Methods and compositions for treating congenital sucrase-isomaltase deficiency

Abstract

Disclosed are recombinant mutant invertase enzymes.  Also disclosed are pharmaceutical compositions comprising an invertase enzyme, e.g., a recombinant mutant invertase enzyme and an optional isomaltase enzyme.  The enzymes and compositions can be used, among other things, to treat sucrase-isomaltase deficiency (e.g., congenital sucrase-isomaltase deficiency or acquired sucrase-isomaltase deficiency).

Description

METHODS AND COMPOSITIONS FOR TREATINGCONGENITAL SUCRASE-ISOMALTASE DEFICIENCYCROSS-REFERENCE TO RELATED APPLICATIONS[1] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 621,997, filed on January 17, 2024; the disclosure of which is hereby incorporated by reference in its entirety for all purposes.SEQUENCE LISTING[2] This application contains a Sequence Listing XML, which has been submitted electronically and is hereby incorporated by reference in its entirety. The XML copy of the Sequence Listing, created on December 30, 2024, is named ANR-008WO_SL.xml and is 98,561 bytes in size.FIELD OF THE INVENTION[3] The invention relates generally to recombinant mutant invertase (sucrase) enzymes and methods and compositions for treating sucrase-isomaltase deficiency.BACKGROUND[4] Congenital sucrose-isomaltase deficiency (CSID) is a rare autosomal recessive inherited disease of the small intestine resulting from genetic mutations in sucrase-isomaltase, an enzyme complex responsible for catalyzing the hydrolysis of dietary sucrose and starch. Ritz et al. Gastroenterology 125(6):1678-85. Decreased or absent invertase (sucrase) and / or isomaltase enzymatic activity has been found in patients with CSID, and investigations at the subcellular and molecular levels in intestinal biopsy specimens have led to the description of several phenotypes, differing in transport efficiency, processing, and sorting of the protein, which result in impaired physiologic functions. Ritz et al. (2003) Gastroenterology 125(6):1678-85, Jacob et al. (2000) J. Clin. Invest. 106(2):281-7, Alfalah et al. (2009) Gastroenterology 136(3):883-92, and Ouwendijk (1996) J. Clin. Invest. 97(3):633-41. The final step of starch digestion typically takes place in the intestinal lumen by α-glucosidases that are localized on the brush border membrane (BBM) of the intestinal epithelium. Human sucrase-isomaltase (SI) is responsible for almost all sucrase activity and about 60 to 80% of maltase activity in the intestinal lumen. Brunner et al. (1979) J. Biol. Chem. 254(6):1821-1828. As a result of the location of sucrase-isomaltase deficiency in the BBM of the intestinal lumen, disaccharidase deficiencies are also common where there is inflammation and has been associated with gastrointestinal complaints in people with gastrointestinal disorders (e.g., abdominal pain, irritable bowel, and Crohn’s disease (CD)). Colombo et al. (2021) Nature Scientific Reports 11:4902, Chumpitazi (2018) Gastroenterol Clin North Am 47(4):715-726, Dbar et al. (2021) World J Clin Cases 9(17): 4178-4187, Friesen et al. (2022) Gastrointest. Disord. 4:1-7.[5] Affected subjects may present with osmotic diarrhea, mild steatorrhea, chronic diarrhea, irritability, and vomiting after consuming sucrose. The difficulty in providing adequate nutrition to these subjects can lead to dehydration, metabolic acidosis, hypercalcemia, failure to thrive, and developmental delay. Cohen (2016) Mol. Cell. Pediatr. 3:5. Failure to absorb dietary disaccharides and starch has implications for the absorption of other nutrients and the hormonal regulation of gastrointestinal function.[6] The prevalence of CSID in the European population has been estimated at 1 in 5,000, but it is higher among the indigenous populations of Alaska, Greenland, and Canada. Marcadier et al. (2015) Canadian Med. Assoc. J. 187(2):102-107. There is also evidence that heterozygous carriers also experience symptoms of CSID, such as chronic diarrhea, abdominal pain, and bloating. It is currently estimated that 2-9% of Americans of European descent may be affected, suggesting that sucrase-isomaltase deficiency has been underrecognized. DeJonge et al. (2014) Gastroenterology 146:S705. Disaccharidase (DS) deficiencies have been reported in patients with inflammatory bowel disease (IBD) including CD, ulcerative colitis, and indeterminant colitis. Sucrase deficiency is common in approximately 14% of subjects. Friesen et al. (2022), supra.[7] Currently, CSID may be treated by the oral administration of a solution of sacrosidase (SUCRAID®). However, sacrosidase does not fully replace the enzymatic activity of the endogenous sucrase-isomaltase enzyme complex. The liquid formulation of sacrosidase in 50% glycerol is thermally unstable and unstable under acidic conditions. As a result, sacrosidase solution must be discarded four weeks after opening and cannot be used in conjunction with warm beverages and infant formula or fruit juices, negatively impacting ease of use and patient compliance.[8] Although developments have been made to date, there is still an ongoing need for new and effective pharmaceutical compositions and therapies for treating and managing sucrase-isomaltase deficiency, such as CSID or acquired sucrase-isomaltase deficiency (ASID). SUMMARY[9] The disclosure is based, in part, upon the discovery of recombinant mutant invertase (sucrase) enzymes that have greater stability and / or activity than naturally occurring enzymes. In particular, the recombinant mutant enzymes described herein exhibit improved pH stability, thermal stability, and / or stability against proteolytic digestion compared to naturally occurring versions of the enzyme. Furthermore, the recombinant mutant enzymes of the invention may have greater specific activity than a wild-type invertase (sucrase) enzyme under physiological conditions, for example, in the stomach and small intestine. Furthermore, it is contemplated that the recombinant mutant enzymes described herein, given their enhanced stability, may be suitable for oral administration and potentially safer, more tolerable, and / or more active than commercially available invertase (sucrase) enzymes. Also provided are pharmaceutical compositions, e.g., solid pharmaceutical compositions, comprising an invertase (sucrase) enzyme described herein and an isomaltase enzyme that are stable at room temperature, allowing for easier distribution, storage, and administration to subjects relative to compositions that are unstable at room temperature and / or require refrigeration. The recombinant enzymes and compositions can be used, among other things, to treat sucrase-isomaltase deficiency (e.g., congenital sucrase-isomaltase deficiency (CSID) or acquired sucrase-isomaltase deficiency (ASID)).

[10] Accordingly, in one aspect, the disclosure provides a recombinant mutant S. cerevisiae invertase enzyme including one or more amino acid substitutions that promote improved pH stability, thermal stability, and / or stability against proteolytic digestion. A recombinant mutant S. cerevisiae invertase enzyme of the disclosure can include one or more amino acid substitutions selected from the group consisting of:(a) a substitution of a T residue at a position corresponding to position 51 of SEQ ID NO: 3;(b) a substitution of an F residue at a position corresponding to position 83 of SEQ ID NO: 3;(c) a substitution of an E residue at a position corresponding to position 293 of SEQ ID NO: 3;(d) a substitution of an N residue at a position corresponding to position 340 of SEQ ID NO: 3;(e) a substitution of a P residue at a position corresponding to position 390 of SEQ ID NO: 3; and(f) a substitution of an A residue at a position corresponding to position 412 of SEQ ID NO: 3; ora combination of any of the foregoing substitutions, and is catalytically active to digest sucrose into glucose and fructose under physiological conditions of the stomach and / or small intestine. For example, in one embodiment, the invertase includes a combination of four of the foregoing substitutions. In another embodiment, the invertase includes a combination of five of the foregoing substitutions. In another embodiment, the invertase comprises all six of the foregoing substitutions..

[11] In certain embodiments, in the invertase,(a) the T residue at a position corresponding to position 51 of SEQ ID NO: 3 is substituted by L (T51L);(b) the F residue at a position corresponding to position 83 of SEQ ID NO: 3 is substituted by Y (F83Y);(c) the E residue at a position corresponding to position 293 of SEQ ID NO: 3 is substituted by Q (E293Q);(d) the N residue at a position corresponding to position 340 of SEQ ID NO: 3 is substituted by A (N340A);(e) the P residue at a position corresponding to position 390 of SEQ ID NO: 3 is substituted by A (P390A);(f) the A residue at a position corresponding to position 412 of SEQ ID NO: 3 is substituted by N (A412N); oror the invertase includes a combination of any of the foregoing substitutions. For example, in one embodiment, the invertase includes a combination of four of the foregoing substitutions. In another embodiment, the invertase includes a combination of five of the foregoing substitutions. In another embodiment, the invertase comprises all six of the foregoing substitutions.

[12] The invertase can include additional substitutions. For example, an invertase of the disclosure can further include a substitution at a D residue at a position corresponding to position 183 of SEQ ID NO: 3, e.g., wherein the D residue at a position corresponding to position 183 of SEQ ID NO: 3 is substituted by an N (D183N). The invertase can include a substitution at an E residue at a position corresponding to position 196 of SEQ ID NO: 3, e.g., wherein the E residue at a position corresponding to position 196 of SEQ ID NO: 3 is substituted by an H (E196H).

[13] The invertase of the disclosure can include four, five, six, seven, eight, nine or ten mutations relative to a corresponding wild-type invertase (e.g., a wild-type invertase of SEQ ID NO: 3). For example, the invertase can include F83Y, N340A, P390A, T51L, E293Q, and A412N substitutions; F83Y, N340A, P390A, T51L, E293Q, A412N, and D183N substitutions; or F83Y, N340A, P390A, T51L, E293Q, A412N, D183N, and E196H substitutions, in each case, relative to SEQ ID NO: 3.

[14] In certain embodiments, the invertase includes the amino acid sequence of any one of SEQ ID NOs: 51-61 or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 51-61. A recombinant mutant S. cerevisiae invertase enzyme of the disclosure can include a substitution or combination of substitutions listed in TABLE 8 or TABLE 12, wherein the invertase enzyme is catalytically active to digest sucrose into glucose and fructose under physiological conditions (e.g., of the stomach and / or small intestine). The invertase can exhibit one or more characteristics indicative of stability or survival in the gastrointestinal tract. For example, the invertase can have a specific activity at about pH 3.5 (e.g., pH 3.6) of at least 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, or 2,000 µmol sucrose consumed per minute per milligram of invertase. In certain embodiments, the invertase has at least 1-fold, 1.5-fold, 2-fold, 2.5-fold, or 3-fold higher activity at about pH 3.5 (e.g., pH 3.6), compared to a corresponding wild-type invertase. In certain embodiments, the invertase has a specific activity at about pH 5.0 (e.g., pH 4.9) of at least 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,100, or 2,200 µmol sucrose consumed per minute per milligram of invertase. In certain embodiments, the invertase has at least 1-fold, 1.5-fold, 2-fold, 2.5-fold, or 3-fold higher activity at about pH 5.0 (e.g., pH 4.9), compared to a corresponding wild-type invertase. In certain embodiments, the invertase has a specific activity at about pH 6.0 or 6.2 of at least 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, or 1,800 µmol sucrose consumed per minute per milligram of invertase. In certain embodiments, the invertase has at least 1-fold, 1.5-fold, 2-fold, 2.5-fold, or 3-fold higher activity at about pH 6.0 or 6.2, compared to a corresponding wild-type invertase. In certain embodiments, the invertase has a specific activity at about pH 7.0 or 7.1 of at least 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, or 1,300 µmol sucrose consumed per minute per milligram of invertase. In certain embodiments, the invertase at least 1-fold, 1.5-fold, 2-fold, 2.5-fold, or 3-fold higher activity at about pH 7.0 or 7.1, compared to a corresponding wild-type invertase. In certain embodiments, the invertase retains at least 80%, 90%, or 95% of activity following incubation at about pH 2.8 for about 2 hours. In certain embodiments, the invertase retains at least 80%, 90%, or 95% of activity following incubation at about 62 ℃ for about 1 hour. In certain embodiments, the invertase retains at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of activity following incubation at about pH 2.5 for about 30 minutes. In certain embodiments, the invertase has at least 1-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, or 5-fold higher stability at about pH 2.5 compared to a corresponding wild-type invertase. In certain embodiments, the invertase has a Tm of at least 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, or 68 ℃. In certain embodiments, the invertase has a Tm that is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ℃ higher than a corresponding wild-type invertase. In certain embodiments, the invertase has higher stability in the presence of pancreatin or pepsin compared to a corresponding wild-type invertase.

[15] In another aspect, the disclosure relates to a nucleic acid encoding an invertase as disclosed herein, an expression vector comprising the nucleic acid, and a cell (e.g., a P. pastoris or S. cerevisiae cell) comprising the expression vector. In another aspect, the disclosure relates to a method of producing a recombinant mutant S. cerevisiae invertase enzyme disclosed herein, the method comprising growing the cell under conditions so that the host cell expresses the invertase, and purifying the invertase.

[16] In another aspect, the disclosure relates to a pharmaceutical composition comprising an invertase as disclosed herein and a pharmaceutically acceptable carrier and / or an excipient. The pharmaceutical composition can, optionally, include an isomaltase enzyme. The invertase and / or the isomaltase can be dried and, for example, formulated for delivery as a powder or compressed into a tablet.

[17] The isomaltase can be a microbial isomaltase, or a functional fragment or variant thereof. The isomaltase can be derived from Saccharomyces cerevisiae, e.g., can include any one of SEQ ID NOs: 37-41, or a functional fragment or variant thereof. The isomaltase can be a recombinant mutant Lactobacillus fermentum isomaltase enzyme (SEQ ID NO: 47), or a functional fragment or variant thereof.

[18] The isomaltase can include the following substitutions using the numbering in SEQ ID NO: 47:(a) K115I, R132K, D226S, E310A, L366M, I421A, A444G, A531D, and F560V substitutions;(b) E93K, K115I, R132K, D226S, E310A, L366M, I421A, A444G, A531D, and F560L substitutions;(c) E93K, K115I, R132K, D226S, E310A, I421A, A444G, E524Q, A531D, and F560L substitutions;(d) E93K, K115I, R132K, D226S, E310A, I421A, A444G, A531D, and F560V substitutions;(e) E93K, K115I, R132K, D226S, L366M, I421A, A444G, E524Q, A531D, and F560L substitutions;(f) E93K, K115I, R132K, D226S, L366M, I421A, A444G, A531D, and F560V substitutions;(g) E93K, K115I, R132K, D226S, I421A, A444G, E524Q, A531D, and F560V substitutions;(h) K115I, R132K, D226S, E310A, L366M, I421A, A444G, E524Q, A531D, and F560L substitutions;(i) K115I, R132K, D226S, E310A, I421A, A444G, E524Q, A531D, and F560V substitutions;(j) K115I, R132K, D226S, L366M, I421A, A444G, E524Q, A531D, and F560V substitutions;(k) E93K, K115I, R132K, A211E, D226S, I421A, A444G, E524Q, A531D, and F560V substitutions;(l) E93K, K115I, R132K, D226S, L366M, I421A, A444G, E524Q, A531D, and F560V substitutions;(m) E93K, K115I, R132K, D226S, E275D, I421A, A444G, E524Q, A531D, and F560V substitutions;(n) T89A, E93K, K115I, R132K, D226S, I421A, A444G, E524Q, A531D, and F560V substitutions;(o) E93K, K115I, R132K, H182R, D226S, I421A, A444G, E524Q, A531D, and F560V substitutions;(p) E93K, K115I, E122D, R132K, D226S, I421A, A444G, E524Q, A531D, and F560V substitutions;(q) E93K, K115I, D226S, I421A, A444G, E524Q, A531D, and F560V substitutions;(r) E93K, K115I, D226S, I421A, A444G, E524Q, A531D, and F560L substitutions;(s) K115I, A211E, D226S, I421A, A444G, A531D, and F560V substitutions;(t) K115I, D226S, L366M, I421A, A444G, E524Q, A531D, and F560L substitutions;(u) K115I, A211E, D226S, I421A, A444G, E524Q, A531D, and F560L substitutions;(v) K115I, R132K, D226S, E310A, L366M, I421A, A444G, A531D, and F560L substitutions;(w) E93K, K115I, D226S, E310A, I421A, A444G, A531D, and F560L substitutions;(x) K115I, D226S, E310A, I421A, A444G, A531D, and F560V substitutions;(y) E93K, K115I, D226S, L366M, I421A, A444G, A531D, and F560L substitutions;(z) K115I, D226S, I421A, A444G, E524Q, A531D, and F560V substitutions;(27) K115I, D226S, E310A, I421A, A444G, E524Q, A531D, and F560L substitutions;(28) K115I, A211E, D226S, L366M, I421A, A444G, A531D, and F560L substitutions;(29) K115I, A211E, D226S, E310A, I421A, A444G, A531D, and F560L substitutions;(30) E93K, K115I, D226S, I421A, A444G, A531D, and F560V substitutions;(31) E93K, K115I, A211E, D226S, I421A, A444G, A531D, and F560L substitutions; or(32) K115I, D226S, L366M, I421A, A444G, A531D, and F560V substitutions.

[19] The invertase included in the pharmaceutical composition can include the amino acid sequence of any one of SEQ ID NOs: 51-61 (e.g., of any one of SEQ ID NOs: 51-53), or a functional fragment thereof.

[20] The pharmaceutical composition can be formulated as an oral dosage form, for example, as a liquid, powder, sachet, granulate, pellet, micropellet, tablet, or minitablet.

[21] In certain embodiments, the pharmaceutical composition has a shelf-life at room temperature of at least 3 months, 6 months, 9 months, 12 months, 15 months, 18 months, 21 months, 24 months, 36 months, 48 months, 60 months, 72 months, 84 months, 96 months, 108 months, or 120 months.

[22] The disclosure further relates to a method of treating sucrase-isomaltase deficiency (e.g., congenital sucrase-isomaltase deficiency (CSID) or acquired sucrase-isomaltase deficiency (ASID)) in a subject in need thereof. The method can include orally administering to the subject an effective amount of the pharmaceutical composition as disclosed herein. The pharmaceutical composition can be administered to the subject together with a meal or snack.

[23] The disclosure further relates to a method of reducing sucrose and branched (1–6 linked) α-limit dextrin concentration in a subject. The method can include orally administering to the subject an effective amount of a pharmaceutical composition disclosed herein. The method can be measured using a hydrogen breath test.

[24] The disclosure further relates to a method of treating sucrase-isomaltase deficiency (e.g., congenital sucrase-isomaltase deficiency (CSID) or acquired sucrase-isomaltase deficiency (ASID)) in a subject in need thereof, the method comprising orally administering to the subject an invertase enzyme of the disclosure and optionally an isomaltase enzyme as disclosed herein.

[25] In certain embodiments, the subject is a mammal, e.g., a human.BRIEF DESCRIPTIONS OF THE DRAWINGS

[26] The invention can be more completely understood with reference to the following drawings.

[27] FIG. 1 is a bar chart showing the specific activities of indicated invertase enzymes as measured by consumption of sucrose at pH 3.6, pH 4.9, pH 6.0, and pH 7.2.

[28] FIGs. 2A and 2B are graphs showing the activities of indicated invertase enzymes following incubation with proteases. FIG. 2A shows the activity of indicated invertases following pretreatment with pepsin at pH 3.5 for indicated time periods to reflect the physiological conditions of the stomach. FIG. 2B shows the activity of indicated invertases following pretreatment with pancreatin at pH 6.0 for 3 hours to reflect fed state conditions of the small intestine.

[29] FIG. 3 is a graph showing the activities of indicated invertase enzymes following incubation at pH 2.8 for indicated time periods to reflect fasted state conditions.

[30] FIG. 4 is a graph showing the activities of indicated invertase enzymes following incubation at 62 °C for indicated time periods. DETAILED DESCRIPTION

[31] The disclosure is based, in part, upon the discovery of recombinant mutant invertase (sucrase) enzymes that have greater stability and / or activity than naturally occurring enzymes. In particular, the recombinant mutant enzymes described herein exhibit improved pH stability, thermal stability, and / or stability against proteolytic digestion compared to naturally occurring versions of the enzyme. Furthermore, the recombinant mutant enzymes of the invention may have greater specific activity than a wild-type invertase (sucrase) enzyme under physiological conditions, for example, in the stomach and small intestine of a subject, e.g., a human subject. Furthermore, it is contemplated that the recombinant mutant enzymes described herein, given their enhanced stability, may be suitable for oral administration, and potentially safer, more tolerable, and / or more active than commercially available invertase (sucrase) enzymes. Also provided are pharmaceutical compositions, e.g., solid pharmaceutical compositions, comprising an invertase (sucrase) enzyme described herein and an isomaltase enzyme that are stable at room temperature, allowing for easier distribution, storage, and administration to subjects relative to compositions that are unstable at room temperature and / or require refrigeration. The recombinant enzymes and compositions can be used, among other things, to treat sucrase-isomaltase deficiency (e.g., CSID or ASID).

[32] Various features and aspects of the invention are discussed in detail below.I. Invertase (Sucrase) Enzymes

[33] Among other things, the disclosure provides recombinant mutant invertase (sucrase) enzymes and pharmaceutical compositions comprising recombinant mutant invertase (sucrase) enzymes that, for example, are useful in treating disorders such as sucrase-isomaltase deficiency (for example, congenital sucrase-isomaltase deficiency (CSID) or acquired sucrase-isomaltase deficiency (ASID)). The enzymes described herein were engineered to work under conditions found in the stomach and small intestines of a human subject. Transit through the stomach is typically about 90% complete in 75 minutes, and typically complete in about 90-120 minutes. Transit through the small intestine is typically complete in 4-6 hours (120-260 minutes). Thus, the enzymes were engineered so that, under stomach conditions, at least 75-85% of the enzyme survives typical fed state conditions of stomach, e.g., pH 3.5 and pepsin, for 75 minutes, and, under fed state conditions of the small intestine, 50% of the enzyme survives fed state small intestine conditions for 180 minutes. The enzymes described herein were also engineered to work briefly under conditions found in the fasted state of the stomach and small intestines.

[34] As used herein, the terms “sucrase” and “invertase” are used interchangeably and refer to any enzyme or a functional fragment thereof, that is capable of catalyzing the hydrolysis of sucrose to fructose and glucose, for example, under physiological conditions, e.g., under the physiological conditions of the stomach and / or small intestine of a subject. Sucrases and invertases are also called, acid invertase, alkaline invertase, β-D-fructofuranosidase, β-fructosidase, β-fructofuranosidase, β-fructofuranoside fructohydrolase, β-fructopyranosidase, β-h-fructosidase, β-invertase, EC 3.2.1.26, exo-β-(2,6)-fructofuraosidase, fructosylinvertase, invertin, glucosucrase, saccharase, and sucrose hydrolase, and, unless indicated otherwise, the terms are used interchangeably herein. The term invertase includes variants having one or more amino acid substitutions, deletions, or insertions relative to a wild-type invertase sequence and / or fusion proteins or conjugates including an invertase.

[35] As used herein, the term “functional fragment” of an invertase refers to fragment of a full-length invertase that retains, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the enzymatic activity of the corresponding full-length, naturally occurring invertase. Invertase enzymatic activity may be assayed by any method known in the art. Exemplary invertase activity assays are described in Bacon (1955) Methods in Enzymology 1: 258-262, Lever (1972) Analytical Biochem. 47: 273-279, U.S. Patent Application Publication No. 2014 / 0250942, on the world wide web at sigmaaldrich.com / technical-documents / protocols / biology / enzymatic-assay-of-invertase.html, and in Example 1 herein.

[36] Exemplary invertase enzymes include invertase enzymes derived from Saccharomyces cerevisiae. The amino acid sequence of an exemplary wild-type invertase enzyme derived from Saccharomyces cerevisiae (including a native signal sequence) is provided as SEQ ID NO: 1, and a nucleotide sequence encoding an exemplary wild-type invertase enzyme derived from Saccharomyces cerevisiae is provided as SEQ ID NO: 35. The amino acid sequence of an exemplary invertase enzyme derived from Saccharomyces cerevisiae (including a heterologous FAKS signal sequence, but otherwise wild-type) is provided as SEQ ID NO: 2. The amino acid sequence of an exemplary wild-type invertase enzyme derived from Saccharomyces cerevisiae (without a signal sequence) is provided as SEQ ID NO: 3.

[37] SEQ ID NO: 1 represents wild-type S. cerevisiae invertase with a native signal sequence where the signal sequence is underlined, and the mature protein sequence is italicized:MLLQAFLFLLAGFAAKISASMTNETSDRPLVHFTPNKGWMNDPNGLWYDEKDAKWHLYFQYNPNDTVWGTPLFWGHATSDDLTHWEDEPIAIAPKRNDSGAFSGSMVVDYNNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAAKSQDYKIEIYSSDDLKSWKLESAFANEGFLGYQYECPGLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYGSALGIAWASNWEYSAFVPTNPWRSSMSLVRKFSLNTEYQANPETELINLKAEPILNISNAGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYAVNTTQTISKSVFPDLSLWFKGLEDPEEYLRMGFEASASSFFLDRGNSKVKFVKENPYFTNRMSVNNQPFKSENDLSYYKVYGLLDQNILELYFNDGDVVSTNTYFMTTGNALGSVNMTTGVDNLFYIDKFQVREVK.

[38] SEQ ID NO: 2 represents wild-type S. cerevisiae invertase with a heterologous FAKS signal sequence, where the signal sequence is underlined, and the mature protein sequence is italicized:MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAEASMTNETSDRPLVHFTPNKGWMNDPNGLWYDEKDAKWHLYFQYNPNDTVWGTPLFWGHATSDDLTHWEDEPIAIAPKRNDSGAFSGSMVVDYNNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAAKSQDYKIEIYSSDDLKSWKLESAFANEGFLGYQYECPGLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYGSALGIAWASNWEYSAFVPTNPWRSSMSLVRKFSLNTEYQANPETELINLKAEPILNISNAGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYAVNTTQTISKSVFPDLSLWFKGLEDPEEYLRMGFEASASSFFLDRGNSKVKFVKENPYFTNRMSVNNQPFKSENDLSYYKVYGLLDQNILELYFNDGDVVSTNTYFMTTGNALGSVNMTTGVDNLFYIDKFQVREVK.

