Biocatalysts for Organic Synthesis

Abstract

The present invention relates to a biocatalyst for organic synthesis, comprising a controlled porosity silica (CPS) as a support material, wherein the pore diameter is from about 20 to about 100 nm, the support material comprises an amino-functionalized surface, and one or more catalytically active enzymes immobilized on the support material. The present invention also relates to the use of such biocatalysts and a method for their production.

Description

Technical Field

[0001] The present invention relates to a biocatalyst for organic synthesis, in which an enzyme is immobilized on controlled porosity silica having an amino-modified surface. The present invention also relates to the use of such a biocatalyst and a method for producing such a biocatalyst.

Background Art

[0002] Enzymes act as biological catalysts in the metabolism of all living cells. Therefore, enzymes can convert organic molecules into different molecules. Due to the specific three-dimensional structure of each particular enzyme, only specific organic molecules interact with the active site of the enzyme such that conversion can occur. Thus, enzymes are usually highly selective catalysts, and for that reason, the use of enzymes as catalysts in synthetic organic chemistry is extremely attractive. However, since enzymes are biological molecules that have evolved for the cellular environment, they are often not suitable for other environments. When used in organic solvents, enzymes tend to aggregate and often precipitate or unfold (i.e., denature). Therefore, immobilizing an enzyme on a solid support and using it as a catalyst in this immobilized state is attractive because it improves the stability of the enzyme, allows reaction conditions that are normally not tolerated by the enzyme, and further facilitates separation from the reaction mixture and recovery of the material. Immobilization also allows the use of immobilized enzymes at much higher concentrations than are possible using free enzymes in solution.

[0003] Desirable industrial-scale synthetic methods using biocatalysts require immobilizing the enzyme on a suitable support and packing it into a fixed-bed reactor suitable for operation in continuous flow mode. This poses additional challenges to the support material used for immobilization in order to withstand pressure and allow sufficient flow rates. Immobilization of enzymes on solid supports has been achieved previously using various techniques and various solid supports.

[0004] PCT / SE2015 / 050108 discloses a biocatalyst by metal chelation, immobilized on a porosity-controlled glass bead or a porous organic bead as a support material by an enzyme. The porosity-controlled glass functions well in terms of activity, but the support material is expensive and brittle.

[0005] Nagy et al. (ChemCatChem 2018, 10, 3490 - 3499) disclose the covalent immobilization of acid phosphatase on silica nanoparticles for biocatalytic applications. For continuous flow applications, due to an exorbitantly high back pressure, the biocatalytic nanoparticles were mixed with large particles of inert silica.

[0006] JP 2002176974 discloses R - hydroxynitrile lyase immobilized by physical adsorption on a porous clay-based sintered carrier or a porous silica-based carrier. The carrier was not surface-modified. In particular, the covalent cross-linking resulted in a significant loss of activity.

[0007] Trevisan et al. (Braz. J. Chem. Eng. 17(1) Mar 2000) disclose a method for preparing silica with a controlled pore diameter for use as a support for enzyme immobilization. The surface was aminopropyl-modified and aminoglucosidase was bound to the surface using glutaraldehyde.

[0008] Nagy et al. (Periodica Polytechnical Chemical Engineering 2019, vol. 63(3), pages 414 - 424) disclose the immobilization of Burkholderia cepacia lipase on hollow silica microspheres (M540) by bisepoxide activation.

[0009] Nagy et al (ChemSusChem 2021, pages 1 - 10 and supporting information pages 1 - 12) disclose the covalent immobilization of lipase B from Candida antarctica (CaLB) onto various functionalized silica nanoparticles. Prior art documents Patent documents Patent document 1 PCT / SE2015 / 050108 Patent document 2 JP 2002176974 Non - patent documents Non - patent document 1 Nagy et al. (ChemCatChem 2018, 10, 3490 - 3499) Non - patent document 2 Trevisan et al. (Braz. J. Chem. Eng. 17(1) Mar 2000) Non - patent document 3 Nagy et al. (Periodica Polytechnical Chemical Engineering 2019, vol.63(3), pages 414 - 424) Non - patent document 4 Nagy et al (ChemSusChem 2021, pages 1 - 10 and supporting information pages 1 - 12)

Summary of the invention

[0010] Accordingly, an object of the present invention is to provide improved and / or alternative means and methods for performing biocatalysis.

Brief description of the drawings

[0011]

Figure 1

Figure 2

Figure 3

Mode for Carrying Out the Invention

[0012] Definitions The term multi-step synthesis refers to a reaction in which a starting material is converted to a product via at least one intermediate. As used in this context, the terms "step" or "steps" refer to one or more of the individual steps that together form all of the steps of a multi-step synthesis. Thus, a "step" of a multi-step synthesis can refer to the conversion of a starting material to a first intermediate, the conversion of one intermediate to the next intermediate, or the conversion of the last intermediate to the product. A multi-step synthesis can be a linear synthesis in which a single starting material is converted to a final product in a linear series of consecutive steps, or a convergent synthesis in which two or more starting materials are converted to a single final product in a series of convergent steps. Each individual enzyme-catalyzed step of the synthesis is catalyzed by at least one of the enzymes immobilized on a support material. Any concurrent reaction that is catalyzed by the immobilized enzyme but does not directly involve the conversion of either the starting material or an intermediate to the product (e.g., a reaction involving the regeneration of a cofactor or coenzyme) is not considered a "step" in the multi-step synthesis herein.

[0013] The term surface as used herein in connection with the biocatalysts of the present invention refers to the entire surface of a porosity-controlled silica (CPS) support material, including the outer surface and the surfaces of the pores. Due to the nature of CPS, most of the surface area is present within the pores.

[0014] The term "sequence identity" as a percentage is defined as a value determined by comparing two optimally aligned sequences over a comparison window, where a portion of the sequences within the comparison window may include additions or deletions (i.e., gaps) as compared to the reference sequence (excluding additions or deletions) for an optimal alignment of the two sequences. This percentage is calculated by determining the number of positions in both sequences where identical amino acid residues exist, obtaining the number of matched positions, dividing the number of matched positions by the total number of positions within the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Unless otherwise indicated, the comparison window is the full length of the reference sequence. In this regard, the optimal alignment is an alignment created by the BLASTP algorithm, which is performed online by the National Center for Biotechnology Information (NCBI Handbook, 2 nd edition [https: / / www.ncbi.nlm.nih.gov / books / NBK143764 / ] (see) using the following input parameters: word length = 3, matrix = BLOSUM62, gap cost = 11, gap extension cost = 1.

[0015] As used herein, the term "about" refers to a value or parameter of this specification that includes (and describes) embodiments directed to the value or parameter itself. For example, a recitation of "about 20" includes a recitation of "20". Numerical ranges include the numbers defining the range. Generally speaking, the term "about" refers to any value of a variable that is either within the experimental error of the indicated value (e.g., within the 95% confidence interval of the mean) or within 10% of the indicated value, whichever is greater.

[0016] The term "continuous flow reactor" refers to a reactor designed to be operated such that one or more reactants are supplied to the reactor substantially continuously in a feed stream, whereby a substantially continuous product stream containing at least one reaction product emerges from the reactor. During operation, at least a portion (preferably all) of the product stream is collected and optionally may be subjected to further processing steps such as purification steps or a second reactor. Any portion of the product stream that is not collected may be recycled to the reactor as part of the feed stream.

[0017] The present invention relates to the following items. The subject matter disclosed in the following items should be regarded as disclosed as if it were disclosed in the claims.

[0018] 1.a. Porosity-controlled silica (CPS) as a support material having a pore diameter of about 20 to about 100 nm, wherein the support material comprises an amino-functionalized surface, b. One or more catalytically active enzymes immobilized on the amino-functionalized surface by a covalent linker comprising a bond selected from amino, amide and imidoamide, comprising a biocatalyst for organic synthesis.

[0019] 2.a. Porosity-controlled silica (CPS) as a support material having a pore diameter of about 20 to about 100 nm, wherein the support material comprises an amino-functionalized surface, b. One or more catalytically active enzymes immobilized on the surface via non-covalent interactions, comprising a biocatalyst for organic synthesis.

[0020] 3.a. Porosity-controlled silica (CPS) as a support material having a pore diameter of about 20 to about 100 nm, wherein the support material surface comprises at least two different coatings, at least one of which provides an amino-functionalized surface, b. Optionally, one or more catalytically active enzymes immobilized on the surface by a covalent linker containing a bond selected from amino, amide, and imidoamide bonds, A biocatalyst for organic synthesis.

[0021] 4.a. Porosity-controlled silica (CPS) as a support material having a pore diameter of about 20 to about 100 nm, wherein the support material includes an amino-functionalized surface, b. Optionally, one or more catalytically active enzymes immobilized on the amino-functionalized surface by a covalent linker containing a bond selected from amino, amide, and imidoamide bonds. The enzyme is selected from List (A), preferably from List (B). A biocatalyst for organic synthesis.

[0022] 5. The biocatalyst according to claim 1, 3, or 4, wherein the covalent linker, when present, includes a bond selected from the group consisting of amino and amide bonds.

[0023] 6. The biocatalyst according to any of the preceding items, wherein the support material has a pore diameter of about 20 to about 60 nm.

[0024] 7. The support material has a surface area of about 50 m 2 / g to about 200 m 2 / g. The biocatalyst according to any of the preceding items.

[0025] 8. The support material has a pore volume of about 0.5 mL / g to about 2.0 mL / g. The biocatalyst according to any of the preceding items.

[0026] 9. The support material has a pore diameter of about 20 to about 60 nm and a surface area of about 50 m 2 / g to about 200 m 2 / g. The biocatalyst according to any of the preceding items.

[0027] 10. The support material has a pore diameter of about 20 to about 60 nm and a surface area of about 50 m 2 / g ~ about 200 m 2 A biocatalyst according to any of the preceding items, having a surface area of / g and a pore volume of about 0.5 mL / g to about 2.0 mL / g.

[0028] 11. A biocatalyst according to item 1, 2, 3 or any item subordinate thereto, wherein the enzyme is selected from list (A), preferably from list (B).

[0029] 12. The support material has a pore diameter of about 20 to about 60 nm and a surface area of about 50 m 2 / g ~ about 200 m 2 / g, and a pore volume of about 0.5 mL / g to about 2.0 mL / g, and the enzyme is a biocatalyst according to any of items 1 to 9, selected from list (A), preferably from list (B).

[0030] 13. A biocatalyst according to any of the preceding items, wherein the surface of the support material includes at least two different coatings, at least one of which provides amino functionalization.

[0031] 14. A biocatalyst according to item 13, wherein the relative amounts of the two coatings are in the range of 9:1 to 1:9, preferably 5:1 to 1:5, more preferably 2:1 to 1:2, and most preferably 1:1 by weight.

[0032] 15. A biocatalyst according to any of items 13 to 14, wherein one of the coatings that does not provide amino functionalization provides a hydrophobic coating.

[0033] 16. A biocatalyst according to item 15, wherein the hydrophobic coating includes an alkyl moiety and / or an aromatic moiety.

[0034] 17. A biocatalyst according to item 16, wherein the hydrophobic coating includes a C 1-12 alkyl group, preferably a C 1-6 alkyl group.

[0035] 18. A biocatalyst according to any of the preceding items, wherein the immobilized enzyme is covalently bonded intermolecularly by a linker containing a bond selected from amino, amide, ester, ether, thioester, imidoamide, imidothioamide, thioether and thioamide.

[0036] 19. A biocatalyst according to item 3, 4 or any item dependent thereon, wherein the enzyme is immobilized on the surface by a covalent linker containing a bond selected from amino, amide and imidoamide.

[0037] 20. The enzyme is immobilized on the surface by a covalent linker containing a bond selected from amino, amide and imidoamide, and before the formation of the bond, the enzyme was immobilized on the surface via non-covalent interactions, A biocatalyst according to item 1, 3, 4 or any item dependent thereon.

[0038] 21. A biocatalyst according to item 20, wherein before the formation of the bond, the enzyme was immobilized on the surface via an interaction mediated by a chelating metal ion.

[0039] 22. A biocatalyst according to item 2, 3, 4 or any item dependent thereon, wherein the enzyme is immobilized on the surface via an interaction mediated by a chelating metal ion.

[0040] 23. The chelating metal ion is Ni 2+ , Cu 2+ , Mg 2+ , Fe 3+ and Zn 2+ A biocatalyst according to item 21 or 22, selected from.

[0041] 24. The metal ion is Zn 2+ ions, a biocatalyst according to item 23.

[0042] 25. A biocatalyst according to any of the preceding items, wherein the covalent linker between the surface and the enzyme, if present, does not contain an imine bond.

[0043] 26. If there is an intermolecular covalent linker, the biocatalyst according to any of the preceding items that does not contain an imine bond.

[0044] 27. If there is a covalent linker, the biocatalyst according to any of the preceding items that contains a crosslink containing 3 to 20 atoms including any combination of C, H, N, and O.

[0045] 28. If there is a covalent linker, the biocatalyst according to any of the preceding items formed using a bifunctional reagent preferably containing at least two reactive groups independently selected from epoxides, esters, anhydrides, N-hydroxysuccinimide esters, imido esters, carbonates, acyl isoureas, carbodiimides, maleimides, haloacetyls, thiosulfonates, isocyanates, and vinyl sulfones.

[0046] 29. The amino-functionalized surface contains a structure of formula (I), (II), or (III):

Chemical formula

[0047] 30. The amino-functionalized surface, in combination with the structure of formula (IV), comprises the structure of formula (I) as defined above:

Chemical formula

[0048] 31. The amino functional group of the surface is not part of a heterocyclic ring, a biocatalyst according to any of the preceding items.

[0049] 32. The surface contains no functional groups capable of chelating metal ions except for amino groups, a biocatalyst according to any of the preceding items.

[0050] 33. The surface comprises an amino-functionalized aliphatic moiety, a biocatalyst according to any of the preceding items.

[0051] 34. The amino-functionalized surface comprises any of the following structures, each W being hydrogen or a covalent linker containing a bond selected from amino, amide and imidoamide, and the catalytically active enzyme is also bound to the linker, a biocatalyst according to any of the preceding items:

Chemical formula

[0052] 35. The amino-functionalized surface comprises structure (I’):

Chemical formula

Chemical formula

[0053] 36. The amino-functionalized surface, where R 4 is W or is phenyl, benzyl, amino-C 2-6 -alkyl, N-(phenyl)amino-C 2-6Alkyl, and N-(benzyl)amino-C 2-6 comprises a structure I' selected from the group consisting of alkyl, or the amino-functionalized surface is R 4 is a biocatalyst according to item 34, comprising a structure I" where W is

[0054] 37. A biocatalyst according to any of the preceding items, wherein the amino-functionalized surface comprises any of the following structures, each W is a covalent linker containing a bond selected from hydrogen, or amino, amide, and imidoamide, and the catalytically active enzyme is also bound to the linker: [Chemical formula]

[0055] 38. A biocatalyst according to any of the preceding items, wherein the amino-functionalized surface comprises any of the following structures, each W is a covalent linker containing a bond selected from hydrogen, or amino, amide, and imidoamide, and the catalytically active enzyme is also bound to the linker: (1) [Chemical formula] (2) [Chemical formula] (3) [Chemical formula] (4) [Chemical formula]

[0056] 39. The immobilized enzyme is a biocatalyst according to any of the preceding items selected from Bifidobacterium adolescentis sucrose phosphorylase (SucP), Candida antarctica lipase (CalB), Thermoanaerobacter brockii secondary alcohol dehydrogenase (TbSADH), aminotransaminase (ATA), Leuconostoc mesenteroides glycosyltransferase (GT), Thermomyces lanuginosus lipase (TLL), and Aspergillus niger amyloglucosidase.

[0057] 40. The biocatalyst according to any of the preceding items, wherein at least one immobilized enzyme retains at least 20% of its activity after immobilization as compared to its state before immobilization.

[0058] 41. The biocatalyst according to any of the preceding items, wherein at least one immobilized enzyme retains at least 50% of its activity after 20 hours under continuous flow conditions as compared to its state before immobilization.

[0059] 42. The biocatalyst according to any of the preceding items, wherein at least two different enzymes are immobilized on a support material.

[0060] 43. The biocatalyst according to item 42, wherein at least two immobilized enzymes can each catalyze different steps of a multi-step organic synthesis.

[0061] 44. The biocatalyst according to item 43, wherein the multi-step synthesis includes at least three intermediates.

[0062] 45. The biocatalyst according to any of the preceding items, wherein at least one of the immobilized enzymes catalyzes the regeneration of a cofactor.

[0063] 46. A lipase (preferably CalB) containing a metal chelate tag such as a His tag is immobilized on a surface containing an amino-functionalized aliphatic moiety via an interaction mediated by chelated Zn 2+ ion, the biocatalyst according to any one of items 1 to 45.

[0064] 47. TbSADH containing a metal chelate tag such as a His tag is immobilized on a surface via an interaction mediated by chelated Zn 2+ ion, the biocatalyst according to any one of items 1 to 45.

[0065] 48. ATA containing a metal chelate tag such as a His tag is immobilized on a surface containing structure (1), (2), (3) or (4) via an interaction mediated by chelated metal ions, the biocatalyst according to any one of items 1 to 45.

[0066] 49. SucP containing a metal chelate tag such as a His tag is immobilized on a surface containing structure (2), the biocatalyst according to any one of items 1 to 45.

[0067] 50. TLL not containing a metal chelate tag such as a His tag is immobilized on a surface, the biocatalyst according to any one of items 1 to 45.

