uniprot reference C6XVU2. SEQ ID NO: 1032: Amino acid sequence for Spirosoma linguale rhamnosidase, Uniparc reference UPI0001A3C989, Uniprot reference D2QUA5. SEQ ID NO: 1033: Amino acid sequence for Pedobacter heparinus rhamnosidase, Uniparc reference UPI00019EE3EB, Uniprot reference C6XU05. SEQ ID NO: 1034: Amino acid sequence for Paenibacillus mucilaginosus rhamnosidase, Uniparc reference UPI0003432C11, Uniprot reference R9ULQ4. SEQ ID NO: 1035: Amino acid sequence for Caulobacter segnis rhamnosidase, Uniparc reference UPI0001BC0C05, Uniprot reference D5VGD9. SEQ ID NO: 1036: Amino acid sequence for Bacteroides cellulosilyticus rhamnosidase, Uniparc reference UPI0001969377, Uniprot reference E2N9B1. SEQ ID NO: 1037: Amino acid sequence for Pedobacter heparinus rhamnosidase, Uniparc reference UPI0001B17C60, Uniprot reference C6Y153. SEQ ID NO: 1038: Amino acid sequence for Formosa agariphila rhamnosidase, Uniparc reference UPI000571C0C2, Uniprot reference T2KNB2. SEQ ID NO: 1039: Amino acid sequence for Lactobacillus acidophilus rhamnosidase, Uniparc reference UPI00004C6D41, Uniprot reference Q5FJ31. SEQ ID NO: 1040: Amino acid sequence for Rhodopirellula baltica rhamnosidase, Uniparc reference UPI00001AC07D, Uniprot reference Q7UYD5.

[0137] SEQ ID NO: 1041: Amino acid sequence for Frankia inefficax rhamnosidase, Uniparc reference UPI0001BF9A6C, Uniprot reference E3IY10. SEQ ID NO: 1042: Amino acid sequence for Streptomyces scabiei rhamnosidase, Uniparc reference UPI0001B7FF91, Uniprot reference C9Z376. SEQ ID NO: 1043: Amino acid sequence for Flavobacterium johnsoniae rhamnosidase, Uniparc reference UPI00006E5F74, Uniprot reference A5FC22. SEQ ID NO: 1044: Amino acid sequence for Streptomyces sp. rhamnosidase, Uniparc reference UPI00034E666D, Uniprot reference S2YWB5. SEQ ID NO: 1045: Amino acid sequence for Acidobacterium capsulatum rhamnosidase, Uniparc reference UPI000198DF25, Uniprot reference C1F149. SEQ ID NO: 1046: Amino acid sequence for Catenovulum agarivorans rhamnosidase, Uniparc reference UPI0003ED82D3, Uniprot reference W7QYP5. SEQ ID NO: 1047: Amino acid sequence for Brachybacterium faecium rhamnosidase, Uniparc reference UPI0001A461B7, Uniprot reference C7MA59. SEQ ID NO: 1048: Amino acid sequence for Klebsiella oxytoca rhamnosidase, Uniparc reference UPI000243A177, Uniprot reference A0A0J9X262. SEQ ID NO: 1049: Amino acid sequence for Chitinophaga pinensis rhamnosidase, Uniparc reference UPI0001B24769, Uniprot reference C7PA70. SEQ ID NO: 1050: Amino acid sequence for Streptomyces bottropensis rhamnosidase, Uniparc reference UPI0002BCAF6B, Uniprot reference M3FYL9.

[0138] SEQ ID NO: 1051: Amino acid sequence for Subdoligranulum variabile rhamnosidase, Uniparc reference UPI0001966B28, Uniprot reference D1PKC7. SEQ ID NO: 1052: Amino acid sequence for Microbacterium testaceum rhamnosidase, Uniparc reference UPI0001F8A51D, Uniprot reference E8NDD8. SEQ ID NO: 1053: Amino acid sequence for Solibacter usitatus rhamnosidase, Uniparc reference UPI000053767B, Uniprot reference Q01V09. SEQ ID NO: 1054: Amino acid sequence for Streptosporangium roseum rhamnosidase, Uniparc reference UPI0001A3EFE6, Uniprot reference D2B240. SEQ ID NO: 1055: Amino acid sequence for Alphaproteobacterium rhamnosidase, Uniparc reference UPI0006CE1E82, Uniprot reference A0A0N1BME3. SEQ ID NO: 1056: Amino acid sequence for Solitalea canadensis rhamnosidase, Uniparc reference UPI000247229E, Uniprot reference H8KPI7. SEQ ID NO: 1057: Amino acid sequence for Parabacteroides goldsteinii rhamnosidase, Uniparc reference UPI0002CB9583, Uniprot reference S0GSF0. SEQ ID NO: 1058: Amino acid sequence for Cyclobacterium marinum rhamnosidase, Uniparc reference UPI00021B9B33, Uniprot reference G0J630. SEQ ID NO: 1059: Amino acid sequence for Solibacter usitatus rhamnosidase, Uniparc reference UPI0000533669, Uniprot reference Q01TX2. SEQ ID NO: 1060: Amino acid sequence for Lunatimonas lonarensis rhamnosidase, Uniparc reference UPI00032D8F6D, Uniprot reference R7ZW70.

[0139] SEQ ID NO: 1061: Amino acid sequence for Rhizobium leguminosarum rhamnosidase, Uniparc reference UPI0000D713F2, Uniprot reference Q1M7P3. SEQ ID NO: 1062: Amino acid sequence for Streptosporangium roseum rhamnosidase, Uniparc reference UPI0001A3EBEB, Uniprot reference D2AYU9. SEQ ID NO: 1063: Amino acid sequence for Parabacteroides distasonis rhamnosidase, Uniparc reference UPI000156F115, Uniprot reference A6LBL4. SEQ ID NO: 1064: Amino acid sequence for a Lachnospiraceae bacterial rhamnosidase, Uniparc reference UPI0003375A10, Uniprot reference R9K6L6. SEQ ID NO: 1065: Amino acid sequence for Chitinophaga pinensis rhamnosidase, Uniparc reference UPI0001A2F0FA, Uniprot reference C7P9Y8. SEQ ID NO: 1066: Amino acid sequence for Caulobacter segnis rhamnosidase, Uniparc reference UPI0001BC0C15, Uniprot reference D5VGC3. SEQ ID NO: 1067: Amino acid sequence for Pedobacter heparinus rhamnosidase, Uniparc reference UPI00019EF6E1, Uniprot reference C6Y145. SEQ ID NO: 1068: Amino acid sequence for Pedobacter heparinus rhamnosidase, Uniparc reference UPI00019EE1A9, Uniprot reference C6Y2X3. SEQ ID NO: 1069: Amino acid sequence for a Deltaproteobacteria bacterial rhamnosidase, Uniparc reference UPI000C8D4928, Uniprot reference A0A2D5SK32. SEQ ID NO: 1070: Amino acid sequence for Thermobaculum terrenum rhamnosidase, Uniparc reference UPI00019BFDCE, Uniprot reference D1CHL4.

[0140] SEQ ID NO: 1071: Amino acid sequence for Opitutus terrae rhamnosidase, Uniparc reference UPI000172B62A, Uniprot reference B1ZY35. SEQ ID NO: 1072: Amino acid sequence for Kribbella flavida rhamnosidase, Uniparc reference UPI00019BFABB, Uniprot reference D2PXQ4. SEQ ID NO: 1073: Amino acid sequence for Streptomyces scabiei rhamnosidase, Uniparc reference UPI0001B80091, Uniprot reference C9Z391. SEQ ID NO: 1074: Amino acid sequence for Actinoplanes sp. rhamnosidase, Uniparc reference UPI00023EC5D1, Uniprot reference G8S540. SEQ ID NO: 1075: Amino acid sequence for Asticcacaulis sp. rhamnosidase, Uniparc reference UPI0003C3CD2B, Uniprot reference V4NSJ1. SEQ ID NO: 1076: Amino acid sequence for Kribbella flavida rhamnosidase, Uniparc reference UPI00019BF65D, Uniprot reference D2PT74. SEQ ID NO: 1077: Amino acid sequence for Bacillus sp. rhamnosidase, Uniparc reference UPI00000BC760, Uniprot reference Q93RE7. SEQ ID NO: 1078: Amino acid sequence for Flavobacterium johnsoniae rhamnosidase, Uniparc reference UPI00006E5FAB, Uniprot reference A5FCG3. SEQ ID NO: 1079: Amino acid sequence for Lunatimonas lonarensis rhamnosidase, Uniparc reference UPI00032EEB9C, Uniprot reference R7ZS84. SEQ ID NO: 1080: Amino acid sequence for Eisenbergiella massiliensis rhamnosidase, Uniparc reference UPI0004B2D794, Uniprot reference A0A3E3IGR6.

[0141] SEQ ID NO: 1081: Amino acid sequence for Catenovulum agarivorans rhamnosidase, Uniparc reference UPI0003ED7515, Uniprot reference W7QF25. SEQ ID NO: 1083: Codon-optimized DNA encoding 6xHis Rhodothermus marinus rhamnosidase, Uniparc reference UPI0001A31108, Uniprot reference D0MFR0. SEQ ID NO: 1084: Codon-optimized DNA encoding 6xHis Streptomyces bingchenggensis rhamnosidase, Uniparc reference UPI0001D90BFE, Uniprot reference D7C463. SEQ ID NO: 1085: Codon-optimized DNA encoding 6xHis Spirosoma linguale rhamnosidase, Uniparc reference UPI0001A3AEAC, Uniprot reference D2QL60. SEQ ID NO: 1086: Codon-optimized DNA encoding 6xHis Roseburia intestinalis rhamnosidase, Uniparc reference UPI0001CD6D48, Uniprot reference D4L2K8. SEQ ID NO: 1087: Codon-optimized DNA encoding 6xHis Draconibacterium orientale rhamnosidase, Uniparc reference UPI000442EF22, Uniprot reference X5DG83. SEQ ID NO: 1088: Codon-optimized DNA encoding 6xHis Catenulispora acidiphila rhamnosidase, Uniparc reference UPI00019E052B, Uniprot reference C7QC24. SEQ ID NO: 1089: Codon-optimized DNA encoding 6xHis Bacteroides thetaiotaomicron rhamnosidase, Uniparc reference UPI000005ADE1, Uniprot reference Q8A916. SEQ ID NO: 1090: Codon-optimized DNA encoding 6xHis Opitutus terrae rhamnosidase, Uniparc reference UPI000172B2E3, Uniprot reference B1ZRE4.

[0142] SEQ ID NO: 1091: Codon-optimized DNA encoding 6xHis Lachnoclostridium phytofermentans rhamnosidase, Uniparc reference UPI00015FE0BE, Uniprot reference A9KJP8. SEQ ID NO: 1092: Codon-optimized DNA encoding 6xHis Rhodanobacter denitrificans rhamnosidase, Uniparc reference UPI00022DA3ED, Uniprot reference M4NH01. SEQ ID NO: 1093: Codon-optimized DNA encoding 6xHis Prevotella ruminicola rhamnosidase, Uniparc reference UPI0001D07633, Uniprot reference D5ETD9. SEQ ID NO: 1094: Codon-optimized DNA encoding 6xHis Aspergillus terreus rhamnosidase, Uniparc reference UPI000259E388, Uniprot reference I0AZ41. SEQ ID NO: 1095: Codon-optimized DNA encoding 6xHis Brachybacterium faecium rhamnosidase, Uniparc reference UPI0001A461B6, Uniprot reference C7MA58. SEQ ID NO: 1096: Codon-optimized DNA encoding 6xHis Flavobacterium johnsoniae rhamnosidase, Uniparc reference UPI00006E4A0D, Uniprot reference A5FCH3. SEQ ID NO: 1097: Codon-optimized DNA encoding 6xHis Rahnella aquatilis rhamnosidase, Uniparc reference UPI000245C507, Uniprot reference H2IYR2. SEQ ID NO: 1098: Codon-optimized DNA encoding 6xHis Bifidobacterium moukalabense rhamnosidase, Uniparc reference UPI0003E4E711, Uniprot reference W4N6H0. SEQ ID NO: 1099: Codon-optimized DNA encoding 6xHis Enterococcus casseliflavus rhamnosidase, Uniparc reference UPI000353BD9F, Uniprot reference S4BBS9. SEQ ID NO: 1100: Codon-optimized DNA encoding 6xHis Geobacillus sp. rhamnosidase, Uniparc reference UPI0001789C0D, Uniprot reference D3EED1.

[0143] SEQ ID NO: 1101: Codon-optimized DNA encoding 6xHis Modestobacter marinus rhamnosidase, Uniparc reference UPI000260A2FE, Uniprot reference I4EYD9. SEQ ID NO: 1102: Codon-optimized DNA encoding 6×His Pedobacter heparinus rhamnosidase, Uniparc reference UPI00019EF0EE, Uniprot reference C6XYM6. SEQ ID NO: 1103: Codon-optimized DNA encoding 6xHis Dyadobacter fermentans rhamnosidase, Uniparc reference UPI00019B5915, Uniprot reference C6VZL3. SEQ ID NO: 1104: Codon-optimized DNA encoding 6xHis Paenibacillus mucilaginosus rhamnosidase, Uniparc reference UPI000212C156, Uniprot reference F8FQQ3. SEQ ID NO: 1105: Codon-optimized DNA encoding 6xHis Paenibacillus sp. rhamnosidase, Uniparc reference UPI0003E2544D, Uniprot reference W4D866. SEQ ID NO: 1106: Codon-optimized DNA encoding 6xHis Bacteroides thetaiotaomicron rhamnosidase, Uniparc reference UPI000005ADD2, Uniprot reference A0A0P0FM19. SEQ ID NO: 1107: Codon-optimized DNA encoding 6xHis Chloroflexus aurantiacus rhamnosidase, Uniparc reference UPI00005BA60B, Uniprot reference A9WDK5. SEQ ID NO: 1108: Codon-optimized DNA encoding 6xHis Thermoclostridium stercorarium rhamnosidase, Uniparc reference UPI00000B098C, Uniprot reference Q9S3L0. SEQ ID NO: 1109: Codon-optimized DNA encoding 6xHis Bifidobacterium moukalabense rhamnosidase, Uniparc reference UPI0003E5C314, Uniprot reference W4N6I2. SEQ ID NO: 1110: Codon-optimized DNA encoding 6xHis Olsenella profusa rhamnosidase, Uniparc reference UPI0003AE032C, Uniprot reference U2USP4.

[0144] SEQ ID NO: 1111: Codon-optimized DNA encoding 6xHis Kribbella flavida rhamnosidase, Uniparc reference UPI00019BDB13, Uniprot reference D2PMT5. SEQ ID NO: 1112: Codon-optimized DNA encoding 6xHis Caulobacter vibrioides rhamnosidase, Uniparc reference UPI00000C7226, Uniprot reference Q9A9K2. SEQ ID NO: 1113: Codon-optimized DNA encoding 6xHis Bacteroides thetaiotaomicron rhamnosidase, Uniparc reference UPI000005BA09, Uniprot reference Q8A076. SEQ ID NO: 1114: Codon-optimized DNA encoding 6xHis Rhodonellum psychrophilum rhamnosidase, Uniparc reference UPI0003745394, Uniprot reference U5BUY4. SEQ ID NO: 1115: Codon-optimized DNA encoding 6xHis Paenibacillus sp. rhamnosidase, Uniparc reference UPI0004F6D660, Uniprot reference A0A089M3T2. SEQ ID NO: 1116: Codon-optimized DNA encoding 6xHis Catenovulum agarivorans rhamnosidase, Uniparc reference UPI0003ED860D, Uniprot reference W7QMH5. SEQ ID NO: 1117: Codon-optimized DNA encoding 6xHis Zobellia galactanivorans rhamnosidase, Uniparc reference UPI000217D8B1, Uniprot reference G0L382. SEQ ID NO: 1118: Codon-optimized DNA encoding 6xHis Bacteroides thetaiotaomicron rhamnosidase, Uniparc reference UPI000005B845, Uniprot reference Q8A1H5. SEQ ID NO: 1119: Codon-optimized DNA encoding 6xHis Bacteroides xylanisolvens rhamnosidase, Uniparc reference UPI0001CD02E9, Uniprot reference D6CYE5. SEQ ID NO: 1120: Codon-optimized DNA encoding 6xHis Pseudarthrobacter chlorophenolicus rhamnosidase, Uniparc reference UPI00018E07C6, Uniprot reference B8HAH3.