[39] SEQ ID NO: 3 represents wild-type S. cerevisiae invertase without a signal sequence:SMTNETSDRPLVHFTPNKGWMNDPNGLWYDEKDAKWHLYFQYNPNDTVWGTPLFWGHATSDDLTHWEDEPIAIAPKRNDSGAFSGSMVVDYNNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAAKSQDYKIEIYSSDDLKSWKLESAFANEGFLGYQYECPGLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYGSALGIAWASNWEYSAFVPTNPWRSSMSLVRKFSLNTEYQANPETELINLKAEPILNISNAGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYAVNTTQTISKSVFPDLSLWFKGLEDPEEYLRMGFEASASSFFLDRGNSKVKFVKENPYFTNRMSVNNQPFKSENDLSYYKVYGLLDQNILELYFNDGDVVSTNTYFMTTGNALGSVNMTTGVDNLFYIDKFQVREVK.

[40] A contemplated invertase enzyme may comprise the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 having at least one (e.g., one, two, three, four, five, six, seven, eight, nine, or ten, 2-10, 3-10, 4-10, 5-10, 6-10, 7-10, 8-10, 9-10, 2-9, 3-9, 4-9, 5-9, 6-9, 7-9, 8-9, 2-8, 3-8, 4-8, 5-8, 6-8, 7-8, 2-6, 3-6, 4-6, or 5-6) mutation(s) and / or may comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[41] Additional exemplary invertase enzymes can be found on the world wide web at brenda-enzymes.org / enzyme.php?ecno=3.2.1.26&onlyTable=Sequence. Additional exemplary invertase enzymes are described in U.S. Patent Application Publication No. 2014 / 0250942.

[42] Among other things, the disclosure provides recombinant mutant invertases that are useful, for example, in treating disorders associated with a reduced ability to digest or absorb dietary sucrose and starches.

[43] In certain embodiments, the invertase comprises: (i) increased activity at acidic pH (e.g., about pH 3.5, 3.6, 4.0, 4.5, 4.9, 5.0, 5.5, 6.0, or 6.5) relative to a corresponding wild-type invertase; (ii) increased activity at neutral pH (e.g., about pH 7.0) relative to a corresponding wild-type invertase; (iii) increased stability at acidic pH (e.g., about pH 2.5) relative to a corresponding wild-type invertase, (iv) increased thermal stability relative to a corresponding wild-type invertase, or any combination of features (i), (ii), (iii), or (iv). Exemplary combinations include (i) and (ii); (i) and (iii); (i) and (iv); (ii) and (iii); (ii) and (iv); (i), (ii), and (iii); (i), (ii), and (iv); (i), (iii), and (iv); (ii), (iii), and (iv); and (i), (ii), (iii), and (iv).

[44] In certain embodiments, the disclosure provides a recombinant mutant S. cerevisiae invertase enzyme comprising one or more amino acid substitutions that promote improved pH stability, thermal stability, and / or stability against proteolytic digestion. A recombinant mutant S. cerevisiae invertase enzyme of the disclosure can comprise one or more amino acid substitutions selected from the group consisting of:(a) a substitution of a T residue at a position corresponding to position 51 of SEQ ID NO: 3;(b) a substitution of an F residue at a position corresponding to position 83 of SEQ ID NO: 3;(c) a substitution of an E residue at a position corresponding to position 293 of SEQ ID NO: 3;(d) a substitution of an N residue at a position corresponding to position 340 of SEQ ID NO: 3;(e) a substitution of a P residue at a position corresponding to position 390 of SEQ ID NO: 3; and(f) a substitution of an A residue at a position corresponding to position 412 of SEQ ID NO: 3; or a combination of any of the foregoing substitutions,and is catalytically active to digest sucrose into glucose and fructose under physiological conditions of the stomach and / or small intestine. For example, in one embodiment, the invertase includes a combination of four of the foregoing substitutions. In another embodiment, the invertase includes a combination of five of the foregoing substitutions. In another embodiment, the invertase comprises all six of the foregoing substitutions.

[45] In certain embodiments of the foregoing, in the invertase,(a) the T residue at a position corresponding to position 51 of SEQ ID NO: 3 is substituted by L (T51L);(b) the F residue at a position corresponding to position 83 of SEQ ID NO: 3 is substituted by Y (F83Y);(c) the E residue at a position corresponding to position 293 of SEQ ID NO: 3 is substituted by Q (E293Q);(d) the N residue at a position corresponding to position 340 of SEQ ID NO: 3 is substituted by A (N340A);(e) the P residue at a position corresponding to position 390 of SEQ ID NO: 3 is substituted by A (P390A);(f) the A residue at a position corresponding to position 412 of SEQ ID NO: 3 is substituted by N (A412N); orthe invertase includes a combination of any of the foregoing substitutions. For example, in one embodiment, the invertase includes a combination of four of the foregoing substitutions. In another embodiment, the invertase includes a combination of five of the foregoing substitutions. In another embodiment, the invertase comprises all six of the foregoing substitutions.

[46] Depending upon the circumstances, the invertase can include additional substitutions. For example, the invertase can further include a substitution at a D residue at a position corresponding to position 183 of SEQ ID NO: 3, e.g., wherein the D residue at a position corresponding to position 183 of SEQ ID NO: 3 is substituted by an N (D183N). The invertase can include a substitution at an E residue at a position corresponding to position 196 of SEQ ID NO: 3, e.g., wherein the E residue at a position corresponding to position 196 of SEQ ID NO: 3 is substituted by an H (E196H).

[47] In certain embodiments, the recombinant mutant invertase comprises at least one (e.g., one, two, three, four, five, six, seven, eight, nine, or ten, 2-10, 3-10, 4-10, 5-10, 6-10, 7-10, 8-10, 9-10, 2-9, 3-9, 4-9, 5-9, 6-9, 7-9, 8-9, 2-8, 3-8, 4-8, 5-8, 6-8, 7-8, 2-6, 3-6, 4-6, or 5-6) mutation(s) at a position corresponding to wild-type S. cerevisiae invertase of SEQ ID NO: 2. In certain embodiments, at least one mutation is selected from a substitution of a D residue at a position corresponding to position 122 of SEQ ID NO: 2; a substitution of a T residue at a position corresponding to position 140 of SEQ ID NO: 2; a substitution of an E residue at a position corresponding to position 156 of SEQ ID NO: 2; a substitution of an F residue at a position corresponding to position 172 of SEQ ID NO: 2; a substitution of a V residue at a position corresponding to position 178 of SEQ ID NO: 2; a substitution of a K residue at a position corresponding to position 259 of SEQ ID NO: 2; a substitution of a D residue at a position corresponding to position 272 of SEQ ID NO: 2; a substitution of a K residue at a position corresponding to position 277 of SEQ ID NO: 2; a substitution of an E residue at a position corresponding to position 285 of SEQ ID NO: 2; a substitution of an A residue at a position corresponding to position 378 of SEQ ID NO: 2; a substitution of an E residue at a position corresponding to position 382 of SEQ ID NO: 2; a substitution of a V residue at a position corresponding to position 399 of SEQ ID NO: 2; a substitution of a G residue at a position corresponding to position 431 of SEQ ID NO: 2; a substitution of a V residue at a position corresponding to position 477 of SEQ ID NO: 2; a substitution of a P residue at a position corresponding to position 479 of SEQ ID NO: 2; a substitution of an A residue at a position corresponding to position 501 of SEQ ID NO: 2; a substitution of an L residue at a position corresponding to position 549 of SEQ ID NO: 2; a substitution of a V residue at a position corresponding to position 581 of SEQ ID NO: 2; a substitution of a G residue at a position corresponding to position 586 of SEQ ID NO: 2; or a substitution of an N residue at a position corresponding to position 589 of SEQ ID NO: 2. In certain embodiments, the recombinant mutant invertase comprises a combination of any of the foregoing substitutions. SEQ ID NO: 2 is the amino acid sequence of an exemplary invertase enzyme derived from Saccharomyces cerevisiae, that is wild-type but for the inclusion of a heterologous FAKS signal sequence.

[48] Also contemplated herein are invertases comprising the foregoing substitutions located at the corresponding positions in SEQ ID NO: 1 (wild-type invertase enzyme derived from Saccharomyces cerevisiae including a native signal sequence) and in SEQ ID NO: 3 (wild-type invertase enzyme derived from Saccharomyces cerevisiae without a signal sequence) (see TABLE 1 below).

[49] In certain embodiments, the invertase comprises: a substitution of a D residue at a position corresponding to position 52 of SEQ ID NO: 1; a substitution of a T residue at a position corresponding to position 70 of SEQ ID NO: 1; a substitution of an E residue at a position corresponding to position 86 of SEQ ID NO: 1; a substitution of an F residue at a position corresponding to position 102 of SEQ ID NO: 1; a substitution of a V residue at a position corresponding to position 108 of SEQ ID NO: 1; a substitution of a K residue at a position corresponding to position 189 of SEQ ID NO: 1; a substitution of a D residue at a position corresponding to position 202 of SEQ ID NO: 1; a substitution of a K residue at a position corresponding to position 207 of SEQ ID NO: 1; a substitution of an E residue at a position corresponding to position 215 of SEQ ID NO: 1; a substitution of an A residue at a position corresponding to position 308 of SEQ ID NO: 1; a substitution of an E residue at a position corresponding to position 312 of SEQ ID NO: 1; a substitution of a V residue at a position corresponding to position 329 of SEQ ID NO: 1; a substitution of a G residue at a position corresponding to position 361 of SEQ ID NO: 1; a substitution of a V residue at a position corresponding to position 407 of SEQ ID NO: 1; a substitution of a P residue at a position corresponding to position 409 of SEQ ID NO: 1; a substitution of an A residue at a position corresponding to position 431 of SEQ ID NO: 1; a substitution of a L residue at a position corresponding to position 479 of SEQ ID NO: 1; a substitution of a V residue at a position corresponding to position 511 of SEQ ID NO: 1; a substitution of a G residue at a position corresponding to position 516 of SEQ ID NO: 1; or a substitution of an N residue at a position corresponding to position 519 of SEQ ID NO: 1. In certain embodiments, the recombinant mutant invertase comprises a combination of any of the foregoing substitutions.

[50] In certain embodiments, the invertase comprises: a substitution of a D residue at a position corresponding to position 33 of SEQ ID NO: 3; a substitution of a T residue at a position corresponding to position 51 of SEQ ID NO: 3; a substitution of an E residue at a position corresponding to position 67 of SEQ ID NO: 3; a substitution of an F residue at a position corresponding to position 83 of SEQ ID NO: 3; a substitution of a V residue at a position corresponding to position 89 of SEQ ID NO: 3; a substitution of a K residue at a position corresponding to position 170 of SEQ ID NO: 3; a substitution of a D residue at a position corresponding to position 183 of SEQ ID NO: 3; a substitution of a K residue at a position corresponding to position 188 of SEQ ID NO: 3; a substitution of an E residue at a position corresponding to position 196 of SEQ ID NO: 3; a substitution of an A residue at a position corresponding to position 289 of SEQ ID NO: 3; a substitution of an E residue at a position corresponding to position 293 of SEQ ID NO: 3; a substitution of a V residue at a position corresponding to position 310 of SEQ ID NO: 3; a substitution of a G residue at a position corresponding to position 342 of SEQ ID NO: 3; a substitution of a V residue at a position corresponding to position 388 of SEQ ID NO: 3; a substitution of a P residue at a position corresponding to position 390 of SEQ ID NO: 3; a substitution of an A residue at a position corresponding to position 412 of SEQ ID NO: 3; a substitution of a L residue at a position corresponding to position 460 of SEQ ID NO: 3; a substitution of a V residue at a position corresponding to position 492 of SEQ ID NO: 3; a substitution of a G residue at a position corresponding to position 497 of SEQ ID NO: 3; or a substitution of an N residue at a position corresponding to position 500 of SEQ ID NO: 3. In certain embodiments, the recombinant mutant invertase comprises a combination of any of the foregoing substitutions.

[51] In certain embodiments, the D residue at a position corresponding to position 122 of SEQ ID NO: 2 is substituted by E (D122E); the T residue at a position corresponding to position 140 of SEQ ID NO: 2 is substituted by L (T140L); the E residue at a position corresponding to position 156 of SEQ ID NO: 2 is substituted by Q (E156Q); the F residue at a position corresponding to position 172 of SEQ ID NO: 2 is substituted by Y (F172Y); the V residue at a position corresponding to position 178 of SEQ ID NO: 2 is substituted by I (V178I); the K residue at a position corresponding to position 259 of SEQ ID NO: 2 is substituted by L (K259L); the D residue at a position corresponding to position 272 of SEQ ID NO: 2 is substituted by N (D272N); the K residue at a position corresponding to position 277 of SEQ ID NO: 2 is substituted by T (K277T); the E residue at a position corresponding to position 285 of SEQ ID NO: 2 is substituted by H (E285H); the A residue at a position corresponding to position 378 of SEQ ID NO: 2 is substituted by T (A378T); the E residue at a position corresponding to position 382 of SEQ ID NO: 2 is substituted by Q (E382Q); the V residue at a position corresponding to position 399 of SEQ ID NO: 2 is substituted by A (V399A); the G residue at a position corresponding to position 431 of SEQ ID NO: 2 is substituted by R (G431R); the V residue at a position corresponding to position 477 of SEQ ID NO: 2 is substituted by D (V477D); the P residue at a position corresponding to position 479 of SEQ ID NO: 2 is substituted by A (P479A); the A residue at a position corresponding to position 501 of SEQ ID NO: 2 is substituted by N (A501N); the L residue at a position corresponding to position 549 of SEQ ID NO: 2 is substituted by I (L549I); the V residue at a position corresponding to position 581 of SEQ ID NO: 2 is substituted by I (V581I); the G residue at a position corresponding to position 586 of SEQ ID NO: 2 is substituted by S (G586S); or the N residue at a position corresponding to position 589 of SEQ ID NO: 2 is substituted by K (N589K); or the invertase comprises a combination of any of the foregoing substitutions. SEQ ID NO: 2 is the amino acid sequence of an exemplary invertase enzyme derived from Saccharomyces cerevisiae, that is wild-type but for the inclusion of a heterologous FAKS signal sequence.

[52] Also contemplated herein are invertases comprising the foregoing substitutions located at the corresponding positions in SEQ ID NO: 1 (wild-type invertase enzyme derived from Saccharomyces cerevisiae including a native signal sequence) and SEQ ID NO: 3 (wild-type invertase enzyme derived from Saccharomyces cerevisiae without a signal sequence) (see TABLE 1 below).

[53] In certain embodiments, in the invertase: the D residue at a position corresponding to position 52 of SEQ ID NO: 1 is substituted by E (D52E); the A residue at a position corresponding to position 53 of SEQ ID NO: 1 is substituted by G (A53G); the T residue at a position corresponding to position 70 of SEQ ID NO: 1 is substituted by L (T70L); the E residue at a position corresponding to position 86 of SEQ ID NO: 1 is substituted by Q (E86Q); the F residue at a position corresponding to position 102 of SEQ ID NO: 1 is substituted by Y (F102Y); the V residue at a position corresponding to position 108 of SEQ ID NO: 1 is substituted by I (V108I); the K residue at a position corresponding to position 189 of SEQ ID NO: 1 is substituted by L (K189L); the D residue at a position corresponding to position 202 of SEQ ID NO: 1 is substituted by N (D202N); the K residue at a position corresponding to position 207 of SEQ ID NO: 1 is substituted by T (K207T); the E residue at a position corresponding to position 215 of SEQ ID NO: 1 is substituted by H (E215H); the A residue at a position corresponding to position 308 of SEQ ID NO: 1 is substituted by T (A308T); the E residue at a position corresponding to position 312 of SEQ ID NO: 1 is substituted by Q (E312Q); the V residue at a position corresponding to position 329 of SEQ ID NO: 1 is substituted by A (V329A); the G residue at a position corresponding to position 361 of SEQ ID NO: 1 is substituted by R (G361R); the V residue at a position corresponding to position 407 of SEQ ID NO: 1 is substituted by D (V407D); the P residue at a position corresponding to position 409 of SEQ ID NO: 1 is substituted by A (P409A); the A residue at a position corresponding to position 431 of SEQ ID NO: 1 is substituted by N (A431N); the L residue at a position corresponding to position 479 of SEQ ID NO: 1 is substituted by I (L479I); the V residue at a position corresponding to position 511 of SEQ ID NO: 1 is substituted by I (V511I); the N residue at a position corresponding to position 519 of SEQ ID NO: 1 is substituted by K (N519K); or the invertase comprises a combination of any of the foregoing substitutions.

[54] In certain embodiments, in the invertase: the D residue at a position corresponding to position 33 of SEQ ID NO: 3 is substituted by E (D33E); the T residue at a position corresponding to position 51 of SEQ ID NO: 3 is substituted by L (T51L); the E residue at a position corresponding to position 67 of SEQ ID NO: 3 is substituted by Q (E67Q); the F residue at a position corresponding to position 83 of SEQ ID NO: 3 is substituted by Y (F83Y); the V residue at a position corresponding to position 89 of SEQ ID NO: 3 is substituted by I (V89I); the K residue at a position corresponding to position 170 of SEQ ID NO: 3 is substituted by L (K170L); the D residue at a position corresponding to position 183 of SEQ ID NO: 3 is substituted by N (D183N); the K residue at a position corresponding to position 188 of SEQ ID NO: 3 is substituted by T (K188T); the E residue at a position corresponding to position 196 of SEQ ID NO: 3 is substituted by H (E196H); the A residue at a position corresponding to position 289 of SEQ ID NO: 3 is substituted by T (A289T); the E residue at a position corresponding to position 293 of SEQ ID NO: 3 is substituted by Q (E293Q); the V residue at a position corresponding to position 310 of SEQ ID NO: 3 is substituted by A (V310A); the G residue at a position corresponding to position 342 of SEQ ID NO: 3 is substituted by R (G342R); the V residue at a position corresponding to position 388 of SEQ ID NO: 3 is substituted by D (V388D); the P residue at a position corresponding to position 390 of SEQ ID NO: 3 is substituted by A (P390A); the A residue at a position corresponding to position 412 of SEQ ID NO: 3 is substituted by N (A412N); the L residue at a position corresponding to position 460 of SEQ ID NO: 3 is substituted by I (L460I); the V residue at a position corresponding to position 492 of SEQ ID NO: 3 is substituted by I (V492I); the G residue at a position corresponding to position 497 of SEQ ID NO: 3 is substituted by S (G497S); the N residue at a position corresponding to position 500 of SEQ ID NO: 3 is substituted by K (N500K); or the invertase comprises a combination of any of the foregoing substitutions.

[55] In certain embodiments, one or more mutations may be conservative substitutions relative to wild-type S. cerevisiae invertase of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, whereas in certain other embodiments, one or more mutations may be non-conservative substitutions relative to wild-type S. cerevisiae invertase of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. As used herein, the term “conservative substitution” refers to a substitution with a structurally and / or functionally similar amino acid.

[56] In certain embodiments, the substitution of a given amino acid is with a hydrophobic amino acid (e.g., A, I, L, M, or V), a positively charged amino acid (e.g., K, R, or H), a negatively charged amino acid (e.g., D or E), a polar neutral amino acid (e.g., N, C, Q, S, or T), an aromatic amino acid (e.g., F, Y, or W) or a bulkier amino acid based on side chain volume or a smaller amino acid based on side chain volume. Conservative substitutions may also be defined by the BLAST (Basic Local Alignment Search Tool) algorithm, the BLOSUM substitution matrix (e.g., BLOSUM 62 matrix), or the PAM substitution:p matrix (e.g., the PAM 250 matrix). Non-conservative substitutions are amino acid substitutions that are not conservative substitutions.

[57] In certain embodiments, the recombinant mutant invertase enzyme comprises one or more substitutions from TABLE 1, wherein the positions of the substitutions are shown relative to SEQ ID NO: 1 (wild-type Saccharomyces cerevisiae invertase enzyme, including a native signal sequence) or SEQ ID NO: 2 (Saccharomyces cerevisiae invertase enzyme including a heterologous FAKS signal sequence, but otherwise wild-type) or SEQ ID NO: 3 (wild-type Saccharomyces cerevisiae invertase enzyme without a signal sequence). The rows in TABLE 1 correspond to a substitution at the same position, but the numbering is based on whether the protein contains a signal sequence (e.g., SEQ ID NO: 1 or SEQ ID NO: 2) or does not contain a signal sequence (e.g., SEQ ID NO: 3).TABLE 1Position relative to SEQ ID NO: 2Position relative to SEQ ID NO: 1Position relative to SEQ ID NO: 3Exemplary SubstitutionsE120E50E31AD122D52D33EA123A53A34GT140T70T51L, QF143F73F54YE156E86E67QF172F102F83YV178V108V89IK259K189K170LD272D202D183NK277K207K188TE285E215E196HL359L289L270FT365T295T276SA378A308A289TE382E312E293QN390N320N301DE415E345E326SL416L346L327QN429N359N340AL459L389L370FT470T400T381SS476S406S387A, NP479P409P390AA501A431A412NN512N442N423GK514K444K425PK519K449K430RS537S467S448DL549L479L460IT573T503T484SV581V511V492IG586G516G497SN589N519N500KF596F526F507IQ597Q527Q508E 

[58] In certain embodiments, the recombinant mutant invertase comprises at least one (e.g., at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or at least eleven) mutation(s) selected from TABLE 1.

[59] In certain embodiments, the recombinant mutant invertase comprises at least three substitutions selected from: the F172Y, N429A, and P479A substitutions relative to SEQ ID NO: 2; the E120A, D122E, and P479A substitutions relative to SEQ ID NO: 2; the K259L, S476N, and K514P substitutions relative to SEQ ID NO: 2; the T140L, E156Q, and L549I substitutions relative to SEQ ID NO: 2; the S476A, P479A, and Q597E substitutions relative to SEQ ID NO: 2; the K277T, E415S, and V581I substitutions relative to SEQ ID NO: 2; the A123G, T140L, and F143Y substitutions relative to SEQ ID NO: 2; the N390D, S476N, and K519R substitutions relative to SEQ ID NO: 2; the E382Q, T573S, and V581I substitutions relative to SEQ ID NO: 2; the D122E, T470S, and S476N substitutions relative to SEQ ID NO: 2; the T140Q, P479A, and K514P substitutions relative to SEQ ID NO: 2; the E120A, V178I, and L549I substitutions relative to SEQ ID NO: 2; the E382Q, S537D, and N589K substitutions relative to SEQ ID NO: 2; the T140L, G586S, and F596I substitutions relative to SEQ ID NO: 2; or the E382Q, L459F, and K519R substitutions relative to SEQ ID NO: 2. It is understood, the recombinant mutant invertase can comprise the same groups of three substitutions noted above with reference to SEQ ID NO: 2, as well as invertases containing the same groups of three substitutions at the corresponding locations SEQ ID NO: 1 or SEQ ID NO: 3 (see TABLE 1).

[60] In certain embodiments, the recombinant mutant invertase comprises at least three substitutions selected from: the F83Y, N340A, and P390A substitutions relative to SEQ ID NO: 3; the E31A, D33E, and P390A substitutions relative to SEQ ID NO: 3; the K170L, S387N, and K425P substitutions relative to SEQ ID NO: 3; the T51L, E67Q, and L460I substitutions relative to SEQ ID NO: 3; the S387A, P390A, and Q508E substitutions relative to SEQ ID NO: 3; the K188T, E326S, and V492I substitutions relative to SEQ ID NO: 3; the A34G, T51L, and F54Y substitutions relative to SEQ ID NO: 3; the N301D, S387N, and K430R substitutions relative to SEQ ID NO: 3; the E293Q, T484S, and V492I substitutions relative to SEQ ID NO: 3; the D33E, T381S, and S387N substitutions relative to SEQ ID NO: 3; the T51Q, P390A, and K425P substitutions relative to SEQ ID NO: 3; the E31A, V89I, and L460I substitutions relative to SEQ ID NO: 3; the E293Q, S448D, and N500K substitutions relative to SEQ ID NO: 3; the T51L, G497S, and F507I substitutions relative to SEQ ID NO: 3; or the E293Q, L370F, and K430R substitutions relative to SEQ ID NO: 3.

[61] In certain embodiments, the invertase comprises the following substitutions at positions corresponding to the amino acids set forth in SEQ ID NO: 1, including: the F102Y, N359A, and P409A substitutions relative to SEQ ID NO: 1; the E50A, D52E, and P409A substitutions relative to SEQ ID NO: 1; the K189L, S406N, and K444P substitutions relative to SEQ ID NO: 1; the T70L, E86Q, and L479I substitutions relative to SEQ ID NO: 1; the S406A, P409A, and Q527E substitutions relative to SEQ ID NO: 1; the K207T, E345S, and V511I substitutions relative to SEQ ID NO: 1; the A53G, T70L, and F73Y substitutions relative to SEQ ID NO: 1; the N320D, S406N, and K449R substitutions relative to SEQ ID NO: 1; the E312Q, T503S, and V511I substitutions relative to SEQ ID NO: 1; the D52E, T400S, and S406N substitutions relative to SEQ ID NO: 1; the T70Q, P409A, and K444P substitutions relative to SEQ ID NO: 1; the E50A, V108I, and L479I substitutions relative to SEQ ID NO: 1; the E312Q, S467D, and N519K substitutions relative to SEQ ID NO: 1; the T70L, G516S, and F526I substitutions relative to SEQ ID NO: 1; or the E312Q, L389F, and K449R substitutions relative to SEQ ID NO: 1.