[0068] 51. A glucosyltransferase containing a metal chelate tag such as a His tag is immobilized on a surface containing structure (1), the biocatalyst according to any one of items 1 to 45.

[0069] 52. Lipase CalB is immobilized on a surface containing structure (1) or (4), preferably non-covalently, and CalB most preferably does not contain a metal chelate tag such as a His tag, the biocatalyst according to any one of items 1 to 45.

[0070] An amyloglucosidase preferably free of metal chelate tags such as His-tags is functionalized by structure (1), (2), (3) or (4) and optionally immobilized on a surface having a different second functionalization which can be structure (1) or an alkyl such as propyl or octyl, a biocatalyst according to any of items 1 to 45.

[0071] 54. Use of a biocatalyst according to any of the preceding items in a reactor for the synthesis of an organic compound.

[0072] 55. Use according to item 54, wherein the reactor is a batch reactor or a continuous flow reactor.

[0073] 56. Use according to item 54, wherein the reactor is a continuous stirred tank reactor (CSTR), a slurry bubble column or a fixed bed reactor.

[0074] 57. Use according to item 55, wherein the reactor is a continuous flow fixed bed reactor.

[0075] 58. Use according to any of items 54 to 57, wherein the synthesis is carried out under aqueous conditions.

[0076] 59. Use according to any of items 54 to 57, wherein the synthesis is carried out in an organic solvent.

[0077] 60. Use according to any of items 54 to 57, wherein the synthesis is carried out in a neat substrate.

[0078] 61. Use according to any of items 54 to 60, wherein the synthesis is a multi-step synthesis.

[0079] 62. Use according to item 61, wherein the multi-step synthesis comprises at least three intermediates.

[0080] 63. Use according to item 61 or 62, wherein the multi-step synthesis is a linear synthesis.

[0081] 64.a.Providing a biocatalyst according to any one of items 1 to 53, disposed within a reactor; b.Supplying a precursor to an organic compound to a flow reactor, whereby an immobilized enzyme catalyzes the reaction to result in the synthesis of an organic compound; and c.Recovering the organic compound, A method for the synthesis of an organic compound, comprising.

[0082] 65.The method according to item 54, wherein the reactor is a batch reactor or a continuous flow reactor.

[0083] 66.The method according to item 65, wherein the reactor is a continuous stirred tank reactor (CSTR), a slurry bubble column or a fixed bed reactor.

[0084] 67.The method according to item 65, wherein the reactor is a continuous flow fixed bed reactor.

[0085] 68.The method according to any one of items 64 to 67, wherein the reaction is carried out under aqueous conditions.

[0086] 69.The method according to any one of items 64 to 67, wherein the reaction is carried out in an organic solvent.

[0087] 70.The method according to any one of items 64 to 67, wherein the reaction is carried out in a pure substrate.

[0088] 71.The method according to any one of items 64 to 70, wherein the synthesis is a multi-step synthesis comprising at least two reaction steps.

[0089] 72.The method according to item 71, wherein the multi-step synthesis comprises at least three intermediates.

[0090] 73.The method according to item 71 or 72, wherein the multi-step synthesis is a linear synthesis.

[0091] 74.a.Providing one or more catalytically active enzymes; b. providing a porosity-controlled silica (CPS) as a support material, wherein the pore diameter is from about 20 to about 100 nm, the support material comprises an amino-functionalized surface, and c. immobilizing one or more enzymes on the support material, A method for producing a biocatalyst according to any one of items 1 to 53, comprising:

[0092] 75. selecting a bifunctional crosslinking reagent capable of forming an amino bond, an amide bond or an imidoamide bond when reacted with an enzyme and / or a surface, and covalently crosslinking the enzyme using the bifunctional reagent, The method according to item 74, further comprising:

[0093] 76. The method according to item 75, wherein the bifunctional crosslinking reagent comprises at least two reactive groups independently selected from epoxides, esters, anhydrides, N-hydroxysuccinimide esters, imido esters, carbonates, acyl isoureas, carbodiimides, maleimides, haloacetyls, thiosulfonates, isocyanates and vinyl sulfones.

[0094] 77. The bifunctional crosslinking reagent is defined by the following formula:

Chemical formula

[0095] 78. The method according to item 77, wherein Y and Z are each independently an epoxide, an N-hydroxysuccinimide ester or an imido ester, preferably Y = Z.

[0096] 79. The method according to item 77, wherein the bifunctional reagent is a bisepoxide reagent, a bifunctional sulfonated N-hydroxysuccinimide ester or a bifunctional imide ester.

[0097] 80. The reagent is a bisepoxide defined by the following formula:

Chemical formula

[0098] 81. The method according to item 77, wherein the reagent is GDE, NPE, PDE, BS3 or DMS.

[0099] 82. The method according to item 77, wherein the bifunctional reagent is a bifunctional sulfonated N-hydroxysuccinimide ester reagent or a bifunctional imide ester.

[0100] 83. The method according to item 82, wherein the reagent is bis(sulfosuccinimidyl)suberate (BS3) or suberimidic acid dimethyl ester (DMS).

[0101] 84. The method according to any one of items 75 to 83, wherein the bifunctional reagent is contacted with the support material before immobilizing the enzyme.

[0102] 85. The method according to any one of items 75 to 84, wherein the bifunctional reagent is contacted with the support material after immobilizing the enzyme.

[0103] 86. The enzyme is a chelating metal ion, preferably Ni 2+ , Cu 2+ , Mg 2+ , Fe 3+ or Zn 2+ , most preferably Zn 2+A method according to item 75 or any item dependent thereon, immobilized on a surface via an interaction mediated by

[0104] 87. A method according to item 75 or any item dependent thereon, wherein at least one of the enzymes comprises a polyhistidine tag.

[0105] 88. A method according to item 75 or any item dependent thereon, wherein the enzyme is selected from list (A), more preferably from list (B).

[0106] 89. A method according to item 75 or any item dependent thereon, wherein at least one of the enzymes is provided in a cell lysate.

[0107] 90. A method according to item 75 or any item dependent thereon, wherein at least one of the enzymes is provided as at least 70% by weight of the total protein in the preparation or as a preparation containing the enzyme.

[0108] The present invention is based on the unexpected discovery that amino-functionalized controlled porosity silica (CPS) is widely useful as a support material for immobilizing enzymes for synthesis and provides better results compared to commercially available support materials (Examples 1-4, 6). The amino-functionalization of the CPS support enables the immobilization of many different types of enzymes with high enzyme loading, high activity retention, and high stability, and is applicable to both His-tagged and non-His-tagged enzymes. The industrial applicability of the amino-functionalized CPS support was further demonstrated in a continuous flow setting (Examples 5, Figure 1).

[0109] In Example 7, various crosslinking reagents were tested using SucP2 immobilized on amino-functionalized CPS as a model, and the superiority of crosslinking agents that provide bonds selected from amino, amide, and imidoamide was demonstrated compared to glutaraldehyde, which provides an imine bond.

[0110] The usefulness of using two different coatings on CPS for enzyme immobilization was examined in Example 8, which provides the ability to adjust the functional groups on the surface.

[0111] The results of Example 9 compared CPS Q30 particles of different sizes (75 - 150 μm, 280 - 500 μm, and 1.18 - 2.36 mm), and even larger categories are feasible, which enables implementation in specific applications that require even higher flow rates or three-phase reactions.

[0112] Example 10 compared two different CPS silicas (Cariact Q30 and Ecovyst) functionalized with propylamine using three different application methods, showing that immobilization with similar performance can be achieved using several different methods and CPS sources.

[0113] Additional advantages may include that the cost of CPS is much lower than other supports such as controlled porosity glass (CPG). Compared to silica nanoparticles, CPS provides beneficial flow characteristics in a packed bed reactor, such as low backpressure at high flow rates. Compared to larger-sized non-porous silica particles, the CPS surface area is much larger, enabling a significantly higher enzyme immobilization yield per weight of the support material. In addition, the CPS support material utilized in the present invention is specially designed for use as a catalyst support, and mechanical properties such as crush strength, abrasion, and wear resistance are characteristics of this material, which means that the catalyst can be utilized in other reactor types such as batch reactors or continuous stirred tank reactors or bubble column reactors. However, despite these attractive features of the catalyst support, the silica surface is known to cause enzyme inactivation, and to date, a high enzyme loading that can maintain a high level of activity on the support surface has not been possible with silica supports, so the silica surface is not generally utilized for enzyme catalysts.

[0114] The amino-functionalized CPS support material is chemically stable, which means that it can be utilized under a wide range of reaction conditions, such as in aqueous solutions, reactions in organic solvents, and reactions without solvents, for example, pure reaction substrates or liquid molten solids.

[0115] Generally, in combination with low cost, a particular balance of properties such as surface area, pore volume, pore diameter, amino-functionalization, chemical stability, and mechanical strength provides an excellent material for biocatalytic applications.

[0116] A further feature of the amino-functionalized CPS material is its ability to facilitate the formation of covalent bonds between the immobilized enzyme and the amino-functionalized coating on the CPS support material. The presence of covalent bonds can be particularly beneficial in reaction media that normally contain components that leach enzymes from the amino-functionalized support material. Furthermore, covalent bonds can be introduced without loss of enzyme activity. This is an important advantage compared to support materials that do not contain an amino-functionalized surface coating.

[0117] Biocatalysts for organic synthesis In a first aspect, the present invention relates to a. Porosity-controlled silica (CPS) as a support material having a pore diameter of about 20 to about 100 nm, wherein the support material comprises an amino-functionalized surface, b. One or more catalytically active enzymes immobilized on the amino-functionalized surface by a covalent linker comprising a bond selected from amino, amide, ester, ether, imidoamide, imidothioamide, thioether, thioester, and thioamide, and relates to a biocatalyst for organic synthesis.

[0118] In a second aspect, the present invention relates to a. Porosity-controlled silica (CPS) as a support material having a pore diameter of about 20 to about 100 nm, wherein the support material comprises an amino-functionalized surface, b. One or more catalytically active enzymes immobilized on the surface via non-covalent interactions, relating to a biocatalyst for organic synthesis.

[0119] In a third aspect, the present invention a. Porosity-controlled silica (CPS) as a support material having a pore size of about 20 to about 100 nm, wherein the surface of the support material comprises at least two different coatings, at least one of which provides an amino-functionalized surface, b. Optionally, one or more catalytically active enzymes immobilized on the surface by a covalent linker comprising a bond selected from amino, amide, ester, ether, thioether, imidoamide, imidothioamide, thioester and thioamide, relating to a biocatalyst for organic synthesis.

[0120] In a fourth aspect, the present invention a. Porosity-controlled silica (CPS) as a support material having a pore size of about 20 to about 100 nm, wherein the support material comprises an amino-functionalized surface, b. Optionally, one or more catalytically active enzymes immobilized on the amino-functionalized surface by a covalent linker comprising a bond selected from amino, amide, ester, ether, thioether, imidoamide, imidothioamide, thioester and thioamide, and the enzyme is selected from the list (A) disclosed below, preferably from the list (B), relating to a biocatalyst for organic synthesis.

[0121] Preferably, the covalent linkers of the first, third and fourth aspects, when present, comprise bonds selected from the group consisting of amino, amide, ester, ether, thioether, thioester and thioamide.

[0122] More preferably, the covalent linkers of the first, third, and fourth aspects, when present, include bonds selected from the group consisting of amino, amide, thioether, thioester, and thioamide. Even more preferably, the covalent linkers of the first, third, and fourth aspects, when present, include bonds selected from the group of amino, amide, or thioamide. Even still more preferably, the covalent linkers of the first, third, and fourth aspects, when present, include bonds selected from the group consisting of amino, amide, and imidoamide.

[0123] Most preferably, the covalent linkers of the first, third, and fourth aspects that immobilize the enzyme on the surface, when present, include bonds selected from amino bonds and amide bonds.

[0124] Without being bound by theory, the covalent linker between the amino-functionalized surface and the enzyme contains (i) a bond formed by a reaction between the amino-functionalized surface and a functional group on the crosslinking reagent used to form the covalent linker, (ii) a bond formed by a reaction between a reactive functional group in the enzyme and a functional group on the crosslinking reagent, and (iii) optionally, any spacer portion present between the functional groups of the crosslinker, preferably containing 3 to 20 atoms including any combination of C, N, H, and O. The bond called (i) preferably includes a bond selected from the group consisting of amino, amide, and imidoamide. The bond called (ii) may include a bond selected from the group consisting of amino, amide, ester, ether, thioether, imidoamide, imidothioamide, thioester, and thioamide.

[0125] Optionally, the immobilized enzyme may be covalently linked intermolecularly by a linker containing a bond selected from amino, amide, ester, ether, thioester, imidoamide, imidothioamide, thioether and thioamide. These bonds are formed by the reaction between a reactive functional group in the enzyme and a functional group on the crosslinking reagent. The intermolecular linker may optionally include a spacer portion present between the functional groups of the crosslinking agent, preferably containing 3 to 20 atoms including any combination of C, N, H and O.

[0126] The amino-functionalized surface contains primary, secondary or tertiary amines.

[0127] The details and preferred features of the biocatalyst are described below.

[0128] Support material The support material according to the first aspect of the present invention is a porosity-controlled silica (CPS) available with several ranges of pore diameters. The untreated silica (SiO 2 ) surface is covered with hydroxyl groups (-OH), but the support material of the present invention is amino-functionalized as described in more detail below.

[0129] The ideal pore-controlled silica material for the present invention is specially designed for use as a catalyst support, for which mechanical properties such as crush strength, abrasion and wear resistance are required characteristics for performance as a catalyst support. The pore-controlled silica material is chemically and thermally stable and also provides a precisely controlled pore diameter and pore size distribution. The particle size and particle size range can be adjusted and optimized according to various industrial reactor types. For example, they can be deployed in a batch reactor or a continuous stirred tank reactor (CSTR) as a slurry catalyst or a slurry bubble column or a fixed bed reactor or a known variant thereof. This is advantageous compared to pore-controlled glass materials that are known to be brittle, prone to abrasion or wear when stirred in a batch reactor or CSTR.

[0130] A suitable pore diameter is an important feature of the support. Pores of a suitable size provide a favorable microenvironment without the complexity due to steric hindrance. The pore structure reduces the solution flow resistance and facilitates the mass transfer of reactants and products throughout the material. The robust non-encapsulated structure of the CPS provides a sturdy non-compressible medium suitable for high-throughput reactor design and linear scale-up at high flow rates. This material exhibits limited swelling in solvents and is chemically and dimensionally stable in most organic media and aqueous environments at pH less than 10.

[0131] Due to the physical properties of the porosity-controlled silica, the porosity-controlled silica becomes suitable for immobilizing enzymes on the carrier surface with a high enzyme loading. Generally, the pore-controlled silica material suitable for the present invention has a pore diameter of about 20 nm to about 100 nm, preferably about 20 to about 60 nm, more preferably about 30 to about 50 nm. Preferably, the surface area is about 50 m 2 / g to about 200 m 2 / g (preferably more than about 60 m 2 / g, for example, more than about 70 m 2 / g). Also, preferably, the pore volume is about 0.5 mL / g to about 2.0 mL / g (more preferably at least about 0.6 mL / g, even more preferably at least about 0.7 mL / g, and most preferably about 0.8 mL / g to about 1.5 mL / g). These properties enable the immobilization of enzymes on the surface of the carrier with a high loading. Preferably, the pore diameter of the support material is about 20 to about 60 nm, and the surface area is about 50 m 2 / g to about 200 m 2 / g. Even more preferably, the support material has a pore diameter of about 20 to about 60 nm, a surface area of about 50 m 2 / g to about 200 m 2 / g, and a pore volume of about 0.5 mL / g to about 2.0 mL / g. Most preferably, the support material has a pore diameter of 20 to 60 nm, a surface area of 50 m 2 / g to 200 m 2 / g, and a pore volume of 0.5 mL / g to 2.0 mL / g. The ranges disclosed herein include the endpoints.

[0132] The CPS support exhibits an enzyme loading capacity that is inversely proportional to its pore diameter. Therefore, a CPS support with a large pore diameter cannot load as much protein as a CPS support with a small pore diameter, which is mainly due to the inverse relationship between the pore diameter and the surface area (i.e., less available surface area for enzyme immobilization).

[0133] The preferred particle size depends on the type of reactor in which the immobilized biocatalyst is deployed. For example, a small particle size, e.g., 75 - 150 μm, is well - suited for the deployment of a batch reactor or a continuous stirred - tank reactor where the immobilized biocatalyst acts as a catalyst slurry in the reactor. When deployed in a fixed - bed reactor, the preferred particle size depends on the chemical reactions and conditions within the reactor. In a liquid - phase fixed - bed reaction, small particles, e.g., 75 - 150 μm, are preferred, but in certain other liquid - phase reactions, as well as in reactions where gases and liquids are deployed together in a fixed - bed reactor, a large particle size, e.g., greater than 200 μm, preferably greater than 500 μm, can be deployed.