[0145] SEQ ID NO: 1121: Codon-optimized DNA encoding 6xHis Dictyoglomus thermophilum rhamnosidase, Uniparc reference UPI0001815896, Uniprot reference B5YC64. SEQ ID NO: 1122: Codon-optimized DNA encoding 6xHis Formosa agariphila rhamnosidase, Uniparc reference UPI00039231C1, Uniprot reference T2KPL4. SEQ ID NO: 1123: Codon-optimized DNA encoding 6xHis Rhodococcus jostii rhamnosidase, Uniparc reference UPI0000DBA6EB, Uniprot reference Q0S9T4. SEQ ID NO: 1124: Codon-optimized DNA encoding 6xHis Lactobacillus crispatus rhamnosidase, Uniparc reference UPI0001D10896, Uniprot reference D5GZ45. SEQ ID NO: 1125: Codon-optimized DNA encoding 6xHis Pedobacter heparinus rhamnosidase, Uniparc reference UPI0001B17DE6, Uniprot reference C6XVU2. SEQ ID NO: 1126: Codon-optimized DNA encoding 6xHis Spirosoma linguale rhamnosidase, Uniparc reference UPI0001A3C989, Uniprot reference D2QUA5. SEQ ID NO: 1127: Codon-optimized DNA encoding 6xHis Pedobacter heparinus rhamnosidase, Uniparc reference UPI00019EE3EB, Uniprot reference C6XU05. SEQ ID NO: 1128: Codon-optimized DNA encoding 6xHis Paenibacillus mucilaginosus rhamnosidase, Uniparc reference UPI0003432C11, Uniprot reference R9ULQ4. SEQ ID NO: 1129: Codon-optimized DNA encoding 6xHis Caulobacter segnis rhamnosidase, Uniparc reference UPI0001BC0C05, Uniprot reference D5VGD9. SEQ ID NO: 1130: Codon-optimized DNA encoding 6xHis Bacteroides cellulosilyticus rhamnosidase, Uniparc reference UPI0001969377, Uniprot reference E2N9B1.

[0146] SEQ ID NO: 1131: Codon-optimized DNA encoding 6xHis Pedobacter heparinus rhamnosidase, Uniparc reference UPI0001B17C60, Uniprot reference C6Y153. SEQ ID NO: 1132: Codon-optimized DNA encoding 6xHis Formosa agariphila rhamnosidase, Uniparc reference UPI000571C0C2, Uniprot reference T2KNB2. SEQ ID NO: 1133: Codon-optimized DNA encoding 6xHis Lactobacillus acidophilus rhamnosidase, Uniparc reference UPI00004C6D41, Uniprot reference Q5FJ31. SEQ ID NO: 1134: Codon-optimized DNA encoding 6xHis Rhodopirellula baltica rhamnosidase, Uniparc reference UPI00001AC07D, Uniprot reference Q7UYD5. SEQ ID NO: 1135: Codon-optimized DNA encoding 6xHis Frankia inefficax rhamnosidase, Uniparc reference UPI0001BF9A6C, Uniprot reference E3IY10. SEQ ID NO: 1136: Codon-optimized DNA encoding 6xHis Streptomyces scabiei rhamnosidase, Uniparc reference UPI0001B7FF91, Uniprot reference C9Z376. SEQ ID NO: 1137: Codon-optimized DNA encoding 6xHis Flavobacterium johnsoniae rhamnosidase, Uniparc reference UPI00006E5F74, Uniprot reference A5FC22. SEQ ID NO: 1138: Codon-optimized DNA encoding 6xHis Streptomyces sp. rhamnosidase, Uniparc reference UPI00034E666D, Uniprot reference S2YWB5. SEQ ID NO: 1139: Codon-optimized DNA encoding 6xHis Acidobacterium capsulatum rhamnosidase, Uniparc reference UPI000198DF25, Uniprot reference C1F149. SEQ ID NO: 1140: Codon-optimized DNA encoding 6xHis Catenovulum agarivorans rhamnosidase, Uniparc reference UPI0003ED82D3, Uniprot reference W7QYP5.

[0147] SEQ ID NO: 1141: Codon-optimized DNA encoding 6xHis Brachybacterium faecium rhamnosidase, Uniparc reference UPI0001A461B7, Uniprot reference C7MA59. SEQ ID NO: 1142: Codon-optimized DNA encoding 6xHis Klebsiella oxytoca rhamnosidase, Uniparc reference UPI000243A177, Uniprot reference A0A0J9X262. SEQ ID NO: 1143: Codon-optimized DNA encoding 6xHis Chitinophaga pinensis rhamnosidase, Uniparc reference UPI0001B24769, Uniprot reference C7PA70. SEQ ID NO: 1144: Codon-optimized DNA encoding 6xHis Streptomyces bottropensis rhamnosidase, Uniparc reference UPI0002BCAF6B, Uniprot reference M3FYL9. SEQ ID NO: 1145: Codon-optimized DNA encoding 6xHis Subdoligranulum variabile rhamnosidase, Uniparc reference UPI0001966B28, Uniprot reference D1PKC7. SEQ ID NO: 1146: Codon-optimized DNA encoding 6xHis Microbacterium testaceum rhamnosidase, Uniparc reference UPI0001F8A51D, Uniprot reference E8NDD8. SEQ ID NO: 1147: Codon-optimized DNA encoding 6xHis Solibacter usitatus rhamnosidase, Uniparc reference UPI000053767B, Uniprot reference Q01V09. SEQ ID NO: 1148: Codon-optimized DNA encoding 6xHis Streptosporangium roseum rhamnosidase, Uniparc reference UPI0001A3EFE6, Uniprot reference D2B240. SEQ ID NO: 1149: Codon-optimized DNA encoding 6xHis alpha Proteobacterium rhamnosidase, Uniparc reference UPI0006CE1E82, Uniprot reference A0A0N1BME3. SEQ ID NO: 1150: Codon-optimized DNA encoding 6xHis Solitalea canadensis rhamnosidase, Uniparc reference UPI000247229E, Uniprot reference H8KPI7.

[0148] SEQ ID NO: 1151: Codon-optimized DNA encoding 6xHis Parabacteroides goldsteinii rhamnosidase, Uniparc reference UPI0002CB9583, Uniprot reference S0GSF0. SEQ ID NO: 1152: Codon-optimized DNA encoding 6xHis Cyclobacterium marinum rhamnosidase, Uniparc reference UPI00021B9B33, Uniprot reference G0J630. SEQ ID NO: 1153: Codon-optimized DNA encoding 6xHis Solibacter usitatus rhamnosidase, Uniparc reference UPI0000533669, Uniprot reference Q01TX2. SEQ ID NO: 1154: Codon-optimized DNA encoding 6xHis Lunatimonas lonarensis rhamnosidase, Uniparc reference UPI00032D8F6D, Uniprot reference R7ZW70. SEQ ID NO: 1155: Codon-optimized DNA encoding 6xHis Rhizobium leguminosarum rhamnosidase, Uniparc reference UPI0000D713F2, Uniprot reference Q1M7P3. SEQ ID NO: 1156: Codon-optimized DNA encoding 6xHis Streptosporangium roseum rhamnosidase, Uniparc reference UPI0001A3EBEB, Uniprot reference D2AYU9. SEQ ID NO: 1157: Codon-optimized DNA encoding 6xHis Parabacteroides distasonis rhamnosidase, Uniparc reference UPI000156F115, Uniprot reference A6LBL4. SEQ ID NO: 1158: Codon-optimized DNA encoding 6xHis Lachnospiraceae bacterial rhamnosidase, Uniparc reference UPI0003375A10, Uniprot reference R9K6L6. SEQ ID NO: 1159: Codon-optimized DNA encoding 6xHis Chitinophaga pinensis rhamnosidase, Uniparc reference UPI0001A2F0FA, Uniprot reference C7P9Y8. SEQ ID NO: 1160: Codon-optimized DNA encoding 6xHis Caulobacter segnis rhamnosidase, Uniparc reference UPI0001BC0C15, Uniprot reference D5VGC3.

[0149] SEQ ID NO: 1161: Codon-optimized DNA encoding 6xHis Pedobacter heparinus rhamnosidase, Uniparc reference UPI00019EF6E1, Uniprot reference C6Y145. SEQ ID NO: 1162: Codon-optimized DNA encoding 6xHis Pedobacter heparinus rhamnosidase, Uniparc reference UPI00019EE1A9, Uniprot reference C6Y2X3. SEQ ID NO: 1163: Codon-optimized DNA encoding 6xHis Deltaproteobacteria bacterial rhamnosidase, Uniparc reference UPI000C8D4928, Uniprot reference A0A2D5SK32. SEQ ID NO: 1164: Codon-optimized DNA encoding 6xHis Thermobaculum terrenum rhamnosidase, Uniparc reference UPI00019BFDCE, Uniprot reference D1CHL4. SEQ ID NO: 1165: Codon-optimized DNA encoding 6xHis Opitutus terrae rhamnosidase, Uniparc reference UPI000172B62A, Uniprot reference B1ZY35. SEQ ID NO: 1166: Codon-optimized DNA encoding 6xHis Kribbella flavida rhamnosidase, Uniparc reference UPI00019BFABB, Uniprot reference D2PXQ4. SEQ ID NO: 1167: Codon-optimized DNA encoding 6xHis Streptomyces scabiei rhamnosidase, Uniparc reference UPI0001B80091, Uniprot reference C9Z391. SEQ ID NO: 1168: Codon-optimized DNA encoding 6xHis Actinoplanes sp. rhamnosidase, Uniparc reference UPI00023EC5D1, Uniprot reference G8S540. SEQ ID NO: 1169: Codon-optimized DNA encoding 6xHis Asticcacaulis sp. rhamnosidase, Uniparc reference UPI0003C3CD2B, Uniprot reference V4NSJ1. SEQ ID NO: 1170: Codon-optimized DNA encoding 6xHis Kribbella flavida rhamnosidase, Uniparc reference UPI00019BF65D, Uniprot reference D2PT74.

[0150] SEQ ID NO: 1171: Codon-optimized DNA encoding 6xHis Bacillus sp. rhamnosidase, Uniparc reference UPI00000BC760, Uniprot reference Q93RE7. SEQ ID NO: 1172: Codon-optimized DNA encoding 6xHis Flavobacterium johnsoniae rhamnosidase, Uniparc reference UPI00006E5FAB, Uniprot reference A5FCG3. SEQ ID NO: 1173: Codon-optimized DNA encoding 6xHis Lunatimonas lonarensis rhamnosidase, Uniparc reference UPI00032EEB9C, Uniprot reference R7ZS84. SEQ ID NO: 1174: Codon-optimized DNA encoding 6xHis Eisenbergiella massiliensis rhamnosidase, Uniparc reference UPI0004B2D794, Uniprot reference A0A3E3IGR6. SEQ ID NO: 1175: Codon-optimized DNA encoding 6xHis Catenovulum agarivorans rhamnosidase, Uniparc reference UPI0003ED7515, Uniprot reference W7QF25. SEQ ID NO: 1176: Codon-optimized DNA encoding 6xHis Streptomyces avermitilis rhamnosidase, Uniparc reference UPI0000184198, Uniprot reference Q82PP4. SEQ ID NO: 1177: N-terminal His tag / linker SEQ ID NO: 1178: C-terminal linker / His tag SEQ ID NO: 1179: Amino acid sequence for variant G1. SEQ ID NO: 1180: Amino acid sequence for mutant G2.

[0151] SEQ ID NO: 1181: Amino acid sequence for mutant G3. SEQ ID NO: 1182: Amino acid sequence for mutant G4. SEQ ID NO: 1183: Amino acid sequence for variant G5. SEQ ID NO: 1184: Codon-optimized DNA encoding the N-terminal 6xHis mutant G1. SEQ ID NO: 1185: Codon-optimized DNA encoding the N-terminal 6xHis mutant G2. SEQ ID NO: 1186: Codon-optimized DNA encoding the N-terminal 6xHis mutant G3. SEQ ID NO: 1187: Codon-optimized DNA encoding the N-terminal 6xHis mutant G4. SEQ ID NO: 1188: Codon-optimized DNA encoding the N-terminal 6xHis mutant G5. SEQ ID NO: 1189: Amino acid sequence for mutant R1. SEQ ID NO: 1190: Amino acid sequence for mutant R2. SEQ ID NO: 1191: Amino acid sequence for mutant R3. SEQ ID NO: 1192: Amino acid sequence for mutant R4. SEQ ID NO: 1193: Amino acid sequence for mutant R5. SEQ ID NO: 1194: Codon-optimized DNA encoding the C-terminal 6xHis mutant R1. SEQ ID NO: 1195: Codon-optimized DNA encoding the C-terminal 6xHis mutant R2. SEQ ID NO: 1196: Codon-optimized DNA encoding the C-terminal 6xHis mutant R3. SEQ ID NO: 1197: Codon-optimized DNA encoding the C-terminal 6xHis mutant R4. SEQ ID NO: 1198: Codon-optimized DNA encoding the C-terminal 6xHis mutant R5.

[0152] Detailed Description of the Invention As previously mentioned, saponins are steroid or terpenoid glycosides with a wide range of uses. Current approaches to obtaining specific saponins in suitable quantities and with suitable purity, such as from plant cell cultures, are limited. The inventors have surprisingly found that enzymatic modification of saponins synthesized and produced by plant cell cultures can facilitate improved availability of the saponins of interest and / or facilitate removal of undesirable saponin components. Accordingly, the present invention provides methods for the enzymatic modification of saponins synthesized and produced by plant cell cultures, products produced by such methods, uses of the products, and related embodiments. In the methods of the present invention, a starting saponin (i.e., the saponin of interest to be enzymatically modified) is converted into a product saponin (i.e., the saponin resulting from the enzymatic modification of the starting saponin).

[0153] The engineered glucosidase polypeptides disclosed herein can be used in methods for the enzymatic modification of saponins of the invention.

[0154] The engineered rhamnosidase polypeptides disclosed herein can be used in methods for the enzymatic modification of saponins of the invention.

[0155] the purpose The methods of the present invention can be applied to achieve multiple objectives, such as (i) improving the yield of a saponin of interest obtainable from a given starting material; (ii) expanding the range of suitable starting materials from which to obtain a saponin of interest; and / or (iii) advantageously removing undesired saponins from the saponin of interest. In the context of the present invention, the term "starting material" refers to material derived from a plant cell culture.

[0156] When the starting material from which the saponin of interest is isolated is in a supply-constrained state, achieving maximum yield of the saponin of interest is clearly important. Independent of the efficiency of extraction and separation processes that would typically be applied to optimally isolate an existing saponin of interest, the present invention can be applied to increase the amount of the saponin of interest that can be obtained from a given starting material. Enzymatic modification of other saponins present in the starting material to form the saponin of interest can enrich the starting material with the saponin of interest, thereby increasing the amount of the saponin of interest that can be obtained.

[0157] Saponins can be obtained from a wide range of sources. The presence and levels of specific saponins in plant material can depend on a wide range of factors, such as plant species, tissue, age, season, environmental conditions, etc. Variation can also be observed between individual plants (e.g., trees) of the same species (see, e.g., WO2018057031). Variation in the levels of specific saponins and their respective ratios can also be observed from one plant cell culture to another. The burden associated with extracting and / or isolating the saponin of interest may mean that certain potential sources of the saponin of interest, such as in vitro plant cell cultures, are not commercially viable due to the relatively low levels of the saponin of interest present. Therefore, enzymatic modification of other saponins present to form the saponin of interest may expand the range of viable sources from which to obtain the saponin of interest.

[0158] It is well understood that different saponins can have different activity profiles, both positive / desired and negative / undesired. Some uses of saponins require a high degree of purification, and separation of the saponin of interest from other saponins, particularly those of similar structure or physical properties, can be laborious. Enzymatic modification of such other saponins may alter their physical properties, thus facilitating their separation from the saponin of interest. Other uses of saponins may not require high purity per se, but may nonetheless be desirable to remove or reduce the amount of a particular saponin component (or components) within a saponin mixture without the need for laborious chromatographic procedures. Enzymatic modification can facilitate the removal or reduction of the level of a particular saponin component within a saponin mixture without the need for chromatographic means.

[0159] saponin The methods of the present invention require a starting saponin (i.e., the saponin that is intended to be enzymatically modified). The starting saponin can be a naturally occurring saponin (i.e., a steroid or terpenoid glycoside found in nature) or an artificially created saponin (i.e., a steroid or terpenoid glycoside not found in nature).

[0160] In some embodiments, the starting saponin is a steroid glycoside, hi other embodiments, the starting saponin is a terpenoid glycoside, particularly a triterpenoid glycoside.

[0161] The naturally occurring starting saponin can be a saponin synthesized by in vitro cultured plant cells from saponin-producing plants, such as those described below.

[0162] Naturally occurring starting saponins include those obtainable from the genera Gypsophilia and Saponaria, such as Saponaria vaccaria or Saponaria officinalis, or from in vitro plant cell cultures derived from the genus Quillaja (Bomford, 1992). Starting saponins obtainable from in vitro plant cell cultures derived from Quillaja species are of particular interest. Specific starting saponins of interest include those obtainable from in vitro plant cell cultures derived from Quillaja brasiliensis or Quillaja saponaria. In one embodiment, the starting saponin is obtained from in vitro plant cell cultures derived from Quillaja saponaria. In one embodiment, the starting saponin is obtained from in vitro plant cell cultures derived from Quillaja brasiliensis.

[0163] In certain embodiments, the starting saponin is a queratic acid glycoside. In certain embodiments, the starting saponin is a phytolaccinic acid glycoside. In certain embodiments, the starting saponin is an echinocystic acid glycoside. In certain embodiments, the starting saponin is a 22-β-hydroxylated queratic acid glycoside. In certain embodiments, the starting saponin is a gypsogenin glycoside.

[0164] Analysis of aqueous / methanol extracts of Quillaja saponaria bark by liquid chromatography / mass spectrometry has revealed over 100 saponins (Nyberg, 2000; Nyberg, 2003; Kite, 2004). Quillaja brasiliensis extracts have also been described, with numerous saponin components in the Quillaja brasiliensis extract corresponding to those found in Quillaja saponaria extracts (Wallace, 2017; Wallace, 2019).