[62] In certain embodiments, the recombinant mutant invertase comprises at least three substitutions selected from: the E31A, D33E, and P390A substitutions relative to SEQ ID NO: 3; the T51L, E67Q, and L460I substitutions relative to SEQ ID NO: 3; the K170L, S387N, and K425P substitutions relative to SEQ ID NO: 3; the S387A, P390A, and Q508E substitutions relative to SEQ ID NO: 3; the K188T, E326S, and V492I substitutions relative to SEQ ID NO: 3; the A34G, T51L, and F54Y substitutions relative to SEQ ID NO: 3; the N301D, S387N, and K430R substitutions relative to SEQ ID NO: 3; the E293Q, T484S, and V492I substitutions relative to SEQ ID NO: 3; the D33E, T381S, and S387N substitutions relative to SEQ ID NO: 3; the T51L, P390A, and K425P substitutions relative to SEQ ID NO: 3; the F83Y, N340A, and P390A substitutions relative to SEQ ID NO: 3; the E31A, V89I, and L460I substitutions relative to SEQ ID NO: 3; the E293Q, S448D, and N500K substitutions relative to SEQ ID NO: 3; the T51L, G497S, and F507I substitutions relative to SEQ ID NO: 3; or the E293Q, L370F, and K430R substitutions relative to SEQ ID NO: 3.

[63] In certain embodiments, the recombinant mutant invertase comprises F172Y, N429A, P479A, and T140L substitutions relative to SEQ ID NO: 2. In certain embodiments, the recombinant mutant invertase comprises F172Y, N429A, P479A, T140L, and D272N substitutions relative to SEQ ID NO: 2. In certain embodiments, the recombinant mutant invertase comprises F172Y, N429A, P479A, T140L, E285H substitutions relative to SEQ ID NO: 2. In certain embodiments, the recombinant mutant invertase comprises F172Y, N429A, P479A, T140L, E382Q substitutions relative to SEQ ID NO: 2.

[64] In certain embodiments, the recombinant mutant invertase comprises F83Y, N340A, P390A, and T51L substitutions relative to SEQ ID NO: 3. In certain embodiments, the recombinant mutant invertase comprises F83Y, N340A, P390A, and T51L, and D183N substitutions relative to SEQ ID NO: 3. In certain embodiments, the recombinant mutant invertase comprises F83Y, N340A, P390A, and T51L, E196H substitutions relative to SEQ ID NO: 3. In certain embodiments, the recombinant mutant invertase comprises F83Y, N340A, P390A, and T51L, E293Q substitutions relative to SEQ ID NO: 3.

[65] In certain embodiments, the recombinant mutant invertase comprises N429A, P479A, and T140L substitutions relative to SEQ ID NO: 2. In certain embodiments, the recombinant mutant invertase comprises F172Y, P479A, and T140L substitutions relative to SEQ ID NO: 2. In certain embodiments, the recombinant mutant invertase comprises F172Y, N429A, and T140L substitutions relative to SEQ ID NO: 2.

[66] In certain embodiments, the recombinant mutant invertase comprises N340A, P390A, and T51L substitutions relative to SEQ ID NO: 3. In certain embodiments, the recombinant mutant invertase comprises F83Y, P390A, and T51L substitutions relative to SEQ ID NO: 3. In certain embodiments, the recombinant mutant invertase comprises F83Y, N340A, and T51L substitutions relative to SEQ ID NO: 3.

[67] In certain embodiments, the recombinant mutant invertase comprises N429A, P479A, T140L, E382Q, and A501N substitutions relative to SEQ ID NO: 2. In certain embodiments, the recombinant mutant invertase comprises F172Y, P479A, T140L, E382Q, and A501N substitutions relative to SEQ ID NO: 2. In certain embodiments, the recombinant mutant invertase comprises F172Y, N429A, T140L, E382Q, and A501N substitutions relative to SEQ ID NO: 2. In certain embodiments, the recombinant mutant invertase comprises F172Y, N429A, P479A, E382Q, and A501N substitutions relative to SEQ ID NO: 2. In certain embodiments, the recombinant mutant invertase comprises F172Y, N429A, P479A, T140L, and A501N substitutions relative to SEQ ID NO: 2. In certain embodiments, the recombinant mutant invertase comprises F172Y, N429A, P479A, T140L, and E382Q, and substitutions relative to SEQ ID NO: 2.

[68] In certain embodiments, the recombinant mutant invertase comprises N340A, P390A, T51L, E293Q, and A412N substitutions relative to SEQ ID NO: 3. In certain embodiments, the recombinant mutant invertase comprises F83Y, P390A, T51L, E293Q, and A412N substitutions relative to SEQ ID NO: 3. In certain embodiments, the recombinant mutant invertase comprises F83Y, N340A, T51L, E293Q, and A412N substitutions relative to SEQ ID NO: 3. In certain embodiments, the recombinant mutant invertase comprises F83Y, N340A, P390A, E293Q, and A412N substitutions relative to SEQ ID NO: 3. In certain embodiments, the recombinant mutant invertase comprises F83Y, N340A, P390A, T51L, and A412N substitutions relative to SEQ ID NO: 3. In certain embodiments, the recombinant mutant invertase comprises F83Y, N340A, P390A, T51L, and E293Q, and substitutions relative to SEQ ID NO: 3.

[69] In certain embodiments, the recombinant mutant invertase comprises at least six substitutions selected from: the F172Y, N429A, P479A, T140L, E382Q, and A501N substitutions relative to SEQ ID NO: 2; the F172Y, N429A, P479A, T140L, D272N, and E285H substitutions relative to SEQ ID NO: 2; the F172Y, N429A, P479A, T140L, D272N, and L549I substitutions relative to SEQ ID NO: 2; the F172Y, N429A, P479A, T140L, D272N, and V581I substitutions relative to SEQ ID NO: 2; the F172Y, N429A, P479A, T140L, E285H, and V581I substitutions relative to SEQ ID NO: 2; the F172Y, N429A, P479A, T140L, E382Q, and A123G substitutions relative to SEQ ID NO: 2; the F172Y, N429A, P479A, T140L, E382Q, and N512G substitutions relative to SEQ ID NO: 2; the F172Y, N429A, P479A, T140L, E382Q, and V178I substitutions relative to SEQ ID NO: 2; and the F172Y, N429A, P479A, T140L, E382Q, and N390D substitutions relative to SEQ ID NO: 2. In certain embodiments, the recombinant mutant invertase comprises the F172Y, N429A, P479A, T140L, E382Q, A501N, and D272N substitutions relative to SEQ ID NO: 2. In certain embodiments, the recombinant mutant invertase comprises the F172Y, N429A, P479A, T140L, E382Q, A501N, D272N, and E285H substitutions relative to SEQ ID NO: 2.

[70] In certain embodiments, the recombinant mutant invertase comprises at least six substitutions selected from: the F83Y, N340A, P390A, T51L, E293Q, and A412N substitutions relative to SEQ ID NO: 3; the F83Y, N340A, P390A, T51L, D183N, and E196H substitutions relative to SEQ ID NO: 3; the F83Y, N340A, P390A, T51L, D183N, and L460I substitutions relative to SEQ ID NO: 3; the F83Y, N340A, P390A, T51L, D183N, and V492I substitutions relative to SEQ ID NO: 3; the F83Y, N340A, P390A, T51L, E196H, and V492I substitutions relative to SEQ ID NO: 3; the F83Y, N340A, P390A, T51L, E293Q, and A34G substitutions relative to SEQ ID NO: 3; the F83Y, N340A, P390A, T51L, E293Q, and N423G substitutions relative to SEQ ID NO: 3; the F83Y, N340A, P390A, T51L, E293Q, and V89I substitutions relative to SEQ ID NO: 3; and the F83Y, N340A, P390A, T51L, E293Q, and N301D substitutions relative to SEQ ID NO: 3. In certain embodiments, the recombinant mutant invertase comprises the F83Y, N340A, P390A, T51L, E293Q, A412N, and D183N substitutions relative to SEQ ID NO: 3. In certain embodiments, the recombinant mutant invertase comprises the F83Y, N340A, P390A, T51L, E293Q, A412N, D183N, and E196H substitutions relative to SEQ ID NO: 3.

[71] In certain embodiments, the recombinant mutant invertase comprises the E120A, D122E, and P479A substitutions (relative to SEQ ID NO: 2). For example, the recombinant mutant invertase may comprise SEQ ID NO: 4, also referred to as V6 herein. In certain embodiments, the recombinant mutant invertase lacks the signal sequence underlined below.

[72] SEQ ID NO: 4MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAEASMTNETSDRPLVHFTPNKGWMNDPNGLWYDAKEAKWHLYFQYNPNDTVWGTPLFWGHATSDDLTHWEDEPIAIAPKRNDSGAFSGSMVVDYNNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAAKSQDYKIEIYSSDDLKSWKLESAFANEGFLGYQYECPGLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYGSALGIAWASNWEYSAFVPTNPWRSSMSLVRKFSLNTEYQANPETELINLKAEPILNISNAGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYAVNTTQTISKSVFADLSLWFKGLEDPEEYLRMGFEASASSFFLDRGNSKVKFVKENPYFTNRMSVNNQPFKSENDLSYYKVYGLLDQNILELYFNDGDVVSTNTYFMTTGNALGSVNMTTGVDNLFYIDKFQVREVK

[73] In certain embodiments, the recombinant mutant invertase comprises the T140L, E156Q, and L549I substitutions relative to SEQ ID NO: 2. For example, the recombinant mutant invertase may comprise SEQ ID NO: 5, also referred to as V9 herein. In certain embodiments, the recombinant mutant invertase lacks the signal sequence underlined below.

[74] SEQ ID NO: 5MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAEASMTNETSDRPLVHFTPNKGWMNDPNGLWYDEKDAKWHLYFQYNPNDTVWGLPLFWGHATSDDLTHWQDEPIAIAPKRNDSGAFSGSMVVDYNNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAAKSQDYKIEIYSSDDLKSWKLESAFANEGFLGYQYECPGLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYGSALGIAWASNWEYSAFVPTNPWRSSMSLVRKFSLNTEYQANPETELINLKAEPILNISNAGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYAVNTTQTISKSVFPDLSLWFKGLEDPEEYLRMGFEASASSFFLDRGNSKVKFVKENPYFTNRMSVNNQPFKSENDLSYYKVYGILDQNILELYFNDGDVVSTNTYFMTTGNALGSVNMTTGVDNLFYIDKFQVREVK.

[75] In certain embodiments, the recombinant mutant invertase comprises the S476A, P479A, and Q597E substitutions relative to SEQ ID NO: 2. For example, the recombinant mutant invertase may comprise SEQ ID NO: 6, also referred to as V14 herein. In certain embodiments, the recombinant mutant invertase lacks the signal sequence underlined below.

[76] SEQ ID NO: 6MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAEASMTNETSDRPLVHFTPNKGWMNDPNGLWYDEKDAKWHLYFQYNPNDTVWGTPLFWGHATSDDLTHWEDEPIAIAPKRNDSGAFSGSMVVDYNNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAAKSQDYKIEIYSSDDLKSWKLESAFANEGFLGYQYECPGLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYGSALGIAWASNWEYSAFVPTNPWRSSMSLVRKFSLNTEYQANPETELINLKAEPILNISNAGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYAVNTTQTISKAVFADLSLWFKGLEDPEEYLRMGFEASASSFFLDRGNSKVKFVKENPYFTNRMSVNNQPFKSENDLSYYKVYGLLDQNILELYFNDGDVVSTNTYFMTTGNALGSVNMTTGVDNLFYIDKFEVREVK.

[77] In certain embodiments, the recombinant mutant invertase comprises the K259L, S476N, and K514P substitutions (relative to SEQ ID NO: 2). For example, the recombinant mutant invertase may comprise SEQ ID NO: 7, also referred to as V22 herein. In certain embodiments, the recombinant mutant invertase lacks the signal sequence underlined below.

[78] SEQ ID NO: 7MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAEASMTNETSDRPLVHFTPNKGWMNDPNGLWYDEKDAKWHLYFQYNPNDTVWGTPLFWGHATSDDLTHWEDEPIAIAPKRNDSGAFSGSMVVDYNNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAALSQDYKIEIYSSDDLKSWKLESAFANEGFLGYQYECPGLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYGSALGIAWASNWEYSAFVPTNPWRSSMSLVRKFSLNTEYQANPETELINLKAEPILNISNAGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYAVNTTQTISKNVFPDLSLWFKGLEDPEEYLRMGFEASASSFFLDRGNSPVKFVKENPYFTNRMSVNNQPFKSENDLSYYKVYGLLDQNILELYFNDGDVVSTNTYFMTTGNALGSVNMTTGVDNLFYIDKFQVREVK.

[79] In certain embodiments, the recombinant mutant invertase comprises the K277T, E415S, and V581I substitutions relative to SEQ ID NO: 2. For example, the recombinant mutant invertase may comprise SEQ ID NO: 8, also referred to as V25 herein. In certain embodiments, the recombinant mutant invertase lacks the signal sequence underlined below.

[80] SEQ ID NO: 8MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAEASMTNETSDRPLVHFTPNKGWMNDPNGLWYDEKDAKWHLYFQYNPNDTVWGTPLFWGHATSDDLTHWEDEPIAIAPKRNDSGAFSGSMVVDYNNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAAKSQDYKIEIYSSDDLKSWTLESAFANEGFLGYQYECPGLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYGSALGIAWASNWEYSAFVPTNPWRSSMSLVRKFSLNTEYQANPETSLINLKAEPILNISNAGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYAVNTTQTISKSVFPDLSLWFKGLEDPEEYLRMGFEASASSFFLDRGNSKVKFVKENPYFTNRMSVNNQPFKSENDLSYYKVYGLLDQNILELYFNDGDVVSTNTYFMTTGNALGSINMTTGVDNLFYIDKFQVREVK.

[81] In certain embodiments, the recombinant mutant invertase comprises the A123G, T140L, and F143Y substitutions relative to SEQ ID NO: 2. For example, the recombinant mutant invertase may comprise SEQ ID NO: 9, also referred to as V32 herein. In certain embodiments, the recombinant mutant invertase lacks the signal sequence underlined below.

[82] SEQ ID NO: 9MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAEASMTNETSDRPLVHFTPNKGWMNDPNGLWYDEKDGKWHLYFQYNPNDTVWGLPLYWGHATSDDLTHWEDEPIAIAPKRNDSGAFSGSMVVDYNNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAAKSQDYKIEIYSSDDLKSWKLESAFANEGFLGYQYECPGLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYGSALGIAWASNWEYSAFVPTNPWRSSMSLVRKFSLNTEYQANPETELINLKAEPILNISNAGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYAVNTTQTISKSVFPDLSLWFKGLEDPEEYLRMGFEASASSFFLDRGNSKVKFVKENPYFTNRMSVNNQPFKSENDLSYYKVYGLLDQNILELYFNDGDVVSTNTYFMTTGNALGSVNMTTGVDNLFYIDKFQVREVK.

[83] In certain embodiments, the recombinant mutant invertase comprises the N390D, S476N, and K519R substitutions relative to SEQ ID NO: 2. For example, the recombinant mutant invertase may comprise SEQ ID NO: 10, also referred to as V38 herein. In certain embodiments, the recombinant mutant invertase lacks the signal sequence underlined below.

[84] SEQ ID NO: 10MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAEASMTNETSDRPLVHFTPNKGWMNDPNGLWYDEKDAKWHLYFQYNPNDTVWGTPLFWGHATSDDLTHWEDEPIAIAPKRNDSGAFSGSMVVDYNNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAAKSQDYKIEIYSSDDLKSWKLESAFANEGFLGYQYECPGLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYGSALGIAWASNWEYSAFVPTDPWRSSMSLVRKFSLNTEYQANPETELINLKAEPILNISNAGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYAVNTTQTISKNVFPDLSLWFKGLEDPEEYLRMGFEASASSFFLDRGNSKVKFVRENPYFTNRMSVNNQPFKSENDLSYYKVYGLLDQNILELYFNDGDVVSTNTYFMTTGNALGSVNMTTGVDNLFYIDKFQVREVK

[85] In certain embodiments, the recombinant mutant invertase comprises the E382Q, T573S, and V581I substitutions relative to SEQ ID NO: 2. For example, the recombinant mutant invertase may comprise SEQ ID NO: 11, also referred to as V60 herein. In certain embodiments, the recombinant mutant invertase lacks the signal sequence underlined below.

[86] SEQ ID NO: 11MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAEASMTNETSDRPLVHFTPNKGWMNDPNGLWYDEKDAKWHLYFQYNPNDTVWGTPLFWGHATSDDLTHWEDEPIAIAPKRNDSGAFSGSMVVDYNNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAAKSQDYKIEIYSSDDLKSWKLESAFANEGFLGYQYECPGLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYGSALGIAWASNWQYSAFVPTNPWRSSMSLVRKFSLNTEYQANPETELINLKAEPILNISNAGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYAVNTTQTISKSVFPDLSLWFKGLEDPEEYLRMGFEASASSFFLDRGNSKVKFVKENPYFTNRMSVNNQPFKSENDLSYYKVYGLLDQNILELYFNDGDVVSTNTYFMSTGNALGSINMTTGVDNLFYIDKFQVREVK.

[87] In certain embodiments, the recombinant mutant invertase comprises the D122E, T470S, and S476N substitutions relative to SEQ ID NO: 2. For example, the recombinant mutant invertase may comprise SEQ ID NO: 12, also referred to as V63 herein. In certain embodiments, the recombinant mutant invertase lacks the signal sequence underlined below.

[88] SEQ ID NO: 12MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAEASMTNETSDRPLVHFTPNKGWMNDPNGLWYDEKEAKWHLYFQYNPNDTVWGTPLFWGHATSDDLTHWEDEPIAIAPKRNDSGAFSGSMVVDYNNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAAKSQDYKIEIYSSDDLKSWKLESAFANEGFLGYQYECPGLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYGSALGIAWASNWEYSAFVPTNPWRSSMSLVRKFSLNTEYQANPETELINLKAEPILNISNAGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYAVNTSQTISKNVFPDLSLWFKGLEDPEEYLRMGFEASASSFFLDRGNSKVKFVKENPYFTNRMSVNNQPFKSENDLSYYKVYGLLDQNILELYFNDGDVVSTNTYFMTTGNALGSVNMTTGVDNLFYIDKFQVREVK.

[89] In certain embodiments, the recombinant mutant invertase comprises the T140Q, P479A, and K514P substitutions relative to SEQ ID NO: 2. For example, the recombinant mutant invertase may comprise SEQ ID NO: 13, also referred to as V70 herein. In certain embodiments, the recombinant mutant invertase lacks the signal sequence underlined below.

[90] SEQ ID NO: 13MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAEASMTNETSDRPLVHFTPNKGWMNDPNGLWYDEKDAKWHLYFQYNPNDTVWGQPLFWGHATSDDLTHWEDEPIAIAPKRNDSGAFSGSMVVDYNNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAAKSQDYKIEIYSSDDLKSWKLESAFANEGFLGYQYECPGLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYGSALGIAWASNWEYSAFVPTNPWRSSMSLVRKFSLNTEYQANPETELINLKAEPILNISNAGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYAVNTTQTISKSVFADLSLWFKGLEDPEEYLRMGFEASASSFFLDRGNSPVKFVKENPYFTNRMSVNNQPFKSENDLSYYKVYGLLDQNILELYFNDGDVVSTNTYFMTTGNALGSVNMTTGVDNLFYIDKFQVREVK.

[91] In certain embodiments, the recombinant mutant invertase comprises the F172Y, N429A, and P479A substitutions (relative to SEQ ID NO: 2). For example, the recombinant mutant invertase may comprise SEQ ID NO: 14, also referred to as V78 herein. In certain embodiments, the recombinant mutant invertase lacks the signal sequence underlined below.

[92] SEQ ID NO: 14MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAEASMTNETSDRPLVHFTPNKGWMNDPNGLWYDEKDAKWHLYFQYNPNDTVWGTPLFWGHATSDDLTHWEDEPIAIAPKRNDSGAYSGSMVVDYNNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAAKSQDYKIEIYSSDDLKSWKLESAFANEGFLGYQYECPGLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYGSALGIAWASNWEYSAFVPTNPWRSSMSLVRKFSLNTEYQANPETELINLKAEPILNISAAGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYAVNTTQTISKSVFADLSLWFKGLEDPEEYLRMGFEASASSFFLDRGNSKVKFVKENPYFTNRMSVNNQPFKSENDLSYYKVYGLLDQNILELYFNDGDVVSTNTYFMTTGNALGSVNMTTGVDNLFYIDKFQVREVK.

[93] In certain embodiments, the recombinant mutant invertase comprises the E120A, V178I, and L549I substitutions relative to SEQ ID NO: 2. For example, the recombinant mutant invertase may comprise SEQ ID NO: 15, also referred to as V83 herein. In certain embodiments, the recombinant mutant invertase lacks the signal sequence underlined below.

[94] SEQ ID NO: 15MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAEASMTNETSDRPLVHFTPNKGWMNDPNGLWYDAKDAKWHLYFQYNPNDTVWGTPLFWGHATSDDLTHWEDEPIAIAPKRNDSGAFSGSMVIDYNNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAAKSQDYKIEIYSSDDLKSWKLESAFANEGFLGYQYECPGLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYGSALGIAWASNWEYSAFVPTNPWRSSMSLVRKFSLNTEYQANPETELINLKAEPILNISNAGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYAVNTTQTISKSVFPDLSLWFKGLEDPEEYLRMGFEASASSFFLDRGNSKVKFVKENPYFTNRMSVNNQPFKSENDLSYYKVYGILDQNILELYFNDGDVVSTNTYFMTTGNALGSVNMTTGVDNLFYIDKFQVREVK.

[95] In certain embodiments, the recombinant mutant invertase comprises the E382Q, S537D, and N589K substitutions relative to SEQ ID NO: 2. For example, the recombinant mutant invertase may comprise SEQ ID NO: 16, also referred to as V87 herein. In certain embodiments, the recombinant mutant invertase lacks the signal sequence underlined below.

[96] SEQ ID NO: 16MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAEASMTNETSDRPLVHFTPNKGWMNDPNGLWYDEKDAKWHLYFQYNPNDTVWGTPLFWGHATSDDLTHWEDEPIAIAPKRNDSGAFSGSMVVDYNNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAAKSQDYKIEIYSSDDLKSWKLESAFANEGFLGYQYECPGLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYGSALGIAWASNWQYSAFVPTNPWRSSMSLVRKFSLNTEYQANPETELINLKAEPILNISNAGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYAVNTTQTISKSVFPDLSLWFKGLEDPEEYLRMGFEASASSFFLDRGNSKVKFVKENPYFTNRMSVNNQPFKDENDLSYYKVYGLLDQNILELYFNDGDVVSTNTYFMTTGNALGSVNMTTGVDKLFYIDKFQVREVK.

[97] In certain embodiments, the recombinant mutant invertase comprises the T140L, G586S, and F596I substitutions relative to SEQ ID NO: 2. For example, the recombinant mutant invertase may comprise SEQ ID NO: 17, also referred to as V89 herein. In certain embodiments, the recombinant mutant invertase lacks the signal sequence underlined below.

[98] SEQ ID NO: 17MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAEASMTNETSDRPLVHFTPNKGWMNDPNGLWYDEKDAKWHLYFQYNPNDTVWGLPLFWGHATSDDLTHWEDEPIAIAPKRNDSGAFSGSMVVDYNNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAAKSQDYKIEIYSSDDLKSWKLESAFANEGFLGYQYECPGLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYGSALGIAWASNWEYSAFVPTNPWRSSMSLVRKFSLNTEYQANPETELINLKAEPILNISNAGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYAVNTTQTISKSVFPDLSLWFKGLEDPEEYLRMGFEASASSFFLDRGNSKVKFVKENPYFTNRMSVNNQPFKSENDLSYYKVYGLLDQNILELYFNDGDVVSTNTYFMTTGNALGSVNMTTSVDNLFYIDKIQVREVK.

[99] In certain embodiments, the recombinant mutant invertase comprises the E382Q, L459F, and K519R substitutions relative to SEQ ID NO: 2. For example, the recombinant mutant invertase may comprise SEQ ID NO: 18, also referred to as V90 herein. In certain embodiments, the recombinant mutant invertase lacks the signal sequence underlined below.

[100] SEQ ID NO: 18MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAEASMTNETSDRPLVHFTPNKGWMNDPNGLWYDEKDAKWHLYFQYNPNDTVWGTPLFWGHATSDDLTHWEDEPIAIAPKRNDSGAFSGSMVVDYNNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAAKSQDYKIEIYSSDDLKSWKLESAFANEGFLGYQYECPGLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYGSALGIAWASNWQYSAFVPTNPWRSSMSLVRKFSLNTEYQANPETELINLKAEPILNISNAGPWSRFATNTTLTKANSYNVDLSNSTGTFEFELVYAVNTTQTISKSVFPDLSLWFKGLEDPEEYLRMGFEASASSFFLDRGNSKVKFVRENPYFTNRMSVNNQPFKSENDLSYYKVYGLLDQNILELYFNDGDVVSTNTYFMTTGNALGSVNMTTGVDNLFYIDKFQVREVK.

[101] In certain embodiments, the recombinant mutant invertase comprises the E31A, D33E, and P390A substitutions (relative to SEQ ID NO: 3). For example, the recombinant mutant invertase may comprise SEQ ID NO: 19, also referred to as V6 herein.