[0134] Certain types of preferred porous silica materials are commercially available. For example, the pore - controlled silica of the CARiACT™ class is available from Fuji Silysia Chemical Ltd (https: / / www.fujisilysia.com / products / cariact / ). These inorganic porous silicas can be produced to have various surface areas and pore diameters. Surprisingly, these materials have been found to be advantageous for immobilizing enzymes on the surface of the carrier with a high enzyme loading. The properties of Fuji CARiACT Q - 30 and Q - 50 make these materials particularly advantageous. The materials of Fuji CARiACT Q - 20C, Q30C, and Q40C are also suitable. According to the manufacturer, the particles have the following characteristics:

Table 1

[0135] Another suitable commercially available material is Ecovyst E30. The particles have the following characteristics:

Table 2

[0136] The porosity-controlled silica of the present invention can be prepared according to the methods described in JP-A-0930809 and JP-A-2020001936. Both methods describe in detail a production method that modifies the properties of silica to provide sufficient control to produce a porosity-controlled silica having the required balance of properties such as the surface area, pore volume, pore diameter, and particle size specified above. In addition, these production methods produce a porosity-controlled silica having the mechanical strength necessary to enable the features of the present invention. Generally, two raw materials, sodium silicate and a mineral acid such as sulfuric acid, are used together in a solution process to produce monomeric silicic acid. The monomeric silicic acid polymerizes to produce primary silica particles called silica sol. These primary particles then aggregate to form a three-dimensional porous structure. The reaction conditions that enable the growth of the primary particles and the conditions for drying the product are used to control and modify physical properties such as the surface area, pore diameter, and pore volume.

[0137] Finally, it should be understood that CPS in the present context is different from the siliceous mesocellular foam (MCF) known in the literature. MCF has a regular 3D cage-like structure with spherical cavities having a diameter of 20 - 40 nm interconnected by pores of a size of about 10 nm, which is smaller than desirable for enzyme immobilization. In contrast, CPS is not a caged structure. In the context of MCF materials, the literature often uses the term pore diameter to define the diameter of the cage, the term pore window to define the cage entrance diameter (or pore diameter), and the window size is typically smaller than the pore diameter. Due to the cage-like structure and small window size, the surface area (m 2 / g) of MCF materials is typically significantly larger than the surface area of CPS (greater than 200 m 2 / g). Since MCF is not currently commercially available on an industrial scale and is prohibitively expensive for industrial applications, it is not a practical alternative to CPS.

[0138] The pore-controlled silica is also different from the hollow microsphere silica described in that document. WO 2013 / 078551 describes hollow microsphere silica as microcapsules containing a silica shell having a thickness of about 50 nm to about 500 μm, the shell having a diameter of about 0.1 μm to about 1500 μm and having a density of about 0.001 g / cm 3 ~ about 1.0 g / cm 3 surrounds a hollow capsule. The hollow characteristics of these materials are clearly shown in FIGS. 2 to 5 of WO 2013 / 078551. These materials are essentially composed of one or more silica-based materials and are surrounded by a shell (generally solid) capped by a functional surface having an affinity or adhesiveness for a matrix of plastic or composite or rubber or fabric, and include a core / g shell / functional surface type reservoir or microcapsule containing a core (gas or hollow). The microcapsules are designed to be introduced into plastic, composite, rubber and fabric products during the processing stage. The gas or hollow microcapsules are dispersed throughout or partially in plastic, composite, rubber and fabric products as density-reducing additives for reducing the density of the final product. The hollow spherical microcapsules of WO 2013 / 078551 are designed as density-reducing additives that can provide the benefits of the material to the formulated product, whereas the pore-controlled silica material of the present invention is specifically designed for use as a catalyst support where mechanical properties such as crush strength, abrasion and wear resistance are required characteristics of the material for performance as a catalyst. Such characteristics would not be provided by such low-density hollow spherical supports having essentially low crush strength. The pore-controlled silica of the present invention is not hollow and includes a three-dimensional silica structure containing accurately controlled pore diameters and pore size distributions throughout the solid, which is well-suited for the efficient and robust synthesis of biocatalysts.

[0139] Immobilized enzyme In principle, any enzyme of interest may be immobilized. Preferably, the enzyme is an esterase, transferase, oxidoreductase, hydrolase, ligase, isomerase or lyase.

[0140] The immobilized enzyme can be selected from the enzymes in List (A) consisting of Items 1 to 62 in Table A below, and enzymatically active sequence variants thereof, preferably variants having similar enzyme activity, most preferably variants having substantially the same enzyme activity.

[0141] It is well known that the amino acid sequence of an enzyme can be significantly changed without significantly affecting enzyme activity. For example, an artificial sequence such as a His tag can be added to enable metal chelation. In other examples, residues that are not important for enzyme activity can be modified to improve properties (such as production stability or efficiency) or simply to provide alternative sequences to avoid patent protection. In some cases, the enzyme sequence can be truncated by omitting portions of the enzyme that are not important for enzyme activity to produce a more compact enzyme. For this reason, enzymatically active sequence variants of the enzymes identified in Table A can also be used and are thus included in List (A).

[0142] Table A: Preferred Immobilized Enzymes

Table 3-1

Table 3-2

[0143] The immobilized enzyme is preferably selected from a list (B) consisting of Thermomyces lanuginosus lipase (TLL, #1 in Table A), Candida antarctica lipase (CalB, #2 in Table A), Bifidobacterium adolescentis sucrose phosphorylase (SucP, #3 in Table A), Thermoanaerobacter brockii secondary alcohol dehydrogenase (TbSADH, #4 in Table A), Arthrobacter sp. aminotransferase (ATA, #60 in Table A), Leuconostoc mesenteroides glucosyltransferase (GT, #18 in Table A), Aspergillus niger-derived amyloglucosidase (#61 in Table A) and aminotransferase (Arthrobacter sp.) (#62 in Table A), and enzymatically active sequence variants thereof, preferably having similar enzymatic activity, most preferably having substantially the same enzymatic activity.

[0144] The enzyme activities relevant to the context of Tables (A) and (B) are shown in Table A by the "EC number" column, which refers to the numerical classification scheme of the Enzyme Commission that classifies enzymes by their catalytic reactions.

[0145] The Uniprot id column in Table (A) refers to the entry on the public Uniprot database of www.uniprot.org as of December 1, 2021. Preferably, each selected enzyme in Table (A) or Table (B) has at least 70% sequence identity, more preferably at least 80%, even more preferably at least 90%, still more preferably at least 95%, and most preferably complete sequence identity with the sequence from the database entry identified by the "Uniprot id" shown in Table A.

[0146] In some embodiments, the enzyme comprises a metal affinity tag, such as a polyhistidine tag (a His tag of 2 to 8 consecutive histidines, preferably 6 histidines), an HQ tag (Godat et al. Promega Notes Number 91 p.17-20), a MAT tag (Watson et al. BioTechniques Vol 42 No 6,2007,p.768) or any other suitable tag known in the art, to facilitate immobilization by metal chelation.

[0147] Amino-functionalized surface The amino-functionalized surface may comprise a structure of formula (I), (II) or (III):

Chemical formula

[0148] In some embodiments, the amino-functionalized surface, in combination with the structure of formula (IV), comprises the structure of formula (I) defined above:

Chemical formula

[0149] Preferably, the amino functional group on the surface is not part of an aromatic heterocycle, and more preferably not part of any heterocycle.

[0150] Preferably, the surface contains no functional groups capable of chelating metal ions other than amino groups.

[0151] More preferably, the surface comprises an amino-functionalized aliphatic moiety.

[0152] In some cases, it is beneficial to have at least two different coatings (at least one of which provides amino-functionalization) on the CPS material. The presence of two different coatings can further optimize the balance of properties suitable for a particular enzyme and result in a more efficient biocatalyst. For example, a particular enzyme may prefer a coating that is inherently highly hydrophobic and may benefit from the presence of amino groups and even metal ions. The presence of at least two coatings, one providing amino-functionalization and the other providing a hydrophobic coating, can accommodate such situations. The hydrophobic coating may include an alkyl moiety and / or an aromatic moiety. Preferably, the hydrophobic coating comprises a C 1-12 alkyl group, preferably a C 1-6 alkyl group and / or a phenyl group.

[0153] For example, one coating may contain an aliphatic chain or phenyl group that provides a hydrophobic environment, and the second coating may contain an amino-functionalized group that may further accommodate metal ions. The relative proportion of the two coatings on the surface can be controlled and optimized to maximize the efficiency of the biocatalyst. The relative amounts of the two coatings can be in the range of 9:1 to 1:9, preferably 5:1 to 1:5, more preferably 2:1 to 1:2, and most preferably 1:1 by weight.

[0154] Furthermore, the presence of the two coatings can benefit the covalent bond between the enzyme and the surface of the CPS material in a preferred situation. The presence of the two coatings enables the adjustment of the surface by the two coatings that can a) provide chemical groups preferred by the enzyme, such as hydrophobic groups like aliphatic chains or phenyl groups, and b) provide amino-functionalized groups that enable covalent bonds between the enzyme and the CPS surface.

[0155] As used herein, the term "C 1-6 alkanediyl" refers to a straight-chain or branched divalent group having 1 to 6 carbon atoms. Examples of C 1-6 alkanediyl include methanediyl, ethanediyl, 1,3-propanediyl, and 2,2-dimethyl-1,4-butanediyl.

[0156] As used herein, the term "C 1-10 alkyl" refers to a straight-chain or branched alkyl group having 1 to 10 carbon atoms, and the term "C 1-4 alkyl" refers to a straight-chain or branched alkyl group having 1 to 4 carbon atoms. Examples of C 1-4 alkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl.

[0157] As used herein, the term "C 5-7"Cycloalkyl" refers to a monocyclic saturated hydrocarbon ring having 5 to 7 carbon atoms. C 5-7 Examples of cycloalkyl include cyclopentyl, cyclohexyl and cycloheptyl.

[0158] As used herein, the term "hydroxy-C 1-4 alkyl" refers to a C 1-4 alkyl group in which one of the hydrogen atoms is replaced by a hydroxy group.

[0159] As used herein, the term "phenyl-C 1-6 alkyl" refers to a C 1-6 alkyl group in which one of the hydrogen atoms is replaced by a phenyl group.

[0160] As used herein, the term "amino-C 2-8 alkyl" refers to a C 2-8 alkyl group in which one of the hydrogen atoms is replaced by an amino group.

[0161] The term "amino" refers to primary, secondary and tertiary amines.

[0162] The term "aminocarbonyl" refers to the group -C(=O)NH 2 .

[0163] As used herein, the terms "N-(phenyl)amino-C 2-8 alkyl" and "N-(phenyl-C 1-6 -alkyl)amino-C 2-8 alkyl" refer to an amino-C 1-6 alkyl group in which one of the hydrogen atoms on the amino group is replaced by a phenyl, or phenyl-C 2-8 alkyl group, respectively.

[0164] Preferably, the amino-functionalized surface comprises any of the following structures, where each W is hydrogen or a covalent linker containing a bond selected from amino, amide, and imidoamide, and the catalytically active enzyme is also bound to the linker:

Chemical formula

[0165] In some embodiments, the amino-functionalized surface comprises structure (I’):

Chemical formula

Chemical formula

[0166] More preferably, the amino-functionalized surface contains any of the following structures, where each W is hydrogen or a covalent linker containing a bond selected from amino, amide and imide amide, and the catalytically active enzyme is also bound to the linker:

Chemical formula

[0167] Most preferably, the amino-functionalized surface contains any of the following structures, where each W is hydrogen or a covalent linker containing a bond selected from amino, amide and imide amide, and the catalytically active enzyme is also bound to the linker: (1) Propylamine

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0168] To avoid misunderstanding, the above names for structures (1)-(4) are derived from the situation where W = H. However, it should be understood that the structure still includes the situation where the covalent linker contains a bond selected from amino, amide, and imidoamide, and the catalytically active enzyme is also bound to the linker.

[0169] Enzyme immobilization In preferred embodiments of the third and fourth aspects, the enzyme is immobilized on the surface by a covalent linker containing a bond selected from amino, amide, and imidoamide, but non-covalent immobilization is also an option.

[0170] In preferred embodiments of the first, third, and fourth aspects, the enzyme is immobilized on the surface by a covalent linker containing a bond selected from amino, amide, and imidoamide. Before the formation of the bond, the enzyme was immobilized on the surface via non-covalent interactions, preferably via interactions mediated by chelated metal ions. Without being bound by theory, the post-non-covalent immobilization may enable more uniform cross-linking formation between the CPS support surface and the enzyme and may improve the retention of activity. In other embodiments, the initial immobilization is via a covalent linker, and optionally followed by intermolecular cross-linking.

[0171] In preferred embodiments of the second, third, and fourth aspects, the enzyme is non-covalently immobilized on the surface via interactions mediated by chelated metal ions.

[0172] The above chelated metal ions are Ni 2+ , Cu 2+ , Mg 2+ , Fe 3+ or Zn 2+ and may be selected from. Preferably, the metal ion is Zn 2+ ions. Such Zn 2+Ionic interactions provide excellent results for certain enzymes such as CalB and TbSADH (see Example 2).

[0173] In certain embodiments of the first, second, third or fourth aspect, the immobilized enzyme is covalently intermolecularly bonded by a linker comprising a bond selected from amino, amide, ester, ether, thioether, imidoamide, imidothioamide, thioester and thioamide.

[0174] Preferably, the covalent linker between the surface and the enzyme, if present, does not contain an imine bond that may be susceptible to cleavage by hydrolysis. Preferably, the intermolecular covalent linker, if present, does not contain an imine bond. Preferably, the covalent linker, if present, comprises a bridge containing 3 to 20 atoms comprising any combination of C, H, N and O.

[0175] In some embodiments, the enzyme comprises a metal affinity tag such as a polyhistidine tag (2 to 8 consecutive histidines, preferably a 6-His tag), an HQ tag (HQHQHQ), a MAT tag (HNHRHKH), or any other suitable tag known in the art to facilitate immobilization by metal chelation. It should be noted that due to the natural metal chelating properties of many enzymes, many industrially relevant enzymes can be immobilized via metal chelation without an affinity tag. However, the advantage of immobilization via metal chelation using an affinity tag is selective enzyme binding compared to physical adsorption, and in addition, the stereochemistry of the immobilized enzyme can be more uniform than in situations where immobilization occurs via non-specific physical adsorption. The affinity tag can also improve the activity after immobilization by promoting immobilization at a controlled position and allowing better and / or more consistent access to the active site. On the other hand, the initial immobilization via a covalent linker can also be applied because it can omit the non-covalent immobilization step, thus obtaining a simplified manufacturing process.

[0176] Covalent crosslinking (between different enzyme molecules and / or between an enzyme molecule and an amino group of an amino-functionalized surface) has certain advantages. Crosslinking allows for better retention of enzyme activity under industrial flow conditions and enables the possibility of recycling and reusing the catalyst in batch operations.

[0177] Crosslinking reagents suitable for the formation of covalent crosslinks are disclosed below.

[0178] Crosslinking reagent Various covalent crosslinking strategies applied to amino-functional groups and enzymes are known in the field of protein chemistry. See, for example, the Crosslinking Technical Handbook published by Thermo Scientific, https: / / tools.thermofisher.com / content / sfs / brochures / 1602163-Crosslinking-Reagents-Handbook.pdf, which is incorporated herein by reference.

[0179] An effective crosslinking reagent for linking an enzyme to the surface of an amino-functionalized support requires at least two (preferably no more than two) reactive groups separated by a linker. An effective crosslinking reagent should contain at least a first reactive group that can react with the amino-functional group bound to the CPS surface to form a stable covalent bond. Furthermore, the crosslinking reagent should contain at least a second reactive group that can react with a functional group present on the enzyme structure to form a stable covalent bond and thus form a covalent bond between the enzyme and the support surface. Alternatively, the reagent can contain at least two reactive groups that can each react with a functional group present on the enzyme structure to form a stable covalent bond and thus form an intermolecular covalent bond between enzyme molecules.

[0180] Functional groups on enzymes suitable for crosslinking include amine groups, carboxylate groups, thiol (sulfhydryl) groups, and hydroxy groups. By appropriate selection of a crosslinking reagent, these functional groups can readily form covalent bonds such as amino, amide, ester, ether, thioether, imidoamide, imidothioamide, thioester, and thioamide. As a specific example, in the case of an epoxide crosslinking reagent, the functional group on the enzyme can react with the epoxide group to generate covalent bonds such as amino bonds, ester bonds, ether bonds, and thioether bonds. For example, in the case of a bisepoxide crosslinking reagent, one of the epoxide groups can react with an amino functional group located on the surface of an amino-functionalized pore control silica support to form an amino covalent bond. The second epoxide group can react with a functional group located on an amino acid residue located on the enzyme. For example, an amino functional group from a lysine amino acid residue can react with the epoxide group to form an amino covalent bond. Alternatively, a thiol functional group from a cysteine amino acid residue can react with the epoxide group to form a thioether covalent bond. In yet a further embodiment, a carboxylate functional group from a glutamate amino acid residue can react with the epoxide group to form an ester covalent bond, or a hydroxyl group from a serine amino acid residue can form an ether covalent bond.

[0181] The crosslinking reagent can likewise be used to form an intermolecular linker between immobilized enzymes, if desired.

[0182] The crosslinking reagent can be homobifunctional or heterobifunctional, which means that the first and second reactive groups may be the same or different. Preferably, the first and second reactive groups are separated by a linker containing 3 to 20 atoms including any combination of C, H, N, and O.

[0183] An effective cross-linking agent should form a covalent bond between the enzyme and the support surface and retain the enzyme activity during cross-linking. Retention of enzyme activity during cross-linking is an important desirable feature of cross-linking reagents that can be adjusted by suitably selecting the reactive group, linker group, and linker length.