[0165] The following text describes specific quinoline-derived starting and product saponins, grouped by "family." Each family has one or more common structural features that distinguish it from other families. Individual members within each family also exhibit specific structural features that distinguish it from other members of the family, including: xylose or rhamnose chemotype—the presence of a xylose or rhamnose residue in the C3 sugar; A or B isomer—A has an acyl chain linked through the 4-position of D-fucose, and B has an acyl chain linked through the 3-position of D-fucose; V1 and V2—the presence of a terminal apiose or xylose residue, respectively, in the C28 sugar (this terminal residue may also be absent in other members of the family). While the text will focus on components typically prominent in Quillaja saponaria aqueous extracts, e.g., bark extracts, it will be understood that (i) other members of the family are also present, and (ii) the ratios of different members of the family can vary both between families and between different saponin sources (Kite, 2004). The specific extraction method used can also affect the ratios of different components obtained. While saponin components have been extensively studied using Quillaja saponaria bark extracts, it is expected that the saponin components synthesized and produced by in vitro plant cell cultures derived from Quillaja saponaria will be the same.

[0166] The A and B isomers may be separable using chromatographic techniques. However, under suitable solvent conditions, these isomers will revert to an equilibrium ratio (see, e.g., Cleland, 1996). The xylose and rhamnose chemotypes typically elute closely. Depending on the chromatographic technique, the rhamnose chemotype may form a minor peak that closely precedes or overlaps the major peak in the family.

[0167] Those skilled in the art will also recognize that the depicted structures contain ionizable groups and may, under appropriate circumstances, exist in dissociated form or as salts. The structures are generally shown with the glucuronic acid moiety in ionized form, and the molecular weights shown are calculated directly from the ions shown (corresponding to the monoisotopic m / z observed using negative mode electrospray mass spectrometry), although all undissociated, dissociated, and salt forms are intended to be encompassed by the recited definitions. Salts are desirably pharmaceutically acceptable, although non-pharmaceutically acceptable salts may nonetheless be useful in the manufacture of pharmaceuticals or for non-pharmaceutical applications.

[0168] Starting saponins obtainable from in vitro cultured plant cells and / or derived from Quillaja saponaria include: QS-18 family components (i.e. triterpenoid glycosides with β-O-glucopyranosylation at the C3 position of the L-rhamnose moiety of QS-21 family components), such as:

[0169] "QS-18 2150 A component," a triterpenoid glycoside identified as part of the QS-18 major peak in Figure 2, has an m / z of 2150 by negative mode electrospray mass spectrometry. The QS-18 2150 A component is believed to be identified in Kite 2004 as peak 76 and corresponds to the A isomer xylose chemotype structures B4 (apiose isomer) and B6 (xylose isomer) characterized in Nyberg 2000 and Nyberg 2003. The QS-18 2150 A component is QS-18 2150 A V1 (i.e., apiose isomer):

[0170] [ka] and / or QS-18 2150 A V2 (i.e., xylose isomers):

[0171] [ka] It can consist of:

[0172] "QS-18 2018 A component," a triterpenoid glycoside identified as part of the QS-18 major peak in Figure 2, has an m / z of 2018 by negative mode electrospray mass spectrometry. The QS-18 A 2018 component is believed to be identified in Kite 2004 as peak 73 and corresponds to the A isomer xylose chemotype structure B2 characterized in Nyberg 2000 and Nyberg 2003. The QS-18 2018 A component is QS-18 2018 A:

[0173] [ka] It can consist of:

[0174] "QS-18 2164 A component," a triterpenoid glycoside identified as part of the QS-18 major peak in Figure 2, has an m / z of 2164 in negative mode electrospray mass spectrometry. The QS-18 2164 A component is believed to be identified in Kite 2004 as peak 74 and corresponds to the A isomer rhamnose chemotype structures B3 (apiose isomer) and B5 (xylose isomer) characterized in Nyberg 2000 and Nyberg 2003. The QS-18 2164 A component is QS-18 2164 A V1 (i.e., apiose isomer):

[0175] [ka] and / or QS-18 2164 A V2 (i.e., xylose isomers):

[0176] [ka] It can consist of:

[0177] "QS-18 2150 B component" is a triterpenoid glycoside with an m / z of 2150 by negative mode electrospray mass spectrometry. QS-18 2150 B component corresponds to the B isomer xylose chemotype structures B4a (apiose isomer) and B6a (xylose isomer) characterized in Nyberg 2000 and Nyberg 2003. QS-18 2150 B component is QS-18 2150 B V1 (i.e., apiose isomer):

[0178] [ka] and / or QS-18 2150 B V2 (i.e., xylose isomers):

[0179] [ka] It can consist of:

[0180] "QS-18 2018 B component" is a triterpenoid glycoside with an m / z of 2018 by negative mode electrospray mass spectrometry. QS-18 2018 B component corresponds to the B isomer xylose chemotype structure B2a characterized in Nyberg 2000 and Nyberg 2003. QS-18 2018 B component is QS-18 2018 B:

[0181] [ka] It can consist of:

[0182] "QS-18 2164 B component" is a triterpenoid glycoside with an m / z of 2164 by negative mode electrospray mass spectrometry. QS-18 2164 B corresponds to the B isomer rhamnose chemotype structures B3a (apiose isomer) and B5a (xylose isomer). QS-18 2164 B component is QS-18 2164 B V1 (i.e., apiose isomer):

[0183] [ka] and / or QS-18 2164 B V2 (i.e., xylose isomers):

[0184] [ka] It can consist of:

[0185] Desglucosyl QS-17 family components (i.e., triterpenoid glycosides that have α-O-rhamnosylation at the C2 position of the arabinofuranose moiety of QS-21 family components but lack the glycosylation of QS-17 family components), such as:

[0186] "Desglucosyl QS-17 2134 A component" is a triterpenoid glycoside with an m / z of 2134 by negative mode electrospray mass spectrometry. The desglucosyl QS-17 2134 A component is believed to be identified in Kite 2004 as peak 75 and corresponds to the A isomer of the xylose chemotype. The desglucosyl QS-17 2134 A component is also known as desglucosyl QS-17 2134 A V1 (i.e., apiose isomer):

[0187] [ka] and / or desglucosyl QS-17 2134 A V2 (i.e., xylose isomer):

[0188] [ka] It can consist of:

[0189] "Desglucosyl QS-17 2002 A component" is a triterpenoid glycoside with an m / z of 2002 by negative mode electrospray mass spectrometry. Desglucosyl QS-17 2002 A component corresponds to the A isomer xylose chemotype, and is:

[0190] [ka] It can consist of:

[0191] "Desglucosyl QS-17 2148 A component" is a triterpenoid glycoside with an m / z of 2148 by negative mode electrospray mass spectrometry. The desglucosyl QS-17 2148 A component is believed to be identified in Kite 2004 as peaks 70 and 72, corresponding to the A isomer rhamnose chemotype. The desglucosyl QS-17 2148 A component is also known as desglucosyl QS-17 2148 A V1 (i.e., apiose isomer):

[0192] [ka] and / or desglucosyl QS-17 2148 A V2 (i.e., xylose isomer):

[0193] [ka] It can consist of:

[0194] "Desglucosyl QS-17 2134 B component" is a triterpenoid glycoside with an m / z of 2134 by negative mode electrospray mass spectrometry. The desglucosyl QS-17 2134 B component is believed to be identified in Kite 2004 as peak 67 and corresponds to the B isomer of the xylose chemotype. The desglucosyl QS-17 2134 B component is also known as desglucosyl QS-17 2134 B V1 (i.e., apiose isomer):

[0195] [ka] and / or desglucosyl QS-17 2134 B V2 (i.e., xylose isomer):

[0196] [ka] It can consist of:

[0197] "Desglucosyl QS-17 2002 B component" is a triterpenoid glycoside with an m / z of 2002 by negative mode electrospray mass spectrometry. Desglucosyl QS-17 2002 B component corresponds to the B isomer of the xylose chemotype, desglucosyl QS-17 2002 B:

[0198] [ka] It can consist of:

[0199] "Desglucosyl QS-17 2148 B component" is a triterpenoid glycoside with an m / z of 2148 by negative mode electrospray mass spectrometry. The desglucosyl QS-17 2148 B component is believed to be identified in Kite 2004 as peak 65 and corresponds to the B isomer of the rhamnose chemotype. The desglucosyl QS-17 2148 B component is also known as desglucosyl QS-17 2148 B V1 (i.e., apiose isomer):

[0200] [ka] and / or desglucosyl QS-17 2148 B V2 (i.e., xylose isomers):

[0201] [ka] It can consist of:

[0202] QS-17 family components (i.e. triterpenoid glycosides with β-O-glucopyranosylation at the C3 position of the L-rhamnose moiety and α-O-rhamnosylation at the C2 position of the arabinofuranose moiety of QS-21 family components), such as:

[0203] "QS-17 2296 A component," a triterpenoid glycoside identified as part of the QS-17 major peak in Figure 2, has an m / z of 2296 in negative mode electrospray mass spectrometry. The QS-17 2296 A component is believed to be identified in Kite 2004 as peak 59 and corresponds to the A isomer of the xylose chemotype structure QS-III. The QS-17 2296 A component is QS-17 2296 A V1 (i.e., apiose isomer):

[0204] [ka] and / or QS-17 2296 A V2 (i.e., xylose isomers):

[0205] [ka] It can consist of:

[0206] "QS-17 2164 A component," a triterpenoid glycoside identified as part of the QS-17 major peak in Figure 2, has an m / z of 2164 in negative mode electrospray mass spectrometry. The QS-17 2164 A component is believed to be identified in Kite 2004 as peak 58, corresponding to the A isomer xylose chemotype. The QS-17 2164 A component is QS-17 2164 A:

[0207] [ka] It can consist of:

[0208] "QS-17 2310 A component," a triterpenoid glycoside identified as part of the QS-17 major peak in Figure 2, has an m / z of 2310 in negative mode electrospray mass spectrometry. The QS-17 2310 A component is believed to be identified in Kite 2004 as peak 57 and corresponds to the A isomer of the rhamnose chemotype. The QS-17 2310 A component is also identified as QS-17 2310 A V1 (i.e., apiose isomer):

[0209] [ka] and / or QS-17 2310 A V2 (i.e., xylose isomers):

[0210] [ka] It can consist of:

[0211] "QS-17 2296 B component" is a triterpenoid glycoside with an m / z of 2296 by negative mode electrospray mass spectrometry. QS-17 2296 B component corresponds to the B isomer of the xylose chemotype structure QS-III in Kite 2004. QS-17 2296 B component is QS-17 2296 B V1 (i.e., apiose isomer):

[0212] [ka] and / or QS-17 2296 B V2 (i.e., xylose isomers):

[0213] [ka] It can consist of:

[0214] "QS-17 2164 B component" is a triterpenoid glycoside with an m / z of 2164 by negative mode electrospray mass spectrometry. QS-17 2164 B component corresponds to the B isomer xylose chemotype. QS-17 2164 B component:

[0215] [ka] It can consist of:

[0216] "QS-17 2310 B component" is a triterpenoid with an m / z of 2310 by negative mode electrospray mass spectrometry. The QS-17 2310 B component corresponds to the B isomer of the rhamnose chemotype. The QS-17 2310 B component is QS-17 2310 B V1 (i.e., apiose isomer):

[0217] [ka] and / or QS-17 2310 B V2 (i.e., xylose isomers):

[0218] [ka] It can consist of:

[0219] Desarabinofuranosyl QS-18 family components (i.e., triterpenoid glycosides that have β-O-glucopyranosylation at the C3 position of the L-rhamnose moiety of QS-21 family components, but lack the arabinofuranose moiety). Desarabinofuranosyl QS-18 family components are present in relatively small amounts in extracts, meaning that they have not been subjected to detailed characterization. Desarabinofuranosyl QS-18 family components can be difficult to isolate from QS-21 family components. Desarabinofuranosyl QS-18 family components include:

[0220] Desarabinofuranosyl QS-18 2018 A component (i.e., the triterpenoid glycoside identified as part of the "2018 peak" in Figure 6). Preferably, the desarabinofuranosyl QS-18 2018 A component has a retention time of approximately 4.5 minutes in the UPLC-UV / MS method described herein, and the major component of the peak has an m / z of 2018 in negative-mode electrospray mass spectrometry. The desarabinofuranosyl QS-18 2018 A component can also be identified in the UPLC-UV method described herein with a retention time of approximately 5.8 minutes. The desarabinofuranosyl QS-18 2018 A component is believed to be identified in Kite 2004 as peak 90, corresponding to the A isomer of the xylose chemotype. A putative structure has been identified for the major desarabinofuranosyl QS-18 2018 A component using MS / MS. The desarabinofuranosyl QS-18 2018 A component is desarabinofuranosyl QS-18 2018 A V1 (i.e., apiose isomer):

[0221] [ka] and / or desarabinofuranosyl QS-18 2018 A V2 (i.e., xylose isomers):

[0222] [ka] It can consist of:

[0223] Desarabinofuranosyl QS-18 1886 A component (i.e., the triterpenoid glycoside identified as part of the "2018 peak" in Figure 6). Preferably, the desarabinofuranosyl QS-18 1886 A component has a retention time of approximately 4.5 minutes in the UPLC-UV / MS method described herein and an m / z of 1886 in negative mode electrospray mass spectrometry. The desarabinofuranosyl QS-18 1886 A component corresponds to the A isomer xylose chemotype and can also be identified by the UPLC-UV method described herein with a retention time of approximately 5.8 minutes. The desarabinofuranosyl QS-18 1886 A component is:

[0224] [ka] It can consist of:

[0225] Desarabinofuranosyl QS-18 2032 A component (i.e., the triterpenoid glycoside identified as part of the "2018 peak" in Figure 6). Preferably, the desarabinofuranosyl QS-18 2032 A component has a retention time of approximately 4.5 minutes in the UPLC-UV / MS method described herein and an m / z of 2032 in negative mode electrospray mass spectrometry. The desarabinofuranosyl QS-18 2032 A component corresponds to the A isomer of the rhamnose chemotype and can also be identified by the UPLC-UV method described herein with a retention time of approximately 5.8 minutes. The desarabinofuranosyl QS-18 2032 A component is desarabinofuranosyl QS-18 2032 A V1 (i.e., the apiose isomer):

[0226] [ka] and / or desarabinofuranosyl QS-18 2032 A V2 (i.e., xylose isomer):

[0227] [ka] It can consist of:

[0228] Desarabinofuranosyl QS-18 2018 B component (i.e., a triterpenoid glycoside with an m / z of 2018 by negative mode electrospray mass spectrometry). The desarabinofuranosyl QS-18 2018 B component corresponds to the B isomer of the xylose chemotype. The desarabinofuranosyl QS-18 2018 B component corresponds to desarabinofuranosyl QS-18 2018 B V1 (i.e., an apiose isomer):

[0229] [ka] and / or desarabinofuranosyl QS-18 2018 B V2 (i.e., xylose isomers):

[0230] [ka] It can consist of:

[0231] Desarabinofuranosyl QS-18 1886 B component (i.e., a triterpenoid glycoside identified by negative mode electrospray mass spectrometry as having an m / z of 1886). The desarabinofuranosyl QS-18 1886 B component corresponds to the B isomer xylose chemotype. The desarabinofuranosyl QS-18 1886 B component is:

[0232] [ka] It can consist of:

[0233] Desarabinofuranosyl QS-18 2032 B component (i.e., a triterpenoid glycoside with an m / z of 2032 by negative mode electrospray mass spectrometry). The desarabinofuranosyl QS-18 2032 B component corresponds to the B isomer of the rhamnose chemotype. The desarabinofuranosyl QS-18 2032 B component corresponds to desarabinofuranosyl QS-18 2032 B V1 (i.e., an apiose isomer):

[0234] [ka] and / or desarabinofuranosyl QS-18 2032 B V2 (i.e., xylose isomer):

[0235] [ka] It can consist of:

[0236] Acetylated desglucosyl QS-17 family components (i.e., triterpenoid glycosides with α-O-rhamnosylation at the C2 position of the arabinofuranose moiety and acetylation at the C3 position of the fucose of QS-21 family components), such as:

[0237] "Acetylated desglucosyl QS-17 2176 A component" is a triterpenoid glycoside with an m / z of 2176 by negative mode electrospray mass spectrometry. The acetylated desglucosyl QS-17 2176 A component corresponds to the A isomer of the xylose chemotype ... apiose chemotype: Acetylated desglucosyl QS-17 2176 A V1 (i.e., apiose isomer):

[0238] [ka] and / or acetylated desglucosyl QS-17 2176 A V2 (i.e., xylose isomer):

[0239] [ka] It can consist of:

[0240] "Acetylated desglucosyl QS-17 2044 A component" is a triterpenoid glycoside with an m / z of 2044 by negative mode electrospray mass spectrometry. The Acetylated desglucosyl QS-17 2044 A component corresponds to the A isomer xylose chemotype. The Acetylated desglucosyl QS-17 2044 A component is:

[0241] [ka] It can consist of:

[0242] "Acetylated desglucosyl QS-17 2190 A component" is a triterpenoid glycoside with an m / z of 2190 by negative mode electrospray mass spectrometry. Acetylated desglucosyl QS-17 2190 A corresponds to the A isomer rhamnose chemotype. The acetylated desglucosyl QS-17 2190 A component is also known as Acetylated desglucosyl QS-17 2190 A V1 (i.e., apiose isomer):

[0243] [ka] and / or acetylated desglucosyl QS-17 2190 A V2 (i.e., xylose isomers):

[0244] [ka] It can consist of:

[0245] Although the starting saponins directly related to the engineered glucosidase polypeptides for use in the methods of the present invention are those that have a cleavable glucose residue, the engineered glucosidase polypeptides can nonetheless be utilized with additional enzymes capable of cleaving other sugar residues. Particular starting saponins related to the engineered glucosidase polypeptides include the following: ·QS-18 family ingredients; QS-17 family ingredients; and Desarabinofuranosyl QS-18 family component.