[102] SEQ ID NO: 19SMTNETSDRPLVHFTPNKGWMNDPNGLWYDAKEAKWHLYFQYNPNDTVWGTPLFWGHATSDDLTHWEDEPIAIAPKRNDSGAFSGSMVVDYNNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAAKSQDYKIEIYSSDDLKSWKLESAFANEGFLGYQYECPGLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYGSALGIAWASNWEYSAFVPTNPWRSSMSLVRKFSLNTEYQANPETELINLKAEPILNISNAGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYAVNTTQTISKSVFADLSLWFKGLEDPEEYLRMGFEASASSFFLDRGNSKVKFVKENPYFTNRMSVNNQPFKSENDLSYYKVYGLLDQNILELYFNDGDVVSTNTYFMTTGNALGSVNMTTGVDNLFYIDKFQVREVK.

[103] In certain embodiments, the recombinant mutant invertase comprises the T51L, E67Q, and L460I substitutions relative to SEQ ID NO: 3. For example, the recombinant mutant invertase may comprise SEQ ID NO: 20, also referred to as V9 herein.

[104] SEQ ID NO: 20SMTNETSDRPLVHFTPNKGWMNDPNGLWYDEKDAKWHLYFQYNPNDTVWGLPLFWGHATSDDLTHWQDEPIAIAPKRNDSGAFSGSMVVDYNNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAAKSQDYKIEIYSSDDLKSWKLESAFANEGFLGYQYECPGLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYGSALGIAWASNWEYSAFVPTNPWRSSMSLVRKFSLNTEYQANPETELINLKAEPILNISNAGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYAVNTTQTISKSVFPDLSLWFKGLEDPEEYLRMGFEASASSFFLDRGNSKVKFVKENPYFTNRMSVNNQPFKSENDLSYYKVYGILDQNILELYFNDGDVVSTNTYFMTTGNALGSVNMTTGVDNLFYIDKFQVREVK.

[105] In certain embodiments, the recombinant mutant invertase comprises the S387A, P390A, and Q508E substitutions relative to SEQ ID NO: 3. For example, the recombinant mutant invertase may comprise SEQ ID NO: 21, also referred to as V14 herein.

[106] SEQ ID NO: 21SMTNETSDRPLVHFTPNKGWMNDPNGLWYDEKDAKWHLYFQYNPNDTVWGTPLFWGHATSDDLTHWEDEPIAIAPKRNDSGAFSGSMVVDYNNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAAKSQDYKIEIYSSDDLKSWKLESAFANEGFLGYQYECPGLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYGSALGIAWASNWEYSAFVPTNPWRSSMSLVRKFSLNTEYQANPETELINLKAEPILNISNAGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYAVNTTQTISKAVFADLSLWFKGLEDPEEYLRMGFEASASSFFLDRGNSKVKFVKENPYFTNRMSVNNQPFKSENDLSYYKVYGLLDQNILELYFNDGDVVSTNTYFMTTGNALGSVNMTTGVDNLFYIDKFEVREVK.

[107] In certain embodiments, the recombinant mutant invertase comprises the K170L, S387N, and K425P substitutions (relative to SEQ ID NO: 3). For example, the recombinant mutant invertase may comprise SEQ ID NO: 22, also referred to as V22 herein.

[108] SEQ ID NO: 22SMTNETSDRPLVHFTPNKGWMNDPNGLWYDEKDAKWHLYFQYNPNDTVWGTPLFWGHATSDDLTHWEDEPIAIAPKRNDSGAFSGSMVVDYNNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAALSQDYKIEIYSSDDLKSWKLESAFANEGFLGYQYECPGLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYGSALGIAWASNWEYSAFVPTNPWRSSMSLVRKFSLNTEYQANPETELINLKAEPILNISNAGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYAVNTTQTISKNVFPDLSLWFKGLEDPEEYLRMGFEASASSFFLDRGNSPVKFVKENPYFTNRMSVNNQPFKSENDLSYYKVYGLLDQNILELYFNDGDVVSTNTYFMTTGNALGSVNMTTGVDNLFYIDKFQVREVK

[109] In certain embodiments, the recombinant mutant invertase comprises the K188T, E326S, and V492I substitutions relative to SEQ ID NO: 3. For example, the recombinant mutant invertase may comprise SEQ ID NO: 23, also referred to as V25 herein.

[110] SEQ ID NO: 23SMTNETSDRPLVHFTPNKGWMNDPNGLWYDEKDAKWHLYFQYNPNDTVWGTPLFWGHATSDDLTHWEDEPIAIAPKRNDSGAFSGSMVVDYNNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAAKSQDYKIEIYSSDDLKSWTLESAFANEGFLGYQYECPGLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYGSALGIAWASNWEYSAFVPTNPWRSSMSLVRKFSLNTEYQANPETSLINLKAEPILNISNAGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYAVNTTQTISKSVFPDLSLWFKGLEDPEEYLRMGFEASASSFFLDRGNSKVKFVKENPYFTNRMSVNNQPFKSENDLSYYKVYGLLDQNILELYFNDGDVVSTNTYFMTTGNALGSINMTTGVDNLFYIDKFQVREVK

[111] In certain embodiments, the recombinant mutant invertase comprises the A34G, T51L, and F54Y substitutions relative to SEQ ID NO: 3. For example, the recombinant mutant invertase may comprise SEQ ID NO: 24, also referred to as V32 herein.

[112] SEQ ID NO: 24SMTNETSDRPLVHFTPNKGWMNDPNGLWYDEKDGKWHLYFQYNPNDTVWGLPLYWGHATSDDLTHWEDEPIAIAPKRNDSGAFSGSMVVDYNNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAAKSQDYKIEIYSSDDLKSWKLESAFANEGFLGYQYECPGLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYGSALGIAWASNWEYSAFVPTNPWRSSMSLVRKFSLNTEYQANPETELINLKAEPILNISNAGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYAVNTTQTISKSVFPDLSLWFKGLEDPEEYLRMGFEASASSFFLDRGNSKVKFVKENPYFTNRMSVNNQPFKSENDLSYYKVYGLLDQNILELYFNDGDVVSTNTYFMTTGNALGSVNMTTGVDNLFYIDKFQVREVK.

[113] In certain embodiments, the recombinant mutant invertase comprises the N301D, S387N, and K430R substitutions relative to SEQ ID NO: 3. For example, the recombinant mutant invertase may comprise SEQ ID NO: 25, also referred to as V38 herein.

[114] SEQ ID NO: 25SMTNETSDRPLVHFTPNKGWMNDPNGLWYDEKDAKWHLYFQYNPNDTVWGTPLFWGHATSDDLTHWEDEPIAIAPKRNDSGAFSGSMVVDYNNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAAKSQDYKIEIYSSDDLKSWKLESAFANEGFLGYQYECPGLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYGSALGIAWASNWEYSAFVPTDPWRSSMSLVRKFSLNTEYQANPETELINLKAEPILNISNAGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYAVNTTQTISKNVFPDLSLWFKGLEDPEEYLRMGFEASASSFFLDRGNSKVKFVRENPYFTNRMSVNNQPFKSENDLSYYKVYGLLDQNILELYFNDGDVVSTNTYFMTTGNALGSVNMTTGVDNLFYIDKFQVREVK.

[115] In certain embodiments, the recombinant mutant invertase comprises the E293Q, T484S, and V492I substitutions relative to SEQ ID NO: 3. For example, the recombinant mutant invertase may comprise SEQ ID NO: 26, also referred to as V60 herein.

[116] SEQ ID NO: 26SMTNETSDRPLVHFTPNKGWMNDPNGLWYDEKDAKWHLYFQYNPNDTVWGTPLFWGHATSDDLTHWEDEPIAIAPKRNDSGAFSGSMVVDYNNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAAKSQDYKIEIYSSDDLKSWKLESAFANEGFLGYQYECPGLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYGSALGIAWASNWQYSAFVPTNPWRSSMSLVRKFSLNTEYQANPETELINLKAEPILNISNAGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYAVNTTQTISKSVFPDLSLWFKGLEDPEEYLRMGFEASASSFFLDRGNSKVKFVKENPYFTNRMSVNNQPFKSENDLSYYKVYGLLDQNILELYFNDGDVVSTNTYFMSTGNALGSINMTTGVDNLFYIDKFQVREVK.

[117] In certain embodiments, the recombinant mutant invertase comprises the D33E, T381S, and S387N substitutions relative to SEQ ID NO: 3. For example, the recombinant mutant invertase may comprise SEQ ID NO: 27, also referred to as V63 herein.

[118] SEQ ID NO: 27SMTNETSDRPLVHFTPNKGWMNDPNGLWYDEKEAKWHLYFQYNPNDTVWGTPLFWGHATSDDLTHWEDEPIAIAPKRNDSGAFSGSMVVDYNNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAAKSQDYKIEIYSSDDLKSWKLESAFANEGFLGYQYECPGLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYGSALGIAWASNWEYSAFVPTNPWRSSMSLVRKFSLNTEYQANPETELINLKAEPILNISNAGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYAVNTSQTISKNVFPDLSLWFKGLEDPEEYLRMGFEASASSFFLDRGNSKVKFVKENPYFTNRMSVNNQPFKSENDLSYYKVYGLLDQNILELYFNDGDVVSTNTYFMTTGNALGSVNMTTGVDNLFYIDKFQVREVK.

[119] In certain embodiments, the recombinant mutant invertase comprises the T51Q, P390A, and K425P substitutions relative to SEQ ID NO: 3. For example, the recombinant mutant invertase may comprise SEQ ID NO: 28, also referred to as V70 herein.

[120] SEQ ID NO: 28SMTNETSDRPLVHFTPNKGWMNDPNGLWYDEKDAKWHLYFQYNPNDTVWGQPLFWGHATSDDLTHWEDEPIAIAPKRNDSGAFSGSMVVDYNNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAAKSQDYKIEIYSSDDLKSWKLESAFANEGFLGYQYECPGLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYGSALGIAWASNWEYSAFVPTNPWRSSMSLVRKFSLNTEYQANPETELINLKAEPILNISNAGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYAVNTTQTISKSVFADLSLWFKGLEDPEEYLRMGFEASASSFFLDRGNSPVKFVKENPYFTNRMSVNNQPFKSENDLSYYKVYGLLDQNILELYFNDGDVVSTNTYFMTTGNALGSVNMTTGVDNLFYIDKFQVREVK.

[121] In certain embodiments, the recombinant mutant invertase comprises the F83Y, N340A, and P390A substitutions (relative to SEQ ID NO: 3). For example, the recombinant mutant invertase may comprise SEQ ID NO: 29, also referred to as V78 herein.

[122] SEQ ID NO: 29SMTNETSDRPLVHFTPNKGWMNDPNGLWYDEKDAKWHLYFQYNPNDTVWGTPLFWGHATSDDLTHWEDEPIAIAPKRNDSGAYSGSMVVDYNNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAAKSQDYKIEIYSSDDLKSWKLESAFANEGFLGYQYECPGLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYGSALGIAWASNWEYSAFVPTNPWRSSMSLVRKFSLNTEYQANPETELINLKAEPILNISAAGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYAVNTTQTISKSVFADLSLWFKGLEDPEEYLRMGFEASASSFFLDRGNSKVKFVKENPYFTNRMSVNNQPFKSENDLSYYKVYGLLDQNILELYFNDGDVVSTNTYFMTTGNALGSVNMTTGVDNLFYIDKFQVREVK.

[123] In certain embodiments, the recombinant mutant invertase comprises the E31A, V89I, and L460I substitutions relative to SEQ ID NO: 3. For example, the recombinant mutant invertase may comprise SEQ ID NO: 30, also referred to as V83 herein.

[124] SEQ ID NO: 30SMTNETSDRPLVHFTPNKGWMNDPNGLWYDAKDAKWHLYFQYNPNDTVWGTPLFWGHATSDDLTHWEDEPIAIAPKRNDSGAFSGSMVIDYNNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAAKSQDYKIEIYSSDDLKSWKLESAFANEGFLGYQYECPGLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYGSALGIAWASNWEYSAFVPTNPWRSSMSLVRKFSLNTEYQANPETELINLKAEPILNISNAGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYAVNTTQTISKSVFPDLSLWFKGLEDPEEYLRMGFEASASSFFLDRGNSKVKFVKENPYFTNRMSVNNQPFKSENDLSYYKVYGILDQNILELYFNDGDVVSTNTYFMTTGNALGSVNMTTGVDNLFYIDKFQVREVK.

[125] In certain embodiments, the recombinant mutant invertase comprises the E293Q, S448D, and N500K substitutions relative to SEQ ID NO: 3. For example, the recombinant mutant invertase may comprise SEQ ID NO: 31, also referred to as V87 herein.

[126] SEQ ID NO: 31SMTNETSDRPLVHFTPNKGWMNDPNGLWYDEKDAKWHLYFQYNPNDTVWGTPLFWGHATSDDLTHWEDEPIAIAPKRNDSGAFSGSMVVDYNNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAAKSQDYKIEIYSSDDLKSWKLESAFANEGFLGYQYECPGLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYGSALGIAWASNWQYSAFVPTNPWRSSMSLVRKFSLNTEYQANPETELINLKAEPILNISNAGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYAVNTTQTISKSVFPDLSLWFKGLEDPEEYLRMGFEASASSFFLDRGNSKVKFVKENPYFTNRMSVNNQPFKDENDLSYYKVYGLLDQNILELYFNDGDVVSTNTYFMTTGNALGSVNMTTGVDKLFYIDKFQVREVK.

[127] In certain embodiments, the recombinant mutant invertase comprises the T51L, G497S, and F507I substitutions relative to SEQ ID NO: 3. For example, the recombinant mutant invertase may comprise SEQ ID NO: 32, also referred to as V89 herein.

[128] SEQ ID NO: 32SMTNETSDRPLVHFTPNKGWMNDPNGLWYDEKDAKWHLYFQYNPNDTVWGLPLFWGHATSDDLTHWEDEPIAIAPKRNDSGAFSGSMVVDYNNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAAKSQDYKIEIYSSDDLKSWKLESAFANEGFLGYQYECPGLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYGSALGIAWASNWEYSAFVPTNPWRSSMSLVRKFSLNTEYQANPETELINLKAEPILNISNAGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYAVNTTQTISKSVFPDLSLWFKGLEDPEEYLRMGFEASASSFFLDRGNSKVKFVKENPYFTNRMSVNNQPFKSENDLSYYKVYGLLDQNILELYFNDGDVVSTNTYFMTTGNALGSVNMTTSVDNLFYIDKIQVREVK.

[129] In certain embodiments, the recombinant mutant invertase comprises the E293Q, L370F, and K430R substitutions relative to SEQ ID NO: 3. For example, the recombinant mutant invertase may comprise SEQ ID NO: 33, also referred to as V90 herein.

[130] SEQ ID NO: 33SMTNETSDRPLVHFTPNKGWMNDPNGLWYDEKDAKWHLYFQYNPNDTVWGTPLFWGHATSDDLTHWEDEPIAIAPKRNDSGAFSGSMVVDYNNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAAKSQDYKIEIYSSDDLKSWKLESAFANEGFLGYQYECPGLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYGSALGIAWASNWQYSAFVPTNPWRSSMSLVRKFSLNTEYQANPETELINLKAEPILNISNAGPWSRFATNTTLTKANSYNVDLSNSTGTFEFELVYAVNTTQTISKSVFPDLSLWFKGLEDPEEYLRMGFEASASSFFLDRGNSKVKFVRENPYFTNRMSVNNQPFKSENDLSYYKVYGLLDQNILELYFNDGDVVSTNTYFMTTGNALGSVNMTTGVDNLFYIDKFQVREVK.

[131] In certain embodiments, the recombinant mutant invertase comprises a non-wild-type invertase with F172Y and N429A mutations relative to SEQ ID NO: 2, without signal sequence. For example, the recombinant mutant invertase may comprise the Sc_S288C invertase with F172Y and N429A mutations (SEQ ID NO: 50)

[132] SEQ ID NO: 50SMTNETSDRPLVHFTPNKGWMNDPNGLWYDEKDAKWHLYFQYNPNDTVWGTPLFWGHATSDDLTNWEDQPIAIAPKRNDSGAYSGSMVVDYNNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAAKSQDYKIEIYSSDDLKSWKLESAFANEGFLGYQYECPGLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYGSALGIAWASNWEYSAFVPTNPWRSSMSLVRKFSLNTEYQANPETELINLKAEPILNISAAGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYAVNTTQTISKSVFADLSLWFKGLEDPEEYLRMGFEVSASSFFLDRGNSKVKFVKENPYFTNRMSVNNQPFKSENDLSYYKVYGLLDQNILELYFNDGDVVSTNTYFMTTGNALGSVNMTTGVDNLFYIDKFQVREVK.

[133] In certain embodiments, the recombinant mutant invertase comprises a non-wild-type invertase with F172Y, N429A, P479A, T140L, E382Q, and A501N mutations relative to SEQ ID NO: 2, without signal sequence. For example, the recombinant mutant invertase may comprise SEQ ID NO: 51, also referred to as VE36 herein.

[134] SEQ ID NO: 51SMTNETSDRPLVHFTPNKGWMNDPNGLWYDEKDAKWHLYFQYNPNDTVWGLPLFWGHATSDDLTHWEDEPIAIAPKRNDSGAYSGSMVVDYNNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAAKSQDYKIEIYSSDDLKSWKLESAFANEGFLGYQYECPGLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYGSALGIAWASNWQYSAFVPTNPWRSSMSLVRKFSLNTEYQANPETELINLKAEPILNISAAGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYAVNTTQTISKSVFADLSLWFKGLEDPEEYLRMGFENSASSFFLDRGNSKVKFVKENPYFTNRMSVNNQPFKSENDLSYYKVYGLLDQNILELYFNDGDVVSTNTYFMTTGNALGSVNMTTGVDNLFYIDKFQVREVK.

[135] In certain embodiments, the recombinant mutant invertase comprises a non-wild-type invertase with F172Y, N429A, P479A, T140L, E382Q, A501N, and D272N mutations relative to SEQ ID NO: 2, without signal sequence. For example, the recombinant mutant invertase may comprise SEQ ID NO: 52, also referred to as VE361 herein.

[136] SEQ ID NO: 52SMTNETSDRPLVHFTPNKGWMNDPNGLWYDEKDAKWHLYFQYNPNDTVWGLPLFWGHATSDDLTHWEDEPIAIAPKRNDSGAYSGSMVVDYNNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAAKSQDYKIEIYSSDNLKSWKLESAFANEGFLGYQYECPGLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYGSALGIAWASNWQYSAFVPTNPWRSSMSLVRKFSLNTEYQANPETELINLKAEPILNISAAGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYAVNTTQTISKSVFADLSLWFKGLEDPEEYLRMGFENSASSFFLDRGNSKVKFVKENPYFTNRMSVNNQPFKSENDLSYYKVYGLLDQNILELYFNDGDVVSTNTYFMTTGNALGSVNMTTGVDNLFYIDKFQVREVK.

[137] In certain embodiments, the recombinant mutant invertase comprises a non-wild-type invertase with F172Y, N429A, P479A, T140L, E382Q, A501N, D272N, and E285H mutations relative to SEQ ID NO: 2, without signal sequence. For example, the recombinant mutant invertase may comprise SEQ ID NO: 53, also referred to as VE362 herein.

[138] SEQ ID NO: 53SMTNETSDRPLVHFTPNKGWMNDPNGLWYDEKDAKWHLYFQYNPNDTVWGLPLFWGHATSDDLTHWEDEPIAIAPKRNDSGAYSGSMVVDYNNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAAKSQDYKIEIYSSDNLKSWKLESAFANHGFLGYQYECPGLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYGSALGIAWASNWQYSAFVPTNPWRSSMSLVRKFSLNTEYQANPETELINLKAEPILNISAAGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYAVNTTQTISKSVFADLSLWFKGLEDPEEYLRMGFENSASSFFLDRGNSKVKFVKENPYFTNRMSVNNQPFKSENDLSYYKVYGLLDQNILELYFNDGDVVSTNTYFMTTGNALGSVNMTTGVDNLFYIDKFQVREVK.

[139] In certain embodiments, the recombinant mutant invertase comprises a non-wild-type invertase with F172Y, N429A, P479A, T140L, D272N, and E285H mutations relative to SEQ ID NO: 2, without signal sequence. For example, the recombinant mutant invertase may comprise SEQ ID NO: 54, also referred to as VE01 herein.

[140] SEQ ID NO: 54SMTNETSDRPLVHFTPNKGWMNDPNGLWYDEKDAKWHLYFQYNPNDTVWGLPLFWGHATSDDLTHWEDEPIAIAPKRNDSGAYSGSMVVDYNNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAAKSQDYKIEIYSSDNLKSWKLESAFANHGFLGYQYECPGLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYGSALGIAWASNWEYSAFVPTNPWRSSMSLVRKFSLNTEYQANPETELINLKAEPILNISAAGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYAVNTTQTISKSVFADLSLWFKGLEDPEEYLRMGFEASASSFFLDRGNSKVKFVKENPYFTNRMSVNNQPFKSENDLSYYKVYGLLDQNILELYFNDGDVVSTNTYFMTTGNALGSVNMTTGVDNLFYIDKFQVREVK.

[141] In certain embodiments, the recombinant mutant invertase comprises a non-wild-type invertase with F172Y, N429A, P479A, T140L, D272N, and L549I mutations relative to SEQ ID NO: 2, without signal sequence. For example, the recombinant mutant invertase may comprise SEQ ID NO: 55, also referred to as VE03 herein.

[142] SEQ ID NO: 55SMTNETSDRPLVHFTPNKGWMNDPNGLWYDEKDAKWHLYFQYNPNDTVWGLPLFWGHATSDDLTHWEDEPIAIAPKRNDSGAYSGSMVVDYNNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAAKSQDYKIEIYSSDNLKSWKLESAFANEGFLGYQYECPGLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYGSALGIAWASNWEYSAFVPTNPWRSSMSLVRKFSLNTEYQANPETELINLKAEPILNISAAGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYAVNTTQTISKSVFADLSLWFKGLEDPEEYLRMGFEASASSFFLDRGNSKVKFVKENPYFTNRMSVNNQPFKSENDLSYYKVYGILDQNILELYFNDGDVVSTNTYFMTTGNALGSVNMTTGVDNLFYIDKFQVREVK.

[143] In certain embodiments, the recombinant mutant invertase comprises a non-wild-type invertase with F172Y, N429A, P479A, T140L, D272N, and V581I mutations relative to SEQ ID NO: 2, without signal sequence. For example, the recombinant mutant invertase may comprise SEQ ID NO: 56, also referred to as VE04 herein.

[144] SEQ ID NO: 56SMTNETSDRPLVHFTPNKGWMNDPNGLWYDEKDAKWHLYFQYNPNDTVWGLPLFWGHATSDDLTHWEDEPIAIAPKRNDSGAYSGSMVVDYNNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAAKSQDYKIEIYSSDNLKSWKLESAFANEGFLGYQYECPGLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYGSALGIAWASNWEYSAFVPTNPWRSSMSLVRKFSLNTEYQANPETELINLKAEPILNISAAGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYAVNTTQTISKSVFADLSLWFKGLEDPEEYLRMGFEASASSFFLDRGNSKVKFVKENPYFTNRMSVNNQPFKSENDLSYYKVYGLLDQNILELYFNDGDVVSTNTYFMTTGNALGSINMTTGVDNLFYIDKFQVREVK.

[145] In certain embodiments, the recombinant mutant invertase comprises a non-wild-type invertase with F172Y, N429A, P479A, T140L, E285H, V581I mutations relative to SEQ ID NO: 2, without signal sequence. For example, the recombinant mutant invertase may comprise SEQ ID NO: 57, also referred to as VE08 herein.

[146] SEQ ID NO: 57SMTNETSDRPLVHFTPNKGWMNDPNGLWYDEKDAKWHLYFQYNPNDTVWGLPLFWGHATSDDLTHWEDEPIAIAPKRNDSGAYSGSMVVDYNNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAAKSQDYKIEIYSSDDLKSWKLESAFANHGFLGYQYECPGLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYGSALGIAWASNWEYSAFVPTNPWRSSMSLVRKFSLNTEYQANPETELINLKAEPILNISAAGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYAVNTTQTISKSVFADLSLWFKGLEDPEEYLRMGFEASASSFFLDRGNSKVKFVKENPYFTNRMSVNNQPFKSENDLSYYKVYGLLDQNILELYFNDGDVVSTNTYFMTTGNALGSINMTTGVDNLFYIDKFQVREVK.

[147] In certain embodiments, the recombinant mutant invertase comprises a non-wild-type invertase with F172Y, N429A, P479A, T140L, E382Q, and A123G mutations relative to SEQ ID NO: 2, without signal sequence. For example, the recombinant mutant invertase may comprise SEQ ID NO: 58, also referred to as VE38 herein.

[148] SEQ ID NO: 58SMTNETSDRPLVHFTPNKGWMNDPNGLWYDEKDGKWHLYFQYNPNDTVWGLPLFWGHATSDDLTHWEDEPIAIAPKRNDSGAYSGSMVVDYNNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAAKSQDYKIEIYSSDDLKSWKLESAFANEGFLGYQYECPGLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYGSALGIAWASNWQYSAFVPTNPWRSSMSLVRKFSLNTEYQANPETELINLKAEPILNISAAGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYAVNTTQTISKSVFADLSLWFKGLEDPEEYLRMGFEASASSFFLDRGNSKVKFVKENPYFTNRMSVNNQPFKSENDLSYYKVYGLLDQNILELYFNDGDVVSTNTYFMTTGNALGSVNMTTGVDNLFYIDKFQVREVK.

[149] In certain embodiments, the recombinant mutant invertase comprises a non-wild-type invertase with F172Y, N429A, P479A, T140L, E382Q, and N512G mutations relative to SEQ ID NO: 2, without signal sequence. For example, the recombinant mutant invertase may comprise SEQ ID NO: 59, also referred to as VE39 herein.