[0184] The preferred reactive groups selected are those capable of reacting with amines, hydroxyls, carboxylates, or sulfhydryls to form stable covalent bonds via amino bonds, amide bonds, ether bonds, ester bonds, imidoamide bonds, imidothioamide bonds, thioether bonds, thioester bonds, or thioamide bonds. Cross-linking reagents capable of cross-linking with the functional groups of the enzyme can be independently selected from epoxides, esters, anhydrides, N-hydroxysuccinimide esters, imido esters, carbonates, acyl isoureas, carbodiimides, maleimides, haloacetyls, thiosulfonates, isocyanates, and vinyl sulfones and contain at least two reactive groups. Reactive groups such as carbodiimides can assist in the formation of covalent bonds, for example, by reacting with carboxylate groups and then themselves undergoing substitution by a second reactive group to form a covalent bond such as an amide bond.

[0185] Preferred cross-linking reagents are defined by the following formula:

Chemical formula

[0186] L represents a linker between the reactive groups containing 3 to 20 atoms including any combination of C, H, N, and O.

[0187] More preferably, the cross-linking reagent has Y = Z and is homobifunctional containing a reactive group selected from epoxides, esters, anhydrides, N-hydroxysuccinimide esters, imido esters, carbonates, acyl isoureas, maleimides, haloacetyls, and vinyl sulfones, most preferably an epoxide, N-hydroxysuccinimide ester, or imido ester.

[0188] Even more preferably, the cross-linking reagent is a bisepoxide reagent, and even more preferably, a bisepoxide defined by the following formula:

Chemical formula

[0189] Most preferably, the bisepoxide reagent is glycerol diglycidyl ether (1,3- or 1,2-substituted glycerol diglycidyl ether, or GDE which can be a mixture of both isomers), neopentyl glycol diglycidyl ether (NPE), poly(tetraethylene oxide) diglycidyl ether (PDE), 1,6-hexanediol diglycidyl ether (HDDE), or glycerol triglycidyl ether (GTGE).

[0190] Another preferred cross-linking reagent includes bifunctional sulfonated N-hydroxysuccinimide ester and bifunctional imido ester, for example, bis(sulfosuccinimidyl) suberate (BS3), or suberimidic acid dimethyl (DMS).

[0191] A known alternative cross-linking agent is glutaraldehyde, but glutaraldehyde is not included in the present invention because it results in the formation of imine bonds. Glutaraldehyde is a particularly reactive cross-linking reagent that causes a significant degree of enzyme inactivation. Since imine bond formation is also reversible, the linkage is not very stable.

[0192] Preferred characteristics of the biocatalyst In a preferred embodiment of the first aspect, the support material has a pore diameter of about 20 to about 60 nm, a surface area of about 50 m 2 / g to about 200 m 2 / g, and a pore volume of about 0.5 mL / g to about 1.5 mL / g, and the enzyme is selected from List (A), more preferably from List (B).

[0193] In a preferred embodiment of the second aspect, the support material has a pore diameter of about 20 to about 60 nm, a surface area of about 50 m 2 / g to about 200 m 2 / g, and a pore volume of about 0.5 mL / g to about 1.5 mL / g, and the enzyme is selected from List (A), more preferably from List (B).

[0194] In a preferred embodiment of the third aspect, the support material has a pore diameter of about 20 to about 60 nm, a surface area of about 50 m 2 / g to about 200 m 2 / g, and a pore volume of about 0.5 mL / g to about 1.5 mL / g, and the enzyme is selected from List (A), more preferably from List (B).

[0195] In some embodiments, the immobilized enzyme retains at least 20%, preferably at least 30%, more preferably at least 40%, even more preferably at least 50%, still more preferably at least 60%, even still more preferably at least 70%, even still more preferably at least 80%, and most preferably at least 90% of its activity after immobilization as compared to its state before immobilization. In rare cases, immobilization can even enhance the activity and result in an apparent retention exceeding 100%. In some embodiments, the immobilized enzyme retains 20% - 100%, preferably 30% - 100%, more preferably 40% - 100%, even more preferably 50% - 100%, still more preferably 60% - 100%, even still more preferably 70% - 100%, even still more preferably 80% - 100%, and most preferably 90% - 100% of its activity after immobilization as compared to its state before immobilization.

[0196] In some embodiments, the immobilized enzyme retains at least 20%, preferably at least 30%, more preferably at least 40%, even more preferably at least 50%, still more preferably at least 60%, yet more preferably at least 70%, even yet more preferably at least 80%, and most preferably at least 90% of its activity after 20 hours under continuous flow conditions as compared to its state before immobilization. In some embodiments, the immobilized enzyme retains from 20% to 100%, preferably from 30% to 100%, more preferably from 40% to 100%, even more preferably from 50% to 100%, still more preferably from 60% to 100%, yet more preferably from 70% to 100%, even yet more preferably from 80% to 100%, and most preferably from 90% to 100% of its activity after 20 hours under continuous flow conditions as compared to its state before immobilization. In some embodiments, the immobilized enzyme retains at least 20%, preferably at least 30%, more preferably at least 40%, even more preferably at least 50%, still more preferably at least 60%, yet more preferably at least 70%, even yet more preferably at least 80%, and most preferably at least 90% of its activity after 70 hours under continuous flow conditions as compared to its state before immobilization. In some embodiments, the immobilized enzyme retains from 20% to 100%, preferably from 30% to 100%, more preferably from 40% to 100%, even more preferably from 50% to 100%, still more preferably from 60% to 100%, yet more preferably from 70% to 100%, even yet more preferably from 80% to 100%, and most preferably from 90% to 100% of its activity after 70 hours under continuous flow conditions as compared to its state before immobilization.

[0197] In some embodiments, the immobilized enzyme retains at least 20%, preferably at least 30%, more preferably at least 40%, still more preferably at least 50%, even more preferably at least 60%, still even more preferably at least 70%, still still more preferably at least 80%, and most preferably at least 90% of its activity after 200 hours under continuous flow conditions as compared to its state before immobilization. In some embodiments, the immobilized enzyme retains from 20% to 100%, preferably from 30% to 100%, more preferably from 40% to 100%, still more preferably from 50% to 100%, even more preferably from 60% to 100%, still even more preferably from 70% to 100%, still still more preferably from 80% to 100%, and most preferably from 90% to 100% of its activity after 200 hours under continuous flow conditions as compared to its state before immobilization.

[0198] In some embodiments, the immobilized enzyme retains at least 20%, preferably at least 30%, more preferably at least 40%, still more preferably at least 50%, even more preferably at least 60%, still even more preferably at least 70%, still still more preferably at least 80%, and most preferably at least 90% of its activity after 500 hours under continuous flow conditions as compared to its state before immobilization. In some embodiments, the immobilized enzyme retains from 20% to 100%, preferably from 30% to 100%, more preferably from 40% to 100%, still more preferably from 50% to 100%, even more preferably from 60% to 100%, still even more preferably from 70% to 100%, still still more preferably from 80% to 100%, and most preferably from 90% to 100% of its activity after 500 hours under continuous flow conditions as compared to its state before immobilization.

[0199] In the above preferred embodiments, the continuous flow conditions can be carried out in an aqueous solution, in an organic solvent, or using a pure substrate.

[0200] In the above preferred embodiments, the continuous flow is carried out in an aqueous solution at a pH of 4.0 to 9.0, or more preferably 6.0 to 7.5.

[0201] In the above preferred embodiment, the continuous flow conditions can include temperatures in the range of 4°C to 100°C, or more preferably 30°C to 70°C.

[0202] It should be noted that a particular process can also accommodate reaction steps that proceed at low conversion rates, for example, at least 20%, or preferably at least 40%, especially when the value of the product of interest is high.

[0203] In some embodiments, at least two different enzymes are immobilized on a support material. More preferably, at least two immobilized enzymes can each catalyze different steps of a multi-step organic synthesis. The multi-step synthesis can include at least 3 intermediates, for example, 3, 4, 5, 6, 7, 8, 9 or 10 intermediates. In some embodiments, the multi-step synthesis includes 3 to 10 steps, for example, 3 to 5 steps, for example, 4 to 6 steps, for example, 5 to 7 steps, for example, 6 to 8 steps, or for example, 4 to 10 steps, for example, 5 to 10 steps, for example, 6 to 10 steps, for example, 7 to 10 steps, for example, 8 to 10 steps, for example, 9 to 10 steps.

[0204] In certain embodiments, at least one of the immobilized enzymes catalyzes the regeneration of a cofactor.

[0205] Most preferred embodiment of the biocatalyst In a preferred embodiment of the second, third or fourth aspect of the present invention, the enzyme is a lipase (preferably CalB) containing a metal chelate tag such as a His tag, and the enzyme is immobilized on a surface containing an amino-functionalized aliphatic moiety via an interaction mediated by chelated Zn 2+ ions.

[0206] In another preferred embodiment of the second, third or fourth aspect, the enzyme is TbSADH containing a metal chelate tag such as a His tag, and the enzyme is immobilized on a surface via an interaction mediated by chelated Zn 2+ ions.

[0207] In a further preferred embodiment of the second, third or fourth aspect, the enzyme is an ATA comprising a metal chelate tag such as a His tag, and the enzyme is immobilized on a surface comprising structure (1), (2), (3) or (4) via an interaction mediated by a chelated metal ion.

[0208] In yet another preferred embodiment of the second, third or fourth aspect, the enzyme is a SucP comprising a metal chelate tag such as a His tag, and the enzyme is immobilized on a surface comprising structure (1).

[0209] In another preferred embodiment of the second, third or fourth aspect, the enzyme is a TLL preferably not comprising a metal chelate tag such as a His tag, and the enzyme is immobilized on a surface comprising structure (1) or (4).

[0210] In another preferred embodiment of the second, third or fourth aspect, the enzyme is a glucosyltransferase comprising a metal chelate tag such as a His tag, and the enzyme is immobilized on a surface comprising structure (1).

[0211] In another preferred embodiment of the first, second, third or fourth aspect, lipase CalB is preferably non-covalently immobilized on a surface comprising structure (1) or (4), and CalB most preferably does not comprise a metal chelate tag such as a His tag.

[0212] In yet another preferred embodiment of the second, third or fourth aspect, the enzyme is CalB (preferably not comprising a metal chelate tag such as a His tag), and the enzyme is immobilized on a surface comprising structure (4).

[0213] In yet another preferred embodiment of the third aspect, the enzyme is an amyloglucosidase (preferably without a metal chelate tag such as a His tag). The enzyme is preferably immobilized on a surface functionalized by structure (1), (2), (3) or (4), optionally with a different second functionalization. The second functionalization can be structure (1), or an alkyl, for example propyl or octyl.

[0214] Use of biocatalyst In a fifth aspect, the present invention provides the use of a biocatalyst according to the first, second, third or fourth aspect in a reactor for synthesizing an organic compound.

[0215] The reactor can be of any industrial reactor type. The reactor can be a batch reactor or a continuous flow reactor. Reactors include continuous stirred tank reactors (CSTRs), slurry bubble columns or fixed bed reactors, and known variations thereof. A continuous flow fixed bed reactor is preferred.

[0216] The synthesis can be carried out under aqueous conditions. Alternatively, the synthesis can be carried out in an organic solvent. The synthesis can also be carried out in pure substrate, which is particularly preferred for lipases. The biocatalyst can also be used in three-phase reactions that require the presence of liquid solutions and gaseous reagents, such as oxidations that utilize oxygen or air as reactants. In such cases, the particle size is preferably adjusted to suit operation within a fixed bed reactor, such as a trickle bed, or a CSTR, or another reactor type specific to three-phase conditions of a bubble column.

[0217] The synthesis in the fifth aspect can be a multi-step synthesis preferably comprising at least three intermediates, for example 3, 4, 5, 6, 7, 8, 9 or 10 intermediates. The multi-step synthesis is preferably a linear synthesis.

[0218] Method for synthesis using biocatalyst In a sixth aspect, the present invention a. Providing a biocatalyst according to the first, second, third or fourth aspect, disposed within a reactor; b. Supplying a precursor to an organic compound to the reactor, whereby the immobilized enzyme catalyzes the reaction and results in the synthesis of the organic compound; and c. Recovering the organic compound, to provide a method for the synthesis of an organic compound.

[0219] The reactor may be of any industrial reactor type. The reactor may be a batch reactor or a continuous flow reactor. Reactors include continuous stirred tank reactors (CSTRs), slurry bubble columns or fixed bed reactors, and known modifications thereof. A continuous flow fixed bed reactor is preferred.

[0220] The reaction can be carried out under aqueous conditions, in an organic solvent, or in a pure substrate. The latter is particularly preferred for lipases. The method can involve three-phase reactions that require the presence of liquid solutions and gaseous reagents, for example, oxidations that utilize oxygen or air as a reactant. In such cases, the particle size is preferably adjusted to suit operation within a fixed bed reactor, such as a trickle bed, or a CSTR, or another reactor type specific to three-phase conditions such as a bubble column.

[0221] The synthesis in the third aspect can be a multi-step synthesis preferably involving at least three intermediates, for example, 3, 4, 5, 6, 7, 8, 9 or 10 intermediates. The multi-step synthesis is preferably a linear synthesis.

[0222] Method for producing a biocatalyst In the seventh aspect, the invention provides a. Providing one or more catalytically active enzymes; b. Providing a controlled porosity silica (CPS) as a support material as defined for the first, second, third or fourth aspect, wherein the pore size is from about 20 to about 100 nm, and the support material comprises an amino-functionalized surface; and c. immobilizing one or more enzymes on a support material, to provide a method for producing a biocatalyst according to the first, second, third or fourth aspect.

[0223] In certain embodiments, the method of the seventh aspect further comprises selecting a bifunctional crosslinking reagent capable of generating an amino bond, an amide bond, an ester bond, an ether bond, a thioether bond, an imidoamide bond, an imidothioamide bond, a thioester bond or a thioamide bond (preferably amino, amide or thioamide, more preferably amino, amide or imidoamide, most preferably amino or amide) when reacted with an enzyme and an amino-functionalized surface, and covalently crosslinking the enzyme using the bifunctional reagent.

[0224] Bifunctional crosslinking reagents suitable for the method of the seventh aspect are described above in connection with the first to fourth aspects.

[0225] The bifunctional reagent can be contacted with the amino-functionalized CPS before, after, or both before and after the enzyme is immobilized. Whether the bifunctional crosslinking reagent is added before and / or after the enzyme immobilization step depends on the aspects and embodiments of the present invention. For example, the bifunctional crosslinking reagent can be added to the amino-functionalized support before enzyme immobilization, whereby one of the reactive groups of the crosslinking reagent reacts with the amino-functionalized surface of the support material. For example, if the crosslinking reagent is a bisepoxide, it is expected that one of the epoxide groups will react with the amino-functionalized CPS surface, while the second epoxide group will react with an amino acid present within the enzyme and is thus available for directly immobilizing the enzyme in a covalent-like manner.

[0226] When added prior to enzyme immobilization, the bifunctional crosslinking reagent can be added in molar excess, molar equivalence, or sub-molar equivalent relative to the number of moles of amino groups on the surface of the CPS. Further, in certain embodiments, the bifunctional crosslinking reagent can be added after the initial non-covalent immobilization if it is predicted that the first reactive group will react with the amino-functionalized CPS surface and the second reactive group will be available to react with an amino acid present within the enzyme.

[0227] Further, in certain embodiments, the crosslinking reagent can be added both before and after immobilization (double crosslinking). The crosslinking reagent can also react to provide a covalent bond between two enzyme structures. The inventors have surprisingly found that use of a bifunctional (preferably, bisepoxide, bifunctional sulfonated N-hydroxysuccinimide ester, or bifunctional imide ester) crosslinking reagent as described enables complete retention of enzyme activity upon crosslinking. Retention of activity upon crosslinking applies across many enzyme classes and thus provides a general protocol for the preparation of stable immobilized biocatalysts. This is in contrast to reports in the literature where loss of activity is a common feature of crosslinking.

[0228] The inventors have also found that attempting immobilization and crosslinking on the CPS surface without amino-functionalization reveals significant loss of activity. It is possible to immobilize an enzyme on the CPS surface without amino-functionalization, but the resulting immobilized biocatalyst has only limited stability, and thus CPS without an amino-functionalized surface does not represent a widely useful support material for immobilizing enzymes for synthesis. See Example 4.3, which shows that biocatalysts produced using the CPS surface without amino-functionalization cannot withstand the presence of a crosslinking reagent without significant loss of activity. Thus, the invention described herein provides a significant advantage compared to situations where immobilization occurs, for example, by non-specific physical adsorption to a surface without an engineered coating.

[0229] Examples 4.1 to 4.4 show complete retention of activity in the presence of a covalent bond. In such embodiments, the immobilized biocatalyst can have additional stability in the presence of the components of the reaction. The additional stability can be manifested as even longer-term stability when operating under continuous flow conditions, such as those operating within a fixed bed reactor. Examples 5.2 to 5.3 show such even longer-term stability within a fixed bed reactor when compared to immobilized enzymes without the presence of a covalent bond.

[0230] The enzyme may be immobilized on the surface via an interaction mediated by a chelating metal ion, preferably Ni 2+ , Cu 2+ , Mg 2+ , Fe 3+ or Zn 2+ , most preferably Zn 2+ by means of an interaction mediated by.

[0231] The enzyme can be selected from list (A), more preferably from list (B). The enzyme can optionally contain a polyhistidine tag.

[0232] At least one of the enzymes can be provided in the cell lysate for the immobilization step. Alternatively, the enzyme can be provided in a purified or isolated form.

[0233] At least one of the enzymes can be provided as a preparation containing at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95%, for example, 70% - 100%, 80% - 100%, 90% - 100%, or 95% - 100% of the enzyme by weight of the total protein in the preparation.