[0246] Although the starting saponins directly related to the engineered rhamnosidase polypeptides for use in the methods of the present invention are those that have a cleavable rhamnose residue, the engineered rhamnosidase polypeptides may nonetheless be utilized in conjunction with additional enzymes capable of cleaving other sugar residues. Particular starting saponins related to the engineered rhamnosidase polypeptides include the following: Desglucosyl QS-17 family components; QS-17 family ingredients; and Acetylated desglucosyl QS-17 family component.

[0247] The methods of the present invention enzymatically modify a starting saponin obtained from a plant cell culture to provide a product saponin (i.e., a saponin resulting from the enzymatic modification).

[0248] The product saponin can be a naturally occurring saponin (i.e., a steroid or terpenoid glycoside found in nature that is itself obtained by the methods of the present invention) or an artificially created saponin (i.e., a steroid or terpenoid glycoside not found in nature).

[0249] In some embodiments, the product saponin is a steroid glycoside, hi other embodiments, the product saponin is a terpenoid glycoside, particularly a triterpenoid glycoside.

[0250] A naturally occurring product saponin may be a saponin synthesized by in vitro cultured plant cells from a saponin-producing plant, such as the plants described below.

[0251] Naturally occurring product saponins include those obtainable from the genera Gypsophilia and Saponaria, such as Saponaria vaccaria or Saponaria officinalis, or from in vitro plant cell cultures derived from the genus Quillaja (Bomford, 1992). Product saponins obtainable from in vitro plant cell cultures derived from Quillaja species are of particular interest. Specific product saponins of interest include those obtainable from in vitro plant cell cultures derived from Quillaja brasiliensis or Quillaja saponaria. In one embodiment, the product saponin is obtained from in vitro plant cell cultures derived from Quillaja saponaria. In one embodiment, the product saponin is obtained from in vitro plant cell cultures derived from Quillaja brasiliensis.

[0252] In certain embodiments, the product saponin is a quinoline glycoside.

[0253] Saponin products obtainable from plant cell cultures derived from Quillaja saponaria include: QS-18 family components (i.e. triterpenoid glycosides with β-O-glucopyranosylation at the C3 position of the L-rhamnose moiety of QS-21 family components), such as: "QS-18 2150 A component." QS-18 2150 A component is QS-18 2150 A V1 (i.e., apiose isomer):

[0254] [ka] and / or QS-18 2150 A V2 (i.e., xylose isomers):

[0255] [ka] It can consist of: "QS-18 2018 A Ingredients". QS-18 2018 A Ingredients are: QS-18 2018 A:

[0256] [ka] It can consist of: "QS-18 2164 A component." QS-18 2164 A component is QS-18 2164 A V1 (i.e., apiose isomer):

[0257] [ka] and / or QS-18 2164 A V2 (i.e., xylose isomers):

[0258] [ka] It can consist of: "QS-18 2150 B component." QS-18 2150 B component is QS-18 2150 B V1 (i.e., apiose isomer):

[0259] [ka] and / or QS-18 2150 B V2 (i.e., xylose isomers):

[0260] [ka] It can consist of: "QS-18 2018 B component". QS-18 2018 B component is QS-18 2018 B:

[0261] [ka] It can consist of: "QS-18 2164 B component." QS-18 2164 B component is QS-18 2164 B V1 (i.e., apiose isomer):

[0262] [ka] and / or QS-18 2164 B V2 (i.e., xylose isomers):

[0263] [ka] It can consist of:

[0264] Desglucosyl QS-17 family components (i.e., triterpenoid glycosides with α-O-rhamnosylation at the C2 position of the arabinofuranose moiety of QS-21 family components), such as:

[0265] "Desglucosyl QS-17 2134 A component." Desglucosyl QS-17 2134 A component is desglucosyl QS-17 2134 A V1 (i.e., apiose isomer):

[0266] [ka] and / or desglucosyl QS-17 2134 A V2 (i.e., xylose isomer):

[0267] [ka] It can consist of:

[0268] "Desglucosyl QS-17 2002 A Ingredient." Desglucosyl QS-17 2002 A Ingredient: Desglucosyl QS-17 2002 A:

[0269] [ka] It can consist of:

[0270] "Desglucosyl QS-17 2148 A component." Desglucosyl QS-17 2148 A component is desglucosyl QS-17 2148 A V1 (i.e., apiose isomer):

[0271] [ka] and / or desglucosyl QS-17 2148 A V2 (i.e., xylose isomer):

[0272] [ka] It can consist of:

[0273] "Desglucosyl QS-17 2134 B component." Desglucosyl QS-17 2134 B component is desglucosyl QS-17 2134 B V1 (i.e., apiose isomer):

[0274] [ka] and / or desglucosyl QS-17 2134 B V2 (i.e., xylose isomer):

[0275] [ka] It can consist of:

[0276] "Desglucosyl QS-17 2002 B component." Desglucosyl QS-17 2002 B component is Desglucosyl QS-17 2002 B:

[0277] [ka] It can consist of:

[0278] "Desglucosyl QS-17 2148 B component." Desglucosyl QS-17 2148 B component is desglucosyl QS-17 2148 B V1 (i.e., apiose isomer):

[0279] [ka] and / or desglucosyl QS-17 2148 B V2 (i.e., xylose isomers):

[0280] [ka] It can consist of:

[0281] QS-21 family ingredients, such as: "QS-21 1988 A component," a triterpenoid glycoside identified as part of the QS-21 major peak in Figure 6, has an m / z of 1988 by negative-mode electrospray mass spectrometry. Preferably, the QS-21 1988 A component in the UPLC-UV / MS method described herein has a retention time of approximately 4.4 minutes and an m / z of 1988 by negative-mode electrospray mass spectrometry. The QS-21 1988 A component is believed to be identified in Kite 2004 as peak 88, and corresponds to the A-isomer xylose chemotype structures S6 (apiose isomer) and S4 (xylose isomer) characterized in Nyberg 2000 and Nyberg 2003. The QS-21 1988 A component is QS-21 1988 A V1 (i.e., apiose isomer):

[0282] [ka] and QS-21 1988 A V2 (i.e., xylose isomers):

[0283] [ka] It can consist of:

[0284] "QS-21 1856 A component," a triterpenoid glycoside identified as part of the QS-21 major peak in Figure 6, has an m / z of 1856 by negative mode electrospray mass spectrometry. Preferably, the QS-21 1856 A component in the UPLC-UV / MS method described herein has a retention time of approximately 4.4 minutes and an m / z of 1856 by negative mode electrospray mass spectrometry. The QS-21 1856 A component is believed to be identified in Kite 2004 as peak 86, and corresponds to the A isomer xylose chemotype structure S2 characterized in Nyberg 2000 and Nyberg 2003. The QS-21 1856 A component is:

[0285] [ka] It can consist of:

[0286] "QS-21 2002 A component," a triterpenoid glycoside identified as part of the QS-21 major peak in Figure 6, has an m / z of 2002 in negative-mode electrospray mass spectrometry. Preferably, the QS-21 2002 A component in the UPLC-UV / MS method described herein has a retention time of approximately 4.4 minutes and an m / z of 2002 in negative-mode electrospray mass spectrometry. The QS-21 2002 A component is believed to be identified in Kite 2004 as peak 85, corresponding to the A-isomer rhamnose chemotype of structures S3 and S5 characterized in Nyberg 2000 and Nyberg 2003. The QS-21 2002 A component is QS-21 2002 A V1 (i.e., an apiose isomer):

[0287] [ka] and QS-21 2002 A V2 (i.e., xylose isomers):

[0288] [ka] It can consist of:

[0289] "QS-21 1988 B component," a triterpenoid glycoside identified as part of the B isomer peak in Figure 6, has an m / z of 1988 by negative-mode electrospray mass spectrometry. Preferably, QS-21 1988 B component in the UPLC-UV / MS method described herein has a retention time of approximately 4.0 minutes and an m / z of 1988 by negative-mode electrospray mass spectrometry. QS-21 1988 B component corresponds to the B isomer xylose chemotype structures S6a (apiose isomer) and S4a (xylose isomer) characterized in Nyberg 2000 and Nyberg 2003. QS-21 1988 B component is QS-21 1988 B V1 (i.e., apiose isomer):

[0290] [ka] and QS-21 1988 B V2 (i.e., xylose isomers):

[0291] [ka] It can consist of:

[0292] "QS-21 1856 B component," a triterpenoid glycoside identified as part of the B isomer peak in Figure 6, has an m / z of 1856 by negative mode electrospray mass spectrometry. QS-21 1856 B component corresponds to the B isomer xylose chemotype structure S2a characterized in Nyberg 2000 and Nyberg 2003. QS-21 1856 B component is:

[0293] [ka] It can consist of:

[0294] "QS-21 2002 B component" is a triterpenoid glycoside with an m / z of 2002 by negative mode electrospray mass spectrometry. QS-21 2002 B component corresponds to the B isomer rhamnose chemotypes of structures S3a and S5a characterized in Nyberg 2000 and Nyberg 2003. QS-21 2002 B component V1 (i.e., apiose isomer):

[0295] [ka] and QS-21 2002 B V2 (i.e., xylose isomers):

[0296] [ka] It can consist of:

[0297] Desarabinofuranosyl QS-21 family components (i.e., triterpenoid glycosides lacking the arabinofuranose moiety of QS-21 family components). Desarabinofuranosyl QS-21 family components are present in relatively small amounts in the extract, meaning that they have not been subjected to detailed characterization. Desarabinofuranosyl QS-21 family components include:

[0298] Desarabinofuranosyl QS-21 1856 A component (i.e., the triterpenoid glycoside identified as part of the "lyophilized peak" in Figure 6). Preferably, the desarabinofuranosyl QS-21 1856 A component has a retention time of approximately 4.7 minutes in the UPLC-UV / MS method described herein, and the major component of the peak has an m / z of 1856 in negative mode electrospray mass spectrometry. The desarabinofuranosyl QS-21 1856 A component is believed to be identified in Kite 2004 as peak 96. A putative structure has been identified for the major desarabinofuranosyl QS-21 1856 A component using MS / MS. The desarabinofuranosyl QS-21 1856 A component is desarabinofuranosyl QS-21 1856 A V1 (i.e., the apiose isomer):

[0299] [ka] and / or desarabinofuranosyl QS-21 1856 A V2 (i.e., xylose isomer):

[0300] [ka] It can consist of:

[0301] Desarabinofuranosyl QS-21 1712 A component (i.e., the triterpenoid glycoside identified as part of the "lyophilized peak" in Figure 6). Preferably, the desarabinofuranosyl QS-21 1712 A component in the UPLC-UV / MS method described herein has a retention time of about 4.7 minutes and an m / z of 1712 in negative mode electrospray mass spectrometry. The desarabinofuranosyl QS-21 1712 A component is desarabinofuranosyl QS-21 1712 A:

[0302] [ka] It can consist of:

[0303] Desarabinofuranosyl QS-21 1870 A component, i.e., a triterpenoid glycoside with an m / z of 1870 by negative mode electrospray mass spectrometry. The desarabinofuranosyl QS-21 1870 A component is desarabinofuranosyl QS-21 1870 A V1 (i.e., apiose isomer):

[0304] [ka] and / or desarabinofuranosyl QS-21 1870 A V2 (i.e., xylose isomer):

[0305] [ka] It can consist of:

[0306] Desarabinofuranosyl QS-21 1856 B component, i.e., a triterpenoid glycoside with an m / z of 1856 by negative mode electrospray mass spectrometry. The desarabinofuranosyl QS-21 1856 B component is desarabinofuranosyl QS-21 1856 B V1 (i.e., an apiose isomer):

[0307] [ka] and / or desarabinofuranosyl QS-21 1856 B V2 (i.e., xylose isomer):

[0308] [ka] It can consist of:

[0309] Desarabinofuranosyl QS-21 1712 B component, i.e., a triterpenoid glycoside with m / z of 1712 in negative mode electrospray mass spectrometry. The desarabinofuranosyl QS-21 1712 B component is desarabinofuranosyl QS-21 1712 B:

[0310] [ka] It can consist of:

[0311] Desarabinofuranosyl QS-21 1870 B component, i.e., a triterpenoid glycoside with an m / z of 1870 by negative mode electrospray mass spectrometry. The desarabinofuranosyl QS-21 1870 B component is desarabinofuranosyl QS-21 1870 B V1 (i.e., an apiose isomer):

[0312] [ka] and / or desarabinofuranosyl QS-21 1870 B V2 (i.e., xylose isomer):

[0313] [ka] It can consist of:

[0314] Acetylated QS-21 family components (i.e., triterpenoid glycosides with acetylation at the C3 position of the fucose of the QS-21 family component), such as:

[0315] "Acetylated QS-21 2030 A component" is a triterpenoid glycoside with an m / z of 2030 by negative mode electrospray mass spectrometry. Acetylated QS-21 2030 A corresponds to the A isomer xylose chemotype. Acetylated QS-21 2030 A component is also known as Acetylated QS-21 2030 A V1 (i.e., apiose isomer):

[0316] [ka] and / or acetylated QS-21 2030 A V2 (i.e., xylose isomers):

[0317] [ka] It can consist of:

[0318] "Acetylated QS-21 1898 A component" is a triterpenoid glycoside with an m / z of 1898 by negative mode electrospray mass spectrometry. Acetylated QS-21 1898 A corresponds to the A isomer xylose chemotype. Acetylated QS-21 1898 A component is:

[0319] [ka] It can consist of:

[0320] "Acetylated QS-21 2044 A component" is a triterpenoid glycoside with an m / z of 2044 by negative mode electrospray mass spectrometry. Acetylated QS-21 2044 A corresponds to the A isomer of the rhamnose chemotype. Acetylated QS-21 2044 A component is also known as Acetylated QS-21 2044 A V1 (i.e., apiose isomer):

[0321] [ka] and / or acetylated QS-21 2044 A V2 (i.e., xylose isomers):

[0322] [ka] It can consist of:

[0323] Product saponins directly related to the engineered glucosidase polypeptides for use in the methods of the present invention are those in which glucose residues have been cleaved compared to the starting saponin. Nevertheless, the engineered glucosidase polypeptides can be utilized with additional enzymes capable of cleaving other sugar residues. Specific product saponins related to the engineered glucosidase polypeptides include the following: Desglucosyl QS-17 family components; QS-21 family ingredients; and Desarabinofuranosyl QS-21 family component.

[0324] Product saponins directly related to the engineered rhamnosidase polypeptides for use in the methods of the present invention are those in which rhamnose residues have been cleaved compared to the starting saponin. Nevertheless, the engineered rhamnosidase polypeptides can be utilized with additional enzymes capable of cleaving other sugar residues. Specific product saponins related to the engineered rhamnosidase polypeptides include the following: ·QS-18 family ingredients; QS-21 family ingredients; and Acetylated QS-21 family component.

[0325] The term "QS-18 family component" as used herein refers to xylose chemotype QS-18 2150 components (A and B isomers, and apiose and xylose isomers: QS-18 2150 A V1, QS-18 2150 A V2, QS-18 2150 B V1, and QS-18 2150 B V2), xylose chemotype QS-18 2018 components (A and B isomers: QS-18 2018 A and QS-18 2018 B), rhamnose chemotype QS-18 2164 components (A and B isomers, and apiose and xylose isomers: QS-18 2164 A V1, QS-18 2164 A V2, QS-18 2164 B V1 and QS-18 2164 B V2).

[0326] The term "desglucosyl QS-17 family component" as used herein refers to xylose chemotype desglucosyl QS-17 2134 components (A and B isomers, and apiose and xylose isomers: desglucosyl QS-17 2134 A V1, desglucosyl QS-17 2134 A V2, desglucosyl QS-17 2134 B V1, and desglucosyl QS-17 2134 B V2), xylose chemotype desglucosyl QS-17 2002 components (A and B isomers: desglucosyl QS-17 2002 A and desglucosyl QS-17 2002 B), rhamnose chemotype desglucosyl QS-17 2148 components (A and B isomers, and apiose and xylose isomers: desglucosyl QS-17 2148 A V1, desglucosyl QS-17 2148 A V2, desglucosyl QS-17 2148 B V1 and desglucosyl QS-17 2148 B V2).

[0327] The term "QS-17 family component" as used herein refers to xylose chemotype QS-17 2296 components (A and B isomers, and apiose and xylose isomers: QS-17 2296 A V1, QS-17 2296 A V2, QS-17 2296 B V1, and QS-17 2296 B V2), xylose chemotype QS-17 2164 components (A and B isomers: QS-17 2164 A and QS-17 2164 B), rhamnose chemotype QS-17 2310 components (A and B isomers, and apiose and xylose isomers: QS-17 2310 A V1, QS-17 2310 A V2, QS-17 2310 B V1 and QS-17 2310 B V2).