[150] SEQ ID NO: 59SMTNETSDRPLVHFTPNKGWMNDPNGLWYDEKDAKWHLYFQYNPNDTVWGLPLFWGHATSDDLTHWEDEPIAIAPKRNDSGAYSGSMVVDYNNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAAKSQDYKIEIYSSDDLKSWKLESAFANEGFLGYQYECPGLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYGSALGIAWASNWQYSAFVPTNPWRSSMSLVRKFSLNTEYQANPETELINLKAEPILNISAAGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYAVNTTQTISKSVFADLSLWFKGLEDPEEYLRMGFEASASSFFLDRGGSKVKFVKENPYFTNRMSVNNQPFKSENDLSYYKVYGLLDQNILELYFNDGDVVSTNTYFMTTGNALGSVNMTTGVDNLFYIDKFQVREVK.

[151] In certain embodiments, the recombinant mutant invertase comprises a non-wild-type invertase with F172Y, N429A, P479A, T140L, E382Q, and V178I mutations relative to SEQ ID NO: 2,without signal sequence. For example, the recombinant mutant invertase may comprise SEQ ID NO: 60, also referred to as VE40 herein.

[152] SEQ ID NO: 60SMTNETSDRPLVHFTPNKGWMNDPNGLWYDEKDAKWHLYFQYNPNDTVWGLPLFWGHATSDDLTHWEDEPIAIAPKRNDSGAYSGSMVIDYNNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAAKSQDYKIEIYSSDDLKSWKLESAFANEGFLGYQYECPGLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYGSALGIAWASNWQYSAFVPTNPWRSSMSLVRKFSLNTEYQANPETELINLKAEPILNISAAGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYAVNTTQTISKSVFADLSLWFKGLEDPEEYLRMGFEASASSFFLDRGNSKVKFVKENPYFTNRMSVNNQPFKSENDLSYYKVYGLLDQNILELYFNDGDVVSTNTYFMTTGNALGSVNMTTGVDNLFYIDKFQVREVK.

[153] In certain embodiments, the recombinant mutant invertase comprises a non-wild-type invertase with F172Y, N429A, P479A, T140L, E382Q, and N390D mutations relative to SEQ ID NO: 2, without signal sequence. For example, the recombinant mutant invertase may comprise SEQ ID NO: 61, also referred to as VE44 herein.

[154] SEQ ID NO: 61SMTNETSDRPLVHFTPNKGWMNDPNGLWYDEKDAKWHLYFQYNPNDTVWGLPLFWGHATSDDLTHWEDEPIAIAPKRNDSGAYSGSMVVDYNNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAAKSQDYKIEIYSSDDLKSWKLESAFANEGFLGYQYECPGLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYGSALGIAWASNWQYSAFVPTDPWRSSMSLVRKFSLNTEYQANPETELINLKAEPILNISAAGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYAVNTTQTISKSVFADLSLWFKGLEDPEEYLRMGFEASASSFFLDRGNSKVKFVKENPYFTNRMSVNNQPFKSENDLSYYKVYGLLDQNILELYFNDGDVVSTNTYFMTTGNALGSVNMTTGVDNLFYIDKFQVREVK.

[155] In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of any one of SEQ ID NOs: 4-18, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 4-18.

[156] In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 4. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 5. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 6. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 7. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 8. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 9. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 10. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 11. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 12. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 13. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 14. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 15. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 16. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 17. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 18.

[157] In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of any one of SEQ ID NOs: 19-33, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 19-33.

[158] In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 19. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 20, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 20. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 21, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 21. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 22. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 23. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 24. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 25. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 26, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 26. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 27. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 28, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 28. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 29, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 29. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 30. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 31, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 31. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 32, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 32. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 33 or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 33.

[159] In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of any one of SEQ ID NOs: 51-61, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 51-53.

[160] In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 51, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 51. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 51 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions.

[161] In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 52, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 52. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 52 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions.

[162] In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 53, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 53. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 53 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions.

[163] In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 54, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 54. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 54 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions.

[164] In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 55, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 55. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 55 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions.

[165] In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 56, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 56. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 56 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions.

[166] In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 57, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 57. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 57 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions.

[167] In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 58, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 58. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 58 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions.

[168] In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 59, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 59. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 59 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions.

[169] In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 60, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 60. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 60 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions.

[170] In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 61, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 61. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 61 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions.

[171] As used herein, percent “identity” between a polypeptide sequence and a reference sequence is defined as the percentage of amino acid residues in the polypeptide sequence that are identical to the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Similarly, percent “identity” between a nucleic acid sequence and a reference sequence is defined as the percentage of nucleotides in the nucleic acid sequence that are identical to the nucleotides in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity (e.g., amino acid sequence identity or nucleic acid sequence identity) can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA, or MUSCLE software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[172] In certain embodiments, a recombinant mutant invertase has increased stability at acidic pH (e.g.,pH 3.0 or 4.0) relative to a corresponding wild-type invertase enzyme. An increased stability at acidic pH may, in certain conditions, allow the recombinant mutant invertase to survive acidic conditions of the digestive system, especially the stomach. An increased stability at acidic pH may, in certain conditions, also allow the recombinant mutant invertase to survive in certain food or beverages, e.g., fruit juice. An increased stability at acidic pH may, in certain conditions, allow the recombinant mutant invertase to survive in fasting conditions.

[173] In certain embodiments, the invertase has a specific activity at about pH 3.5 (e.g., 3.6) of at least 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, or 2,000 µmol sucrose consumed per minute per milligram of invertase. In certain embodiments, the invertase has at least 1-fold, 1.5-fold, 2-fold, 2.5-fold, or 3-fold higher activity at about pH 3.5 (e.g., 3.6), compared to a corresponding wild-type invertase. In certain embodiments, the invertase has a specific activity at about pH 5.0 (e.g., pH 4.9) of at least 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,100, or 2,200 µmol sucrose consumed per minute per milligram of invertase. In certain embodiments, the invertase has at least 1-fold, 1.5-fold, 2-fold, 2.5-fold, or 3-fold higher activity at about pH 5.0 (e.g., pH 4.9), compared to a corresponding wild-type invertase. In certain embodiments, the invertase has a specific activity at about pH 6.0 or 6.2 of at least 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, or 1,800 µmol sucrose consumed per minute per milligram of invertase. In certain embodiments, the invertase has at least 1-fold, 1.5-fold, 2-fold, 2.5-fold, or 3-fold higher activity at about pH 6.0 or 6.2, compared to a corresponding wild-type invertase. In certain embodiments, the invertase has a specific activity at about pH 7.0 or 7.1 of at least 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, or 1,300 µmol sucrose consumed per minute per milligram of invertase. In certain embodiments, the invertase has at least 1-fold, 1.5-fold, 2-fold, 2.5-fold, or 3-fold higher activity at about pH 7.0 or 7.1, compared to a corresponding wild-type invertase. In certain embodiments, the invertase retains at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of activity following incubation at about pH 2.5 for about 30 minutes. In certain embodiments, the invertase has at least 1-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, or 5-fold higher stability at about pH 2.5 compared to a corresponding wild-type invertase. In certain embodiments, the invertase is deglycosylated. In certain embodiments, the invertase is glycosylated.

[174] Methods for testing the stability and activity of an invertase are known in the art and can include, for example, the methods described in Example 1 herein. In certain embodiments, stability of an invertase in low pH is determined using by exposing the invertase to a specific pH, and using the dinitro salicylate (DNS) colorimetric method to monitor reducing sugar formation upon hydrolysis of sucrose.

[175] In certain embodiments, a recombinant mutant invertase has increased stability at higher temperature (e.g.,Tm of 56-68℃) relative to a corresponding wild-type invertase enzyme. In certain embodiments, the invertase has a Tm of at least 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, or 68 ℃. In certain embodiments the invertase has a Tm that is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ℃ higher than a corresponding wild-type invertase. Increased thermostability may allow the invertase to be stable when manufactured under conditions, for example, when dried (e.g., spray dried, lyophilized) to form a powder or when compressed under high temperatures as a tablet.

[176] Methods for testing the thermostability and activity of an invertase are known in the art and can include, for example, the methods described in Example 1 herein. In certain embodiments, stability and activity of an invertase at high temperature is determined by treatment of invertase samples at high temperature prior to assaying residual activity using the DNS method as described above.

[177] In certain embodiments, the invertase has increased stability in the presence of a protease (e.g., a serine protease and / or an aspartic protease) relative to the corresponding wild-type invertase enzyme. Increased stability in the presence of a protease may, in certain conditions, allow a recombinant mutant invertase to survive the physiological conditions of the gastrointestinal lumen (e.g., stomach and / or small intestine) in a fed or fasted state. In certain embodiments, a recombinant mutant invertase has increased stability in the presence of pancreatin or pepsin relative to a corresponding wild-type invertase enzyme. Without wishing to be bound by theory, it is contemplated that the increased stability allows the invertase enzyme to be immediately active to degrade sucrose prior to uptake in the brush borders of the upper small intestine.

[178] In certain embodiments, a disclosed invertase enzyme retains at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of activity following incubation with pepsin (e.g., incubation with about 10 mg / ml pepsin at about at 37 °C in simulated gastric fluid (SGF) for about 2 hours). In certain embodiments, a disclosed invertase enzyme retains at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of activity following incubation with pancreatin (e.g., incubation with about 3.4 mg / ml pancreatin at about at 37 °C in simulated intestinal fluid (SIF) for about 2 hours).

[179] Methods for testing the proteolytic stability and activity of an invertase are known in the art and can include, for example, the methods described in Example 1 herein. In certain embodiments, stability and activity of an invertase in the presence of pepsin or pancreatin is determined by incubation of invertase with pepsin or pancreatin prior to assaying residual activity using the DNS method as described above.II. Isomaltase Enzymes

[180] Among other things, the invention provides pharmaceutical compositions optionally further comprising an isomaltase enzyme that, for example, are useful in treating disorders such as congenital sucrase-isomaltase deficiency (CSID).

[181] As used herein, the term “isomaltase” refers to any enzyme, or a functional fragment thereof, that is capable of catalyzing the cleavage of branched (1–6 linked) α-limit dextrins (starches). Isomaltases are also called α-glucosidase, oligo-1,6-glucosidase, and EC 3.2.1.10, and, unless indicated otherwise, the terms are used interchangeably herein. The term isomaltase includes variants having one or more amino acid substitutions, deletions, or insertions relative to a wild-type isomaltase sequence, and / or fusion proteins or conjugates including a isomaltase. As used herein, the term “functional fragment” of a isomaltase refers to fragment of a full-length isomaltase that retains, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the enzymatic activity of the corresponding full-length, naturally occurring isomaltase. Exemplary isomaltase activity assays are described in Noguchi et al. (2003) J. Biochem. 134(4):543-50 and Schonert et al. (1998) J. Bacteriol. 180(9):2574-8. Additionally, because glucose is a product of the enzymatic reactions catalyzed by both invertase and isomaltase, it is understood that an assay based on the detection of glucose may in certain embodiments be used to measure the activity of both an invertase enzyme and an isomaltase enzyme.

[182] Exemplary isomaltase enzymes include isomaltase enzymes derived from Saccharomyces cerevisiae. The amino acid sequences of exemplary wild-type isomaltase enzymes derived from Saccharomyces cerevisiae are provided as SEQ ID NOs: 37-41 and nucleotide sequences encoding exemplary wild-type isomaltase enzymes derived from Saccharomyces cerevisiae are provided as SEQ ID NOs: 42-46.

[183] An exemplary wild-type isomaltase enzyme derived from L. fermentum is provided as SEQ ID NO: 47.

[184] SEQ ID NO: 47MIYTPKWWWQNSVVYQVYPRSFQDSNHDGIGDLKGIISRLDYIKKLGADIIWLNPIYRSPNVDNGYDISDYRAIDPTFGSLTDFKELLTKAHELGLKIMMDLVVNHSSDENEWFKQSRQGKENPYRDYYIWRDPVDGHEPTNWGSYFSGSAWQYDETSGQYYLHLFAVKQPDLNWENEAVRHSVYDIMNWWADLGVDGFRMDVINLISKPAVYKDVPTAPGMQYGDVEPVVANGHRMHEFLQEMHQAVMAKHDLVTVGETPGATTDDAKKYANLEQTELNMVFEFEHVGLDGNDNPALGKWSDKKVSLPELRDNLVKWQTQLNGKAWNSLYWNNHDQPRVVSRFGNDDPKYRVVSAKMLATMLHCLQGTPYIYAGEELGMTNTTFNSLSDYRDLESINAYHQLVDEEHLVDGKTMSRYLAIHSRDNARTPMQWDDSKNAGFSDAEPWIAVNPNYSEINAKAALADPSSVFYHYQKLIQMRHDLPVMTEGKFALVNGNELDEQVFAYTRDDGETTLLVVANFTKETIKREYAAGQGKLLLSNYEDDMGETLRPYEAKVYEFSSKR.

[185] An exemplary isomaltase enzyme variant may comprise an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 47. In certain embodiments, the isomaltase enzyme variant comprises at least one amino acid substitution (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid substitutions) and an amino acid sequence that has at least 98%, 98.5%, 99% or 99.5% sequence identity to SEQ ID NO: 47. In certain embodiments, the isomaltase comprises substitutions at positions corresponding to the amino acids sequence set forth in SEQ ID NO: 47, including: the E93K, K115I, R132K, D226S, E310A, L366M, I421A, A444G, A531D, and F560L substitutions; the E93K, K115I, R132K, D226S, E310A, I421A, A444G, E524Q, A531D, and F560L substitutions; the E93K, K115I, R132K, D226S, E310A, I421A, A444G, A531D, and F560V substitutions; the E93K, K115I, R132K, D226S, L366M, I421A, A444G, E524Q, A531D, and F560L substitutions; the E93K, K115I, R132K, D226S, L366M, I421A, A444G, A531D, and F560V substitutions; the E93K, K115I, R132K, D226S, I421A, A444G, E524Q, A531D, and F560V substitutions; the K115I, R132K, D226S, E310A, L366M, I421A, A444G, E524Q, A531D, and F560L substitutions; the K115I, R132K, D226S, E310A, L366M, I421A, A444G, A531D, and F560V substitutions; the K115I, R132K, D226S, E310A, I421A, A444G, E524Q, A531D, and F560V substitutions; the K115I, R132K, D226S, L366M, I421A, A444G, E524Q, A531D, and F560V substitutions; the E93K, K115I, R132K, A211E, D226S, I421A, A444G, E524Q, A531D, and F560V substitutions; the E93K, K115I, R132K, D226S, L366M, I421A, A444G, E524Q, A531D, and F560V substitutions; the E93K, K115I, R132K, D226S, E275D, I421A, A444G, E524Q, A531D, and F560V substitutions; the T89A, E93K, K115I, R132K, D226S, I421A, A444G, E524Q, A531D, and F560V substitutions; the E93K, K115I, R132K, H182R, D226S, I421A, A444G, E524Q, A531D, and F560V substitutions; the E93K, K115I, E122D, R132K, D226S, I421A, A444G, E524Q, A531D, and F560V substitutions; the E93K, K115I, D226S, I421A, A444G, E524Q, A531D, and F560V substitutions; the E93K, K115I, D226S, I421A, A444G, E524Q, A531D, and F560L substitutions; the K115I, A211E, D226S, I421A, A444G, A531D, and F560V substitutions; the K115I, D226S, L366M, I421A, A444G, E524Q, A531D, and F560L substitutions; the K115I, A211E, D226S, I421A, A444G, E524Q, A531D, and F560L substitutions; the K115I, R132K, D226S, E310A, L366M, I421A, A444G, A531D, and F560L substitutions; the E93K, K115I, D226S, E310A, I421A, A444G, A531D, and F560L substitutions; the K115I, D226S, E310A, I421A, A444G, A531D, and F560V substitutions; the E93K, K115I, D226S, L366M, I421A, A444G, A531D, and F560L substitutions; the K115I, D226S, I421A, A444G, E524Q, A531D, and F560V substitutions; the K115I, D226S, E310A, I421A, A444G, E524Q, A531D, and F560L substitutions; the K115I, A211E, D226S, L366M, I421A, A444G, A531D, and F560L substitutions; the K115I, A211E, D226S, E310A, I421A, A444G, A531D, and F560L substitutions; the E93K, K115I, D226S, I421A, A444G, A531D, and F560V substitutions; the E93K, K115I, A211E, D226S, I421A, A444G, A531D, and F560L substitutions; or the K115I, D226S, L366M, I421A, A444G, A531D, and F560V substitutions.

[186] Additional exemplary isomaltase enzymes can be found on the world wide web at brenda-enzymes.org / enzyme.php?ecno=3.2.1.10&onlyTable=Sequence.

[187] A contemplated isomaltase enzyme may comprise the amino acid sequence of any one of SEQ ID NOs: 37-41, or an amino acid sequence that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 42-46. Other isomaltase enzymes that may be used in combination with the recombinant invertase enzymes described herein are described in International Patent Application Publication No. WO 2024 / 015943, which claims priority to U.S. Provisional Patent Application No. 63 / 388,845, filed on July 13, 2022.

[188] In certain embodiments, the isomaltase enzyme comprises one or more conservative substitutions relative to isomaltase enzyme disclosed herein. In other embodiments, the isomaltase enzyme comprises one or more non-conservative substitutions relative to isomaltase enzyme disclosed herein.

[189] In certain embodiments, the composition comprises (i) an invertase comprising the amino acid sequence of SEQ ID NO: 14 (variant V78 with a FAKS signal sequence) or SEQ ID NO: 29 (variant 78 without a signal sequence) and (ii) one or more of the isomaltase enzymes described in this Section II.

[190] In certain embodiments, the composition comprises (i) an invertase comprising the amino acid sequence of SEQ ID NO: 49 (Sc_S288C invertase with F172Y and N429A mutations relative to SEQ ID NO: 2, with wild-type signal sequence) or SEQ ID NO: 50 (Sc_S288C invertase with F172Y and N429A mutations relative to SEQ ID NO: 2 without a signal sequence) and (ii) one or more of the isomaltase enzymes described in this Section II.III. Enzyme Production

[191] Methods for producing invertase and / or isomaltase enzymes are known in the art. For example, DNA molecules encoding an invertase and / or isomaltase enzyme can be chemically synthesized using the sequence information provided herein. Synthetic DNA molecules can be ligated to other appropriate nucleotide sequences, including, e.g., expression control sequences, to produce conventional gene expression constructs encoding the desired invertase and / or isomaltase enzyme.

[192] Nucleic acids encoding desired invertase and / or isomaltase enzymes can be incorporated (ligated) into expression vectors, which can be introduced into host cells through conventional transfection or transformation techniques. Transformed host cells can be grown under conditions that permit the host cells to express the genes that encode the invertase and / or isomaltase enzyme.

[193] An exemplary DNA sequence encoding a wild-type S. cerevisiae invertase is provided as SEQ ID NO: 35.

[194] SEQ ID NO: 35 (wild-type S. cerevisiae invertase DNA sequence):ATGCTTTTGCAAGCTTTCCTTTTCCTTTTGGCTGGTTTTGCAGCCAAAATATCTGCATCAATGACAAACGAAACTAGCGATAGACCTTTGGTCCACTTCACACCCAACAAGGGCTGGATGAATGACCCAAATGGGTTGTGGTACGATGAAAAAGATGCCAAATGGCATCTGTACTTTCAATACAACCCAAATGACACCGTATGGGGTACGCCATTGTTTTGGGGCCATGCTACTTCCGATGATTTGACTCATTGGGAAGATGAACCCATTGCTATCGCTCCCAAGCGTAACGATTCAGGTGCTTTCTCTGGCTCCATGGTGGTTGATTACAACAACACGAGTGGGTTTTTCAATGATACTATTGATCCAAGACAAAGATGCGTTGCGATTTGGACTTATAACACTCCTGAAAGTGAAGAGCAATACATTAGCTATTCTCTTGATGGTGGTTACACTTTTACTGAATACCAAAAGAACCCTGTTTTAGCTGCCAACTCCACTCAATTCAGAGATCCAAAGGTGTTCTGGTATGAACCTTCTCAAAAATGGATTATGACGGCTGCCAAATCACAAGACTACAAAATTGAAATTTACTCCTCTGATGACTTGAAGTCCTGGAAGCTAGAATCTGCATTTGCTAATGAAGGTTTCTTAGGCTACCAATATGAATGTCCAGGTTTGATTGAAGTCCCAACTGAGCAAGATCCTTCCAAATCCTATTGGGTCATGTTTATTTCTATCAATCCAGGTGCACCTGCTGGCGGTTCCTTCAACCAATATTTTGTTGGATCCTTCAATGGTACTCATTTTGAAGCGTTTGACAATCAATCTAGAGTGGTAGATTTTGGTAAGGACTACTATGCCTTGCAAACTTTCTTCAACACAGACCCAACGTACGGTTCAGCATTAGGTATTGCCTGGGCTTCAAACTGGGAGTACAGTGCCTTTGTCCCAACTAACCCATGGAGATCATCCATGTCTTTGGTCCGCAAGTTTTCTTTGAACACTGAATATCAAGCTAATCCAGAGACTGAATTGATCAATTTGAAAGCCGAACCAATATTGAACATTAGTAATGCTGGTCCCTGGTCTCGTTTTGCTACTAACACAACTCTAACTAAGGCCAATTCTTACAATGTCGATTTGAGCAACTCGACTGGTACCCTAGAGTTTGAGTTGGTTTACGCTGTTAACACCACACAAACCATATCCAAATCCGTCTTTCCCGACTTATCACTTTGGTTCAAGGGTTTAGAAGATCCTGAAGAATATTTAAGAATGGGTTTTGAAGCCAGTGCTTCTTCCTTCTTTTTGGACCGTGGTAACTCTAAGGTCAAGTTTGTCAAGGAGAACCCATATTTCACAAACAGAATGTCTGTCAACAACCAACCATTCAAGTCTGAGAACGACCTAAGTTACTATAAAGTGTACGGCCTACTGGATCAAAACATCTTGGAATTGTACTTCAACGATGGAGATGTGGTTTCTACAAATACCTACTTCATGACCACCGGTAACGCTCTAGGATCTGTGAACATGACCACTGGTGTCGATAATTTGTTCTACATTGACAAGTTCCAAGTAAGGGAAGTAAAATAG

[195] An exemplary DNA sequence encoding a wild-type L. fermentum isomaltase is provided in SEQ ID NO: 48.

[196] SEQ ID NO: 48 (wild-type L. fermentum isomaltase DNA sequence):ATGATATATACACCAAAATGGTGGTGGCAAAATTCAGTTGTCTACCAAGTCTATCCACGGAGTTTTCAAGACAGCAATCATGATGGCATTGGTGATTTAAAAGGAATCATCAGTCGGCTTGACTATATTAAAAAACTAGGTGCTGATATTATCTGGCTCAATCCAATCTACCGTTCGCCAAACGTTGATAATGGGTATGACATCAGTGATTATCGGGCAATTGATCCAACTTTTGGCTCATTGACCGACTTTAAGGAGCTGCTGACAAAGGCTCATGAACTTGGATTAAAAATAATGATGGATCTGGTGGTCAATCACTCATCAGATGAAAATGAATGGTTTAAGCAAAGCCGCCAAGGAAAAGAAAATCCATACCGTGACTATTATATCTGGCGTGATCCAGTCGATGGCCATGAACCAACTAATTGGGGATCATACTTCTCTGGTTCAGCTTGGCAGTACGATGAGACAAGCGGTCAATACTACCTTCACCTGTTCGCTGTAAAGCAGCCGGATCTAAACTGGGAAAATGAAGCCGTTCGTCATTCGGTATACGACATCATGAATTGGTGGGCAGATCTCGGTGTCGATGGATTTCGAATGGATGTCATCAACTTAATTTCAAAACCGGCTGTCTACAAAGACGTTCCAACCGCGCCCGGAATGCAGTATGGCGATGTCGAGCCGGTTGTTGCCAATGGTCATCGGATGCACGAATTCCTTCAAGAAATGCATCAAGCTGTAATGGCCAAGCATGATTTGGTAACAGTTGGCGAAACACCTGGTGCTACAACAGATGATGCCAAGAAGTACGCCAACCTTGAACAGACTGAGCTGAACATGGTTTTTGAGTTTGAGCATGTCGGCTTAGACGGTAACGACAACCCAGCTTTAGGAAAATGGAGCGACAAAAAAGTCAGTCTGCCAGAATTGCGAGATAACCTGGTGAAATGGCAGACTCAGCTGAATGGTAAGGCTTGGAACTCGCTTTACTGGAACAACCATGATCAGCCACGCGTCGTATCCCGTTTTGGCAATGATGATCCAAAATATCGTGTCGTTTCGGCTAAAATGTTGGCAACAATGCTGCACTGTCTGCAAGGCACGCCTTATATCTACGCAGGTGAGGAACTGGGAATGACCAACACGACCTTTAACTCTTTGTCTGACTATCGTGATTTGGAGAGCATTAATGCCTACCATCAGCTGGTTGACGAAGAGCATCTAGTTGATGGAAAGACAATGAGCAGATATCTGGCAATTCATTCTCGGGACAATGCCAGAACGCCAATGCAATGGGATGACAGCAAGAATGCCGGATTTTCGGATGCTGAACCTTGGATCGCGGTCAATCCCAACTATTCAGAGATCAACGCAAAGGCGGCACTGGCGGATCCATCATCCGTCTTCTATCACTACCAGAAACTCATTCAAATGCGCCATGACTTGCCAGTAATGACAGAAGGAAAGTTTGCGTTGGTCAATGGCAACGAATTGGATGAGCAGGTCTTTGCTTACACGCGTGATGATGGAGAAACAACGCTGCTTGTAGTAGCCAACTTTACTAAGGAAACAATCAAGCGAGAATACGCGGCTGGTCAAGGCAAGCTCTTATTAAGCAACTACGAGGATGACATGGGAGAAACTCTGCGTCCATATGAAGCTAAAGTATATGAATTCAGTTCAAAGAGGTAA

[197] Nucleic acids encoding a recombinant mutant invertase and / or isomaltase may be generated by mutating a nucleotide sequence encoding a wild-type invertase, e.g., SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or a wild-type isomaltase, e.g., SEQ ID NOs: 37-41, using methods known in the art. SEQ ID Nos: 1 and 2 represent isomaltase protein sequences containing N-terminal signal sequences that can be removed post-translationally. SEQ ID NO: 3 represents a corresponding mature isomaltase protein without an -N-terminal signal sequence. Furthermore, nucleic acids encoding such recombinant invertase and / or isomaltase enzymes may be codon optimized for expression in a heterologous cell, e.g., an Escherichia coli, Saccharomyces cerevisiae, or Pichia pastoris cell, using methods known in the art.