[0234] The present disclosure also provides a process for optimizing a biocatalyst comprising a catalytically active enzyme immobilized on an amino-functionalized CPS support (see Figure 3). First, one or more catalytically active enzymes to be immobilized are selected and provided (100).

[0235] Second, two, three, four, five or more different controlled porosity silica (CPS) support materials are provided that meet the criteria provided under the above section entitled Support Material. In summary, the pore size is from about 20 to about 100 nm and the support material includes an amino-functionalized surface. Preferably, the support material is as defined above for the first, second, third or fourth aspect of the invention. The support material may be amino-functionalized, for example, by propylamine, propyl NH-N-aminoethyl, phenethyl-methyl-NH-N-aminoethyl, or propyl-NH-N-aminohexyl. The support material may contain immobilized metal chelate groups. To identify suitable support materials, immobilization of one or more enzymes onto the support materials is performed and the immobilized enzyme activity between the support materials is compared (200). Various immobilization conditions may be tested. Initial conditions may include the absence and presence of chelating metal ions such as Ni 2+ , Cu 2+ , Mg 2+ , Fe 3+ and Zn 2+ . Immobilization conditions may include, for example, a buffer having a pH of 6-8, a salt concentration of 0-300 mM, a temperature of 20-30 °C, and / or an incubation time of 2-24 hours.

[0236] Optionally, further immobilization conditions may be screened to maximize the immobilized enzyme activity by immobilizing at at least two different values of at least one parameter of the best initial conditions, i.e., initial enzyme concentration, metal ion concentration, buffer type, pH, salt concentration, additives, time and temperature, and subsequently comparing the immobilized enzyme activity between the immobilization conditions and selecting the best optimized immobilization conditions (300).

[0237] As described above, covalent crosslinking can be useful for certain applications. Optionally, the crosslinking conditions can be screened to maximize the immobilized enzyme activity and / or stability by testing at least two values of at least one parameter of crosslinking agent type, crosslinking agent amount, crosslinking agent concentration, crosslinking solution composition, crosslinking time, and crosslinking temperature. The crosslinking agent meets the criteria provided under the above section entitled crosslinking reagents. The crosslinking agent type can include at least one bisepoxide, at least one bifunctional sulfonated N-hydroxysuccinimide ester, and / or at least one bifunctional imide ester. Activity is typically compared to non-crosslinked immobilized enzyme. The crosslinking agent concentration can be from 0.1 to 3% (v / v), the crosslinking temperature can be from 0 to 25 °C, the crosslinking time can be from 2 to 24 hours, and / or the crosslinking solution can have a pH of 6 to 8. The immobilized enzyme activities between crosslinking conditions can be compared, and subsequently, the best crosslinking conditions can be selected (400).

[0238] Optionally, one or more of the above steps (200)-(400) can be repeated with additional variations to further optimize the results (500). By this process, at least one immobilized enzyme can retain at least 20% (e.g., 20% to 100%, preferably 50%, or 50% to 100%) of its activity after immobilization, preferably after 20 hours under continuous flow conditions, compared to its state before immobilization. The optimized biocatalyst is then scaled up and manufactured as needed (600).

[0239] General description related to the present disclosure The term "comprising" should be interpreted as including, but not limited to. All references are incorporated herein by reference. The placement of the present disclosure into sections with headings and subheadings is for ease of reading only and should in no way be construed as limiting, in particular, the sections are in no way intended to exclude or limit the combination of features under different headings and subheadings with each other.

Examples

[0240] The following examples should not be regarded as limiting. For further information regarding the details of the experiments, those skilled in the art are referred to another section entitled Materials and Methods.

[0241] Example 1 Preparation of Supports 1.1 Supports Both the pore-controlled glass (CPG) amino-functionalized support and the pore-controlled silica (CPS) amino-functionalized support used for immobilization were synthesized in-house. The CPG core (particle size 70 - 125 μm, pore diameter 50 nm, surface area 60 m 2 ) was purchased from Schott AG. The CPS cores (Cariact Q30: particle size 70 - 150 μm, pore diameter 30 nm, surface area 99 m 2 , and Cariact Q50: particle size 70 - 150 μm, pore diameter 50 nm, surface area 80 m 2 ) were purchased from Fuji Silysia Chemical. The polymer beads used for comparison with CPG and CPS were purchased from commercial suppliers. ReliZyme EA403 / S (ethylamine functionalization on a cross-linked copolymer of methacrylate) and ReliZyme HA403 / S (hexylamine functionalization on a cross-linked copolymer of methacrylate) were purchased from Biokal. Dowex® 66 free base (polyamine groups on a styrene - divinylbenzene matrix) was purchased from Sigma - Aldrich.

[0242] 1.2 Synthesis of Amino-Functionalized Supports The pore control core (CPG or CPS) (5 g) was transferred to a jacketed glass reactor equipped with an overhead stirrer, and toluene was added (100 mL). The core was amino-functionalized with four different silanes (Table 1). Each silane (15 mL) was added to the reactor and incubated for 22 h (250 - 290 rpm, 80 °C). The solid was filtered under vacuum, washed with toluene (2 × 200 mL), and then washed with 99.7% EtOH (2 × 200 mL). After the washing step, the solid was filtered under vacuum for 60 min using a glass filter funnel. The amino-functionalized support was transferred to a crystallization dish and dried overnight at 120 °C under vacuum.

[0243] Table 1. Silanes for the amino-functionalization of pore control glass and pore control silica (CPG and CPS).

Table 4

[0244] 1.3 Chelating agent binding To a 250 mL Erlenmeyer flask containing methanol (16 mL), 2’,4’-dihydroxyacetophenone (500 mg, 3.3 mmol) was added. An amino-functionalized support (2 g) was added to the mixture and incubated for 2 h (150 rpm, 25 °C). Sodium borohydride (600 mg, 15.9 mmol) was slowly added to the flask and incubated for an additional 2 h (150 rpm, 25 °C). The slurry containing the support was washed stepwise as follows: The support slurry was transferred to a glass filter and washed with deionized water (40 mL) under vacuum. Deionized water (10 mL) was added and added dropwise without applying vacuum. Deionized water (40 mL) was added and vacuum was applied. Deionized water (10 mL) was added and added dropwise without applying vacuum. Thereafter, the pH of each support was evaluated and if the pH exceeded 7, additional deionized water (45 mL) was added and vacuum was applied. After reaching the desired pH of 7 by deionized water washing, the support was washed with ethanol (50 mL) under vacuum, followed by washing with acetone (25 mL) under vacuum. The support to which the chelating agent was bound was dried overnight in a desiccator under vacuum.

[0245] 1.4 Metal stripping from the EziG support CPG support LCAA CPG (Fe 3+ ), HybCPG VBC (Fe 3+ ), and HybCPG copo (Fe 3+ )(see International Publication No. 2015 / 115993) were also used as comparative examples of the immobilization techniques described in the present invention. To evaluate the effect of various metals on the CPG support, chelated Fe 3+It was peeled off from the support. The CPG material (5 g) was transferred to a 100 mL Duran bottle, and 2 M HCl (50 mL) was added. The flask was left on a tilt / roller mixer for 35 minutes (70 rpm, 21 °C). This procedure was repeated for a further 3 cycles, and between each cycle, the HCl was removed, fresh 2 M HCl (50 mL) was added, and it was incubated for 35 minutes (70 rpm, 21 °C). The solid was filtered under vacuum using a glass filter funnel, washed with deionized water (500 mL), followed by 25 mM sodium phosphate (NaP), pH 7.3 (300 mL), and further washed with deionized water (300 mL) and acetone (50 mL). The peeled support was dried overnight in a desiccator under vacuum.

[0246] 1.5 Metal Deposition Each immobilized support (100 mg) was weighed and placed into a 15 mL Falcon tube, and deionized water (2 mL) was added. The mixture was used as a support slurry to fill a maximum of 6 wells on the plate, which was prepared according to the support required for each assay. To each well in a 96-well deep well plate, the slurry containing the support (200 μL) was transferred to reach approximately 10 mg / well of the support. For the deposition of metal ions onto the amino-functionalized support, two stock solutions of each metal salt (FeCl 3 , ZnCl 2 , NiCl 2 , MgCl 2 , CuCl 2, 1.55 and 0.26 mM) were prepared. For CalB immobilization, a 1.55 mM metal stock solution (300 μL) was added to the support to reach a final concentration of 0.93 mM in the well (Table 2). For TbSADH immobilization, a 0.26 mM metal stock solution (300 μL) was added to the support to reach a final concentration of 0.15 mM in the well (Table 2). Plates containing the support and the metal solution were incubated for 30 minutes (1300 rpm, 21 °C). Subsequently, the metal solution was removed, and the support was washed with deionized water (4 × 1 mL), followed by washing with buffer or deionized water (1 × 1 mL) (Table 2). For comparison purposes, formulations without the presence of metal were also included in the reaction screening of each enzyme.

[0247] Table 2. Metal deposition conditions on amino-functionalized supports for various enzymes.

Table 5

[0248] 1.6 Zinc deposition on a gram scale An immobilized support (1 g) was weighed and placed in a 100 mL Duran bottle, and deionized water (10.4 mL) was added. ZnCl 2 Stock solutions were prepared for metal ion deposition and added to the support according to the required final metal concentration. The bottle was left on a tilt / roller mixer for 1 hour (20 rpm, 21 °C). The slurry containing the support was transferred to a glass filter column, washed with deionized water (60 mL), and subsequently washed with acetone (30 mL) under vacuum. The support was transferred to a 15 mL Falcon tube and dried overnight in a desiccator under vacuum. This material was used in Examples 4.1 - 4.5 and 5.1 - 5.3.

[0249] 1.7 Enzymes Lipase from Candida antarctica B (CalB) with an N-terminal His-tag, alcohol dehydrogenase from Thermoanaerobacter brockii (TbSADH) with an N-terminal His-tag, wild-type sucrose phosphorylase from Bifidobacterium adolescentis (SucP1) with an N-terminal His-tag, a mutant of sucrose phosphorylase from Bifidobacterium adolescentis (SucP2, see International Publication No. 2016 / 075219 and J. Agric. Food Chem. 2017, 65, 6030 - 6041) with an N-terminal His-tag, and glycosyltransferase from Leuconostoc mesenteroides with an N-terminal His-tag were generated in-house using known culture procedures. Lipases from Candida species and lipases from Aspergillus oryzae were purchased from Sigma-Aldrich. Lipozyme® TL 100 L and Lipozyme® TL IM were purchased from Novozymes.

[0250] 1.8 Analytical methods H 2 (Flow rate 2 mL / min) was used as the carrier gas, and gas chromatography chiral analysis was performed using an Agilent 6890N GC system equipped with a flame ionization detector (FID) and an Agilent J&W CP-Chirasil Dex CB capillary column (dimensions: 25 m × 0.25 mm × 0.25 μm; stationary phase: cyclodextrin bonded to dimethylpolysiloxane).

[0251] GC-FID method for the reaction using lipase: Injector temperature: 220 °C; Detector temperature: 250 °C; Injection volume: 1 μL, and split ratio: 20:1. Oven temperature program: Starting at 115 °C for 9 minutes; Total run time: 9 minutes. Retention times: Dodecane 4.3 minutes; (R)-1-phenylethyl acetate 5.4 minutes; (R)-1-phenylethanol 7.5 minutes; (S)-1-phenylethanol 8.2 minutes.

[0252] GC-FID method for the reaction using TbSADH: Injector temperature: 200 °C; Detector temperature: 275 °C; Injection volume: 1 μL; Split ratio: 20:1. Oven temperature program: Starting at 100 °C for 2 minutes; Rate 15 °C / min to 195 °C for 2 minutes; Total run time: 11 minutes. Retention time: Phenoxy-2-propanone 5.7 minutes; (S)-1-Phenoxy-2-propanol 6.3 minutes; (R)-1-Phenoxy-2-propanol 6.4 minutes.

[0253] HPLC-RID method for the reaction using SucP1: Oven temperature: 30 °C; Detector temperature: 30 °C; Injection volume: 10 μL; Flow rate: 0.8 mL / min; Total run time: 12 minutes. Retention time: Phosphorylated sugar 3.8 minutes; Sucrose 4.5 minutes; Glucose 5.4 minutes; Fructose 5.9 minutes.

[0254] High performance liquid chromatography for the reaction using SucP2 was performed using an Agilent Infinity II HPLC system equipped with an ESA Corona charged aerosol detector (CAD) and a Shodex HILICpak VG-50 4E column (Dimensions: 4.6 mm × 250 mm; Stationary phase: Polymer-based containing chemically bonded amino groups and hydrophilic groups), with acetonitrile:methanol:water (75:10:15) as the mobile phase in isocratic mode.

[0255] HPLC-CAD method for the reaction using SucP2: Oven temperature: 50 °C; Injection volume: 10 μL; Flow rate: 0.8 mL / min; Total run time: 14 minutes. Retention time: Fructose 6.1 minutes; Glucose 7.2 minutes; Sucrose 9.4 minutes; Cordybiose 11.6 minutes.

[0256] High-performance liquid chromatography for the reaction using glycosyltransferase analysis was performed using a Rigol L3400 HPLC system equipped with a Rigol L3500 UV-Vis detector and Merck SeQuant® ZIC®-HILIC 3.5 μm, 150 Å (dimensions: 4.6 mm × 150 mm; stationary phase: polyetheretherketone), with acetonitrile and 50 mM ammonium acetate, pH 4.5 (pH adjusted with formic acid) (75:25) as the mobile phase in isocratic mode.

[0257] HPLC-UV-Vis method for the reaction using glycosyltransferase: Oven temperature: 40 °C; injection volume: 2 μL; flow rate: 0.75 mL / min; total run time: 6 min. Retention time: hydroquinone 1.4 min; α-arbutin 2.0 min.

[0258] Example 2 Reaction screening using an amino-functionalized support 2.1 Immobilization of CalB 2.1.1 Immobilization The cell-free extract containing CalB-N-His in 20 mM MOPS, pH 7.5 (50 mL) was clarified by centrifugation for 60 min (11 000 rpm, 4 °C), followed by filtration using a 0.45 μm PVDF filter. The pH of the clarified cell-free extract was adjusted to 7.5 using 0.1 M NaOH. The cell-free extract (500 μL) was transferred to a 96-well deep-well plate containing a freshly deposited metal-containing support and incubated for 2 h (1200 rpm, 25 °C). The supernatant was removed and the immobilized support was washed with 250 mM MOPS, pH 7.5 (2 × 1 mL). The catalyst was dried overnight in a desiccator under vacuum.

[0259] 2.1.2 Determination of immobilization yield The cell-free extract and supernatant from immobilization were diluted 400-fold with 20 mM MOPS, pH 7.5. Aliquots (30 μL) of the diluted enzyme solution and supernatant were mixed with a reaction mixture (170 μL) containing 20 mM MOPS, pH 7.5 and 3 mM 4-nitrophenyl acetate in acetonitrile. Using a plate reader, the formation of 4-nitrophenol was measured by the absorbance (A 410 ) at 410 nm every 10 seconds for 3 minutes). From the linear regression of the data points, the reaction rates with each supernatant from the immobilized and fresh enzyme solutions were extracted (A 410 / min). The immobilization yield was calculated by determining the percentage of enzyme activity remaining in the supernatant after immobilization compared to the enzyme activity in the cell-free extract (Table 3). All measurements were performed in duplicate. Results are shown as the average of individual samples.

Chem.

[0260] Scheme 1. Hydrolysis of 4-nitrophenyl acetate (3 mM) by lipase for determination of immobilization yield.

[0261] 2.1.3 Activity of the immobilized catalyst in organic solvents The activity of the heterogeneous catalyst was determined by its ability to convert (±)-1-phenylethanol and vinyl acetate to (R)-phenylethyl acetate (Scheme 2). A reaction mixture (1 mL) containing 2 M (±)-1-phenylethanol, 1.2 M vinyl acetate and 1% (v / v) dodecane (internal standard) in MTBE was added to the immobilized catalyst and the mixture was incubated for 30 minutes (1200 rpm, 25 °C). After mixing was stopped and the catalyst was allowed to settle, an aliquot (10 μL) of the supernatant was added to EtOAc (990 μL) and analyzed by GC-FID to determine the conversion of (±)-1-phenylethanol (Table 3). All measurements were performed in duplicate. Results are shown as the average of individual samples.

Chem.

[0262] Scheme 2. Transesterification of (±)-1-phenylethanol (2 M) with vinyl acetate (1.2 M) catalyzed by immobilized CalB.

[0263] Table 3. Performance of immobilized CalB on amino-functionalized supports chelated with various metals. The immobilization yield was based on the activity of the supernatant after immobilization regarding the hydrolysis of 4-nitrophenyl acetate, and the conversion was based on the activity of the immobilized catalyst regarding the transesterification of (±)-1-phenylethanol with vinyl acetate. The CPS used in Table 3 was CPS Q30.

Table 6-1

Table 6-2

Table 6-3

Table 6-4

[0264] 2.2 Immobilization of TbSADH-N-His 2.2.1 Immobilization The lyophilized powder of TbSADH-N-His (300 mg) was rehydrated with deionized water (8.61 mL) and diluted with 50 mM MOPS, 150 mM NaCl, pH 7 (21.39 mL) to obtain a cell-free extract containing 10 mg / mL (lyophilized powder / total volume of solution). The cell-free extract (500 μL) was transferred to a 96-well deep well plate containing a freshly deposited support with metal and incubated for 2 h (1200 rpm, 25 °C). The supernatant was removed, and the immobilized support was washed with 20 mM MOPS, pH 7.5 (2 × 1 mL), and used immediately after removing the remaining buffer in the well.