[0328] The term "QS-21 family component" as used herein refers to xylose chemotype QS-21 1988 components (A and B isomers, and apiose and xylose isomers: QS-21 1988 A V1, QS-21 1988 A V2, QS-21 1988 B V1, and QS-21 1988 B V2), xylose chemotype QS-21 1856 components (A and B isomers: QS-21 1856 A and QS-21 1856 B), rhamnose chemotype QS-21 2002 components (A and B isomers, and apiose and xylose isomers: QS-21 2002 A V1, QS-21 2002 A V2, QS-21 2002 B V1 and QS-21 2002 B V2).

[0329] The term "desarabinofuranosyl QS-18 family component" as used herein refers to xylose chemotype desarabinofuranosyl QS-18 2018 components (A and B isomers, and apiose and xylose isomers: desarabinofuranosyl QS-18 2018 A V1, desarabinofuranosyl QS-18 2018 A V2, desarabinofuranosyl QS-18 2018 B V1, and desarabinofuranosyl QS-18 2018 B V2), xylose chemotype desarabinofuranosyl QS-18 1886 components (A and B isomers: desarabinofuranosyl QS-18 1886 A and desarabinofuranosyl QS-18 1886 B), rhamnose chemotype desarabinofuranosyl QS-18 2032 components (A and B isomers, and apiose and xylose isomers: desarabinofuranosyl QS-18 2032 A V1, desarabinofuranosyl QS-18 2032 A V2, desarabinofuranosyl QS-18 2032 B V1 and desarabinofuranosyl QS-18 2032 B V2).

[0330] The term "acetylated desglucosyl QS-17 family component" as used herein refers to the xylose chemotype acetylated desglucosyl QS-17 2176 component (apiose and xylose isomers: acetylated desglucosyl QS-17 2176 A V1 and acetylated desglucosyl QS-17 2176 A V2), the xylose chemotype acetylated desglucosyl QS-17 2044 A component, and the rhamnose chemotype acetylated desglucosyl QS-17 2190 component (apiose and xylose isomers: acetylated desglucosyl QS-17 2190 A V1 and acetylated desglucosyl QS-17 2190 A V2).

[0331] The term "desarabinofuranosyl QS-21 family component" as used herein refers to xylose chemotype desarabinofuranosyl QS-21 1856 components (A and B isomers, and apiose and xylose isomers: desarabinofuranosyl QS-21 1856 A V1, desarabinofuranosyl QS-21 1856 A V2, desarabinofuranosyl QS-21 1856 B V1, and desarabinofuranosyl QS-21 1856 B V2), xylose chemotype desarabinofuranosyl QS-21 1712 components (A and B isomers: desarabinofuranosyl QS-21 1712 A and desarabinofuranosyl QS-21 1712 B), rhamnose chemotype desarabinofuranosyl QS-21 By this reference is meant the 1870 components (A and B isomers, as well as apiose and xylose isomers: desarabinofuranosyl QS-21 1870 A V1, desarabinofuranosyl QS-21 1870 A V2, desarabinofuranosyl QS-21 1870 B V1 and desarabinofuranosyl QS-21 1870 B V2).

[0332] The term "acetylated QS-21 family component" as used herein refers to the xylose chemotype acetylated QS-21 2030 component (apiose and xylose isomers: acetylated QS-21 2030 A V1 and acetylated QS-21 2030 A V2), the xylose chemotype acetylated QS-21 1898 A component, and the rhamnose chemotype acetylated QS-21 2044 component (apiose and xylose isomers: acetylated QS-21 2044 A V1 and acetylated QS-21 2044 A V2).

[0333] plant cell culture The term "plant cell culture" is understood as an in vitro culture of any plant tissue or any plant cells derived from any tissue from a plant that produces saponin, such as the plants described and referenced above. The tissue or cells from the plant cell culture are capable of synthesizing saponin. The term "capable of synthesizing saponin" in the sense of the present invention refers to the ability of the cells to synthesize and produce saponin. Depending on various factors, such as the origin of the plant cell culture and its cell culture conditions, saponin synthesis and its production must be triggered. In the sense of the present invention, the term "under conditions conducive to the synthesis of saponin" refers to conditions that cause and result in the efficient synthesis and production of saponin.

[0334] In the sense of the present invention, plant cell cultures can be obtained and produced according to any known method. Lambert et al. (2011) provide a review of the in vitro culture of common saponin-producing plants. WO 94 / 10291 (incorporated herein by reference) discloses how to obtain in vitro cultures of plant cells from the species Quillaja saponaria, which are capable of synthesizing and producing saponins.

[0335] Methods for culturing plant cells and general culturing conditions for plant cells are known in the art. Conventional culture media known for plant cell culture, such as classical Murashige-Skoog (MS) medium, White's medium, or Linsmeyer-Skoog medium, can be used in the methods of the present invention. Typically, these media contain at least one or more macronutrients, such as selected from NH4NO3, KNO3, CaCl2, MgSO4, KH2PO4, NH4Cl or KCl; at least one or more micronutrients, such as selected from KI, H3BO3, MnSO4, ZnSO4, Na2MoO4, CuSO4, CoCl2, DeSO4 or Na2EDTA; at least one or more vitamins, such as selected from myoinisotol, nicotinic acid, pyridixine-HCl or thiamine-HCl, for example, in a total concentration of between 0.01 and 3 g / L, for example between 50 and 150 mg / L; at least one or more amino acids, such as glycine; at least one or more carbon sources, such as selected from sucrose, glucose or fructose; and at least one or more plant hormones, such as selected from one or more cytokinins or one or more auxins, such as 2,4-D and / or NAA.

[0336] The plant cells used in the method of the present invention can be derived from various tissues and organs of a given plant body, such as primordia, leaves, stems, hairy roots, internodes, cambium, whether cultured in suspension, in liquid medium, or on solid medium, for example, callus.In one embodiment, the plant cells used in the method of the present invention are derived from the cambium, for example, cambium meristem cells (CMC).In an alternative embodiment, the plant cells are derived from hairy roots.

[0337] The plant cells used in the methods of the present invention can be callus, e.g., callus derived from the cambium of a plant. Typically, "callus" is defined as a cluster of dedifferentiated cells cultured on a solidified medium. Callus generation can be achieved from any plant tissue explant by any method known to those skilled in the art, such as those described in International Publication No. WO 94 / 10291, U.S. Patent Application Publication No. 2019 / 0134128, or International Publication No. WO 15 / 082978. Briefly, small-sized plant-derived tissue explants can be surface-sterilized, for example, by thoroughly washing with clean water, using a disinfectant such as hypochlorite, a wetting agent such as Tween or Triton, an antibacterial agent, and / or an antifungal agent. The surface-sterilized explants are then typically placed on the surface of a solidified medium such as agar and incubated in a sterile environment until a mass of undifferentiated cells develops near the plant source material (typically for 2 to 12 weeks, e.g., 8 weeks). Callus can be gradually purified and further propagated by repeating the same solid medium culture, i.e., by inoculating small pieces of callus formed in the previous solid medium culture onto fresh solid medium, for example, every four weeks. Callus culture conditions, including medium components such as MS medium, pH range, carbon source, nitrogen source, macro- and micro-salts, vitamins, and growth regulators, are well known to those skilled in the art.

[0338] The callus thus formed on solid medium and refined by subculture can be inoculated into a liquid medium and cultivated to obtain a suspension cell culture. The terms "suspension plant culture" and "plant cell suspension" are interchangeable and refer to an in vitro culture of plant cells dispersed in a liquid medium and growing in suspension. In one embodiment, the plant cells used in the method of the present invention are grown in suspension in a liquid medium. To prepare plant cells into a suspension culture, for example, the cells are removed from the callus and transferred to a sterile culture vessel containing a nutrient culture medium. It is understood that a medium optimized for a suspension cell system may differ from that optimal for a callus. Determining a suitable and optimal culture medium is within the skill of a person skilled in the art.

[0339] The transition from callus to suspension cell culture is also known to those skilled in the art (e.g., as described in International Publication No. 94 / 10291 or U.S. Patent Application Publication No. 2019 / 0134128). Suspension culture conditions, such as classical MS medium, including medium components, pH range, carbon source, nitrogen source, macro- and micro-salts, vitamins, and growth regulators, are well known to those skilled in the art. Once growth in suspension has been initiated and adapted, the suspension cells can be subcultured and expanded, for example, by dilution, for example, every four weeks.

[0340] Saponin synthesis by plant cells used in the methods of the present invention (i.e., capable of synthesizing saponin) can be optimized by inducing the cells with an inducer. Suitable inducers for use in the methods of the present invention are monococarboxylic compound-type inducers, such as 5-chlorosalicyclic acid, salicylic acid, acetylsalicyclic acid, methyl esters such as methyl jasmonate (MeJa), or chemically synthesized 2-HEJ. In one embodiment, the inducer is MeJa.

[0341] Typically, the concentration of an inducer is stated by reference to the volume of culture medium, but an alternative way to define the concentration is by reference to PCV%. The term "PCV" stands for Packed Cell Volume and refers to the volume occupied by cells in the culture medium. It can be calculated as follows: PCV (%) = (volume of cell pellet / volume of sample) × 100. A suitable range for an inducer, such as MeJa, is 0.5-10 μM / PCV%.

[0342] The inducer can be added directly to the culture medium, or the culture medium can be replaced with culture medium containing the inducer.

[0343] The induction time can be adjusted depending on the plant cell under consideration. Determining the optimal duration is within the skill of the art. Preferably, induction can be carried out for 1 to 14 days, more preferably for 2 to 10 days, and even more preferably for 3 to 8 days.

[0344] Alternatively, or in addition to induction, saponin synthesis by the plant cells used in the methods of the present invention can be further optimized by depleting the cells of nitrogen, e.g., before induction. Nitrogen depletion refers to reducing the levels of all nitrogen sources present in the culture medium. Preferably, e.g., before induction, the cell culture medium of the cells is removed and replaced with culture medium containing no nitrogen source. The preferred range for the duration of nitrogen depletion (before induction) is 1 to 9 days, preferably 2 to 7 days, and more preferably 3 to 6 days.

[0345] Induction and / or nitrogen depletion are non-limiting examples of "conditions that lead to the synthesis of saponins" in the sense of the present invention.

[0346] The saponin synthesized by the plant cells used in the methods of the present invention can either be secreted extracellularly into the culture medium and / or remain intracellularly located. Thus, the starting saponin intended to be enzymatically modified or converted within the meaning of the present invention can be contained in the cell culture medium. In other words, the cell culture medium of plant cells capable of synthesizing saponin can be used as the starting material and subjected to enzymatic treatment. In some embodiments, glycosidase is optionally added to the culture medium after nitrogen has been depleted from the culture medium as described above and / or the cells have been induced with an inducer as described above. Alternatively, the starting saponin intended to be enzymatically modified or converted within the meaning of the present invention can be recovered from the intracellular contents of the plant cells. For example, the plant cells can be lysed (by any technique known in the art) and the resulting intracellular contents can be subjected to enzymatic treatment.

[0347] In the context of the present invention, the term "plant cell culture extract" is understood to mean any extract or material obtained from an in vitro plant cell culture containing saponins, including starting and / or product saponins, whether synthesized and / or recovered. For example, the plant cell culture extract may be plant cells or lysed plant cells. The plant cell culture extract may be crude or partially or completely purified. The plant cell culture extract may be obtained from a plant cell culture extract obtained from the genus Gypsophilia, Saponaria, e.g., Saponaria vaccaria or Saponaria officinalis, or the genus Quillaja (Bomford, 1992), e.g., Quillaja species. Specific plant cell culture extracts include those obtained from Quillaja brasiliensis or Quillaja saponaria. In one embodiment, the plant cell culture extract is obtained from Quillaja saponaria. In one embodiment, the plant cell culture extract is obtained from Quillaja brasiliensis.

[0348] The term "recovered" is understood as recovering a saponin, for example, a saponin synthesized by a plant cell or plant cell culture. The saponin may be recovered from the culture medium. Alternatively, the saponin may be recovered from the plant cell, for example, by extraction.

[0349] Any known extraction method is suitable, such as extraction with non-aqueous polar solvents, with acidic or basic media, or by mechanically disrupting the plant cells, such as by ball milling or sonication (e.g., as described in WO 94 / 10291). Alternatively, saponins can be extracted by freezing the cell pellet obtained after centrifugation of the cell culture (resulting in cell lysis). Preferably, the cell pellet is frozen at -20°C, more preferably -70°C, for example, for at least 24 hours.

[0350] Extraction can be carried out using water or a lower alcohol (e.g., methanol or ethanol) as the solvent, including mixtures thereof. In one embodiment, the starting saponin is obtained by aqueous extraction (e.g., using a solvent comprising at least 80% v / v water, particularly at least 90% v / v water, for example at least 95% v / v water). In one embodiment, the starting saponin is obtained by methanol extraction (e.g., using a solvent comprising at least 80% v / v methanol, particularly at least 90% v / v methanol, for example at least 95% v / v methanol). In one embodiment, the starting saponin is obtained by ethanol extraction (e.g., using a solvent comprising at least 80% v / v ethanol, particularly at least 90% v / v ethanol, for example at least 95% v / v ethanol). In one embodiment, the starting saponin is obtained by methanol / ethanol extraction (e.g., using a solvent comprising at least 20% v / v methanol, particularly at least 30% v / v methanol, for example at least 40% v / v methanol, and at least 20% v / v ethanol, particularly at least 30% v / v ethanol, for example at least 40% v / v ethanol). In one embodiment, the starting saponin is obtained by water / ethanol extraction (e.g., using a solvent comprising at least 20% v / v water, particularly at least 30% v / v water, for example at least 40% v / v water, and at least 20% v / v ethanol, particularly at least 30% v / v ethanol, for example at least 40% v / v ethanol). In one embodiment, the starting saponin is obtained by water / methanol extraction (e.g. using a solvent comprising at least 20% v / v water, particularly at least 30% v / v water, for example at least 40% v / v water, and at least 20% v / v methanol, particularly at least 30% v / v methanol, for example at least 40% v / v methanol).

[0351] The methods of the present invention can be applied to starting saponins in a range of contexts. The starting saponin can be in the form of a minor component in a saponin-containing composition (ignoring solvent, if present), such as a minor component of a plant cell culture extract. The starting saponin can be in the form of a major component in a saponin-containing composition, such as a major component of a plant cell culture extract. The starting saponin can be in the form of a minor component in a processed, e.g., partially purified, plant cell culture extract. The starting saponin can be in the form of a major component in a processed, e.g., partially purified, plant cell culture extract. In some embodiments, the starting saponin is substantially purified at the time of enzymatic modification. In further embodiments, the starting saponin is contained in a crude material, such as a crude plant cell culture extract.

[0352] Purification refers to the isolation of a component from other components. Thus, partial purification refers to the isolation of a component to some extent from other components. Substantial purification refers to the substantial isolation of a component from other components, such as when the component comprises at least 50% w / w, particularly at least 70%, particularly at least 80%, e.g., at least 90% of the component content (50%, 70%, 80%, and 90% purity, respectively). Partial purification, with respect to a plant cell culture extract, refers to the isolation of the starting saponin to some extent from other extracted components. Substantially purified, with respect to a plant cell culture extract, refers to the substantial isolation of the starting saponin from other extracted components, such as when the starting saponin comprises at least 50% w / w, particularly at least 70%, particularly at least 80%, e.g., at least 90% of the extracted component content. Partial or substantial purification can be achieved via a variety of means, including chromatography, filtration through semipermeable membranes, treatment with selective adsorbents such as polyvinylpolypyrrolidone (PVPP), etc.

[0353] While the starting saponin can be a specific chemical, in many situations, such as saponins obtained by extraction, there may be multiple starting saponins, which are enzymatically modified to provide their corresponding product saponins. As noted above with respect to individual saponins, the present invention is applicable to multiple starting saponins in a range of contexts. Multiple starting saponins, including closely related starting saponins, can undergo equivalent enzymatic modifications simultaneously. Multiple starting saponins, including distinguishable starting saponins, can undergo different enzymatic modifications simultaneously (in the presence of two or more enzymes) or sequentially (sequential treatment with distinct enzymes). Multiple starting saponins can contain both closely related and distinguishable starting saponins.

[0354] The method of the present invention can be applied to starting saponins in the form of the following components: crude plant cell culture extracts, e.g. water and / or lower alcohol extracts, in particular aqueous cell extracts; partially purified plant cell culture extracts, for example aqueous and / or lower alcohol plant cell culture extracts, in particular aqueous plant cell culture extracts; PVPP-treated plant cell culture extracts, for example PVPP-treated aqueous and / or lower alcohol plant cell culture extracts, in particular PVPP-treated aqueous plant cell culture extracts; ·Plant cell culture fraction equivalent to Quil A; · Plant cell culture fraction corresponding to fraction A; · a plant cell culture fraction corresponding to fraction B (see Nyberg 2003); · Plant cell culture fraction corresponding to fraction C; · QS-7 fraction; · QS-17 fraction; QS-18 fraction; or ·QS-21 fraction.