[198] In one embodiment, the disclosure relates to a nucleotide sequence encoding a recombinant mutant invertase that comprises the following substitutions: E120A, D122E, and P479A, relative to SEQ ID NO: 2, e.g., a recombinant mutant invertase referred to as V6 herein.

[199] In one embodiment, the disclosure relates to a nucleotide sequence encoding a recombinant mutant invertase that comprises the following substitutions: T140L. E156Q, and L549I relative to SEQ ID NO: 2, e.g., a recombinant mutant invertase referred to as V9 herein.

[200] In one embodiment, the disclosure relates to a nucleotide sequence encoding a recombinant mutant invertase that comprises the following substitutions: S476A, P479A, and Q597E relative to SEQ ID NO: 2, e.g., a recombinant mutant invertase referred to as V14 herein.

[201] In one embodiment, the disclosure relates to a nucleotide sequence encoding a recombinant mutant invertase that comprises the following substitutions: K259L, S476N, and K514P relative to SEQ ID NO: 2, e.g., a recombinant mutant invertase referred to as V22 herein.

[202] In one embodiment, the disclosure relates to a nucleotide sequence encoding a recombinant mutant invertase that comprises the following substitutions: K277T, E415S, and V581I relative to SEQ ID NO: 2, e.g., a recombinant mutant invertase referred to as V25 herein.

[203] In one embodiment, the disclosure relates to a nucleotide sequence encoding a recombinant mutant invertase that comprises the following substitutions: A123G, T140L. and F143Y relative to SEQ ID NO: 2, e.g., a recombinant mutant invertase referred to as V32 herein.

[204] In one embodiment, the disclosure relates to a nucleotide sequence encoding a recombinant mutant invertase that comprises the following substitutions: N390D, S476N, and K519R relative to SEQ ID NO: 2, e.g., a recombinant mutant invertase referred to as V38 herein.

[205] In one embodiment, the disclosure relates to a nucleotide sequence encoding a recombinant mutant invertase that comprises the following substitutions: E382Q, T573S, and V581I relative to SEQ ID NO: 2, e.g., a recombinant mutant invertase referred to as V60 herein.

[206] In one embodiment, the disclosure relates to a nucleotide sequence encoding a recombinant mutant invertase that comprises the following substitutions: D122E, T470S, and S476N relative to SEQ ID NO: 2, e.g., a recombinant mutant invertase referred to as V63 herein.

[207] In one embodiment, the disclosure relates to a nucleotide sequence encoding a recombinant mutant invertase that comprises the following substitutions: T140Q, P479A, and K514P relative to SEQ ID NO: 2, e.g., a recombinant mutant invertase referred to as V70 herein.

[208] In one embodiment, the disclosure relates to a nucleotide sequence encoding a recombinant mutant invertase that comprises the following substitutions: F172Y, N429A, and P479A relative to SEQ ID NO: 2, e.g., a recombinant mutant invertase referred to as V78 herein.

[209] In one embodiment, the disclosure relates to a nucleotide sequence encoding a recombinant mutant invertase that comprises the following substitutions: E120A, V178I, and L549I relative to SEQ ID NO: 2, e.g., a recombinant mutant invertase referred to as V83 herein.

[210] In one embodiment, the disclosure relates to a nucleotide sequence encoding a recombinant mutant invertase that comprises the following substitutions: E382Q, S537D, and N589K relative to SEQ ID NO: 2, e.g., a recombinant mutant invertase referred to as V87 herein.

[211] In one embodiment, the disclosure relates to a nucleotide sequence encoding a recombinant mutant invertase that comprises the following substitutions: T140L, G586S, and F596I relative to SEQ ID NO: 2, e.g., a recombinant mutant invertase referred to as V89 herein.

[212] In one embodiment, the disclosure relates to a nucleotide sequence encoding a recombinant mutant invertase that comprises the following substitutions: E382Q, L459F, and K519R relative to SEQ ID NO: 2, e.g., a recombinant mutant invertase referred to as V90 herein.

[213] In one embodiment, the disclosure relates to a nucleotide sequence encoding a recombinant mutant invertase that comprises the following substitutions: F172Y, N429A, P479A, T140L, E382Q, and A501N relative to SEQ ID NO: 2, e.g., a recombinant mutant invertase referred to as VE36 herein.

[214] In one embodiment, the disclosure relates to a nucleotide sequence encoding a recombinant mutant invertase that comprises the following substitutions: F172Y, N429A, P479A, T140L, D272N, and E285H relative to SEQ ID NO: 2, e.g., a recombinant mutant invertase referred to as VE01 herein.

[215] In one embodiment, the disclosure relates to a nucleotide sequence encoding a recombinant mutant invertase that comprises the following substitutions: F172Y, N429A, P479A, T140L, D272N, and L549I relative to SEQ ID NO: 2; e.g., a recombinant mutant invertase referred to as VE03 herein.

[216] In one embodiment, the disclosure relates to a nucleotide sequence encoding a recombinant mutant invertase that comprises the following substitutions: F172Y, N429A, P479A, T140L, D272N, and V581I substitutions relative to SEQ ID NO: 2; e.g., a recombinant mutant invertase referred to as VE04 herein.

[217] In one embodiment, the disclosure relates to a nucleotide sequence encoding a recombinant mutant invertase that comprises the following substitutions: F172Y, N429A, P479A, T140L, E285H, and V581I substitutions relative to SEQ ID NO: 2; e.g., a recombinant mutant invertase referred to as VE08 herein.

[218] In one embodiment, the disclosure relates to a nucleotide sequence encoding a recombinant mutant invertase that comprises the following substitutions: F172Y, N429A, P479A, T140L, E382Q, and A123G substitutions relative to SEQ ID NO: 2; e.g., a recombinant mutant invertase referred to as VE38 herein.

[219] In one embodiment, the disclosure relates to a nucleotide sequence encoding a recombinant mutant invertase that comprises the following substitutions: F172Y, N429A, P479A, T140L, E382Q, and N512G substitutions relative to SEQ ID NO: 2; e.g., a recombinant mutant invertase referred to as VE39 herein.

[220] In one embodiment, the disclosure relates to a nucleotide sequence encoding a recombinant mutant invertase that comprises the following substitutions: F172Y, N429A, P479A, T140L, E382Q, and V178I substitutions relative to SEQ ID NO: 2; e.g., a recombinant mutant invertase referred to as VE40 herein.

[221] In one embodiment, the disclosure relates to a nucleotide sequence encoding a recombinant mutant invertase that comprises the following substitutions: the F172Y, N429A, P479A, T140L, E382Q, and N390D substitutions relative to SEQ ID NO: 2, e.g., a recombinant mutant invertase referred to as VE44 herein.

[222] In one embodiment, the disclosure relates to a nucleotide sequence encoding a recombinant mutant invertase that comprises the following substitutions: F172Y, N429A, P479A, T140L, E382Q, A501N, and D272N substitutions relative to SEQ ID NO: 2, e.g., a recombinant mutant invertase referred to as VE361 herein.

[223] In certain embodiments, the recombinant mutant invertase comprises the F172Y, N429A, P479A, T140L, E382Q, A501N, D272N, and E285H substitutions relative to SEQ ID NO: 2, e.g., a recombinant mutant invertase referred to as VE362 herein.

[224] An exemplary DNA sequence encoding a recombinant mutant invertase referred to as VE362 herein is provided in SEQ ID NO: 63.

[225] SEQ ID NO: 63 (DNA sequence encoding VE362 invertase):ATGAGATTCCCATCTATTTTCACCGCTGTCTTGTTCGCTGCCTCCTCTGCATTGGCTGCCCCTGTTAACACTACCACTGAAGACGAGACTGCTCAAATTCCAGCTGAAGCAGTTATCGGTTACTCTGACCTTGAGGGTGATTTCGACGTCGCTGTTTTGCCTTTCTCTAACTCCACTAACAACGGTTTGTTGTTCATTAACACCACTATCGCTTCCATTGCTGCTAAGGAAGAGGGTGTCTCTCTCGAGAAAAGAGAGGCCGAAGCTTCTATGACCAATGAAACTTCTGACAGACCATTGGTTCACTTCACACCCAACAAGGGTTGGATGAACGACCCAAACGGACTTTGGTATGATGAGAAAGATGCAAAATGGCATCTGTATTTCCAGTACAATCCTAATGACACGGTCTGGGGTTTGCCACTGTTTTGGGGTCACGCAACTTCTGATGACTTAACTCATTGGGAAGATGAGCCAATCGCTATCGCTCCTAAGCGTAACGATTCAGGTGCCTACTCCGGATCCATGGTTGTCGATTATAATAACACTAGTGGTTTCTTCAACGATACAATTGATCCCAGACAGCGATGTGTGGCAATTTGGACCTACAATACACCAGAATCAGAGGAACAATACATTTCGTACTCCTTGGATGGTGGTTACACGTTCACTGAATATCAGAAAAACCCTGTCTTGGCTGCGAATAGTACACAATTTAGAGATCCGAAAGTTTTCTGGTATGAGCCTTCACAAAAATGGATCATGACTGCTGCAAAGTCCCAGGACTACAAGATTGAGATTTACTCTTCAGACAATTTGAAGTCATGGAAATTGGAGAGCGCTTTTGCCAATCATGGATTCCTGGGCTATCAATATGAGTGCCCTGGATTGATAGAAGTACCAACAGAACAAGATCCATCCAAGTCTTACTGGGTGATGTTTATCAGCATTAATCCTGGTGCTCCCGCTGGCGGATCTTTTAACCAATACTTTGTGGGTTCCTTCAACGGTACACATTTTGAAGCATTTGATAACCAATCCAGAGTTGTTGACTTTGGTAAAGACTATTATGCTTTACAAACTTTCTTCAACACCGACCCCACTTACGGTTCTGCATTGGGTATAGCTTGGGCAAGCAATTGGCAATACTCCGCATTCGTCCCTACTAATCCATGGCGTTCCTCTATGAGTCTGGTCCGTAAGTTTTCTCTCAATACTGAGTATCAGGCCAACCCAGAGACAGAACTGATCAACTTAAAAGCTGAGCCTATTCTGAATATCTCTGCTGCCGGTCCATGGTCAAGATTCGCAACAAATACAACCCTGACTAAAGCTAATAGCTACAATGTGGATCTTAGCAATAGTACGGGTACCCTGGAATTCGAATTGGTTTATGCAGTAAACACAACTCAGACCATCTCGAAATCCGTATTCGCTGACTTATCCCTATGGTTTAAGGGTCTTGAGGATCCAGAGGAATACTTGAGAATGGGTTTTGAGAATAGTGCCTCCTCGTTCTTTTTGGACAGGGGAAATTCGAAAGTGAAGTTCGTTAAAGAGAATCCATATTTCACTAATAGAATGAGCGTGAATAATCAACCGTTTAAGTCTGAAAATGACTTGTCCTACTACAAAGTATATGGCTTGTTGGACCAAAACATCCTTGAACTATACTTTAATGATGGGGATGTTGTCTCAACTAATACTTACTTTATGACCACAGGAAACGCTTTGGGATCAGTTAACATGACAACGGGTGTTGATAATCTTTTCTATATTGACAAGTTTCAAGTTAGGGAAGTTAAGTAATAG.

[226] Specific expression and purification conditions will vary depending upon the expression system employed. For example, if a gene is to be expressed in E. coli, S. cerevisiae, or P. pastoris, it can be cloned into an expression vector by positioning the engineered gene downstream from a suitable bacterial or yeast promoter, e.g., Trp or Tac, and a prokaryotic or eukaryotic signal sequence. In yeast, it may be advantageous to introduce the coding gene into the yeast genome (such as by homologous recombination) for higher expression. The expressed protein accumulates in refractile or inclusion bodies or is secreted, and can be harvested after disruption of the cells by French press or sonication, or by simply collecting the supernatant if secreted. The refractile bodies then are solubilized, and the proteins refolded and cleaved by methods known in the art. Secreted proteins can be further purified using methods known in the art such as ion exchange, affinity chromatography, or salt precipitation.

[227] An invertase and / or isomaltase enzyme can be produced by growing (culturing) a host cell transfected with an expression vector encoding such invertase and / or isomaltase enzyme, under conditions that permit expression of the invertase and / or isomaltase enzyme. Following expression, the invertase and / or isomaltase enzyme can be harvested and purified or isolated using techniques known in the art, e.g., affinity tags such as glutathione-S-transferase (GST) and histidine tags.

[228] Exemplary methods for recombinant expression and purification of invertase enzymes are described in Mohandesi et al. (2016) Biotech 6(2):129. Exemplary methods for recombinant expression and purification of isomaltase enzymes are described in Deng et al. (2014) FEBS Open Bio 4:200–212.

[229] In certain embodiments, an invertase and / or isomaltase enzyme is dried, e.g., spray-dried or lyophilized. Pharmaceutical proteins may be dried in many ways, e.g., by removal of water, organic solvent or liquid polymer by means including drying with N2, air or inert gases, vacuum oven drying, lyophilization, washing with a volatile organic solvent followed by evaporation of the solvent, evaporation in a fume hood, tray drying, fluid bed drying, spray drying, vacuum drying, or roller drying.

[230] Spray drying invertase and / or isomaltase enzymes allows water to be separated from the invertase and / or isomaltase enzyme preparation, allowing for continuous production of dry solids in powder, granulate, or agglomerate form from liquid feedstocks such as emulsions and pumpable suspensions. Spray drying involves the atomization of a liquid feedstock comprising invertase and / or isomaltase enzyme into a spray of droplets and contacting the droplets with hot air or gas in a drying chamber. The atomization process may be conducted using a two-fluid atomizer that mixes the liquid feedstock with a drying gas such as compressed air or nitrogen. Operating conditions and dryer design are selected according to the drying characteristics of the invertase and / or isomaltase enzyme and the desired powder qualities. Exemplary methods for spray drying enzymes are described in United States Patent Application Publication No. 2015 / 0353913. The invertase and isomaltase enzyme may be spray dried separately or combined and spray dried together.

[231] It is contemplated that a disclosed invertase and / or isomaltase enzyme may be modified, engineered or chemically conjugated. For example, it is contemplated that a disclosed invertase and / or isomaltase enzyme can be conjugated to an effector agent using standard in vitro conjugation chemistries. If the effector agent is a polypeptide, the invertase and / or isomaltase enzyme can be chemically conjugated to the effector or joined to the effector as a fusion protein. Construction of fusion proteins is within ordinary skill in the art.

[232] In certain embodiments, depending upon a particular mode of administration or site of activity, a disclosed invertase and / or isomaltase enzyme can be modified with a moiety that improves its stabilization and / or retention in circulation, e.g., in blood, serum, or other tissues. For example, a disclosed invertase and / or isomaltase enzyme may be conjugated to a polymer, e.g., a substantially non-antigenic polymer, such as a polyalkylene oxide or a polyethylene oxide. In certain embodiments, a disclosed invertase and / or isomaltase enzyme is conjugated to a water soluble polymer, e.g., a hydrophilic polyvinyl polymer, e.g., polyvinylalcohol or polyvinylpyrrolidone. Examples of such polymers include polyalkylene oxide homopolymers such as polyethylene glycol (PEG) or polypropylene glycols, polyoxyethylenated polyols, copolymers thereof and block copolymers thereof. Additional useful polymers include polyoxyalkylenes such as polyoxyethylene, polyoxypropylene, and block copolymers of polyoxyethylene and polyoxypropylene, polymethacrylates, carbomers, and branched or unbranched polysaccharides.IV. Pharmaceutical Compositions

[233] For therapeutic use, an invertase and / or isomaltase enzyme described herein preferably is combined with a pharmaceutically acceptable carrier and / or an excipient. The term “pharmaceutically acceptable” as used herein refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[234] The term “pharmaceutically acceptable carrier” as used herein refers to buffers, carriers, and excipients suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable carriers include any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions (e.g., such as an oil / water or water / oil emulsions), and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see, e.g., see e.g., Adeboye Adejare, Remington: The Science and Practice of Pharmacy (23rd ed. 2020). Pharmaceutically acceptable carriers include buffers, solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art.

[235] In another aspect, the disclosure relates to a pharmaceutical composition comprising: an invertase enzyme, e.g., a recombinant mutant invertase as described herein; a separate isomaltase enzyme; and a pharmaceutically acceptable carrier and / or an excipient. In certain embodiments, the invertase and / or the isomaltase is dried (e.g., spray-dried or lyophilized).

[236] In certain embodiments of the pharmaceutical compositions disclosed herein (i) the invertase is a microbial invertase (e.g., derived from Saccharomyces cerevisiae), or a functional fragment or variant thereof, (ii) the invertase comprises a sequence of any one of SEQ ID NOs: 1-33 or 51-61, or a functional fragment or variant thereof, (iii) the invertase is a recombinant mutant S. cerevisiae invertase as described herein, (iv) the isomaltase is a microbial isomaltase (e.g., derived from S. cerevisiae or L. fermentum, or a functional fragment or variant of either of the foregoing, (v) the isomaltase comprises any one of SEQ ID NOs: 37-41, or a functional fragment or variant thereof, (vi) the isomaltase comprises a sequence of SEQ ID NO: 47, (vii) the isomaltase comprises substitutions at positions corresponding to the amino acids sequence set forth in SEQ ID NO: 47, including: the E93K, K115I, R132K, D226S, E310A, L366M, I421A, A444G, A531D, and F560L substitutions; the E93K, K115I, R132K, D226S, E310A, I421A, A444G, E524Q, A531D, and F560L substitutions; the E93K, K115I, R132K, D226S, E310A, I421A, A444G, A531D, and F560V substitutions; the E93K, K115I, R132K, D226S, L366M, I421A, A444G, E524Q, A531D, and F560L substitutions; the E93K, K115I, R132K, D226S, L366M, I421A, A444G, A531D, and F560V substitutions; the E93K, K115I, R132K, D226S, I421A, A444G, E524Q, A531D, and F560V substitutions; the K115I, R132K, D226S, E310A, L366M, I421A, A444G, E524Q, A531D, and F560L substitutions; the K115I, R132K, D226S, E310A, L366M, I421A, A444G, A531D, and F560V substitutions; the K115I, R132K, D226S, E310A, I421A, A444G, E524Q, A531D, and F560V substitutions; the K115I, R132K, D226S, L366M, I421A, A444G, E524Q, A531D, and F560V substitutions; the E93K, K115I, R132K, A211E, D226S, I421A, A444G, E524Q, A531D, and F560V substitutions; the E93K, K115I, R132K, D226S, L366M, I421A, A444G, E524Q, A531D, and F560V substitutions; the E93K, K115I, R132K, D226S, E275D, I421A, A444G, E524Q, A531D, and F560V substitutions; the T89A, E93K, K115I, R132K, D226S, I421A, A444G, E524Q, A531D, and F560V substitutions; the E93K, K115I, R132K, H182R, D226S, I421A, A444G, E524Q, A531D, and F560V substitutions; the E93K, K115I, E122D, R132K, D226S, I421A, A444G, E524Q, A531D, and F560V substitutions; the E93K, K115I, D226S, I421A, A444G, E524Q, A531D, and F560V substitutions; the E93K, K115I, D226S, I421A, A444G, E524Q, A531D, and F560L substitutions; the K115I, A211E, D226S, I421A, A444G, A531D, and F560V substitutions; the K115I, D226S, L366M, I421A, A444G, E524Q, A531D, and F560L substitutions; the K115I, A211E, D226S, I421A, A444G, E524Q, A531D, and F560L substitutions; the K115I, R132K, D226S, E310A, L366M, I421A, A444G, A531D, and F560L substitutions; the E93K, K115I, D226S, E310A, I421A, A444G, A531D, and F560L substitutions; the K115I, D226S, E310A, I421A, A444G, A531D, and F560V substitutions; the E93K, K115I, D226S, L366M, I421A, A444G, A531D, and F560L substitutions; the K115I, D226S, I421A, A444G, E524Q, A531D, and F560V substitutions; the K115I, D226S, E310A, I421A, A444G, E524Q, A531D, and F560L substitutions; the K115I, A211E, D226S, L366M, I421A, A444G, A531D, and F560L substitutions; the K115I, A211E, D226S, E310A, I421A, A444G, A531D, and F560L substitutions; the E93K, K115I, D226S, I421A, A444G, A531D, and F560V substitutions; the E93K, K115I, A211E, D226S, I421A, A444G, A531D, and F560L substitutions; or the K115I, D226S, L366M, I421A, A444G, A531D, and F560V substitutions, (viii) the isomaltase is a recombinant mutant isomaltase set forth in International Patent Application No. PCT / US2023 / 070180, filed on July 13, 2023, or (ix) or a combination of any one of features (i) – (viii). In certain embodiments, the invertase comprises the amino acid sequence of SEQ ID NO: 51, or a functional fragment thereof. In certain embodiments, the invertase comprises the amino acid sequence of SEQ ID NO: 52, or a functional fragment thereof. In certain embodiments, the invertase comprises the amino acid sequence of SEQ ID NO: 53, or a functional fragment thereof.

[237] In certain embodiments, the composition is formulated as an oral dosage form. In certain embodiments, the composition is formulated as a liquid, powder, sachet, granulate, pellet, micropellet, tablet, or minitablet. In certain embodiments, the composition is formulated as a liquid, powder, sachet, or tablet. In certain embodiments, the composition has a shelf-life at room temperature of at least 3 months, 6 months, 9 months, 12 months, 15 months, 18 months, 21 months, 24 months, 36 months, 48 months, 60 months, 72 months, 84 months, 96 months, 108 months, or 120 months.

[238] In certain embodiments, the invertase and / or isomaltase enzymes can be formulated, or co-administered (either at the same time or sequentially), for example, by an enteral route (e.g., orally), with a pH increasing agent, for example, a protein pump inhibitor (PPI), to enhance the stability of the invertase and / or isomaltase enzyme, for example, in an acidic environment, for example, in the gastrointestinal tract.

[239] Proton pump inhibitors are a group of drugs whose main action is pronounced and long-lasting reduction of gastric acid production. Proton pump inhibitors act by blocking the hydrogen / potassium adenosine triphosphatase enzyme system (the H+ / K+ ATPase, or more commonly just gastric proton pump) of the gastric parietal cell. The proton pump is the terminal stage in gastric acid secretion, being directly responsible for secreting H+ ions into the gastric lumen, making it an ideal target for inhibiting acid secretion. Examples of proton pump inhibitors include: Omeprazole (brand names: LOSEC®, PRILOSEC®, ZEGERID®); Lansoprazole (brand names: PREVACID®, ZOTON®, INHIBITOL®); Esomeprazole (brand names: NEXIUM®); and Pantoprazole (brand names: PROTONIX®, SOMAC®, PANTOLOC®).

[240] Pharmaceutical compositions containing a recombinant invertase and / or isomaltase enzyme disclosed herein can be presented in a dosage unit form and can be prepared by any suitable method. A pharmaceutical composition should be formulated to be compatible with its intended route of administration. The pharmaceutical compositions may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions, dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form will depend upon the intended mode of administration and therapeutic application. In certain embodiments, the composition is formulated as an oral dosage form. The oral dosage form may, for example, be formulated as a liquid, powder, sachet, granulate, pellet, micropellet, tablet, or minitablet. In some embodiments, the composition is formulated as a liquid, powder, sachet, or tablet.

[241] Although the compositions preferably are formulated for administration enterally (for example, orally), such compositions can be administered by a parenteral mode (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular injection). The phrases "parenteral administration" and "administered parenterally" as used herein mean modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and infrasternal injection and infusion.

[242] The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for stable storage at high concentration. Sterile injectable solutions can be prepared by incorporating an agent described herein in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating an agent described herein into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying that yield a powder of an agent described herein plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[243] Depending upon the mode of administration, for example, by parenteral administration, it may be desirable to produce a pharmaceutical formulation that is sterile. Sterilization can be accomplished by any suitable method, e.g., filtration through sterile filtration membranes. Where the composition is lyophilized, filter sterilization can be conducted prior to or following lyophilization and reconstitution.

[244] In certain embodiments, a disclosed composition comprises a polyionic reagent which may, e.g., coat the invertase and / or isomaltase enzyme (e.g., the composition comprises a polyionic coating). Exemplary polyionic reagents include PSS (poly(Sodium 4-styrenesulfonate), PAA (poly Acrylic acid sodium salt), PMG (poly(methylene-co-guanidine) hydrochloride), DS (dextran sulfate), PMA (poly(methyl acrylate)), or PVS (polyvinylsiloxane).