[0265] 2.2.2 Determination of immobilization yield The cell-free extract and supernatant from immobilization were diluted 8-fold with 50 mM MOPS, 150 mM NaCl, pH 7. Aliquots (20 μL) of the diluted enzyme solution and supernatant were mixed with a reaction mixture (180 μL) containing 50 mM MOPS, pH 8, 1 mM NADP + and 100 mM 2-propanol. Using a plate reader, the formation of NADPH was measured by the absorbance (A 340 ) at 340 nm every 10 seconds for 3 minutes (Scheme 3). From the linear regression of the data points, the reaction rates with each supernatant from the immobilized and fresh enzyme solutions were extracted (A 340 / min). The immobilization yield was calculated by determining the percentage of the enzyme activity remaining in the supernatant after immobilization compared to the enzyme activity in the cell-free extract (Table 4). All measurements were performed in duplicate. Results are shown as the average of individual samples.

Chemical formula

[0266] Scheme 3. Oxidation of 2-propanol (100 mM) and NADPH formation by TbSADH for determination of immobilization yield.

[0267] 2.2.3 Activity of the immobilized catalyst in an aqueous medium The activity of the heterogeneous catalyst was determined by its ability to convert phenoxy-2-propanone to 1-phenoxy-2-propanol in the presence of NADPH as a hydride donor. 2-Propanol was added to regenerate NADP + to NADPH (Scheme 4). 20 mM phenoxy-2-propanone and 1 mM NADP +A reaction mixture (1 mL) containing 2 SO 4 and 10% (v / v) 2-propanol was added to the immobilized catalyst, and the mixture was incubated for 1 h (1500 rpm, 30 °C). Mixing was stopped, and after allowing the catalyst to sediment, an aliquot (500 μL) of the supernatant was added to EtOAc (500 μL), and the reaction product was extracted over 5 min (1500 rpm, 25 °C). A 96-well deep well plate was allowed to stand for 5 min to facilitate phase separation. The organic layer (200 μL) was transferred to another plate and dried over

Chemical formula

[0268] Scheme 4. Reduction of phenoxy-2-propanone (20 mM) catalyzed by immobilized TbSADH using 2-propanol (10% v / v) for cofactor regeneration.

[0269] Table 4. Performance of immobilized TbSADH-N-His on amino-functionalized supports chelated with various metals. The immobilization yield is based on the activity of the supernatant after immobilization with respect to the oxidation of 2-propanol, and the conversion is based on the activity of the immobilized catalyst with respect to the reduction of phenoxy-2-propanone. The CPS used in Table 4 is CPS Q30.

Table 7-1

Table 7-2

Table 7-3

[0270] 2.4 Immobilization of non-His-tagged enzyme (lipase from Candida species) 2.4.1 Immobilization A commercially available lipase solution from Candida species was diluted 4-fold with 20 mM MOPS pH 7.5. The enzyme solution (500 μL) was transferred to a 96-well deep well plate containing a freshly deposited support with metal and incubated for 2 hours (1200 rpm, 25 °C). The supernatant was removed and the immobilized support was washed with 20 mM MOPS, pH 7.5 (2 × 1 mL). The catalyst was dried overnight in a desiccator under vacuum.

[0271] 2.4.2 Determination of immobilization yield The enzyme solution and supernatant from immobilization were diluted 400-fold with 20 mM MOPS, pH 7.5. Aliquots (30 μL) of the diluted enzyme solution and supernatant were mixed with a reaction mixture (170 μL) containing 20 mM MOPS, pH 7.5 and 3 mM 4-nitrophenyl acetate in acetonitrile. Using a plate reader, the formation of 4-nitrophenol was measured by absorbance at 410 nm every 10 seconds for 3 minutes (Scheme 1). From the linear regression of the data points, the reaction rates with each supernatant from immobilization and the fresh enzyme solution were extracted (A 410 / min). The immobilization yield was calculated by determining the percentage of enzyme activity remaining in the supernatant after immobilization compared to the enzyme activity in the cell-free extract (Table 5). All measurements were performed in duplicate. Results are shown as the average of individual samples. 410 / min). The immobilization yield was calculated by determining the percentage of enzyme activity remaining in the supernatant after immobilization compared to the enzyme activity in the cell-free extract (Table 5). All measurements were performed in duplicate. Results are shown as the average of individual samples.

[0272] 2.4.3 Activity of the immobilized catalyst in organic solvents (±)-1-Phenylethanol and vinyl acetate were converted to (R)-phenylethyl acetate to determine the activity of the heterogeneous catalyst (Scheme 5). A reaction mixture (1 mL) containing 2 M (±)-1-phenylethanol, 1.2 M vinyl acetate, and 1% (v / v) dodecane (internal standard) in MTBE was added to the immobilized catalyst, and the mixture was incubated for 3 h (1200 rpm, 25 °C). After mixing was stopped and the catalyst was allowed to settle, an aliquot (10 μL) of the supernatant was added to EtOAc (990 μL) and analyzed by GC-FID to determine the conversion of (±)-1-phenylethanol (Table 5). All measurements were performed in duplicate. Results are shown as the average of individual samples. For comparison purposes, a support without amino functionalization was also included in the reaction screening of this enzyme.

Chemical Structure

[0273] Scheme 5. Transesterification of (±)-1-phenylethanol (2 M) with vinyl acetate (1.2 M) catalyzed by immobilized lipase from Candida species.

[0274] Table 5. Performance of immobilized lipase from Candida species on amino-functionalized supports chelated with various metals. The immobilization yield is based on the activity of the supernatant after immobilization regarding the hydrolysis of 4-nitrophenyl acetate, and the conversion rate is based on the activity of the immobilized catalyst regarding the transesterification of (±)-1-phenylethanol with vinyl acetate. The CPS used in Table 6 is CPS Q30.

Table 8-1

Table 8-2

[0275] 2.5 Immobilization of non-His-tagged enzyme (lipase from Aspergillus oryzae) 2.5.1 Immobilization A commercially available lipase solution derived from Aspergillus oryzae was diluted 2-fold with deionized water, and the enzyme solution (500 μL) was transferred to a 96-well deep well plate containing a newly deposited support with metal and incubated for 2 hours (1200 rpm, 25 °C). The supernatant was removed, and the immobilized support was washed with 50 mM HEPES, 50 mM ammonium acetate, 10 mM CaCl 2 , pH 7 solution (2 × 1 mL), and subsequently washed with 50 mM HEPES, 50 mM ammonium acetate, 10 mM CaCl 2 , 130 mM sucrose, pH 7 solution (1 × 1 mL). The catalyst was dried overnight under vacuum in a desiccator.

[0276] 2.5.2 Determination of immobilization yield The enzyme solution and supernatant from immobilization were diluted 10-fold with deionized water. Using a plate reader, the absorbance (A 280 ) of aliquots (200 μL) of the diluted enzyme solution and supernatant was measured at 280 nm. The immobilization yield was calculated by determining the absorbance in the supernatant after immobilization (correlating to the amount of protein present in the solution) compared to the absorbance in the initial enzyme solution (Table 6).

[0277] 2.5.3 Activity of the immobilized catalyst in organic solvents The activity of the heterogeneous catalyst was determined by its ability to convert (±)-1-phenylethanol and vinyl acetate to (R)-phenylethyl acetate (Scheme 6). A reaction mixture (1 mL) containing 2 M (±)-1-phenylethanol. 1.2 M vinyl acetate in MTBE and 1% (v / v) dodecane (internal standard) were added to the immobilized catalyst, and the mixture was incubated for 2 hours (1200 rpm, 25 °C). After stopping the mixing and allowing the catalyst to settle, an aliquot (10 μL) of the supernatant was added to EtOAc (990 μL) and analyzed by GC-FID to determine the conversion of (±)-1-phenylethanol (Table 6). All measurements were performed in duplicate. Results are shown as the average of individual samples. For comparison purposes, a support without amino functionalization was also included in the reaction screening of this enzyme. [Chemical]

[0278] Scheme 6. Transesterification of (±)-1-phenylethanol (2 M) with vinyl acetate (1.2 M) catalyzed by immobilized lipase from Aspergillus oryzae.

[0279] Table 6. Performance of immobilized lipase from Aspergillus oryzae on amino-functionalized supports chelated with various metals. The immobilization yield is based on the absorbance (A 280 ) of the supernatant after immobilization, and the conversion rate is based on the activity of the immobilized catalyst for the transesterification of (±)-1-phenylethanol with vinyl acetate. The CPS used in Table 7 is CPS Q30.

Table 9-1

Table 9-2

Table 9-3

[0280] 2.6 Immobilization of non-His-tagged enzyme (Lipozyme® TL 100L) 2.6.1 Immobilization of TLL on CPS propylamine A solution of Lipozyme® TL 100L containing Thermomyces lanuginosus lipase (TLL) for immobilization was prepared by diluting a commercially available enzyme (60% v / v) with deionized water (final solution at pH 6.7). The diluted enzyme (250 mL) was transferred to a 500 mL Duran bottle containing 50 g of a support (CPS Q30 or Q50 functionalized with propylamine). The sample was incubated on a tilt / roller mixer for 1.5 h (70 rpm, 21 °C). After immobilization, the supernatant was removed and the immobilized support was washed with 50 mM HEPES, 50 mM ammonium acetate, 10 mM CaCl 2 , pH 7 solution (1 × 250 mL), followed by washing with 50 mM HEPES, 50 mM ammonium acetate, 10 mM CaCl 2 , 130 mM sucrose, pH 7 solution (1 × 250 mL). For each washing step, the immobilized catalyst was mixed with the washing solution on a tilt / roller mixer for 3 min (70 rpm, 21 °C). The catalyst was filtered under vacuum to remove most of the washing solution (6 min), transferred to a 3 L glass flask, and dried using a rotary evaporator for 115 - 120 min (40 mbar, 40 °C).

[0281] 2.6.2 Immobilization of TLL on CPS phenethyl-methyl-NH-N-aminoethyl A solution of Lipozyme® TL 100L containing Thermomyces lanuginosus lipase (TLL) for immobilization was prepared by diluting a commercially available enzyme (60% v / v) with deionized water (final solution at pH 6.7). The diluted enzyme (100 mL) was transferred to a 250 mL Duran bottle containing 20 g of a support (CPS Q50 functionalized with phenethyl-methyl-NH-N-aminoethyl). The sample was incubated on a tilt / roller mixer for 1.5 h (70 rpm, 21 °C). After immobilization, the supernatant was removed and the immobilized support was washed with multiple solutions to obtain the most active immobilized preparation of 50 mM HEPES, 50 mM ammonium acetate, 9 mM CaCl 2, a pH 7 solution (3 × 100 mL) was found. For each washing step, the immobilized catalyst was mixed with the washing solution on a tilt / roller mixer for 3 minutes (70 rpm, 21 °C). The catalyst was filtered under vacuum to remove most of the washing solution (45 minutes), transferred to a crystallizing dish, and dried under vacuum in a desiccator for 26 hours.

[0282] 2.6.3 Determination of immobilization yield The enzyme solution and supernatant from immobilization were diluted 50-fold and 10-fold respectively with deionized water. Using a plate reader, the absorbance (A 280 ) of aliquots (200 μL) of the diluted enzyme solution and supernatant was measured at 280 nm. The immobilization yield was calculated by determining the absorbance in the supernatant after immobilization (correlating to the amount of protein present in the solution) compared to the absorbance in the initial enzyme solution (Table 7).

[0283] 2.6.4 Activity of the immobilized catalyst in organic solvents The activity of the heterogeneous catalyst was determined by its ability to convert (±)-1-phenylethanol and vinyl acetate to (R)-phenylethyl acetate. A reaction mixture (1 mL) containing 2 M (±)-1-phenylethanol, 1.2 M vinyl acetate, and 1% (v / v) dodecane (internal standard) in MTBE was added to a 1.5 mL Eppendorf tube containing the immobilized catalyst (10 mg), and the mixture was incubated for 30 minutes (1500 rpm, 35 °C). After stopping the mixing and allowing the catalyst to settle, an aliquot (10 μL) of the supernatant was added to MTBE (990 μL) and analyzed by GC-FID to determine the conversion of (±)-1-phenylethanol (Table 7). All measurements were performed in duplicate. Results are shown as the average of individual samples. The activity of the immobilized catalyst was compared to a commercially available immobilized catalyst Lipozyme® TL IM from Novozymes (Table 7).

[0284] Table 7. Performance of immobilized Lipozyme® TL 100L on the amino-functionalized support. The immobilization yield is the absorbance (A 280) Based on , the conversion rate is based on the activity of the immobilized catalyst for the transesterification of (±)-1-phenylethanol using vinyl acetate.

Table 10

[0285] 2.6.5 Activity of the immobilized catalyst for the transesterification of blended soybean oil To obtain a product with a low final melting temperature, the activity of the immobilized catalyst was also evaluated for the transesterification of a soybean oil blend composed of 60% refined and bleached soybean oil and 40% fully hydrogenated soybean oil supplied by Bunge (BG F41120-000 SOY SHTG). The melted soybean oil blend (500 μL) was added to a 2 mL Eppendorf tube containing immobilized TLL (15 mg) and incubated for 30 minutes (1500 rpm, 70 °C). After stopping the mixing and allowing the catalyst to sediment, an aliquot of the oil (350 μL) was transferred to a 2 mL Eppendorf tube, placed in ice-cold water to solidify the fat, and then stored in a freezer (-20 °C) for at least 1 hour before analysis. The endpoint of the melting range of the sampled reaction was analyzed using a Melting Point System MP55 (Mettler Toledo) equipped with a capillary (Hirschmann, L 75 mm, I.D 0.95 mm, O.D. 1.35 mm) at a heating rate of 2 °C / min from 34 °C to 67 °C. The endpoint of the melting temperature was recorded as the temperature at which the intensity signal (transmittance) reached a plateau and no further increase in intensity was detected (Table 8). The endpoint of the melting range of the initial soybean oil blend was 64.7 °C. The lower end of the melting range indicates a high degree of the transesterification reaction that occurred, thereby indicating high enzyme activity.

[0286] Table 8. Performance of immobilized Lipozyme® TL 100L with respect to amino-functionalized supports and Lipozyme® TL IM. The efficiency of the catalyst in the transesterification of blended soybean oil is based on the endpoint of the melting range of the soybean oil blend after the reaction.

Table 11

[0287] Example 3 Screening of various silanes for pore-controlled silica functionalization 3.1 Synthesis of various amino-functionalized supports using pore-controlled silica The synthesis of various amino-functionalized supports using pore-controlled silica was carried out as described above. As shown in Table 9, a structurally diverse group of amino-silanes was used.

[0288] Table 9. Silanes for amino-functionalization of pore-controlled silica.

Table 12

[0289] 3.2 Metal deposition and immobilization The procedure for metal deposition for the screening of various supports was carried out as described above. In this series of experiments, the presence or absence of Zn 2+ as a metal was evaluated. For CalB-N-His immobilization, a 1.55 mM metal stock solution (300 μL) was added to the support to reach a final concentration of 0.93 mM in the well (Table 2). For the immobilization of lipase from Candida species and TbSADH, a 0.26 mM metal stock solution (300 μL) was added to the support to reach a final concentration of 0.16 mM in the well (Table 2). As described in the previous section, the immobilization of CalB-N-His, lipase from Candida species, and TbSADH was carried out (results are shown in Tables 10 - 13). For comparison purposes, CPS without amino-functionalization was also included in the reaction screening of this series of experiments.

[0290] Table 10. Performance of immobilized CalB-N-His on amino-functionalized controlled-porosity silica The immobilization yield is based on the activity of the supernatant after immobilization with respect to the hydrolysis of 4-nitrophenyl acetate, and the conversion rate is based on the activity of the immobilized catalyst with respect to the transesterification of (±)-1-phenylethanol using vinyl acetate. The CPS used in Table 11 is CPS Q30.

Table 13

[0291] Table 11. Performance of immobilized lipase derived from Candida species on amino-functionalized controlled-porosity silica. The immobilization yield is based on the activity of the supernatant after immobilization regarding the hydrolysis of 4-nitrophenyl acetate, and the conversion rate is based on the activity of the immobilized catalyst regarding the transesterification of (±)-1-phenylethanol using vinyl acetate. The CPS used in Table 12 is CPS Q30.

Table 14

[0292] Table 12. Performance of immobilized TbSADH-N-His on amino-functionalized controlled-porosity silica. The immobilization yield is based on the activity of the supernatant after immobilization regarding the oxidation of 2-propanol, and the conversion rate is based on the activity of the immobilized catalyst regarding the reduction of phenoxy-2-propanone. The CPS used in Table 13 is CPS Q30.