[0355] The method of the present invention can be applied to starting saponins in the following compositions: ·QS-7 family ingredients and QS-18 family ingredients; ·QS-7 family ingredients and QS-17 family ingredients; ·QS-17 family ingredients and QS-18 family ingredients; QS-7 family ingredients, QS-17 family ingredients and QS-18 family ingredients.

[0356] Like other QS families, QS-7 family members contain several closely related structures, including xylose and rhamnose chemotypes, xylose and apiose isomers, and A and B isomers:

[0357] [ka] Some QS-7 family members may lack the glucose or rhamnose linked to β-D-fuc.

[0358] enzymatic modification The present invention provides enzymatic modification of saponins obtained from in vitro plant cell cultures. Enzymatic modifications contemplated by the present invention include conversion of a starting saponin to a product saponin by removal of one or more sugar residues from the starting saponin. Preferably, the enzymatic modification contemplated by the present invention is conversion of a starting saponin to a product saponin by removal of one or more sugar residues from the starting saponin.

[0359] In certain embodiments, the enzymatic modification involves the removal of a single sugar residue from the starting saponin, i.e., removal of a terminal sugar residue (an "exo" action). In other embodiments, the enzymatic conversion involves the removal of multiple sugar residues from the starting saponin, i.e., cleavage at a saccharide bond other than the terminal position (an "endo" action), resulting in the removal of multiple sugar residues (such as two, three, or four sugar residues) linked via that saccharide bond.

[0360] Specific sugar residues that can be removed include (e.g. consist of): glucose, in particular terminal glucose, in particular β-glucose, for example β-glucose from chiral acid glycosides, for example the β-D-glucose residues highlighted below:

[0361] [ka]

[0362] Rhamnose, in particular terminal rhamnose, in particular α-rhamnose, for example α-rhamnose from chelate glycosides, for example the α-L-rhamnose residues highlighted below:

[0363] [ka] especially,

[0364] [ka]

[0365] Specific single sugar enzymatic conversions of interest include: QS-18 family ingredients to QS-21 family ingredients, such as: QS-18 2150 components (i.e., QS-18 2150 A and / or QS-18 2150 B to QS-21 1988 components, e.g.: QS-18 2150 A component to QS-21 1988 A component, for example: QS-18 2150 A V1 to QS-21 1988 A V1 ·QS-18 2150 A V2 to QS-21 1988 A V2 QS-18 2150 B component to QS-21 1988 B component, for example: QS-18 2150 B V1 to QS-21 1988 B V1 QS-18 2150 B V2 to QS-21 1998 B V2 QS-18 2150 V1 components (i.e., QS-18 2150 A V1 and / or QS-18 2150 B V1) to QS-21 1988 V1 components, for example: QS-18 2150 A V1 to QS-21 1988 A V1 QS-18 2150 B V1 to QS-21 1988 B V1 QS-18 2150 V2 components (i.e., QS-18 2150 A V2 and / or QS-18 2150 B V2) to QS-21 1988 V2 components, for example: ·QS-18 2150 A V2 to QS-21 1988 A V2 QS-18 2150 B V2 to QS-21 1988 B V2 QS-18 2018 ingredients (i.e., QS-18 2018 A and / or QS-18 2018 B) to QS-21 1856 ingredients, for example: QS-18 2018 A component to QS-21 1856 A component QS-18 2018 B component to QS-21 1856 B component QS-18 2164 components (i.e., QS-18 2164 A and / or QS-18 2164 B) to QS-21 2002 components, for example: QS-18 2164 A component to QS-21 2002 A component, for example: QS-18 2164 A V1 to QS-21 2002 A V1 ·QS-18 2164 A V2 to QS-21 2002 A V2 QS-18 2164 B component to QS-21 2002 B component, for example: QS-18 2164 B V1 to QS-21 2002 B V1 QS-18 2164 B V2 to QS-21 2002 B V2 QS-18 2164 V1 components (i.e., QS-18 2164 A V1 and / or QS-18 2164 B V1) to QS-21 2002 V1 components, for example: QS-18 2164 A V1 to QS-21 2002 A V1 QS-18 2164 B V1 to QS-21 2002 B V1 QS-18 2164 V2 components (i.e., QS-18 2164 A V2 and / or QS-18 2164 B V2) to QS-21 2002 V2 components, for example: ·QS-18 2164 A V2 to QS-21 2002 A V2 QS-18 2164 B V2 to QS-21 2002 B V2

[0366] Desglucosyl QS-17 family components to QS-21 family components, such as: Desglucosyl QS-17 2134 components (i.e., desglucosyl QS-17 2134 A and / or desglucosyl QS-17 2134 B) to QS-21 1988 components, such as: Desglucosyl QS-17 2134 A components to QS-21 1988 A components, for example: Desglucosyl QS-17 2134 A V1 to QS-21 1988 A V1 Desglucosyl QS-17 2134 A V2 to QS-21 1988 A V2 Desglucosyl QS-17 2134 B components to QS-21 1988 B components, for example: Desglucosyl QS-17 2134 B V1 to QS-21 1988 B V1 Desglucosyl QS-17 2134 B V2 to QS-21 1988 B V2 Desglucosyl QS-17 2134 V1 components (i.e., desglucosyl QS-17 2134 A V1 and / or desglucosyl QS-17 2134 B V1) to QS-21 1988 V1 components, such as: Desglucosyl QS-17 2134 A V1 to QS-21 1988 A V1 Desglucosyl QS-17 2134 B V1 to QS-21 1988 B V1 Desglucosyl QS-17 2134 V2 components (i.e., desglucosyl QS-17 2134 A V2 and / or desglucosyl QS-17 2134 B V2) to QS-21 1988 V2 components, such as: Desglucosyl QS-17 2134 A V2 to QS-21 1988 A V2 Desglucosyl QS-17 2134 B V2 to QS-21 1988 B V2 Desglucosyl QS-17 2002 components (i.e., desglucosyl QS-17 2002 A and / or desglucosyl QS-17 2002 B) to QS-21 1856 components, such as: Desglucosyl QS-17 2002 A component to QS-21 1856 A component Desglucosyl QS-17 2002 B component to QS-21 1856 B component Desglucosyl QS-17 2148 components (i.e., desglucosyl QS-17 2148 A and / or desglucosyl QS-17 2148 B) to QS-21 2002 components, such as: Desglucosyl QS-17 2148 A components to QS-21 2002 A components, for example: Desglucosyl QS-17 2148 A V1 to QS-21 2002 A V1 Desglucosyl QS-17 2148 A V2 to QS-21 2002 A V2 Desglucosyl QS-17 2148 B components to QS-21 2002 B components, for example: Desglucosyl QS-17 2148 B V1 to QS-21 2002 B V1 Desglucosyl QS-17 2148 B V2 to QS-21 2002 B V2 Desglucosyl QS-17 2134 V1 components (i.e., desglucosyl QS-17 2134 A V1 and / or desglucosyl QS-17 2134 B V1) to QS-21 1988 V1 components, such as: Desglucosyl QS-17 2148 A V1 to QS-21 2002 A V1 Desglucosyl QS-17 2148 B V1 to QS-21 2002 B V1 Desglucosyl QS-17 2134 V2 components (i.e., desglucosyl QS-17 2134 A V2 and / or desglucosyl QS-17 2134 B V2) to QS-21 1988 V1 components, such as: Desglucosyl QS-17 2148 A V2 to QS-21 2002 A V2 Desglucosyl QS-17 2148 B V2 to QS-21 2002 B V2

[0367] QS-17 family ingredients to QS-18 family ingredients, such as: QS-17 2296 components (i.e., QS-17 2296 A and / or QS-17 2296 B) to QS-18 2150 components, for example: QS-17 2296 A component to QS-18 2150 A component, for example: QS-17 2296 A V1 to QS-18 2150 A V1 ·QS-17 2296 A V2 to QS-18 2150 A V2 QS-17 2296 B component to QS-18 2150 B component, for example: QS-17 2296 B V1 to QS-18 2150 B V1 QS-17 2296 B V2 to QS-18 2150 B V2 QS-17 2296 V1 components (i.e., QS-17 2296 A V1 and / or QS-17 2296 B V1) to QS-18 2150 V1 components, for example: QS-17 2296 A V1 to QS-18 2150 A V1 QS-17 2296 B V1 to QS-18 2150 B V1 QS-17 2296 V2 components (i.e., QS-17 2296 A V2 and / or QS-17 2296 B V1) to QS-18 2150 V2 components, for example: ·QS-17 2296 A V2 to QS-18 2150 A V2 QS-17 2296 B V2 to QS-18 2150 B V2 QS-17 2164 components (i.e., QS-17 2164 A and / or QS-17 2164 B) to QS-18 2018 components, for example: QS-17 2164 A component to QS-18 2018 A component QS-17 2164 B component to QS-18 2018 B component QS-17 2310 components (i.e., QS-17 2310 A and / or QS-17 2310 B) to QS-18 2164 components, for example: QS-17 2310 A component to QS-18 2164 A component, for example: QS-17 2310 A V1 to QS-18 2164 A V1 ·QS-17 2310 A V2 to QS-18 2164 A V2 QS-17 2310 B component to QS-18 2164 B component, for example: QS-17 2310 B V1 to QS-18 2164 B V1 QS-17 2310 B V2 to QS-18 2164 B V2 QS-17 2310 V1 components (i.e., QS-17 2310 A V1 and / or QS-17 2310 B V1) to QS-18 2164 V1 components, for example: QS-17 2310 A V1 to QS-18 2164 A V1 QS-17 2310 B V1 to QS-18 2164 B V1 QS-17 2310 V2 components (i.e., QS-17 2310 A V2 and / or QS-17 2310 B V2) to QS-18 2164 V2 components, for example: ·QS-17 2310 A V2 to QS-18 2164 A V2 QS-17 2310 B V2 to QS-18 2164 B V2

[0368] QS-17 family components, including desglucosyl QS-17 family components, such as: QS-17 2296 components (i.e., QS-17 2296 A and / or QS-17 2296 B) to desglucosyl QS-17 2134 components, such as: QS-17 2296 A component to desglucosyl QS-17 2134 A component, for example: QS-17 2296 A V1 to Desglucosyl QS-17 2134 A V1 QS-17 2296 A V2 to Desglucosyl QS-17 2134 A V2 QS-17 2296 B component to desglucosyl QS-17 2134 B component, for example: QS-17 2296 B V1 to Desglucosyl QS-17 2134 B V1 QS-17 2296 B V2 to Desglucosyl QS-17 2134 B V2 QS-17 2296 V1 components (i.e., QS-17 2296 A V1 and / or QS-17 2296 B V1) to desglucosyl QS-17 2134 V1 components, such as: QS-17 2296 A V1 to Desglucosyl QS-17 2134 A V1 QS-17 2296 B V1 to Desglucosyl QS-17 2134 B V1 QS-17 2296 V2 components (i.e., QS-17 2296 A V2 and / or QS-17 2296 B V1) to desglucosyl QS-17 2134 V2 components, such as: QS-17 2296 A V2 to Desglucosyl QS-17 2134 A V2 QS-17 2296 B V2 to Desglucosyl QS-17 2134 B V2 QS-17 2164 components (i.e., QS-17 2164 A and / or QS-17 2164 B) to desglucosyl QS-17 2002 components, such as: QS-17 2164 A component to desglucosyl QS-17 2002 A QS-17 2164 B component to desglucosyl QS-17 2002 B QS-17 2310 components (i.e., QS-17 2310 A and / or QS-17 2310 B) to desglucosyl QS-17 2148 components, such as: QS-17 2310 A component to desglucosyl QS-17 2148 A component, for example: QS-17 2310 A V1 to Desglucosyl QS-17 2148 A V1 QS-17 2310 A V2 to Desglucosyl QS-17 2148 A V2 QS-17 2310 B component to QS-21, for example: QS-17 2310 B V1 to Desglucosyl QS-17 2148 B V1 QS-17 2310 B V2 to Desglucosyl QS-17 2148 B V2 QS-17 2310 V1 components (i.e., QS-17 2310 A V1 and / or QS-17 2310 B V1) to desglucosyl QS-17 2148 V1 components, such as: QS-17 2310 A V1 to Desglucosyl QS-17 2148 A V1 QS-17 2310 B V1 to Desglucosyl QS-17 2148 B V1 QS-17 2310 V2 components (i.e., QS-17 2310 A V2 and / or QS-17 2310 B V2) to desglucosyl QS-17 2148 V2 components, such as: QS-17 2310 A V2 to Desglucosyl QS-17 2148 A V2 QS-17 2310 B V2 to Desglucosyl QS-17 2148 B V2.

[0369] Other monosaccharide enzymatic transformations of interest include: Desarabinofuranosyl QS-18 family compounds to desarabinofuranosyl QS-21 family compounds, such as: Desarabinofuranosyl QS-18 2018 components (i.e., desarabinofuranosyl QS-18 2018 A and / or desarabinofuranosyl QS-18 2018 B) to desarabinofuranosyl QS-21 1856 components, such as: Desarabinofuranosyl QS-18 2018 A component to desarabinofuranosyl QS-21 1856 A component, for example: Desarabinofuranosyl QS-18 2018 A V1 to Desarabinofuranosyl QS-21 1856 A V1 Desarabinofuranosyl QS-18 2018 A V2 to Desarabinofuranosyl QS-21 1856 A V2 Desarabinofuranosyl QS-18 2018 B component to desarabinofuranosyl QS-21 1856 B component, for example: Desarabinofuranosyl QS-18 2018 B V1 to Desarabinofuranosyl QS-21 1856 B V1 Desarabinofuranosyl QS-18 2018 B V2 to Desarabinofuranosyl QS-21 1856 B V2 Desarabinofuranosyl QS-18 2018 V1 components (i.e., desarabinofuranosyl QS-18 2018 A V1 and / or desarabinofuranosyl QS-18 2018 B V1) to desarabinofuranosyl QS-21 1856 V1 components, such as: Desarabinofuranosyl QS-18 2018 A V1 to Desarabinofuranosyl QS-21 1856 A V1 Desarabinofuranosyl QS-18 2018 B V1 to Desarabinofuranosyl QS-21 1856 B V1 Desarabinofuranosyl QS-18 2018 V2 components (i.e., desarabinofuranosyl QS-18 2018 A V2 and / or desarabinofuranosyl QS-18 2018 B V2) to desarabinofuranosyl QS-21 1856 V2 components, such as: Desarabinofuranosyl QS-18 2018 A V2 to Desarabinofuranosyl QS-21 1856 A V2 Desarabinofuranosyl QS-18 2018 B V2 to Desarabinofuranosyl QS-21 1856 B V2 Desarabinofuranosyl QS-18 1886 components (i.e., desarabinofuranosyl QS-18 1886 A and / or desarabinofuranosyl QS-18 1886 B) to desarabinofuranosyl QS-21 1712 components, such as: Desarabinofuranosyl QS-18 1886 A component to desarabinofuranosyl QS-21 1712 A Desarabinofuranosyl QS-18 1886 B component to desarabinofuranosyl QS-21 1712 B Desarabinofuranosyl QS-18 2032 components (i.e., desarabinofuranosyl QS-18 2032 A and / or desarabinofuranosyl QS-18 2032 B) to desarabinofuranosyl QS-21 1870 components, such as: Desarabinofuranosyl QS-18 2032 A component to desarabinofuranosyl QS-21 1870 A component, for example: Desarabinofuranosyl QS-18 2032 A V1 to Desarabinofuranosyl QS-21 1870 A V1 Desarabinofuranosyl QS-18 2032 A V2 to Desarabinofuranosyl QS-21 1870 A V2 Desarabinofuranosyl QS-18 2032 B components to QS-21, for example: Desarabinofuranosyl QS-18 2032 B V1 to Desarabinofuranosyl QS-21 1870 B V1 QS-17 Desarabinofuranosyl QS-18 2032 B V2 to Desarabinofuranosyl QS-21 1870 B V2 Desarabinofuranosyl QS-18 2032 V1 components (i.e., desarabinofuranosyl QS-18 2032 A V1 and / or desarabinofuranosyl QS-18 2032 B V1) to desarabinofuranosyl QS-21 1870 V1 components, such as: Desarabinofuranosyl QS-18 2032 A V1 to Desarabinofuranosyl QS-21 1870 A V1 Desarabinofuranosyl QS-18 2032 B V1 to Desarabinofuranosyl QS-21 1870 B V1 Desarabinofuranosyl QS-18 2032 V2 components (i.e., desarabinofuranosyl QS-18 2032 A V2 and / or desarabinofuranosyl QS-18 2032 B V2) to desarabinofuranosyl QS-21 1870 V2 components, such as: Desarabinofuranosyl QS-18 2032 A V2 to Desarabinofuranosyl QS-21 1870 A V2 Desarabinofuranosyl QS-18 2032 B V2 to Desarabinofuranosyl QS-21 1870 B V2

[0370] Acetylated desglucosyl QS-17 components to acetylated QS-21 family components, for example: Acetylated desglucosyl QS-17 2176 A to acetylated QS-21 2030 A, for example: Acetylated desglucosyl QS-17 2176 A V1 to Acetylated QS-21 2030 A V1 Acetylated desglucosyl QS-17 2176 A V2 to Acetylated QS-21 2030 A V2 Acetylated desglucosyl QS-17 2044 A to Acetylated QS-21 1898 A Acetylated desglucosyl QS-17 2190 A to Acetylated QS-21 2044 A, for example: Acetylated desglucosyl QS-17 2190 A V1 to Acetylated QS-21 2044 A V1 Acetylated desglucosyl QS-17 2190 A V2 to acetylated QS-21 2044 A V2.