[245] In certain embodiments, a disclosed composition and / or dosage comprises: (i) about 750 to about 75,000, about 750 to about 60,000, about 750 to about 45,000, about 750 to about 30,000, about 750 to about 15,000, about 750 to about 10,000, about 750 to about 7500, about 750 to about 5,000, about 750 to about 2500, about 750 to about 1,000, about 1,000 to about 75,000, about 1,000 to about 60,000, about 1,000 to about 45,000, about 1,000 to about 30,000, about 1,000 to about 15,000, about 1,000 to about 10,000, about 1,000 to about 7,500, about 1,000 to about 5,000, about 1,000 to about 2,500, about 2,500 to about 75,000, about 2,500 to about 60,000, about 2,500 to about 45,000, about 2,500 to about 30,000, about 2,500 to about 15,000, about 2,500 to about 10,000, about 2,500 to about 7,500, about 2,500 to about 5,000, about 5,000 to about 75,000, about 5,000 to about 60,000, about 5,000 to about 45,000, about 5,000 to about 30,000, about 5,000 to about 15,000, about 5,000 to about 10,000, about 5,000 to about 7,500, about 7,500 to about 75,000, about 7,500 to about 60,000, about 7,500 to about 45,000, about 7,500 to about 30,000, about 7,500 to about 15,000, about 7,500 to about 10,000, about 10,000 to about 75,000, about 10,000 to about 60,000, about 10,000 to about 45,000, about 10,000 to about 30,000, about 10,000 to about 15,000, about 15,000 to about 75,000, about 15,000 to about 60,000, about 15,000 to about 45,000, about 15,000 to about 30,000, about 30,000 to about 75,000, about 30,000 to about 60,000, about 30,000 to about 45,000, about 45,000 to about 75,000, about 45,000 to about 60,000, or about 60,000 to about 75,000 international units (I.U.) of invertase enzyme; and / or (ii) about 750 to about 75,000, about 750 to about 60,000, about 750 to about 45,000, about 750 to about 30,000, about 750 to about 15,000, about 750 to about 10,000, about 750 to about 7,500, about 750 to about 5,000, about 750 to about 2,500, about 750 to about 1,000, about 1,000 to about 75,000, about 1,000 to about 60,000, about 1,000 to about 45,000, about 1,000 to about 30,000, about 1,000 to about 15,000, about 1,000 to about 10,000, about 1,000 to about 7,500, about 1,000 to about 5,000, about 1,000 to about 2,500, about 2,500 to about 75,000, about 2,500 to about 60,000, about 2,500 to about 45,000, about 2,500 to about 30,000, about 2,500 to about 15,000, about 2,500 to about 10,000, about 2,500 to about 7,500, about 2,500 to about 5,000, about 5,000 to about 75,000, about 5,000 to about 60,000, about 5,000 to about 45,000, about 5,000 to about 30,000, about 5,000 to about 15,000, about 5,000 to about 10,000, about 5,000 to about 7,500, about 7,500 to about 75,000, about 7,500 to about 60,000, about 7,500 to about 45,000, about 7,500 to about 30,000, about 7,500 to about 15,000, about 7,500 to about 10,000, about 10,000 to about 75,000, about 10,000 to about 60,000, about 10,000 to about 45,000, about 10,000 to about 30,000, about 10,000 to about 15,000, about 15,000 to about 75,000, about 15,000 to about 60,000, about 15,000 to about 45,000, about 15,000 to about 30,000, about 30,000 to about 75,000, about 30,000 to about 60,000, about 30,000 to about 45,000, about 45,000 to about 75,000, about 45,000 to about 60,000, or about 60,000 to about 75,000 international units (I.U.) of isomaltase enzyme.

[246] In certain embodiments, a disclosed composition and / or dosage comprises: (i) about 5,000 to about 12,000, about 5,000 to about 11,000, about 5,000 to about 10,000, about 5,000 to about 9,000, about 5,000 to about 8,000, about 5,000 to about 7,000, about 5,000 to about 6,000, about 6,000 to about 12,000, about 6,000 to about 11,000, about 6,000 to about 10,000, about 6,000 to about 9,000, about 6,000 to about 8,000, about 6,000 to about 7,000, about 7,000 to about 12,000, about 7,000 to about 11,000, about 7,000 to about 10,000, about 7,000 to about 9,000, about 7,000 to about 8,000, about 8,000 to about 12,000, about 8,000 to about 11,000, about 8,000 to about 10,000, about 8,000 to about 9,000, about 9,000 to about 12,000, about 9,000 to about 11,000, about 9,000 to about 10,000, about 10,000 to about 12,000, about 10,000 to about 11,000, about 11,000 to about 12,000, about 5,000, about 6,000, about 7,000, about 8,000, about 8,500, about 9,000, about 10,000, about 11,000, or about 12,000 international units (I.U.) of invertase enzyme; and / or (ii) about 5,000 to about 12,000, about 5,000 to about 11,000, about 5,000 to about 10,000, about 5,000 to about 9,000, about 5,000 to about 8,000, about 5,000 to about 7,000, about 5,000 to about 6,000, about 6,000 to about 12,000, about 6,000 to about 11,000, about 6,000 to about 10,000, about 6,000 to about 9,000, about 6,000 to about 8,000, about 6,000 to about 7,000, about 7,000 to about 12,000, about 7,000 to about 11,000, about 7,000 to about 10,000, about 7,000 to about 9,000, about 7,000 to about 8,000, about 8,000 to about 12,000, about 8,000 to about 11,000, about 8,000 to about 10,000, about 8,000 to about 9,000, about 9,000 to about 12,000, about 9,000 to about 11,000, about 9,000 to about 10,000, about 10,000 to about 12,000, about 10,000 to about 11,000, about 11,000 to about 12,000, about 5,000, about 6,000, about 7,000, about 8,000, about 8,500, about 9,000, about 10,000, about 11,000, or about 12,000 international units (I.U.) of isomaltase enzyme.

[247] In certain embodiments, a disclosed invertase and / or isomaltase enzyme or composition is administered to a subject together with a meal or snack. In certain embodiments, a disclosed invertase and / or isomaltase enzyme or composition is administered to a subject together with each meal or snack that the subject eats. In certain embodiments, a disclosed invertase and / or isomaltase enzyme or composition is administered to a subject once every 7 days, once every 6 days, once every 5 days, once every 4 days, once every 3 days, once every 2 days, once every day, 2 times every day, 3 times every day, 4 times every day, 5 times every day, 6 times every, or more than 6 times every day.

[248] Depending upon the circumstances, the composition can be formulated as a powder, granulate, pellet, micropellet, or a minitablet. The composition can be encapsulated in a capsule, e.g., a hydroxypropyl methylcellulose (HPMC) capsule, soft gelatin capsule, or a hard gelatin capsule. Alternatively, the composition can be formulated as a tablet dosage form. The dosage form, e.g., granules, tablets, minitablets, pellets, micropellets, capsules can be enterically coated using, e.g., one or more methacrylic polymers, such as methacrylic acid methylmethacrylate copolymers (e.g., Eudragit L and S), and methacrylic acid ethyl acrylate copolymer (Eudragit L30D), hydroxyl propyl methyl cellulose acetate, hydroxy propyl methyl cellulose phthalate succinate and others.

[249] In certain embodiments, the composition comprises less than 50%, less than 40%, less than 30%, less than 20%, less than 10% or less than 5% glycerol. In certain embodiments, the composition does not comprise glycerol.

[250] In certain embodiments, a disclosed pharmaceutical composition has a shelf-life (e.g., retains at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of its biological activity) at room temperature of at least 3 months, 6 months, 9 months, 12 months, 15 months, 18 months, 21 months, 24 months, 36 months, 48 months, 60 months, 72 months, 84 months, 96 months, 108 months, or 120 months. In certain embodiments, a disclosed pharmaceutical composition has a shelf-life at room temperature of from about 3 to about 120 months, from about 3 to about 96 months, from about 3 to about 72 months, from about 3 to about 48 months, from about 3 to about 24 months, from about 3 to about 21 months, from about 3 to about 18 months, from about 3 to about 15 months, from about 3 to about 12 months, from about 3 to about 9 months, from about 3 to about 6 months, from about 6 to about 120 months, from about 6 to about 96 months, from about 6 to about 72 months, from about 6 to about 48 months, from about 6 to about 24 months, from about 6 to about 21 months, from about 6 to about 18 months, from about 6 to about 15 months, from about 6 to about 12 months, from about 6 to about 9 months, from about 9 to about 120 months, from about 9 to about 96 months, from about 9 to about 72 months, from about 9 to about 48 months, from about 9 to about 24 months, from about 9 to about 21 months, from about 9 to about 18 months, from about 9 to about 15 months, from about 9 to about 12 months, from about 12 to about 120 months, from about 12 to about 96 months, from about 12 to about 72 months, from about 12 to about 48 months, from about 12 to about 24 months, from about 12 to about 21 months, from about 12 to about 18 months, from about 12 to about 15 months, from about 15 to about 120 months, from about 15 to about 96 months, from about 15 to about 72 months, from about 15 to about 48 months, from about 15 to about 24 months, from about 15 to about 21 months, from about 15 to about 18 months, from about 18 to about 120 months, from about 18 to about 96 months, from about 18 to about 72 months, from about 18 to about 48 months, from about 18 to about 24 months, from about 18 to about 21 months, from about 21 to about 120 months, from about 21 to about 96 months, from about 21 to about 72 months, from about 21 to about 48 months, from about 21 to about 24 months, from about 24 to about 120 months, from about 24 to about 96 months, from about 24 to about 72 months, from about 24 to about 48 months, from about 48 to about 120 months, from about 48 to about 96 months, from about 48 to about 72 months, from about 72 to about 120 months, from about 72 to about 96 months, or from about 96 to about 120 months. In certain embodiments, a disclosed pharmaceutical composition retains at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of its biological activity after 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks at 40 °C and 75% relative humidity (RH).V. Therapeutic Uses

[251] Themethods and compositions disclosed herein can be used to treat diseases or disorders. For example, the disclosure provides a method of treating sucrase-isomaltase deficiency (e.g., congenital sucrase isomaltase deficiency (CSID) or acquired sucrase-isomaltase deficiency (ASID)) in a subject. The method comprises administering (e.g., orally administering) to the subject an effective amount of (i) an invertase enzyme (e.g., a recombinant mutant invertase enzyme described herein), and / or (ii) an isomaltase enzyme. For example, the method may comprise administering a disclosed pharmaceutical composition comprising (i) a dried (e.g., spray-dried or lyophilized) invertase enzyme (e.g., a recombinant mutant invertase enzyme described herein), and / or (ii) a dried (e.g., spray-dried or lyophilized) isomaltase enzyme. In certain embodiments, the pharmaceutical composition is administered to the subject together with a meal or snack.

[252] Under certain conditions, the method reduces abdominal pain, bloating, and / or nausea in the subject, and as a result, the method reduces a total symptom score (for example, measured by abdominal pain, bloating and nausea) for the subject. Furthermore, the invention permits greater diet liberalization in the subject (e.g., allows the subject to better tolerate certain foods, e.g., grains (e.g., wheat, potatoes, corn, rice, and bread), fruits (e.g., apples, bananas, apricots, and oranges) and / or vegetables (e.g., beets, carrots, and black beans)).

[253] The term “effective amount” as used herein refers to the amount of an active agent (e.g., a disclosed invertase and / or isomaltase enzyme) sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route.

[254] As used herein, “treat”, “treating” and “treatment” mean the treatment of a disease in a subject, e.g., in a human. This includes: (a) inhibiting the disease, i.e., arresting its development; and (b) relieving the disease, i.e., causing regression of the disease state. As used herein, the terms “subject” and “patient” refer to an organism to be treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and more preferably includes humans.

[255] The disclosure also provides a method of reducing an amount of sucrose and branched (1–6 linked) α-limit dextrin in a subject, for example, a subject with sucrase-isomaltase deficiency (e.g., congenital sucrase-isomaltase deficiency (CSID) or acquired sucrase-isomaltase deficiency (ASID)). The method comprises administering (e.g., orally administering) to the subject an effective amount of (i) an invertase enzyme (e.g., a recombinant mutant invertase enzyme described herein), and / or (ii) an isomaltase. For example, the method may comprise administering a disclosed pharmaceutical composition comprising (i) a dried (e.g., spray-dried or lyophilized) invertase (e.g., a recombinant mutant invertase described herein), and (ii) a dried (e.g., spray-dried or lyophilized) isomaltase. An amount of sucrose and branched (1–6 linked) α-limit dextrin in a subject may refer to an amount of sucrose and branched (1–6 linked) α-limit dextrin measured by a hydrogen breath test, e.g., a sucrose methane hydrogen breath test or a 13C-sucrose breath test. An amount of sucrose and branched (1–6 linked) α-limit dextrin in a subject may refer to an amount of sucrose and branched (1–6 linked) α-limit dextrin in a body fluid (e.g., blood, plasma, serum, or urine), tissue and / or cell in the subject.

[256] The methods and compositions described herein can be used alone or in combination with other therapeutic agents and / or modalities. The term administered “in combination,” as used herein, is understood to mean that two (or more) different treatments are delivered to the subject during the course of the subject’s affliction with the disorder, such that the effects of the treatments on the patient overlap at a point in time. In certain embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as “simultaneous” or “concurrent delivery”. In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In certain embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In certain embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.

[257] In certain embodiments, a method or composition described herein, is administered in combination with one or more fructose degrading enzymes. Exemplary fructose degrading enzymes include glucose (xylose) isomerases.

[258] Throughout the description, where compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present disclosure that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present disclosure that consist essentially of, or consist of, the recited processing steps. Similarly, throughout the description, where compositions are described as consisting essentially of specific components, or where processes and methods are described as consisting essentially of specific steps, it is contemplated that, additionally, there are compositions of the present disclosure that consist of the recited components, and that there are processes and methods according to the present disclosure that consist of the recited processing steps.

[259] In the application, where an element or component is said to be included in and / or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components.

[260] Further, it should be understood that elements and / or features of a composition or a method described herein can be combined in a variety of ways without departing from the spirit and scope of the present invention, whether explicit or implicit herein. For example, where reference is made to a particular compound, that compound can be used in various embodiments of compositions of the present invention and / or in methods of the present invention, unless otherwise understood from the context. In other words, within this application, embodiments have been described and depicted in a way that enables a clear and concise application to be written and drawn, but it is intended and will be appreciated that embodiments may be variously combined or separated without parting from the present teachings and invention(s). For example, it will be appreciated that all features described and depicted herein can be applicable to all aspects of the invention(s) described and depicted herein.

[261] It should be understood that the expression “at least one of” includes individually each of the recited objects after the expression and the various combinations of two or more of the recited objects unless otherwise understood from the context and use. The expression “and / or” in connection with three or more recited objects should be understood to have the same meaning unless otherwise understood from the context.

[262] The use of the term “include,” “includes,” “including,” “have,” “has,” “having,” “contain,” “contains,” or “containing,” including grammatical equivalents thereof, should be understood generally as open-ended and non-limiting, for example, not excluding additional unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[263] Where the use of the term “about” is before a quantitative value, the present invention also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term “about” refers to a ±10% variation from the nominal value unless otherwise indicated or inferred.

[264] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the present invention remain operable. Moreover, two or more steps or actions may be conducted simultaneously.

[265] The use of any and all examples, or exemplary language herein, for example, “such as” or “including,” is intended merely to illustrate better the present invention and does not pose a limitation on the scope of the invention unless claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the present invention.EXAMPLES

[266] The following Examples are merely illustrative and are not intended to limit the scope or content of the invention in any way.Example 1: Invertase Selection and Engineering

[267] This Example describes the design of recombinant mutant S. cerevisiae invertases with improved tolerance of low pH, resistance to digestion by pepsin and pancreatin, improved thermostability, and high catalytic activity.Invertase Expression

[268] Expression constructs encoding recombinant S. cerevisiae invertase (GenBank: AFN08663.1, SEQ ID NO: 1) were constructed for expression in the yeast expression strains Pichia pastoris and S. cerevisiae. Expression constructs were designed with or without a C-terminal or N-terminal 6x-His affinity tag (SEQ ID NO: 62) and were tested for expression and tolerance of the added His tag. Further expression constructs were designed with either the natural invertase secretion leader intact (SEQ ID NO: 1) or with a substituted alpha-mating factor (FAKS) leader (SEQ ID NO: 2). The nucleic acid sequences of each expression construct were optimized for high expression in the corresponding host. The synthesized nucleic acid sequences encoding for the invertases were cloned into pD1204 (S. cerevisiae) or pD902 (P. pastoris).

[269] For expression in S. cerevisiae, the strain CEN.PK2-1C was transformed chemically using the lithium acetate method (Ito et al. (1983) J. Bacteriol. 153(1):163-168). Cells harboring plasmid were selected by plating on CM-URA media (MP Biomedicals, Santa Ana, CA) over 3 days at 30 °C. Three individual colonies for each construct were isolated and used to inoculate 500 µL of CM-URA media. The inoculated culture was incubated at 30 °C with shaking at 1,000 rpm for 16 hours. 40 µL of overnight culture was then used to inoculate 500 µL of YP induction media (1% yeast extract, 2% peptone, 0.1 M potassium phosphate, 2% raffinose, and 2% galactose). The induction culture was then incubated at 30 °C with shaking at 1,000 rpm for 72 hours. The induction culture was supplemented with 50 µL 20% galactose every 24 hours after induction. The supernatant was clarified by centrifugation and secreted invertase expression was analyzed by PAGE (4-12% Bis-tris, MOPS-buffered; Invitrogen).

[270] For expression in P. pastoris, 20 µg of expression plasmid was linearized by digestion with PmeI. The DNA was isolated by precipitation with ethanol and reconstituted in Qiagen EB buffer. For expression studies, the strain BG10 was transformed by electroporation. For protein engineering, the strain BG10 was transformed chemically using the lithium acetate method (Ito et al.,supra). Cells harboring plasmid were selected by plating on YPD media with 250 µg / mL zeocin (Teknova) over 3 days at 30 °C. Three individual colonies for each construct were isolated and used to inoculate 350 µl of BYPG media with 250 µg / mL zeocin in a deep-well microplate. The inoculated culture was then incubated at 30 °C with shaking at 1,000 rpm for 60 hours. 250 µL of the overnight culture was then added to 250 µL BYPM induction media (BYP media supplemented with 20% methanol). The induction culture was then incubated at 30 °C with shaking at 1,000 rpm for 72 hours. Cultures were supplemented with 50 µL BYP with 10% methanol every 12 hours after induction. Supernatants were clarified from cells by centrifugation and secreted invertase expression was analyzed by PAGE.

[271] Expression yields were determined by PAGE densitometry (after deglycosylation with PNGaseF, if deglycosylated). For experiments using deglycosylated enzyme, culture supernatant samples were treated with PNGaseF after denaturation according to manufacturer’s protocol (NEB) and the samples were analyzed by PAGE. Full-length glycosylated or deglycosylated invertase band intensities were determined and intensity relative to BSA standards was used to estimate protein concentration.

[272] Expression and yield of S. cerevisiae invertase in P. pastoris was typically better than in S. cerevisiae.Invertase Activity and Stability Assays

[273] In the initial stages, when the resulting proteins tested without purification, specific activities were approximated by assaying units per amount of deglycosylated or glycosylated protein (1 unit of enzyme consumes 1 µmole sucrose per minute at 37 °C).

[274] Activity assays were performed using the dinitro salicylate (DNS) colorimetric method to monitor reducing sugar formation upon hydrolysis of sucrose. Briefly, the DNS method detects the reducing sugars liberated by the action of hydrolase enzymes on carbohydrates, under specific pH and temperatures (Bailey (1988) Applied Microbiology and Biotechnology 29: 494–496). Upon reaction with a reducing sugar DNS is reduced to 3-amino-5-nitro salicylic acid and the reaction product can be monitored at 540 nm.

[275] Culture supernatants were diluted 1 / 20 into reaction buffer containing 237 mM NaCl and either: A) 50 mM glycine, pH 3.5 (buffer A); B) 50 mM sodium acetate, pH 4.7 (buffer B); C) 50 mM MES, pH 5.8 (buffer C); or D) 50 mM sodium phosphate, pH 6.8 (buffer D). Diluted enzymes were then further diluted 10-fold into 10 mM sucrose in reaction buffer pre-equilibrated at 37 °C. Reaction progress was then estimated by the DNS method. To stop the reaction, 20 µL of reaction was removed to 30 µL DNS reagent (10 mg / mL dinitrosalicylic acid, 300 mg / mL tartrate, 0.4 N NaOH) and the mixture was immediately heated to 99 °C for 5 minutes. 40 µL of the DNS reaction was diluted with 160 µL water and the absorbance was read at 540 nm. Reducing sugars present were determined relative to 1:1 glucose:fructose standards.

[276] Thermostability was assayed by treatment of invertase samples at 56.5 °C for 30 minutes prior to assaying residual activity at pH 4.7 (in reaction buffer B, described above) using the DNS method as described above.

[277] Low pH tolerance assays were performed as follows. Culture supernatants including the invertase enzymes were diluted 1 / 20 into 237 mM NaCl, 50 mM glycine pH 2.8 and preincubated at 37 °C for 30 minutes. Preincubations were then diluted into 10 mM sucrose in reaction buffer at a final of pH 4.7 and activity was assayed using the DNS method as described above.

[278] Resistance to pepsin was assayed by treating the invertase enzymes with 10 mg / mL soluble pepsin from porcine gastric mucosa (Sigma-Aldrich, St. Louis, MO) in 237 mM NaCl, 50 mM NaOAc pH 4 at 37 °C. Culture supernatants including the invertase enzymes were diluted 20-fold into 10 mg / mL pepsin and incubated for 2 hours at 37 °C. Residual invertase activity was then assayed by diluting the pepsin treatment reaction 20-fold into 10 mM sucrose in reaction buffer at a final pH of 4.7 and activity was assayed using the DNS method as described above. The overexpressed S. cerevisiae invertase and most invertase variants showed insignificant degradation under these conditions.

[279] Resistance to pancreatin was assayed by treating the invertases with 3.4 mg / mL soluble porcine pancreatin (Sigma-Aldrich, St. Louis, MO) in 237 mM NaCl, 50 mM MES pH 6 at 37 °C. Culture supernatants were diluted 20-fold into 3.4 mg / mL pancreatin and incubated for 2 hours at 37 °C. Residual invertase activity was then assayed by diluting the pancreatin treatment reaction 20-fold into 10 mM sucrose in reaction buffer at a final pH of 4.7 and activity was assayed using the DNS method as described above.Invertase Engineering

[280] 95 recombinant S. cerevisiae invertase variants (denoted as V1-V95) were created using protein engineering principles, each containing three substitutions relative to the wild-type natural protein S. cerevisiae invertase (denoted as V0). TABLE 2 shows certain exemplary amino acid mutations in S. cerevisiae invertase (SEQ ID NO: 2) that were analyzed in the invertase engineering program. The variants along with the wild-type (wt) S. cerevisiae invertase were expressed in P. pastoris and tested as culture supernatants for activity and stability. Each variant was tested for seven properties using the assays described above, including: 1) expression level, 2) activity at pH 3.5, 3) activity at pH 7, 4) thermostability, 5) stability at pH 2.8, 6) resistance to pepsin, and 7) resistance to pancreatin.TABLE 2E120AK277TN429AK519RD122EK259LL459FS537DA123GL359FT470SL549IT140LT365ST573SV581IT140QA378TS476NG586SF143YE382QS476AN589KE156QN390DP479AF596IF172YE415SN512GQ597EV178IL416QK514P  

[281] Overall, 94 invertase variants were expressed and tested (one variant did not express) and compared against wild-type enzyme. In general, 13 invertase variants showed an improvement pH 2.8 stability, thermostability, pH 3.5 stability, and pH 7.0 activity over the parent invertase, 35 invertase displayed mixed properties, and 46 invertase variants displayed worse properties than the parent invertase. Exemplary results for the engineered invertase variants compared to parental, wild-type invertase (S. cerevisiae invertase) are shown in TABLE 3 and the substitutions present in the various invertase variants (relative to S. cerevisiae invertase (SEQ ID NO: 2) listed in TABLE 3 are shown in TABLE 4. TABLE 3 includes the properties of the 13 invertase variants (V6, V9, V14, V25, V32, V38, V60, V63, V70, V78, V83, V87, V89, and V90), that showed one or more improved properties relative to parental invertase, and three exemplary invertase variants (V3, V75, and V82) that performed worse than the parental invertase.TABLE 3InvertaseStability at pH2.8 a)Thermostability a)Activity at pH3.5 a)Activity at pH7.0a)Parental invertase (V0)~~~~V6+++~+V9+++~~V14+++~+V22+---V25~~~+V32+++~V38~+~+V60++~~V63~+~~V70~++~+V78++++++V83+~~~V87++~~V89+~~+V90++~~V3---+V75----V82----aRelative impacts of the substitutions are indicated as strongly positive (++), moderately positive (+), moderately negative (-) or neutral (~) relative to the parent invertase.TABLE 4NameSubstitution 1Substitution 2Substitution 3SEQ ID NO:V6E120AD122EP479ASEQ ID NO: 4V9T140LE156QL549ISEQ ID NO: 5V14S476AP479AQ597ESEQ ID NO: 6V22K259LS476NK514PSEQ ID NO: 7V25K277TE415SV581ISEQ ID NO: 8V32A123GT140LF143YSEQ ID NO: 9V38N390DS476NK519RSEQ ID NO: 10V60E382QT573SV581ISEQ ID NO: 11V63D122ET470SS476NSEQ ID NO: 12V70T140QP479AK514PSEQ ID NO: 13V78F172YN429AP479ASEQ ID NO: 14V83E120AV178IL549ISEQ ID NO: 15V87E382QS537DN589KSEQ ID NO: 16V89T140LG586SF596ISEQ ID NO: 17V90E382QL459FK519RSEQ ID NO: 18V3K277TT365SN512G V75D122EL359FA378T V82E120AL416QS537D 

[282] The relative impact of each substitution on each target property was estimated and is set forth in TABLE5 (amino acid numbering is relative toS. cerevisiae invertase (SEQ ID NO: 2). In particular, the impact of certain amino acid substitutions relative to wild-type S. cerevisiae invertase (SEQ ID NO: 2) that showed significant contribution to at least one of three key invertase properties: pH 7.0 activity, thermostability, or low pH survival, are summarized in TABLE 5.TABLE 5Amino Acid SubstitutionActivity at pH7.0a)Low pH Stability a)Thermostability a)A378T+- --A501N ~~+D272N+ ~~E156Q ~+ E285H ~~++E382Q ~++F172Y++~ G431R ~+-G586S+ ~~K259L-++~K277T+~-L549I+~+N589K ~++P479A+++++T140L ~+++V178I ~+-V399A+-+V477D+ ~~ V581I ~++D122E-++aRelative impacts of substitutions are indicated as strongly positive (++), moderately positive (+), moderately negative (-) or neutral (~).Further Characterization of Certain Invertase Variants

[283] A number of invertase variants showing improved properties relative to the parental invertase in terms of activity and / or stability were further characterized. In particular, two studies were performed on independent preparations of the selected variants and the results are summarized in TABLES6 and 7. TABLE 6 summarizes the characteristics of three variants compared against wild-type parental invertase.TABLE 6 Activity (U / mg protein)aStabilityInvertase VariantpH 3.5pH 5.0pH 6.2pH 7.1pH 2.5bThermo-stabilityc(Tm, °C)PepsindPancreatindV0 (Parent)7689566601711655>80>80V781288152010993116362>80>80V227701029637967654>80>80V6103711888302476061>80>80aSpecific activity was estimated as µmole sucrose consumed per minute per mg of deglycosylated invertase in reactions (single point measurements from single variant preparations for each reaction pH).bStability at pH 2.5 was measured as the percent residual activity remaining after pretreatment at 37 °C for 30 minutes.cThermostability was estimated as the observed temperature of 50% loss of activity after 30 minutes of pretreatment.dPercent residual activity after protease pretreatment. Protease stability was assessed at 37 °C at 10 mg / mL pepsin in SGF, pH 4 or 3.4 mg / mL pancreatin in SIF, pH 6. All variants showed <20% loss of activity under the assay conditions after 2 hours pretreatment. 