Table 15

[0293] Example 4 Crosslinking of Enzymes to Amino-Functionalized CPS Supports 4.1 Crosslinking of SucP2 Immobilized on Amino-Functionalized Pore-Controlled Silica 4.1.1 Immobilization The lyophilized powder of SucP2 (760 mg) was rehydrated in 150 mM MOPS buffer, pH 7 (19 mL) to obtain a cell-free extract concentration of 40 mg / mL (lyophilized powder / buffer solution). The prepared cell-free extract was resuspended on a tilt / roller mixer for 10 min (60 rpm, 21 °C), and then centrifuged for 10 min (8000 rpm, 21 °C). After centrifugation, 20 mg of the support (CPS Q30 functionalized with propylamine, and Zn from a solution with a final concentration of 0.15 mM 2+The cell-free extract (1 mL) was transferred to a 1.5 mL Eppendorf tube containing the

[0294] 4.1.2 Crosslinking treatment The crosslinking reagents used were glutaraldehyde (GA), and bisepoxide neopentyl glycol diglycidyl ether (NPE), glycerol diglycidyl ether (GDE), and polyethylene glycol diglycidyl ether (PDE). A solution of 2% (v / v) of each crosslinking reagent and 20% (v / v) ethanol was prepared in 150 mM MOPS buffer, pH 7 and added to the immobilized catalyst (1 mL). 150 mM MOPS buffer, pH 7 (1 mL) was added to the immobilized SucP2 to prepare a control catalyst (non-crosslinked). Both the control catalyst and the crosslinked catalyst were incubated overnight on a tube rotator (15 rpm, 2 °C). The buffer / crosslinking solution was removed and the catalyst was washed with 150 mM MOPS buffer, pH 7 (3 × 1 mL). The crosslinking treatment under different conditions was performed in duplicate.

[0295] 4.1.3 Activity of the immobilized catalyst in an aqueous medium The ability to convert sucrose and glucose to kojibiose was used to determine the activity of the cross-linked heterogeneous catalyst (with and without leaching treatment) (Scheme 7). A reaction mixture (1 mL) containing 1 M sucrose and 1 M glucose in deionized water was added to the immobilized catalyst. The mixture was incubated (1200 rpm, 55 °C), and samples of the supernatant (10 μL) were taken after 1 h, 2 h, 3 h, and 4 h to determine the productivity of the catalyst. The samples were diluted with deionized water (190 μL), and 100 mM glycerol (internal standard) in acetonitrile:deionized water (85:15) (200 μL) was added. The mixture was quenched (2 min) by placing the Eppendorf tube on ice and then centrifuged for 2 min (15000 rpm, 21 °C) for debris separation. The supernatant was transferred to a plastic inlet vial and analyzed by HPLC-CAD to determine the conversion of sucrose and glucose to kojibiose (Table 13). [Chemical Formula]

[0296] Scheme 7. Glycosyl transfer of sucrose (1 M) and glucose (1 M) catalyzed by immobilized SucP2 to produce kojibiose

[0297] Table 13. Performance of immobilized SucP2 on propylamine-functionalized controlled pore silica (Q30) and subsequent cross-linking treatment. Productivity is based on the activity of the immobilized catalyst for the glycosyl transfer of sucrose and glucose to produce kojibiose as a function of time and support amount. The productivity retention is based on the retention of the productivity of the cross-linked catalyst compared to the control catalyst without cross-linking. [Table 16]

[0298] 4.2 Effect of pH on the cross-linking of immobilized SucP2 4.2.1 Immobilization The lyophilized powder of SucP2 (840 mg) was rehydrated in 150 mM MOPS buffer, pH 7 (10 mL) to obtain a cell-free extract concentration of 40 mg / mL (lyophilized powder / buffer solution). The prepared cell-free extract was resuspended on a tilt / roller mixer for 10 min (60 rpm, 21 °C) and then centrifuged for 10 min (8000 rpm, 21 °C). After centrifugation, the cell-free extract (1 mL) was transferred to a 1.5 mL Eppendorf tube containing 20 mg of the support (CPS Q30 functionalized with propylamine and Zn 2+ deposition) from a solution with a final concentration of 0.15 mM. The sample was incubated for 1 h (15 rpm, 21 °C), and after immobilization, the supernatant was removed. The immobilized support was washed with 150 mM MOPS buffer, pH 7 (2 × 1 mL).

[0299] 4.2.2 Crosslinking treatment The bisepoxides used as crosslinking reagents were glycerol diglycidyl ether (GDE) and polyethylene glycol diglycidyl ether (PDE). Solutions of 2% (v / v) of each bisepoxide and 20% (v / v) ethanol were prepared in 150 mM MOPS buffers, pH 7 and pH 8, and 150 mM Tris / HCl buffer pH 9 and added to the immobilized catalyst (1 mL). 150 mM MOPS buffers, pH 7 and pH 8, and 150 mM Tris / HCl buffer pH 9 (1 mL) were added to the immobilized SucP2 to prepare a control catalyst (non-crosslinked). Both the control catalyst and the crosslinked catalyst were incubated overnight on a tube rotator (15 rpm, 25 °C). The buffer / crosslinking solution was removed, and the catalyst was washed with 150 mM MOPS buffer, pH 7 (2 × 1 mL). The crosslinking treatment was performed in duplicate under different conditions.

[0300] 4.2.3 Leaching treatment As a protocol for determining the efficiency of crosslinking, a leaching test was performed on the catalyst. One sample from each different crosslinking condition (different crosslinking reagents and pH) was subjected to the leaching process, and samples from the same conditions were kept separately and used as controls to enable the determination of the productivity of the immobilized catalyst with and without using the leaching protocol. 0.5 M sodium phosphate buffer, pH 7 (1 mL) was added to the immobilized catalyst and incubated on a tube rotator for 2 hours (15 rpm, 21 °C) for leaching. After the leaching protocol, the catalyst was washed with 150 mM MOPS buffer, pH 7 (2 × 1 mL). According to the control test, treating the immobilized catalyst with 0.5 M sodium phosphate is an effective method for leaching enzymes that have not undergone crosslinking from the immobilized catalyst.

[0301] 4.2.4 Activity of the immobilized catalyst in an aqueous medium The activity (with and without leaching treatment) of the heterogeneous catalyst after crosslinking was determined by its ability to convert sucrose and glucose to kojibiose (Scheme 9). A reaction mixture (1 mL) containing 1 M sucrose and 1 M glucose in deionized water was added to the immobilized catalyst. The mixture was incubated (1200 rpm, 55 °C), and samples of the supernatant (10 μL) were taken after 1 hour, 2 hours, 3 hours, and 4 hours to determine the productivity of the catalyst. The samples were diluted with deionized water (190 μL), and 100 mM glycerol (internal standard) in acetonitrile:deionized water (85:15) (200 μL) was added. The mixture was quenched (2 minutes) by placing the Eppendorf tube on ice and then centrifuged for 2 minutes (15000 rpm, 21 °C) for debris separation. The supernatant was transferred to a plastic inlet vial and analyzed by HPLC-CAD to determine the conversion of sucrose and glucose to kojibiose (Table 14).

[0302] Table 14. Performance of immobilized SucP2 on propylamine-functionalized controlled-porosity silica (Q30) and subsequent cross-linking treatment at different pH values. Productivity is based on the activity of the immobilized catalyst for glycosyl transfer of sucrose and glucose to produce kojibiose as a function of time and support amount. The productivity retention rate is based on the retention rate of the productivity of the catalyst after leaching treatment compared to the sample without leaching treatment.

Table 17

[0303] 4.3 Cross-linking treatment of immobilized SucP2 with and without amino-functionalization of the CPS support 4.3.1 Immobilization The lyophilized powder of SucP2 was rehydrated in 150 mM MOPS buffer, pH 7 (10 mL) to obtain a cell-free extract concentration of 120 mg / mL (lyophilized powder / buffer solution). The prepared cell-free extract was resuspended on a tilt / roller mixer for 10 minutes (70 rpm, 21 °C) and then centrifuged for 10 minutes (8000 rpm, 21 °C). After centrifugation and separation of cell debris, the cell-free extract (1 mL) was added to 50 mg of the support (CPS Q30 functionalized with propylamine and Zn 2+ deposited from a solution with a final concentration of 0.15 mM, or non-functionalized CPS Q30 and Zn 2+ deposited from a solution with a final concentration of 0.15 mM). The enzyme was left immobilized on the support on a tilt / roller mixer (70 rpm, 21 °C) for 1 hour, and after immobilization, the supernatant was removed. The immobilized support was washed with 150 mM MOPS buffer, pH 7 (2 × 1 mL) and used as a wet catalyst for cross-linking.

[0304] 4.3.2 Cross-linking treatment A cross-linking solution was prepared using 2% (v / v) glycerol diglycidyl ether (GDE) and 20% (v / v) ethanol in 150 mM Tris / HCl buffer, pH 9. The cross-linking solution (1 mL) was added to the immobilized catalyst and incubated overnight on a tilt / roller mixer (70 rpm, 21 °C). The cross-linking solution was removed and the catalyst was washed with 150 mM MOPS buffer, pH 7 (2 × 1 mL).

[0305] 4.3.3 Activity of the immobilized catalyst in an aqueous medium The activity of the heterogeneous catalyst (cross-linked or non-cross-linked) was determined by its ability to convert sucrose and glucose to kojibiose (Scheme 9). A reaction mixture (1 mL) containing 1 M sucrose and 1 M glucose in deionized water was added to the immobilized catalyst. The mixture was incubated (1200 rpm, 55 °C) and aliquots (5 μL) of the reaction mixture were taken after 30, 50, 70, and 90 minutes to determine the productivity of the catalyst. The aliquots taken were diluted with deionized water (95 μL) containing 13.5 mM ribose (internal standard), and then acetonitrile (200 μL) was added. The mixture was quenched (2 minutes) by placing the Eppendorf tube on ice and then centrifuged for 2 minutes for solid separation (15000 rpm, 21 °C). The supernatant was transferred to a plastic inlet vial and analyzed by HPLC-CAD to determine the conversion of sucrose and glucose to kojibiose (Table 15).

[0306] Table 15. Performance of immobilized SucP2 on propylamine-functionalized controlled pore silica (Q30) or propylamine non-functionalized controlled pore silica (Q30). Productivity is based on the activity of the immobilized catalyst for glycosyl transfer of sucrose and glucose to produce kojibiose as a function of time and support amount. The productivity retention rate is based on the retention rate of the productivity of the catalyst after cross-linking treatment compared to the sample without cross-linking treatment.

Table 18

[0307] 4.4 Modification of CPS-Q30 propylamine with bisepoxide In addition to the stabilization of the enzyme immobilized on CPS-Q30 via crosslinking treatment with bisepoxide, the modification of amino-functionalized CPS with bisepoxide before enzyme immobilization was also demonstrated. Glycerol diglycidyl ether (GDE) was used to perform the support modification with bisepoxide. In a 2 mL Eppendorf tube, CPS-Q30 propylamine, and Zn from a solution (10 mg) with a final concentration of 0.15 mM 2+ deposition was wetted with deionized water (1 mL), and then the bisepoxide solution was added. 15% (v / v) GDE and 20% (v / v) ethanol in 150 mM Tris / HCl buffer, pH 9 (500 μL) were added to the support and incubated overnight (1200 rpm, 30 °C). The bisepoxide solution was removed, and the catalyst was washed with deionized water (2 × 1 mL), followed by washing with 25 mM sodium phosphate buffer, 150 mM NaCl, pH 8 (1 × 1 mL).

[0308] 4.4.1 Immobilization The lyophilized powder of glycosyltransferase was prepared in 25 mM sodium phosphate buffer, 150 mM NaCl, pH 8 to obtain a cell-free extract concentration of 14 mg / mL (lyophilized powder / buffer solution). 10 mg of the support (as described above, CPS-Q30 propylamine, and Zn from a solution with a final concentration of 0.15 mM 2+ deposition, and CPS-Q30 propylamine, and Zn from a GDE-modified solution with a final concentration of 0.15 mM 2+ deposition) in a 2 mL Eppendorf tube was transferred with the prepared cell-free extract (1 mL). The sample was incubated on a tube rotator for 2 hours (20 rpm, 21 °C), and after immobilization, the supernatant was removed. The immobilized support was washed with 50 mM MOPS buffer, pH 7.2 (3 × 1 mL).

[0309] 4.4.2 Determination of immobilization yield An aliquot of the initial enzyme solution or an aliquot of the supernatant from immobilization (250 μL) was mixed with a reaction mixture (750 μL) containing 0.67 M sucrose, 0.13 M hydroquinone, and 0.013 M ascorbic acid in 50 mM MOPS buffer, pH 7.2, and incubated for 1 h (600 rpm, 30 °C). An aliquot of the mixture (25 μL) was diluted with deionized water (55 μL), and then acetonitrile (100 μL) was added. The formation of α-arbutin was measured by HPLC UV-Vis (Scheme 8). The immobilization yield was calculated by determining the percentage of enzyme activity remaining in the supernatant after immobilization compared to the enzyme activity in the cell-free extract (Table 16).

[0310] 4.4.3 Crosslinking and double crosslinking treatment A solution of 6% (v / v) GDE and 20% (v / v) ethanol was prepared in 25 mM sodium phosphate buffer, pH 7, and added to the catalyst immobilized on CPS-Q30 propylamine + Zn 2+ (crosslinking) and CPS-Q30 propylamine + Zn 2+ GDE modification (double crosslinking) (1 mL). 25 mM sodium phosphate buffer, pH 7 (1 mL) was added to the immobilized glycosyltransferase to prepare a control catalyst (non-crosslinked). Both the control catalyst and the crosslinked catalyst were incubated overnight on a tube rotator (15 rpm, 25 °C). The buffer / crosslinking solution was removed, and the catalyst was washed with 50 mM MOPS buffer, pH 7.2 (3 × 1 mL). The crosslinking treatment was carried out in duplicate under different conditions, and one of the replicates containing the non-crosslinked catalyst was subjected to the leaching treatment.

[0311] 4.4.4 Leaching treatment (CPS CPS-Q30 propylamine + Zn 2+ or CPS-Q30 propylamine + Zn 2+One sample from each of the different conditions (with and without cross-linking treatment for GDE modification) was subjected to the leaching protocol, keeping samples from the same conditions separately and using them as controls to enable the determination of the productivity of the immobilized catalysts with and without the leaching protocol. 0.5 M sodium phosphate buffer, pH 7 (1 mL) was added to the immobilized catalyst and incubated on a tube rotator for 2 h (20 rpm, 21 °C), and the leaching protocol was carried out. After leaching, the supernatant was removed and kept for evaluating the enzyme leached from the catalyst. The leached support was washed with 50 mM MOPS buffer, pH 7.2 (3 × 1 mL).

[0312] 4.4.5 Determination of leached proteins The supernatant from the leaching solution was tested for proteins leached from the support using the bathocuproine disulfonate (BCS) assay. Bovine serum albumin (BSA) protein standard and reagents for protein determination were purchased from ThermoFisher as a commercial kit. According to the instructions provided by the BCS kit, BSA solutions for the standard protein curve and samples from the incubation of the leaching solution were prepared. Using a plate reader, the protein concentration was measured by the absorbance (A 562 ) at 562 nm. A 562 vs BSA [mg / ml] The amount of leached protein in mg / mL was extrapolated from the BSA calibration curve constructed by linear interpolation of the linear region of the plot (y = 1.6285x + 0.1335, R 2 = 99.8%).

[0313] 4.4.6 Activity of the immobilized catalyst The activity of the heterogeneous catalyst was determined by its ability to convert sucrose and hydroquinone into α-arbutin and fructose in the presence of ascorbic acid as an antioxidant (Scheme 8). A reaction mixture (1 mL) containing 0.5 M sucrose, 0.1 M hydroquinone, and 0.01 M ascorbic acid in 50 mM MOPS pH 7.2 was added to the immobilized catalyst, and the mixture was incubated for 1 h (1300 rpm, 30 °C). Mixing was stopped and the catalyst was allowed to sediment. An aliquot (25 μL) of the supernatant was diluted with deionized water (55 μL), and then acetonitrile (100 μL) was added. The mixture was transferred to a plastic inlet vial and analyzed by HPLC UV-Vis to determine the conversion of sucrose and hydroquinone to α-arbutin.

Chemical formula

[0314] Scheme 8. Glycosyltransferase-catalyzed glycosyl transfer reaction to evaluate the immobilization yield on the tested support and the performance of the different obtained immobilized catalysts.

[0315] Table 16. Performance of immobilized glycosyltransferase on propylamine-functionalized controlled porosity silica (Q30) (with and without bisepoxide modification) and subsequent crosslinking treatment. Productivity is based on the activity of the immobilized catalyst that produces α-arbutin. The productivity retention rate is based on the relative productivity of the treated catalyst (support modification / x-linking) compared to the untreated catalyst (control).

Table 19

[0316] Example 5 Catalyst Stability under Continuous Flow Conditions 5.1 Stability of CalB Immobilized on Amino-Functionalized Pore-Controlled Silica in Organic Solvents under Continuous Flow 5.1.1 Immobilization The cell-free extract containing CalB-N-His in 20 mM MOPS, pH 7.5 (2 × 40 mL) was clarified by centrifugation for 60 minutes (11,000 rpm, 4 °C), followed by filtration using a 0.45 μm PVDF filter. The pH of the clarified cell-free extract was adjusted to 7.5 using 0.1 M NaOH. After preparation, the cell-free extract (70 mL) was transferred to a 50 mL Falcon tube containing 700 mg of support (CPS-Q30 functionalized with propylamine and Zn 2+ deposition) from a solution with a final concentration of 140 mM. The enzyme was left for 3 hours on a tilt / roller mixer (70 rpm, 25 °C) for immobilization on the support. The supernatant was removed and the immobilized support was transferred to a glass filter and washed with 250 mM MOPS, pH 7.5 (3 × 20 mL). The catalyst was transferred to a 15 mL Falcon tube and dried overnight under vacuum in a desiccator.