[0371] The enzymatic conversion can be applied to a single starting saponin or to multiple starting saponins in parallel. It will be understood that the process can include or consist of the conversions specified above, depending on the composition of the starting material and the enzymes used. Furthermore, while a process may be limited to the use of a single enzyme intended to remove a particular sugar residue or group of sugar residues from (i) a single starting saponin, (ii) a family of starting saponins, or (iii) multiple families of starting saponins, the process can also employ multiple enzymes intended to remove multiple sugar residues from (i) a single starting saponin, (ii) a family of starting saponins, or (iii) multiple families of starting saponins. Processes involving multiple enzymes can be performed sequentially (i.e., a single enzyme is applied to the saponin material at any one time) or in parallel (i.e., two or more enzymes, e.g., two or three enzymes, particularly two enzymes, are applied to the saponin material at any one time), or a combination thereof.

[0372] A process involving the removal of multiple sugar residues can include the removal of a single (but different) sugar residue from multiple starting saponins and / or the removal of multiple sugar residues (e.g., 2, 3 or 4 residues, particularly 2 or 3, especially 2 residues) from a particular starting saponin. The removal of multiple sugar residues from a particular starting saponin can include any combination of the removal of a single residue and / or the removal of multiple residues in a single cleavage.

[0373] An exemplary process can include (e.g., consist of) the removal of glucose and rhamnose, particularly α-rhamnose and β-glucose residues, e.g., α-L-rhamnose and β-D-glucose residues, from chiral acid glycosides:

[0374] [ka]

[0375] Specific multisaccharide enzymatic transformations of interest include: QS-17 family ingredients to QS-21 family ingredients, such as: QS-17 2296 components (i.e., QS-17 2296 A and / or QS-17 2296 B) to QS-21 1988 components, such as: QS-17 2296 A component to QS-21 1988 A component, for example: QS-17 2296 A V1 to QS-21 1988 A V1 ·QS-17 2296 A V2 to QS-21 1988 A V2 QS-17 2296 B component to QS-21 1988 B component, for example: QS-17 2296 B V1 to QS-21 1988 B V1 QS-17 2296 B V2 to QS-21 1988 B V2 QS-17 2296 V1 components (i.e., QS-17 2296 A V1 and / or QS-17 2296 B V1) to QS-21 1988 V1 components, for example: QS-17 2296 A V1 to QS-21 1988 A V1 QS-17 2296 B V1 to QS-21 1988 B V1 QS-17 2296 V2 components (i.e., QS-17 2296 A V2 and / or QS-17 2296 B V2) to QS-21 1988 V2 components, for example: ·QS-17 2296 A V2 to QS-21 1988 A V2 QS-17 2296 B V2 to QS-21 1988 B V2 QS-17 2164 components (i.e., QS-17 2164 A and / or QS-17 2164 B) to QS-21 1856 components, such as: QS-17 2164 A component to QS-21 1856 A component QS-17 2164 B component to QS-21 1856 B component QS-17 2310 components (i.e., QS-17 2310 A and / or QS-17 2310 B) to QS-21 2002 components, for example: QS-17 2310 A component to QS-21 2002 A component, for example: QS-17 2310 A V1 to QS-21 2002 A V1 ·QS-17 2310 A V2 to QS-21 2002 A V2 QS-17 2310 B component to QS-21 2002 B component, for example: QS-17 2310 B V1 to QS-21 2002 B V1 QS-17 2310 B V2 to QS-21 2002 B V2 QS-17 2310 V1 ingredients (i.e., QS-17 2310 A V1 and / or QS-17 2310 B V1) to QS-21 2002 V1, for example: QS-17 2310 A V1 to QS-21 2002 A V1 QS-17 2310 B V1 to QS-21 2002 B V1 QS-17 2310 V2 components (i.e., QS-17 2310 A V2 and / or QS-17 2310 B V2) to QS-21 2002 V2, for example: ·QS-17 2310 A V2 to QS-21 2002 A V2 QS-17 2310 B V2 to QS-21 2002 B V2.

[0376] Plant cell culture extracts can contain complex mixtures of saponin components and, as a result, can undergo multiple transformations when multiple enzymes are present. For example, a starting mixture containing QS-17, QS-18, and desglucosyl QS-17 components treated in parallel with appropriate β-glucosidases and α-rhamnosidases can undergo transformations including: QS-18 family components to QS-21 family components, especially QS-18 2150 components to QS-21 1988 components; Desglucosyl QS-17 family components to QS-21 family components, especially desglucosyl QS-17 2134 components to QS-21 1988 components; QS-17 family components to desglucosyl QS-17 family components to QS-21 components, in particular QS-17 2296 components to desglucosyl QS-17 2134 components to QS-21 1988 components; and - QS-17 family components to QS-18 family components to QS-21 family components, especially QS-17 2296 components to QS-18 2150 components to QS-21 1988 components.

[0377] Enzyme selection Large protein or DNA databases of natural and artificial glycosidases are available. Candidate enzymes can be selected and screened to assess their suitability for achieving a particular transformation under specific reaction conditions. The suitability of an enzyme will depend on many factors, including: Target sugars (e.g., glucose, rhamnose) · Target sugar anomer (α or β); · Target sugar enantiomer (D or L); Target sugar configuration (endo or exo); and · Target sugar environment (e.g., chemical / physical, affecting accessibility and reactivity).

[0378] Additional factors that promote efficient conversion include: Conversion speed; Environmental sensitivity – including pH, temperature, substrate, product and contaminant concentration tolerance; and Specificity for the target sugar, including with respect to other sugar residues, other anomers, other sugar residue configurations, and between different residues of the same sugar anomer and configuration within the substrate (when multiple such residues are present).

[0379] Those skilled in the art will understand that the level and type of specificity required from an enzyme will depend on the objectives to be achieved and the overall situation.

[0380] The conversion of QS-18 family components to QS-21 family components requires an enzyme that exhibits β-exoglucosidase activity. Conversion of QS-17 family components to desglucosyl QS-17 family components requires an enzyme that exhibits β-exoglucosidase activity. The conversion of desglucosyl QS-17 family components to QS-21 family components requires an enzyme that exhibits α-exorhamnosidase activity. The conversion of QS-17 family components to QS-18 family components requires an enzyme that exhibits α-exorhamnosidase activity.

[0381] It can be noted that many of the target Quillaja saponaria starting saponins contain only one glucose residue. Many of the target Quillaja saponaria starting saponins contain multiple rhamnose residues, and therefore, selectivity for a particular rhamnose residue is relatively important in practice. For example, conversion of desglucosyl QS-17 family components to QS-21 components or QS-17 family components to QS-18 family components requires specificity for exorhamnosidase action over endorhamnosidase action. Furthermore, rhamnosidase specificity for α-O-rhamnosylation at the C2 position of the arabinofuranose moiety over other terminal rhamnose residues (e.g., in rhamnose chemotype components) may also be desirable. In certain embodiments, removal of terminal rhamnose from rhamnose chemotype components (either alone or in conjunction with either α-O-rhamnosylation at the C2 position of the arabinofuranose moiety) may be desirable to better facilitate their chromatographic separation from xylose chemotype components.

[0382] In one embodiment, the enzymatic conversion is carried out by a single enzyme. The single enzyme may be a glucosidase, particularly a β-exoglucosidase. The single enzyme glucosidase may be an engineered glucosidase polypeptide described in the present invention. Alternatively, the single enzyme is a rhamnosidase, particularly an α-exorhamnosidase. The single enzyme rhamnosidase may be an engineered rhamnosidase polypeptide described in the present invention.

[0383] Preferred enzymes are those that efficiently enzymatically convert the starting saponin to the desired product saponin with limited or no undesired conversion of other saponin components present.

[0384] In one embodiment, the enzymatic conversion is carried out by two or more enzymes, for example by two or three enzymes, in particular by two enzymes. The enzymatic modification (or conversion) by two or more enzymes can comprise sequential / series enzymatic modification. Alternatively, the enzymatic modification (or conversion) by two or more enzymes can comprise simultaneous / parallel enzymatic modification. The enzymatic modification (or conversion) by at least three enzymes can comprise a combination of sequential / series (modification by one enzyme) and simultaneous / parallel (modification by at least two other enzymes) enzymatic modification, in any order. When multiple enzymes are provided, they can exist as different proteins or can be in the form of one or more fusion proteins.

[0385] An enzyme of interest is a glucosidase, such as a β-exoglucosidase. The glucosidase can be an engineered glucosidase polypeptide of the invention. Another enzyme of interest is a rhamnosidase, such as an α-exorhamnosidase. The rhamnosidase can be an engineered rhamnosidase polypeptide of the invention. Enzyme combinations of interest include those comprising, e.g., consisting of, a glucosidase and a rhamnosidase, particularly a β-exoglucosidase and an α-exorhamnosidase. Enzymatic modifications involving glucosidases and rhamnosidases, particularly β-exoglucosidases and α-exorhamnosidases, can be carried out sequentially with a glucosidase (e.g., β-exoglucosidase) and then a rhamnosidase (e.g., α-exorhamnosidase), sequentially with a rhamnosidase (e.g., α-exorhamnosidase) and then a glucosidase (e.g., β-exoglucosidase), or, advantageously, simultaneously with both a glucosidase (e.g., β-exoglucosidase) and a rhamnosidase (e.g., α-exorhamnosidase). Enzyme combinations of particular interest comprise, e.g., consist of, an engineered glucosidase of the invention and an engineered rhamnosidase polypeptide of the invention.

[0386] The enzymes utilized are typically exogenous to the saponin material, i.e., will not be found naturally within the source of the saponin obtained by extraction. The enzyme can be a native, i.e., naturally occurring glycosidase, or alternatively, a non-naturally occurring glycosidase. In one embodiment, the glucosidase enzyme is a naturally occurring glucosidase (e.g., an exoglucosidase such as β-exoglucosidase). In a second embodiment, the glucosidase enzyme is a non-naturally occurring glucosidase (e.g., an exoglucosidase such as β-exoglucosidase). In one embodiment, the rhamnosidase enzyme is a naturally occurring rhamnosidase (e.g., an exorhamnosidase such as α-exorhamnosidase). In a second embodiment, the rhamnosidase enzyme is a non-naturally occurring rhamnosidase (e.g., an exorhamnosidase such as α-exorhamnosidase).

[0387] The enzyme can be modified ("engineered") compared to a reference enzyme. Point mutations introduced by genetic engineering, alone or in combination, can provide advantages such as increased activity, increased specificity, increased stability, increased expression, etc. Assays for confirming enzyme properties are well known to those skilled in the art. For example, activity can be quantified by methods such as those shown in the Examples (see Examples 4-7) or by similar methods.

[0388] Different enzymes may exhibit different sensitivities to environmental conditions such as pH, temperature, substrate concentration, product concentration, solvent composition, presence of contaminants, etc. Such parameters can be taken into consideration during screening of candidate enzymes for the desired activity.

[0389] Candidate enzymes having β-glucosidase activity include those in EC 3.2.1.21.

[0390] β-exoglucosidases of interest include those set forth in Table 7, particularly SEQ ID NOs: 262, 208, 63, 229, 250, 5, 101, 207, 169, 247, 302, 324, 319, 9, 240, 325, and 338, and functional variants thereof. Particular β-exoglucosidases of interest include SEQ ID NOs: 262, 208, 63, 229, 250, 5, 101, 207, 169, 247, 302, 324, and 319, and functional variants thereof, such as SEQ ID NOs: 262, 208, 63, 229, 250, 5, 101, and 207, and functional variants thereof.

[0391] Another group of β-exoglucosidases of interest include those set forth in Table 9, particularly SEQ ID NOs: 850, 879, 868, 826, 804, 888, 881, 891, 816, 827, 857, 853, 842, 814, 886, 885, 838, 829, 808, 828, 870, 873, 844, 882, 874, 825, 824, 823, 810, 894, 849, 803, 890, 841, 832, 830, 845, 871, 837, 883 and 809, and functional variants thereof. Specific β-exoglucosidases of interest include those listed in SEQ ID NOs: 850, 879, 868, 826, 804, 888, 881, 891, 816, 827, 857, 853, 842, 814, 886, 885, 838, 829, 808, 828, 870, 873, 844, 882, 874, 825, 824, 823, 810, 894, 849, 803, 890, and 841 , and functional variants thereof, for example, SEQ ID NOs: 850, 879, 868, 826, 804, 888, 881, 891, 816, 827, 857, 853, 842, 814, 886, 885, 838, 829, 808, 828, 870, 873, 844, 882, 874, 825, 824, 823, 810 and 894, and functional variants thereof.

[0392] SEQ ID NO: 262 and functional variants thereof are particularly desirable β-exoglucosidases. In one embodiment, the β-exoglucosidase comprises, e.g., consists of, (i) SEQ ID NO: 262; or (ii) a functional variant thereof having at least 80% identity to SEQ ID NO: 262, particularly at least 90%, particularly at least 95%, such as at least 96%, at least 97%, at least 98%, for example at least 99% identity; or (iii) a functional fragment of at least 100, particularly at least 200, particularly at least 300, such as at least 400, for example at least 500 consecutive amino acids of SEQ ID NO: 262.

[0393] Candidate enzymes with α-rhamnosidase activity include those in EC 3.2.1.40.

[0394] Alpha-exorhamnosidases of interest include SEQ ID NOs: 992, 1003, 1052, 1073, 1017, 1055, 1075, 1001, 1007, 1061, 1079, 1027, 1039, 1041, 989, 1053, 1018, 1066, 1082, 1076, 993, 1077, 1046, 1015, 1063, 1054, 1074, 1067, and 1033, and functional variants thereof. Particular alpha-exorhamnosidases of interest include SEQ ID NOs: 992, 1003, 1052, 1073, 1017, 1055, 1075, 1001, 1007, 1061, 1079, 1027, 1039, 1041, 989, 1053, 1018, 1066, 1082, 1076, 993 and 1077, and functional variants thereof, such as SEQ ID NOs: 992, 1003, 1052, 1073, 1017, 1055, 1075, 1001, 1007, 1061, 1079, 1027, 1039, 1041 and 989, and functional variants thereof.

[0395] SEQ ID NO: 1017 and functional variants thereof are particularly desirable exorhamnosidases. In one embodiment, the α-exorhamnosidase comprises, e.g., consists of, (i) SEQ ID NO: 1017; or (ii) a functional variant thereof having at least 80% identity to SEQ ID NO: 1017, particularly at least 90%, particularly at least 95%, such as at least 96%, at least 97%, at least 98%, for example at least 99% identity; or (iii) a functional fragment of at least 100, particularly at least 200, particularly at least 300, such as at least 400, for example at least 500 consecutive amino acids of SEQ ID NO: 1017.

[0396] Functional variants of interest in the present application include (i) sequences having at least 80% identity to a reference sequence, particularly at least 90%, particularly at least 95%, such as at least 96%, at least 97%, at least 98%, for example at least 99% identity; or (ii) sequences comprising, e.g. consisting of, a fragment of at least 100, particularly at least 200, particularly at least 300, such as at least 400, for example at least 500 consecutive amino acids of the reference sequence.

[0397] Specific desirable functional variants of interest include, for example, sequences having 1 to 20 additions, deletions and / or substitutions, particularly 1 to 15 additions, deletions and / or substitutions, particularly 1 to 10 additions, deletions and / or substitutions, for example 1 to 5 additions, deletions and / or substitutions, relative to the reference sequence, and for example, those consisting of such sequences.

[0398] The degree of sequence identity can be determined using the Needleman and Wunsch homology alignment algorithm, the ClustalW program or the BLASTP algorithm, using default settings. Algorithms using global alignments (Needleman and Wunsch) are preferred.

[0399] "Percentage of sequence identity," "percent identity," and "percent identity" are used herein to refer to a comparison between polynucleotide or polypeptide sequences and are determined by comparing two optimally aligned sequences over a comparison window, where a portion of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps) when compared to a reference sequence due to optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where identical nucleic acid bases or amino acid residues exist in both sequences, or by aligning nucleic acid bases or amino acid residues with gaps to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Optimal alignment and percent sequence identity are determined using the BLAST and BLAST 2.0 algorithms (see, for example, Altschul, 1990; Altschul, 1997). Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information website. Briefly, BLAST analysis involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in a query sequence that, when aligned with words of the same length in a database sequence, match or meet some positive threshold score, T. T is referred to as the neighborhood word score threshold (Altschul, supra). These initial neighborhood word hits serve as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. For nucleotide sequences, cumulative scores are calculated using the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate cumulative scores.Extension of word hits in each direction is stopped when the cumulative alignment score falls below its maximum achieved value by an amount X; when the cumulative score falls below zero due to the accumulation of one or more negatively scoring residue alignments; or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff, 1989).

[0400] Many other algorithms are available that function similarly to BLAST in providing percent identity between two sequences. Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith, 1981, the homology alignment algorithm of Needleman, 1970, the search for similarity method of Pearson, 1988, computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin software package), or by visual inspection (see generally Current Protocols in Molecular Biology, 1995). In addition, sequence alignment and determination of percent sequence identity can be performed using the BESTFIT or GAP program in the GCG Wisconsin software package (Accelrys, Madison, WI) using the default parameters provided. The ClustalW program is also suitable for determining identity.