[284] Strong improvements in activity, low pH stability and thermostability were seen for variants V78 (F172Y, N429A, P479A; SEQ ID NO: 14) and V6 (E120A, D122E, P479A; SEQ ID NO: 4). Both variants V6 and V78 contained the broadly beneficial substitution P479A and at least one other substitution positive for one or more critical properties. V22 (K259L, S476N, K514P; SEQ ID NO: 7) showed an improvement in low pH stability. V22 contained the K259L substitution which was found to be beneficial for low pH stability but not high pH activity.

[285] To further compare enzymes that were expressed under similar conditions, Sc_S288C (SEQ ID NO: 36) invertase was expressed in P. pastoris and assayed under identical conditions as certain of the better variants. Further variants of Sc_S288C invertase were tested that included the two additional substitutions found in V78 (F172Y and N429A) to assess their impact in the context of the Sc_S288C invertase sequence. The results of the comparison are summarized in TABLE 7.TABLE 7 Activity (U / mg protein)aStability VariantpH 3.5pH 5pH 6pH 7pH 2.7bThermo-stabilitycPepsindPancreatindV0 (Parent)960 10208003002855>80>80V781540167014708208262>80>80ScS288C1230133011075808964ndndScS288C + F172Y + 429A1240137011906909765ndndaSpecific activity was estimated as µmole sucrose consumed per minute per mg of deglycosylated invertase in reactions. Expression and reactions were performed in triplicate.bStability at pH 2.7 was measured as the percent residual activity remaining after pretreatment at 37 °C for 30 minutes.cThermostability was estimated as the observed temperature of 50% loss of activity after 30 minutes of pretreatment.dPercent residual activity after protease pretreatment. Protease stability was assessed at 37 °C at 10 mg / mL pepsin in SGF, pH 4 or 3.4 mg / mL pancreatin in SIF, pH 6. All variants showed <20% loss of activity under the assay conditions after 2 hours pretreatment.

[286] The activity profile for the Sc_S288C invertase was more comparable to that of the parent S. cerevisiae invertase, but similar differences were observed in low pH and thermostability. This was consistent with the expected impact of the P479A substitution on invertase activity. The variant V78 showed improvement in activity relative to Sc_S288C invertase but slightly poorer thermostability.

[287] Together, these studies identified several substitutions that can be used alone or in combination to positively impact activity, low pH stability and thermostability of S. cerevisiae invertase. Variant V78 shows all-around significant improvement relative to the parental invertase.Example 2: Engineering of Enhanced Invertase Variants

[288] This Example describes the enhancement of a recombinant mutant S. cerevisiae invertase identified in Example 1 with improved tolerance of low pH, improved thermostability, and high catalytic activity.Invertase Engineering

[289] Recombinant S. cerevisiae invertase variants were created based on variant V78, each containing the three substitutions of V78 and three additional mutations relative to the wild-type S. cerevisiae invertase. TABLE 8 shows amino acid substitutions in each of the variants, where the locations of the substitutions are made with reference to SEQ ID NO: 2. During expression, the signal sequences are removed.

[290] The variants and the wild-type (wt) S. cerevisiae invertase were each expressed in P. pastoris and culture supernatants were tested for activity and stability. Each variant was tested for various properties using the assays described in Example 1 above, including: (i) activity at pH 3.6, 4.8, 6, and 7.1, (ii) thermostability, (iii) stability at pH 2.8, (iv) resistance to pepsin, and (v) resistance to pancreatin.TABLE 8VariantSub 1Sub 2Sub 3Sub 4Sub 5Sub 6SEQ ID NOV78F172YN429AP479A---29VE01F172YN429AP479AT140LD272NE285H54VE03F172YN429AP479AT140LD272NL549I55VE04F172YN429AP479AT140LD272NV581I 56VE08F172YN429AP479AT140LE285HV581I 57VE36F172YN429AP479AT140LE382QA501N51VE38F172YN429AP479AT140LE382QA123G58VE39F172YN429AP479AT140LE382QN512G59VE40F172YN429AP479AT140LE382QV178I60VE44F172YN429AP479AT140LE382QN390D61 

[291] The new variants were expressed, tested, and compared against the parental V78 enzyme. In general, all nine invertase variants showed an improvement in pH 2.8 stability, thermostability, pH 3.6 activity, pH 4.8 activity, and pH 6 activity, with four of the variants further showing improved pH 7.1 activity over the parent invertase. Exemplary activity results for the engineered invertase variants compared to the parental invertase (V78) are shown in TABLE 9and stability results are shown in TABLE 10. TABLE 9 Specific Activity (µmole / min / mg @ 10 mM sucrose, 37°C) SGF, pH 3.6aSGF, pH 4.8 aSIF, pH 6 aSIF, pH 7.1 aV78~~~~VE01++++VE03++++VE04++++VE08++++VE36+++~VE38+++~VE39+++~VE40+++~VE44+++~a Relative impacts of substitutions are indicated as positive (+) or neutral (~) relative to the parental invertase V78.TABLE 10 Pepsin Stability (t1 / 2, min) aPancreatin Stability (t1 / 2, min) aThermostability (Residual @ 60 min) apH Stability (t1 / 2, min) a 32 mg / ml, pH 3.5 5 mg / ml, pH 6.062°C, SIF pH 5.0pH 2.8V78~~~~VE01~~++++++VE03~~+++++VE04~~+++++VE08~~++++++VE36~~++++++VE38~~+++++VE39~~++++VE40~~+++++VE44~~+++++a Relative impacts of substitutions are indicated as very strongly positive (+++), strongly positive (++), moderately positive (+), or neutral (~) relative to the parental invertase V78.

[292] The relative impact of each substitution on each target property was estimated and is set forth in TABLE11 (amino acid numbering is relative toS. cerevisiae invertase (SEQ ID NO: 2). In particular, the impact of certain amino acid substitutions relative to wild-type S. cerevisiae invertase (SEQ ID NO: 2) that showed significant contribution to at least one of three key invertase properties: pH 7.0 activity, or low pH stability, or thermostability, are summarized in TABLE 11.TABLE 11Amino Acid SubstitutionActivity at pH 7.0 aLow pH Stability aThermostability aF172Y++~ P479A+++++N429A ~~+L549I+ ~~T470S ~+ E120A ~~++N512G ~++N390D+~+A123G ~++T140L ~+++E382Q ~+-V581I ~++D272N~ ++~E285H ~~+++a Relative impacts of substitutions are indicated as very strongly positive (+++), strongly positive (++), moderately positive (+), moderately negative (-) or neutral (~). 

[293] Together, these studies identified further substitutions and combinations of substitutions that can be used to positively impact activity, low pH stability and thermostability of S. cerevisiae invertase. Variant VE36 showed all-around significant improvement relative to the parental invertase. Example 3: Engineering of Enhanced Invertase Variants for Enhanced Thermostability and Stability at Low pH

[294] This Example describes the enhancement of a recombinant mutant S. cerevisiae invertase identified in Example 2 with improved tolerance of low pH and improved thermostability.Invertase Engineering

[295] Additional recombinant S. cerevisiae invertase variants were created based on variant VE36 identified in Example 2, each containing the six substitutions of VE36 and one or two further mutations relative to the wild-type S. cerevisiae invertase. TABLE 12 shows amino acid substitutions in each of the two variants VE361 and VE362 as well as variant VE36, where the locations of the substitutions are made with reference to SEQ ID NO: 2. During expression the signal sequences are removed.

[296] The variants VE361 and VE362 along with the wild-type (wt) S. cerevisiae invertase, V78 (variant from Example 1), and VE36 (variant from Example 2) were each expressed in P. pastoris and tested as culture supernatants for activity and stability. Each variant was tested for various properties using the assays described in Example 1 above, including: 1) activity at pH 3.6, 4.9, 6, and 7, 2) thermostability, 3) stability at pH 2.8, 4) resistance to pepsin, and 5) resistance to pancreatin.TABLE 12VariantSub 1Sub 2Sub 3Sub 4Sub 5Sub 6Sub 7Sub 8SEQ ID NOVE36F172YN429AP479AT140LE382QA501N  51VE361F172YN429AP479AT140LE382QA501ND272N 52VE362F172YN429AP479AT140LE382QA501ND272NE285H53 

[297] Two invertase variants were expressed, tested, and compared against the wild-type, sc_S288C, V78, and VE36 enzymes. Exemplary activity results for the engineered invertase variants compared to the parental invertase (V78) are shown in TABLE 9 and FIG. 1, and stability results are shown in TABLE 10. Graphs detailing the resistance of variants to pepsin and pancreatin are shown in FIGs. 2A and 2B, respectively. The stability of the variants at pH 2.8 is shown in FIG. 3, and the thermostability of variants at 62°C is shown in FIG. 4. In general, the variants VE361 and VE362 had improved stability at pH 2.8 and increased thermostability compared to variants VE36 and V78, and variant VE36 also showed improved stability at pH 2.8 and increased stability relative to variant V78 from Example 1.TABLE 13 Specific Activity (µmole / min / mg @ 10 mM sucrose, 37°C) SGF, pH 3.6SGF, pH 4.9SIF, pH 6SIF, pH 7Wild-typeDEDASc_S288CGHFBV78GHGCVE36IIGBVE361HHFBVE362IIFBA=Specific Activity below 300; B= Specific Activity between 300-500; C=Specific Activity between 500-700; D=Specific Activity between 800-1000; E=Specific Activity between 1001-1200; F=Specific Activity between 1200-1400; G=Specific Activity between 1401-1600; H=Specific Activity between 1601-1800; I=Specific Activity above 1800TABLE 14 Thermostability (t1 / 2, min)pH Stability (t1 / 2, min)Pepsin Stability (t1 / 2, min)Pancreatin Stability (t1 / 2, min) 62°C, SIF pH 5.0pH 2.832 mg / ml, pH 3.5 5 mg / ml, pH 6.0Wild-typeAC>120>120Sc_S288CFD>120>120V78BD>120>120VE36HH>120>120VE361HH>120>120VE362HH>120>120A=t1 / 2below 10; B= t1 / 2between 10-50; C= t1 / 2between 51-100; D= t1 / 2 between 101-200; E= t1 / 2between 201-300; F= t1 / 2between 301-400; G= t1 / 2between 401-500; H= t1 / 2above 500.INCORPORATION BY REFERENCE

[298] The entire disclosure of each of the patent and scientific documents referred to herein is incorporated by reference for all purposes.EQUIVALENTS

[299] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.  

Claims

1. A recombinant mutant S. cerevisiae invertase enzyme, wherein the invertase comprises one or more amino acid substitutions selected from the group consisting of:(a) a substitution of a T residue at a position corresponding to position 51 of SEQ ID NO: 3;(b) a substitution of an F residue at a position corresponding to position 83 of SEQ ID NO: 3;(c) a substitution of an E residue at a position corresponding to position 293 of SEQ ID NO: 3;(d) a substitution of an N residue at a position corresponding to position 340 of SEQ ID NO: 3;(e) a substitution of a P residue at a position corresponding to position 390 of SEQ ID NO: 3; and(f) a substitution of an A residue at a position corresponding to position 412 of SEQ ID NO: 3; ora combination of any of the foregoing substitutions, and is catalytically active to digest sucrose into glucose and fructose under physiological conditions (e.g., of the stomach and / or small intestine).

2. The invertase of claim 1, wherein, in the invertase:(a) the T residue at a position corresponding to position 51 of SEQ ID NO: 3 is substituted by L (T51L);(b) the F residue at a position corresponding to position 83 of SEQ ID NO: 3 is substituted by Y (F83Y);(c) the E residue at a position corresponding to position 293 of SEQ ID NO: 3 is substituted by Q (E293Q);(d) the N residue at a position corresponding to position 340 of SEQ ID NO: 3 is substituted by A (N340A);(e) the P residue at a position corresponding to position 390 of SEQ ID NO: 3 is substituted by A (P390A);(f) the A residue at a position corresponding to position 412 of SEQ ID NO: 3 is substituted by N (A412N); orthe invertase comprises a combination of any of the foregoing substitutions.

3. The invertase of claim 1 or 2, wherein the invertase further comprises a substitution at a D residue at a position corresponding to position 183 of SEQ ID NO: 3.

4. The invertase of claim 3, wherein the D residue at the position corresponding to position 183 of SEQ ID NO: 3 is substituted by an N (D183N).

5. The invertase of any one of claims 1-4, wherein the invertase further comprises a substitution at an E residue at a position corresponding to position 196 of SEQ ID NO: 3.

6. The invertase of claim 5, wherein the E residue at the position corresponding to position 196 of SEQ ID NO: 3 is substituted by an H (E196H).

7. The invertase of any one of claims 1-6, wherein the invertase comprises four, five, six, seven, or eight mutations relative to the corresponding wild-type invertase.

8. The invertase of any one of claims 1-7, wherein the invertase comprises F83Y, N340A, P390A, T51L, E293Q, and A412N substitutions.

9. The invertase of any one of claims 1-7, wherein the invertase comprises F83Y, N340A, P390A, T51L, E293Q, A412N, and D183N substitutions.

10. The invertase of any one of claims 1-7, wherein the invertase comprises F83Y, N340A, P390A, T51L, E293Q, A412N, D183N, and E196H substitutions. 11. The invertase of any one of claims 1-10, wherein the invertase comprises the amino acid sequence of any one of SEQ ID NOs: 53, 51-52, or 54-61 or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 53, 51-52, or 54-61.

12. A recombinant mutant S. cerevisiae invertase enzyme comprising a substitution or combination of substitutions listed in TABLE 8or TABLE12, wherein the invertase enzyme is catalytically active to digest sucrose into glucose and fructose under physiological conditions (e.g., of the stomach and / or small intestine).

13. The invertase of any one of claims 1-12, wherein the invertase has a specific activity at about pH 3.5 (e.g., pH 3.6) of at least 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, or 2,000 µmol sucrose consumed per minute per milligram of invertase.

14. The invertase of any one of claims 1-13, wherein the invertase has at least 1-fold, 1.5-fold, 2-fold, 2.5-fold, or 3-fold higher activity at about pH 3.5 (e.g., pH 3.6), compared to a corresponding wild-type invertase.

15. The invertase of any one of claims 1-14, wherein the invertase has a specific activity at about pH 5.0 (e.g., pH 4.9) of at least 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,100, or 2,200 µmol sucrose consumed per minute per milligram of invertase.

16. The invertase of any one of claims 1-15, wherein the invertase has at least 1-fold, 1.5-fold, 2-fold, 2.5-fold, or 3-fold higher activity at about pH 5.0 (e.g., pH 4.9), compared to a corresponding wild-type invertase.

17. The invertase of any one of claims 1-16, wherein the invertase has a specific activity at about pH 6.0 or 6.2 of at least 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, or 1,800 µmol sucrose consumed per minute per milligram of invertase.

18. The invertase of any one of claims 1-17, wherein the invertase has at least 1-fold, 1.5-fold, 2-fold, 2.5-fold, or 3-fold higher activity at about pH 6.0 or 6.2, compared to a corresponding wild-type invertase.

19. The invertase of any one of claims 1-18, wherein the invertase has a specific activity at about pH 7.0 or 7.1 of at least 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, or 1,300 µmol sucrose consumed per minute per milligram of invertase.

20. The invertase of any one of claims 1-19, wherein the invertase has at least 1-fold, 1.5-fold, 2-fold, 2.5-fold, or 3-fold higher activity at about pH 7.0 or 7.1, compared to a corresponding wild-type invertase.

21. The invertase of any one of claims 1-20, wherein the invertase retains at least 80%, 90%, or 95% of activity following incubation at about pH 2.8 for about 2 hours.

22. The invertase of any one of claims 1-21, wherein the invertase retains at least 80%, 90%, or 95% of activity following incubation at about 62 ℃ for about 1 hour.

23. The invertase of any one of claims 1-22, wherein the invertase retains at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of activity following incubation at about pH 2.5 for about 30 minutes.

24. The invertase of any one of claims 1-23, wherein the invertase has at least 1-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, or 5-fold higher stability at about pH 2.5 compared to a corresponding wild-type invertase.

25. The invertase of any one of claims 1-24, wherein the invertase has a melting temperature (Tm) of at least 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, or 68 ℃.

26. The invertase of any one of claims 1-25, wherein the invertase has a Tm that is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ℃ higher than a corresponding wild-type invertase.

27. The invertase of any one of claims 1-26, wherein the invertase has higher stability in the presence of pancreatin or pepsin compared to a corresponding wild-type invertase.

28. A nucleic acid encoding the invertase of any one of claims 1-27.

29. An expression vector comprising the nucleic acid of claim 28.

30. A cell comprising the expression vector of claim 29.

31. The cell of claim 30, wherein the cell is a P. pastoris or S. cerevisiae cell.

32. A method of producing a recombinant mutant S. cerevisiae invertase enzyme, the method comprising: %2) growing the cell of claim 30 or 31 under conditions so that the cell expresses the invertase, and%2) purifying the invertase.

33. A pharmaceutical composition comprising the invertase of any one of claims 1-27 and a pharmaceutically acceptable carrier and / or an excipient.

34. A pharmaceutical composition comprising the invertase enzyme of any one of claims 1-27, optionally an isomaltase enzyme, and a pharmaceutically acceptable carrier and / or an excipient.

35. The pharmaceutical composition of claim 34, wherein the invertase is dried.

36. The pharmaceutical composition of claim 34 or 35, wherein the composition is formulated for delivery as a powder or compressed into a tablet.

37. The pharmaceutical composition of claim 34 or 35, wherein the isomaltase is dried.

38. The pharmaceutical composition of claim 37, wherein the composition is formulated for delivery as a powder or compressed into a tablet.

39. The pharmaceutical composition of any one of claims 33-38, wherein the isomaltase is a microbial isomaltase, or a functional fragment or variant thereof.

40. The pharmaceutical composition of any one of claims 33-39, wherein the isomaltase is derived from Saccharomyces cerevisiae.

41. The pharmaceutical composition of claim 40, wherein the isomaltase comprises the amino acid sequence of any one of SEQ ID NOs: 37-41 or a functional fragment or variant thereof.

42. The pharmaceutical composition of any one of claims 33-41, wherein the isomaltase comprises a recombinant mutant Lactobacillus fermentum isomaltase (SEQ ID NO: 47) or a functional fragment or variant thereof.

43. The pharmaceutical composition of claim 42, wherein the isomaltase further comprises:(a) K115I, R132K, D226S, E310A, L366M, I421A, A444G, A531D, and F560V substitutions; (b) E93K, K115I, R132K, D226S, E310A, L366M, I421A, A444G, A531D, and F560L substitutions; (c) E93K, K115I, R132K, D226S, E310A, I421A, A444G, E524Q, A531D, and F560L substitutions; (d) E93K, K115I, R132K, D226S, E310A, I421A, A444G, A531D, and F560V substitutions; (e) E93K, K115I, R132K, D226S, L366M, I421A, A444G, E524Q, A531D, and F560L substitutions;(f) E93K, K115I, R132K, D226S, L366M, I421A, A444G, A531D, and F560V substitutions; (g) E93K, K115I, R132K, D226S, I421A, A444G, E524Q, A531D, and F560V substitutions; (h) K115I, R132K, D226S, E310A, L366M, I421A, A444G, E524Q, A531D, and F560L substitutions; (i) K115I, R132K, D226S, E310A, I421A, A444G, E524Q, A531D, and F560V substitutions; (j) K115I, R132K, D226S, L366M, I421A, A444G, E524Q, A531D, and F560V substitutions; (k) E93K, K115I, R132K, A211E, D226S, I421A, A444G, E524Q, A531D, and F560V substitutions; (l) E93K, K115I, R132K, D226S, L366M, I421A, A444G, E524Q, A531D, and F560V substitutions; (m) E93K, K115I, R132K, D226S, E275D, I421A, A444G, E524Q, A531D, and F560V substitutions; (n) T89A, E93K, K115I, R132K, D226S, I421A, A444G, E524Q, A531D, and F560V substitutions; (o) E93K, K115I, R132K, H182R, D226S, I421A, A444G, E524Q, A531D, and F560V substitutions; (p) E93K, K115I, E122D, R132K, D226S, I421A, A444G, E524Q, A531D, and F560V substitutions; (q) E93K, K115I, D226S, I421A, A444G, E524Q, A531D, and F560V substitutions;(r) E93K, K115I, D226S, I421A, A444G, E524Q, A531D, and F560L substitutions; (s) K115I, A211E, D226S, I421A, A444G, A531D, and F560V substitutions; (t) K115I, D226S, L366M, I421A, A444G, E524Q, A531D, and F560L substitutions;(u) K115I, A211E, D226S, I421A, A444G, E524Q, A531D, and F560L substitutions;(v) K115I, R132K, D226S, E310A, L366M, I421A, A444G, A531D, and F560L substitutions; (w) E93K, K115I, D226S, E310A, I421A, A444G, A531D, and F560L substitutions; (x) K115I, D226S, E310A, I421A, A444G, A531D, and F560V substitutions; (y) E93K, K115I, D226S, L366M, I421A, A444G, A531D, and F560L substitutions;(z) K115I, D226S, I421A, A444G, E524Q, A531D, and F560V substitutions; (27) K115I, D226S, E310A, I421A, A444G, E524Q, A531D, and F560L substitutions;(28) K115I, A211E, D226S, L366M, I421A, A444G, A531D, and F560L substitutions;(29) K115I, A211E, D226S, E310A, I421A, A444G, A531D, and F560L substitutions;(30) E93K, K115I, D226S, I421A, A444G, A531D, and F560V substitutions; (31) E93K, K115I, A211E, D226S, I421A, A444G, A531D, and F560L substitutions; or (32) K115I, D226S, L366M, I421A, A444G, A531D, and F560V substitutions.

44. The pharmaceutical composition of claim 42 or 43, wherein the invertase comprises the amino acid sequence of any one of SEQ ID NOs: 51-61 or a functional fragment thereof.

45. The pharmaceutical composition of claim 42 or 43, wherein the invertase comprises the amino acid sequence of any one of SEQ ID NOs: 51-53 or a functional fragment thereof.

46. The pharmaceutical composition of any one of claims 33-45, wherein the composition is formulated as an oral dosage form.

47. The pharmaceutical composition of claim 46, wherein the composition is formulated as a liquid, powder, sachet, granulate, pellet, micropellet, tablet, or minitablet.

48. The pharmaceutical composition of claim 47, wherein the composition is formulated as a liquid, powder, sachet, or tablet.

49. The pharmaceutical composition of any one of claims 33-48, wherein the composition has a shelf-life at room temperature of at least 3 months, 6 months, 9 months, 12 months, 15 months, 18 months, 21 months, 24 months, 36 months, 48 months, 60 months, 72 months, 84 months, 96 months, 108 months, or 120 months.

50. A method of treating sucrase-isomaltase deficiency (e.g., congenital sucrase-isomaltase deficiency (CSID) or acquired sucrase-isomaltase deficiency (ASID)) in a subject in need thereof, the method comprising orally administering to the subject an effective amount of the pharmaceutical composition of any one of claims 33-49.

51. The method of claim 50, wherein the pharmaceutical composition is administered to the subject together with a meal or snack.

52. A method of reducing sucrose and branched (1–6 linked) α-limit dextrin concentration in a subject, the method comprising orally administering to the subject an effective amount of the pharmaceutical composition of any one of claims 33-49.

53. The method of claim 52, wherein the method reduces sucrose and branched (1–6 linked) α-limit dextrin concentration in the subject as measured by a hydrogen breath test.

54. A method of treating sucrase-isomaltase deficiency (e.g., congenital sucrase-isomaltase deficiency (CSID) or acquired sucrase-isomaltase deficiency (ASID)) in a subject in need thereof, the method comprising orally administering to the subject an invertase enzyme of any one of claims 1-27 and optionally an isomaltase enzyme.

55. The method of claim 54, wherein the isomaltase is the recombinant mutant isomaltase of any one of claims 39-43.

56. The method of any one of claims 50-55, wherein the subject is a mammal.

57. The method of any one of claims 50-56, wherein the subject is a human. 

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