[0401] Modestobacter marinus glucosidase (Uniparc Reference UPI000260A2FA, Uniprot Reference I4EYD5; SEQ ID NO: 262 herein) is a naturally occurring glucosidase that exhibits β-exoglucosidase activity, for example, converting QS-18 family members to QS-21 family members. Despite its potent activity, we have found that the properties of wild-type Modestobacter marinus glucosidase can be altered by introducing one or more mutations.

[0402] The present invention describes an engineered glycosidase polypeptide for use in the methods of the invention, comprising, e.g., consisting of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 262, or a functional fragment thereof, wherein the engineered glycosidase polypeptide is: F44Y; V60L; G117A; F170N; V263G or V263L; N351H or N351Q; A355H, A355I, A355L, A355M, A355R, A355T or A355W; A356P; R357A, R357C, R357K, R357M or R357Q; G362C; T365A, T365N or T365S; L367C; V394R; V395Y; Q396E, Q396G, Q396N, Q396P, Q396R, Q396S or Q396Y; F430W; R435F; V438T; V440F; F442M or F442Q; G444T; A473F or A473R; L474C, L474I or L474V; I475F; L492C, L492G, L492H, L492I, L492N, L492Q, L492V, L492W or L492Y; Q493F or Q493H; P494H or P494I; S495I, S495K or S495Q; G496P or G496W; D498A, D498E, D498F, D498I, D498K, D498L, D498N, D498P, D498R, D498S, D498T or D498V; A502R; M504G or M504R; L507A or L507R; T508M; L529M; F535P; A536D or A536E; A537R; F541A, F541I, F541L, F541M or F541V; L542I; Q543G or Q543L; E547L; and Y585W It contains at least one residue substitution from

[0403] The glucosidase may include 1 to 42 substitutions, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26-30 or 31-43 substitutions.

[0404] The present invention also describes an engineered glucosidase polypeptide for use in the methods of the invention, comprising, e.g., consisting of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 262, or a functional fragment thereof, wherein the engineered glucosidase polypeptide is F44Y; V263L; N351H; A355H, A355M or A355W; R357M; T365N; L367C; Q396R; V438T; F442Q; L474C; I475F; L492V, L492N or L492H, M504R; L507R; and F541I It contains at least one residue substitution from

[0405] The glucosidase may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all 16 substitutions.

[0406] The engineered glucosidase polypeptide can comprise, e.g., consist of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 262, or a functional fragment thereof, wherein the engineered glucosidase polypeptide comprises at least one residue substitution from F44Y, V263L, A355W, R357M, T365N, L367C, Q396R, F442Q, L474C, I475F, and F541I.

[0407] Suitably, the engineered glucosidase polypeptide comprises, e.g., consists of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 262, or a functional fragment thereof, wherein the engineered glucosidase polypeptide comprises the following residue substitutions: F44Y, V263L, A355W, R357M, T365N, L367C, Q396R, F442Q, L474C, I475F, and F541I.

[0408] The present invention provides (i) F44Y (ii) V263L (iii) N351H (iv) A355H, A355I, A355L, A355M, A355R, A355T or A355W (v) A356P (vi) R357M (vii) T365N (viii) L367C (ix) F442Q (x) G443D (xi) A473F (xii) L474C (xiii) I475F (xiv) L492H, L492N, L492V (xv) P494I (xvi) G496P (xvii) D498P (xviii) M504R (xix) L507R (xx) F535P (xxi) A537R (xxii) F541I (xxiii) L542I (xxiv) E547L and (xxv) E588K The present invention describes a polypeptide for use in the methods of the present invention, comprising the amino acid sequence of SEQ ID NO: 262, comprising 1 to 25 mutations selected from the list consisting of:

[0409] The variant glucosidase may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or all 25 mutations.

[0410] In some embodiments, the engineered glucosidase is (i) F44Y (ii) V263L (iii) N351H (iv) A355H, A355I, A355L, A355M, A355R, A355T or A355W (v) A356P (vi) R357M (vii) T365N (viii) L367C (ix) F442Q (x) G443D (xi) A473F (xii) L474C (xiii) I475F (xiv) L492H, L492N, L492V (xv) P494I (xvi) G496P (xvii) D498P (xviii) M504R (xix) L507R (xx) F535P (xxi) A537R (xxii) F541I (xxiii) L542I (xxiv) E547L and (xxv) E588K and the polypeptide is not a polypeptide comprising the amino acid sequence of SEQ ID NO: 262 containing 1 to 25 mutations selected from the list consisting of:

[0411] The above engineered glucosidase polypeptides may also be referred to herein as examples of "variant glucosidases." The mutant glucosidase can include F44Y. The mutant glucosidase can include V60L. The mutant glucosidase can include G117A. The mutant glucosidase can include F170N. The variant glucosidase may comprise V263G or V263L, particularly V263L. The mutant glucosidase may comprise N351H or N351Q, particularly N351H. The variant glucosidase can comprise A355H, A355I, A355L, A355M, A355R, A355T, or A355W. In some embodiments, the variant glucosidase comprises A355H. In some embodiments, the variant glucosidase comprises A355I. In some embodiments, the variant glucosidase comprises A355L. In some embodiments, the variant glucosidase comprises A355M. In some embodiments, the variant glucosidase comprises A355R. In some embodiments, the variant glucosidase comprises A355T. In some embodiments, the variant glucosidase comprises A355W.

[0412] The mutant glucosidase can include A356P. The mutant glucosidase may comprise R357A, R357C, R357K, R357M or R357Q, particularly R357M. The mutant glucosidase can include G362C. The mutant glucosidase may comprise T365A, T365N or T365S, particularly T365N. The mutant glucosidase may comprise L367C. The mutant glucosidase can include V394R. The mutant glucosidase can include V395Y. The mutant glucosidase may comprise Q396E, Q396G, Q396N, Q396P, Q396R, Q396S or Q396Y, in particular Q396R. The mutant glucosidase can include F430W. The mutant glucosidase can include R435F. The variant glucosidase can include V438T. The mutant glucosidase can include V440F. The mutant glucosidase may comprise F442M or F442Q, particularly F442Q. The mutant glucosidase can include G443D. The mutant glucosidase can include G444T. The mutant glucosidase may comprise A473F or A473R, particularly A473F. The mutant glucosidase may comprise L474C, L474I or L474V, particularly L474C. The mutant glucosidase may comprise I475F.

[0413] The mutant glucosidase can comprise L492C, L492G, L492H, L492I, L492N, L492Q, L492V, L492W, or L492Y, particularly L492H, L492N, or L492V. In some embodiments, the mutant glucosidase comprises L492H. In some embodiments, the mutant glucosidase comprises L492N. In some embodiments, the mutant glucosidase comprises L492V. The mutant glucosidase can include Q493F or Q493H. The variant glucosidase may comprise P494H or P494I, particularly P494I. The mutant glucosidase can include S495I, S495K, or S495Q. The mutant glucosidase may comprise G496P or G496W, particularly G496P. The variant glucosidase may comprise D498A, D498E, D498F, D498I, D498K, D498L, D498N, D498P, D498R, D498S, D498T or D498V, particularly D498P. The mutant glucosidase can include A502R. The mutant glucosidase may comprise M504G or M504R, particularly M504R. The mutant glucosidase may comprise L507A or L507R, particularly L507R. The mutant glucosidase can include T508M. The mutant glucosidase can include L529M. The mutant glucosidase can include F535P. The mutant glucosidase can include A536D or A536E. The mutant glucosidase can include A537R.

[0414] The mutant glucosidase may comprise F541A, F541I, F541L, F541M or F541V, particularly F541I. The mutant glucosidase can include L542I. The variant glucosidase can include Q543G or Q543L. The mutant glucosidase can include E547L. The mutant glucosidase can include Y585W. The mutant glucosidase can include E588K. The mutant glucosidase can include R357M, T365N, A473F, L474C and I475F. The mutant glucosidase can include F44Y, R357M, T365N, F442Q, A473F, L474C, and I475F. The mutant glucosidase can include F44Y, V263L, R357M, T365N, F442Q, A473F, L474C, I475F and F541I. The mutant glucosidase can include F44Y, V263L, A355W, R357M, T365N, L367C, Q396R, F442Q, L474C, I475F and F541I.

[0415] The mutant glucosidases were F44Y, V263L, R357M, T365N, F442Q, L474C, I475F, F541I, and: (iii) N351H (iv) A355H, A355I, A355L, A355M, A355R, A355T or A355W (v) A356P (viii) L367C (x) G443D (xi) A473F (xiv) L492H, L492N, L492V (xv) P494I (xvi) G496P (xvii) D498P (xviii) M504R (xix) L507R (xx) F535P (xxi) A537R (xxiii) L542I (xxiv) E547L and (xxv) E588K The mutations may include 0 to 17 mutations selected from the list consisting of:

[0416] A mutant glucosidase can include a "tag," which is a sequence of amino acids that allows for isolation and / or identification of the polypeptide. For example, the addition of an affinity tag can be useful for purification. Exemplary affinity tags that can be used include a histidine (HIS) tag (e.g., a hexahistidine tag or a 6xHis tag), a FLAG tag, and an HA tag. The tag can be located at the N-terminus or C-terminus and can be directly linked or linked via a linking sequence. SEQ ID NO: 1177 provides the sequence for an exemplary 6xHis tag, including a linker sequence, that can be attached to the N-terminus. SEQ ID NO: 1178 provides the sequence for an exemplary 6xHis tag, including a linker sequence, that can be attached to the C-terminus.

[0417] In certain embodiments, tags as used herein are removable, e.g., capable of being removed by chemical agents or enzymatic means when they are no longer needed, e.g., after the polypeptide has been purified.

[0418] The variant glucosidase may comprise 1000 or fewer residues, particularly 950 or fewer residues, especially 900 or fewer residues, for example 850 or fewer residues. The mutant glucosidase is (i) F44Y (ii) V263L (iii) N351H (iv) A355H, A355I, A355L, A355M, A355R, A355T or A355W (v) A356P (vi) R357M (vii) T365N (viii) L367C (ix) F442Q (x) G443D (xi) A473F (xii) L474C (xiii) I475F (xiv) L492H, L492N, L492V (xv) P494I (xvi) G496P (xvii) D498P (xviii) M504R (xix) L507R (xx) F535P (xxi) A537R (xxii) F541I (xxiii) L542I (xxiv) E547L and (xxv) E588K The amino acid sequence may consist of the amino acid sequence of SEQ ID NO: 262, containing 1 to 25 mutations selected from the list consisting of:

[0419] The variant glucosidase desirably exhibits an FIOP (fold improvement over parent) relative to SEQ ID NO: 262 of at least 1.05, particularly at least 2, particularly at least 10, such as at least 50. FIOP can be determined by the method described in Example 4.

[0420] Kluyvera flavida rhamnosidase (Uniparc Reference UPI00019BDB13, Uniprot Reference D2PMT5; SEQ ID NO: 1017 herein) is a naturally occurring rhamnosidase that exhibits α-exorhamnosidase activity, including the conversion of desglucosyl QS-17 family components to QS-21 family components. Despite its potent activity, we have found that the properties of wild-type Kluyvera flavida rhamnosidase can be altered by introducing one or more mutations.

[0421] The present application describes an engineered rhamnosidase polypeptide for use in the methods of the invention, comprising, e.g., consisting of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 1017, or a functional fragment thereof, wherein the engineered rhamnosidase polypeptide is: (i) A56C (ii) A143P (iii) Q181H, Q181R, or Q181S (iv) L214M (v) G215S (vi...

Claims

1. Steps below: (i) providing a plant cell culture extract containing saponins; and (ii) enzymatically converting the starting saponin from the plant cell culture extract into a product saponin.

2. A method for producing the product saponin, comprising:

2. Steps below: (i) culturing a plant cell capable of synthesizing saponin under conditions conducive to the synthesis of saponin; (ii) recovering the saponin from the plant cell culture; and (iii) enzymatically converting the starting saponin from the recovered saponin into a product saponin.

2. A method for producing the product saponin, comprising:

3. Steps below: (i) culturing a plant cell capable of synthesizing saponin under conditions conducive to the synthesis of saponin; (ii) enzymatically converting the starting saponin derived from the synthesized saponin into a product saponin; and (iii) recovering the saponin from the plant cell culture 2. A method for producing the product saponin, comprising:

4. Steps below: (i) culturing a plant cell capable of synthesizing saponin under conditions conducive to the synthesis of saponin; (ii) recovering the saponin from the plant cell culture; and (iii) enzymatically converting the starting saponin from the recovered saponin into a product saponin.

1. A method for increasing the amount of product saponin obtainable from a plant cell culture, comprising:

5. Steps below: (i) culturing a plant cell capable of synthesizing saponin under conditions conducive to the synthesis of saponin; (ii) enzymatically converting the starting saponin derived from the synthesized saponin into a product saponin; and (iii) recovering the saponin from the plant cell culture 1. A method for increasing the amount of product saponin obtainable from a plant cell culture, comprising:

6. Steps below: (i) culturing a plant cell capable of synthesizing saponin under conditions conducive to the synthesis of saponin; (ii) recovering the saponin; and (iii) enzymatically converting the starting saponin from the recovered saponin into a product saponin.

1. A method for reducing the amount of starting saponin obtainable from a plant cell culture, comprising:

7. Steps below: (i) culturing a plant cell capable of synthesizing saponin under conditions conducive to the synthesis of saponin; (ii) enzymatically converting the starting saponin derived from the synthesized saponin into a product saponin; and (iii) recovering the saponin from the plant cell culture 1. A method for reducing the amount of starting saponin obtainable from a plant cell culture, comprising:

8. Steps below: (i) culturing plant cells capable of synthesizing saponins under conditions conducive to the synthesis of saponins; and (ii) recovering the saponin 1. A method for producing saponin by plant cell culture, comprising: The yield of product saponin is increased by enzymatically converting the starting saponin from the recovered saponin to said product saponin. The above method.

9. 9. The method according to any one of claims 1 to 8, wherein the saponin is a queronic acid glycoside.

10. 10. The method according to any one of claims 1 to 9, wherein the plant cell culture extract or the plant cells are from Quillaja Saponaria.

11. 11. The method of claim 10, wherein the starting saponin is a QS-18 family member.

12. 11. The method of claim 10, wherein the starting saponin is a desglucosyl QS-17 family member.

13. 11. The method of claim 10, wherein the starting saponin is a QS-17 family member.

14. 11. The method of claim 10, wherein the starting saponin is a desarabinofuranosyl QS-18 family member.

15. 11. The method of claim 10, wherein the starting saponin is an acetylated desglucosyl QS-17 family component.

16. 13. The method of claim 11 or 12, wherein the product saponin is a QS-21 family component.

17. 14. The method of claim 13, wherein the product saponin is a QS-18 family component.

18. 14. The method of claim 13, wherein the product saponin is a desglucosyl QS-17 family member.

19. 15. The method of claim 14, wherein the product saponin is a desarabinofuranosyl QS-21 family member.

20. 16. The method of claim 15, wherein the product saponin is an acetylated QS-21 family component.

21. 21. The method of any one of claims 1 to 20, wherein a single starting saponin is converted to a single product saponin.

22. 21. The method of any one of claims 1 to 20, wherein a plurality of starting saponins is converted into a plurality of product saponins.

23. 23. The method of any one of claims 1 to 22, wherein the enzymatic conversion comprises removal of β-glucose residues by glucosidase.

24. The glucosidase is selected from the group consisting of SEQ ID NOs: 262, 208, 63, 229, 250, 5, 101, 207, 169, 247, 302, 324, 319, 9, 240, 325, 338, 850, 879, 868, 826, 804, 888, 881, 891, 816, 827, 857, 853, 842, 814, 886, 885, 838 24. The method of claim 23, comprising, e.g. consisting of, an amino acid sequence according to: 829, 808, 828, 870, 873, 844, 882, 874, 825, 824, 823, 810, 894, 849, 803, 890, 841, 832, 830, 845, 871, 837, 883 or 809 or a functional variant thereof.

25. 25. The method of any one of claims 1 to 24, wherein the enzymatic conversion comprises removal of α-rhamnose residues by rhamnosidase.

26. 26. The method of claim 25, wherein the rhamnosidase comprises, e.g. consists of, an amino acid sequence according to SEQ ID NO: 992, 1003, 1052, 1073, 1017, 1055, 1075, 1001, 1007, 1061, 1079, 1027, 1039, 1041, 989, 1053, 1018, 1066, 1082, 1076, 993, 1077, 1046, 1015, 1063, 1054, 1074, 1067 or 1033, or a functional variant thereof.

27. A saponin prepared by the method of any one of claims 1 to 26.

28. 27. Use of a saponin prepared by the method of any one of claims 1 to 26 in the manufacture of an adjuvant.

29. 28. An adjuvant composition comprising the saponin of claim 27.

30. 28. An immunogenic composition comprising the saponin of claim 27 and an antigen or a polynucleotide encoding the antigen.

31. (i) a saponin according to claim 27 and (ii) an antigen or a polynucleotide encoding the antigen Includes a kit of parts.

Images