[0317] 5.1.2 Continuous flow runs and sample analysis The dried catalyst (300 mg) was packed into a Uniqsis column reactor (1 cm OD). A reaction mixture for the feed stream was prepared using 2 M (±)-1-phenylethanol, 1.2 M vinyl acetate, and 1% (v / v) dodecane in MTBE (1 L). The packed reactor was assembled into a Uniqsis FlowSyn system. The reaction mixture was continuously fed to the reactor (0.61 mL / min, 30 °C) and samples were collected periodically. Samples (10 μL) from the outlet were diluted with EtOAc (990 μL) in a glass vial and analyzed by GC-FID to determine the conversion of (±)-1-phenylethanol. The results are shown in Table 17, where stable operation over a continuous flow of more than 73 hours is demonstrated.

[0318] Table 17. Stability in the flow of CalB immobilized on CPS-Q30 propylamine with 140 mM Zn 2+ The conversion rate is based on the activity of the immobilized catalyst for the transesterification of (±)-1-phenylethanol and vinyl acetate.

Table 20

[0319] 5.2 Stability of SucP1 in continuous flow with and without bridge treatment 5.2.1 Immobilization The purified SucP1 was diluted with 50 mM MOPS buffer, pH 8 to obtain an enzyme solution concentration of 0.6 mg / mL (enzyme / buffer solution). 10 mL of the prepared enzyme solution was transferred to a 15 mL Falcon tube containing 300 mg of support (CPS-Q30 functionalized with propylamine and Zn 2+ deposited) from a solution with a final concentration of 0.15 mM. The sample was left on a tilt / roller mixer for 1 hour (60 rpm, 21 °C), and after immobilization, the supernatant was removed. The immobilized support was washed with 50 mM MOPS buffer, pH 8 (2 × 10 mL). This procedure was repeated twice, and one of the immobilized catalysts was subjected to bridge treatment.

[0320] 5.2.2 Bridge treatment A crosslinking solution was prepared using 2% (v / v) neopentyl glycol diglycidyl ether (NPE) and 20% (v / v) ethanol in 50 mM MOPS buffer, pH 8. The crosslinking solution (30 mL) was added to the immobilized catalyst and incubated overnight on a tilt / roller mixer (60 rpm, 21 °C). The solution was removed, and the catalyst was washed with 50 mM MOPS buffer, pH 8 (2 × 10 mL).

[0321] 5.2.3 Continuous flow run and sample analysis Immediately after immobilization or crosslinking treatment, a Uniqsis column reactor (1 cm OD) was packed with a wet catalyst (300 mg). A reaction mixture for the feed stream was prepared using 2 M sucrose in 100 mM sodium phosphate buffer, pH 7.8 (1 L). Multiple batches of the reaction mixture were prepared as required for reactor feeding. The packed reactor was assembled into a Uniqsis FlowSyn system. The reaction mixture was continuously fed into the reactor (0.2 mL / min, 25 °C), and samples were collected periodically. Samples from the outlet (50 μL) were quenched with 1 M HCl (50 μL), diluted with deionized water (100 μL), and centrifuged for 2 minutes (15000 rpm, 21 °C) for debris separation. The supernatant was transferred to a plastic inlet vial and analyzed by HPLC-RID to determine the conversion of phosphate to glucose 1-phosphate (G1P) (Scheme 9) (Table 18).

Chemical formula

[0322] Scheme 9. Conversion of sucrose (1 M) to G1P and fructose catalyzed by immobilized SucP1.

[0323] Table 18. Comparison of the stability of immobilized SucP1 in flow with and without crosslinking treatment. The conversion rate is based on the activity of the immobilized catalyst with respect to the production of G1P at production time.

Table 21

[0324] 5.3 Stability of SucP2 in continuous flow with and without crosslinking treatment 5.3.1 Preparation of non-crosslinked catalyst, and continuous flow run 5.3.1.1 Immobilization The lyophilized powder of SucP2 was rehydrated in 150 mM MOPS buffer, pH 7 (50 mL) to obtain a cell-free extract concentration of 40 mg / mL (lyophilized powder / buffer solution). The prepared cell-free extract was resuspended on a tilt / roller mixer for 10 minutes (70 rpm, 21 °C) and then centrifuged for 10 minutes (8000 rpm, 21 °C). After centrifugation and separation of cell debris, the cell-free extract was transferred to a 50 mL Falcon tube containing 3 g of support (CPS-Q30 functionalized with propylamine having 0.15 mM Zn 2+ deposition). The enzyme was left immobilized on the support on a tilt / roller mixer (70 rpm, 21 °C) for 1 hour, and after immobilization, the supernatant was removed. The immobilized support was washed with 150 mM MOPS buffer, pH 7 (3 × 45 mL) and used as a wet catalyst for flow runs.

[0325] 5.3.1.2 Continuous flow runs and sample analysis The wet catalyst (4.5 g) was packed into a Uniqsis column reactor (1 cm OD). A reaction mixture for the feed stream was prepared using 1 M sucrose and 1 M glucose in deionized water (1 L). Multiple batches of the reaction mixture were prepared as required for reactor feeding. The packed reactor was assembled into a Uniqsis FlowSyn system. The reaction mixture was first fed (0.125 mL / min, 25 °C) until the product could be detected at the reactor outlet. Then the temperature was adjusted to 55 °C and the flow was maintained at 0.125 mL / min. Samples were collected periodically and the productivity was calculated in kg kojibiose / kg support*time. Samples (5 μL) from the outlet were diluted with deionized water (95 μL) and 100 mM glycerol (internal standard) in acetonitrile:deionized water (85:15) (100 μL) was added. The mixture was quenched (2 minutes) by placing the Eppendorf tube on ice and then centrifuged for 2 minutes (15000 rpm, 21 °C) for debris separation. The supernatant was transferred to a plastic inlet vial and analyzed by HPLC-CAD to determine the conversion of sucrose and glucose to kojibiose (Table 19).

[0326] 5.3.2 Preparation of the cross-linking catalyst and continuous flow operation 5.3.2.1 Immobilization The lyophilized powder of SucP2 was rehydrated in 150 mM MOPS buffer, pH 7 (2 × 50 mL) to obtain a cell-free extract concentration of 40 mg / mL (lyophilized powder / buffer solution). The prepared cell-free extract was resuspended on a tilt / roller mixer for 10 minutes (70 rpm, 21 °C), and then centrifuged for 10 minutes (8000 rpm, 21 °C). After centrifugation and separation of cell debris, two tubes containing the cell-free extract and a 100 mL Duran bottle containing 10.5 g of a support (CPS functionalized with propylamine having Zn 2+ deposits) were combined. The enzyme was left immobilized on the support on a tilt / roller mixer (70 rpm, 21 °C) for 1 hour, and after immobilization, the supernatant was removed. The immobilized support was washed with 150 mM MOPS buffer, pH 7 (3 × 90 mL).

[0327] 5.3.2.2 Cross-linking treatment A cross-linking solution was prepared using 2% (v / v) glycerol diglycidyl ether (GDE) and 20% (v / v) ethanol in 150 mM Tris / HCl buffer, pH 9. The cross-linking solution (100 mL) was added to the immobilized catalyst and incubated overnight on a tilt / roller mixer (70 rpm, 21 °C). The solution was removed, and the catalyst was washed with 150 mM MOPS buffer, pH 7 (1 × 90 mL), and 150 mM MOPS buffer pH 7, 250 mM sucrose (1 × 90 mL). The catalyst was filtered under vacuum to remove the excess buffer and then dried overnight under vacuum in a desiccator.

[0328] 5.3.3 Continuous flow operation and sample analysis The dried catalyst (2.6 g) was packed into two Uniqsis column reactors (1 cm OD) connected in series. A reaction mixture for the feed stream was prepared using 1 M sucrose and 1 M glucose in deionized water (1 L). Multiple batches were prepared as required for reactor feeding. The packed reactors were assembled into a Uniqsis FlowSyn system. The reaction mixture was first fed (0.125 mL / min, 25 °C) until products could be detected at the reactor outlet. Subsequently, the temperature was adjusted to 55 °C and the flow was maintained at 0.125 mL / min. Samples were collected periodically and productivity was calculated as kg kojibiose / kg support * time. Samples from the outlet (5 μL) were diluted with deionized water (95 μL) and 100 mM glycerol (internal standard) in acetonitrile:deionized water (85:15) (100 μL) was added. The mixture was quenched (2 min) by placing the Eppendorf tube on ice and then centrifuged for 2 min (15000 rpm, 21 °C) for debris separation. The supernatant was transferred to a plastic inlet vial and analyzed by HPLC-CAD to determine the conversion of sucrose and glucose to kojibiose (Table 19).

[0329] Table 19. Comparison of the stability in flow of immobilized SucP2 with and without cross-linking treatment. Since the catalyst was packed as a wet formulation, productivity in flow is represented by the production of kojibiose as a function of the amount of support per hour by immobilized SucP2 during production [kg コージビオース / kg 支持体 * time].

Table 22

[0330] 5.4 Long-term continuous flow runs using cross-linked SucP2 catalyst 5.4.1 Zn deposition CPS Q30 functionalized with propylamine (5 g) was weighed and placed into a 50 mL Falcon tube, and deionized water (46.45 mL) was added. 36.7 mM ZnCl 2Add the stock solution (1.05 mL) and leave the tube on a tilt / roller mixer for 1 h (70 rpm, 21 °C). Wash the support with deionized water (3 × 50 mL) and 150 mM MOPS buffer, pH 7 (1 × 50 mL). Zn 2+ Immediately after the deposition procedure, the support was used for immobilization.

[0331] 5.4.2 Immobilization Rehydrate the lyophilized powder of SucP2 (6 g) in 150 mM MOPS buffer, pH 7 (50 mL) to obtain a cell-free extract concentration of 120 mg / mL (lyophilized powder / buffer solution). Resuspend the prepared cell-free extract on a tilt / roller mixer for 10 min (70 rpm, 21 °C), then centrifuge for 15 min (8000 rpm, 21 °C). After centrifugation and separation of cell debris, add the whole cell-free extract preparation to a 50 mL Falcon tube containing the support with fresh Zn 2+ deposition. Immobilize the enzyme on the support on a tube rotator (20 rpm, 21 °C) for 90 min, and after immobilization, remove the supernatant. Wash the immobilized support with 150 mM MOPS buffer, pH 7 (2 × 50 mL).

[0332] 5.4.3 Crosslinking treatment Prepare a crosslinking solution using 4% (v / v) glycerol diglycidyl ether (GDE) and 20% (v / v) ethanol in 150 mM Tris / HCl buffer, pH 9. Add the crosslinking solution (100 mL) to the immobilized catalyst and incubate overnight on a tube rotator (20 rpm, 21 °C). Remove the solution and wash the catalyst with 150 mM MOPS buffer, pH 7 (1 × 10 mL) and 400 mM sucrose, 50 mg / mL maltodextrin in 150 mM MOPS buffer, pH 7 (1 × 90 mL). Filter the catalyst under vacuum to remove the excess buffer, then dry it under vacuum in a desiccator for 6 h.

[0333] 5.4.4 Continuous flow run and sample analysis Two Uniqsis column reactors (1 cm OD) were packed with the dried catalyst (2 g). A reaction mixture for the feed stream was prepared using 1 M sucrose and 1 M glucose in deionized water (1 L). Multiple batches were prepared as required for reactor feeding. The packed reactors were assembled into the Uniqsis FlowSyn system. The reaction mixture was first fed (0.25 mL / min, 45 °C), then the flow was decreased and maintained at 0.1 mL / min. Samples were collected periodically and productivity was calculated in kg cellobiose / kg support * time. Samples from the outlet (5 μL) were diluted with deionized water containing 13.5 mM ribose (internal standard) (95 μL) and acetonitrile (100 μL) was added. The mixture was quenched by placing the Eppendorf tube on ice (2 min), then centrifuged for 2 min (15,000 rpm, 21 °C) for debris separation. The supernatant was transferred to a plastic inlet vial and analyzed by HPLC-CAD to determine the conversion of sucrose and glucose to cellobiose (Table 20).

[0334] Table 20. Production time of immobilized SucP2 using cross-linking treatment. Productivity is based on the production of cellobiose as a function of the amount of support per hour of the immobilized SucP2 catalyst.

Table 23-1

Table 23-2

[0335] Example 6 Comparison of CPS Support with Other Supports Using the reaction that converts sucrose and glucose shown in Scheme 7 to kojibiose, the activity of SucP2 enzyme immobilized on the pore-controlled silica supports (Q30 and Q50 supplied by Fuji) of the present invention was evaluated by comparison with the activity of SucP2 enzyme immobilized on other commercially available porous silica supports and pore-controlled glass supports. The following commercially available silica supports were used: 1) MCM-48 (supplied by Sigma Aldrich), a mesoporous silica having a regular cage structure essentially similar to the mesoporous cellular foam (MCF) structure described above in the present disclosure, SBA-15 (supplied by Sigma Aldrich), a mesoporous silica containing regular hexagonal pores, and Q15 (supplied by Fuji), a pore-controlled silica containing pores with a diameter of 15 nm (pore volume 1.00 ml / g, surface area 200 m 2 / g). Further, an example of an amino-functionalized pore-controlled glass material carrier (LCAA CPG(Zn 2+ )) was included in the comparative test. Each silica support was first treated with (3-aminopropyl)trimethoxysilane by the method described in Section 1.2 to produce an amino-functionalized silica support, and subsequently further treated with Zn 2+ by the method described in Section 1.5 to produce a porous silica functionalized with propylamine containing Zn 2+ . Without using crosslinking, the immobilization of SucP2 onto the porous silica and LCAA CPG(Zn 2+ ) was carried out by the method described in Section 4.1.1. The immobilization yield was determined as described in Section 2.5.2, and the activity of the immobilized SucP2 catalyst for the target reaction was measured as described in Section 3.1.3. All experiments were performed in duplicate. Figure 2 shows the results of the comparative test, clearly demonstrating the excellent performance of the pore-controlled silica of the present invention.

[0336] Example 7 Crosslinking of SucP2 Immobilized on Amino-Functionalized Pore-Controlled Silica 7.1. Immobilization The freeze-dried powder of SucP2 (500 mg) was rehydrated in 150 mM MOPS buffer, pH 7 (50 mL) to obtain a cell-free extract concentration of 10 mg / mL (freeze-dried powder / buffer solution). The prepared cell-free extract was resuspended on a tilt / roller mixer for 10 minutes (60 rpm, 21 °C) and then centrifuged for 10 minutes (8000 rpm, 21 °C). After centrifugation, 10 mg of the support (propylamine, copolymer propyl + propyl NH-N-aminoethyl, propyl-3N-(4,5-dihydroimidazole), phenethyl-methyl-NH-N-aminoethyl functionalized CPS Q30, and Zn from a solution with a final concentration of 0.105 g / L 2+ deposition) was transferred to a 1.5 mL Eppendorf tube containing the cell-free extract (1 mL). The sample was incubated for 2 hours (20 rpm, 21 °C), and after immobilization, the supernatant was removed. The immobilized support was washed with 150 mM MOPS buffer, pH 7 (2 × 1 mL).

[0337] 7.2 Determination of immobilization yield An aliquot of the initial enzyme solution or an aliquot of the supernatant from immobilization (100 μL) was mixed with a reaction mixture (400 μL) containing 1 M sucrose and 1 M glucose in deionized water and incubated for 2 hours (600 rpm, 55 °C). An aliquot of the mixture ((10 μL) was taken, diluted with deionized water (190 μL), and 100 mM glycerol (internal standard) in acetonitrile:deionized water (85:15) (200 μL) was added. The mixture was quenched by placing the Eppendorf tube on ice (2 minutes) and then centrifuged for 2 minutes for debris separation (15000 rpm, 21 °C). The supernatant was transferred to a plastic inlet vial and analyzed by HPLC-CAD to determine the conversion of sucrose and glucose to kojibiose. The immobilization yield was calculated by determining the percentage of enzyme activity remaining in the supernatant after immobilization compared to the enzyme activity in the cell-free extract (21)

[0338] 7.3 Crosslinking treatment The crosslinking reagents used were glutaraldehyde (GA), dimethyl suberimidate (DMS), bis(sulfosuccinimidyl) suberate (BS3), and glycerol diglycidyl ether (GDE) (see Table 23). Solutions of each crosslinking reagent were prepared in 150 mM MOPS buffer according to Table 21, and subsequently 0.5 mL was added to the immobilized catalyst. 150 mM MOPS buffer, pH 7 (0.5 mL) was added to the immobilized SucP2 to prepare a control catalyst (non-crosslinked). Both the control catalyst and the crosslinked catalyst were incubated overnight on a tube rotator (20 rpm, 21 °C). The buffer / crosslinking solution was removed, and the catalysts treated with DMS and BS3 were washed once with 1 mL of 20 mM TRIS, pH 7, and then all the catalysts were washed with 150 mM MOPS buffer, pH 7 (2 × 1 mL). The crosslinking treatment was carried out in duplicate under different conditions.

[0339] Table 21. Solutions used for crosslinking treatment of immobilized SucP2

Table 24

[0340] 7.4 Leaching treatment As a protocol for determining the efficiency of crosslinking, a leaching test was performed on the catalyst. One sample from each different crosslinking condition (different crosslinking reagents) was subjected to the leaching treatment, and samples from the same conditions were kept separately and used as controls to enable the determination of the productivity of the immobilized catalyst with and without using the leaching protocol. 0.5 M sodium phosphate buffer, pH 7 (1 mL) was added to the immobilized catalyst and incubated on a tube rotator for 2 h (20 rpm, 21 °C) for leaching. After the leaching protocol, the catalyst was washed with 150 mM MOPS buffer, pH 7 (2 × 1 mL). According to the control test, treating the immobilized catalyst with 0.5 M sodium phosphate is an effective method for leaching the enzyme not crosslinked from the immobilized catalyst.

[0341] 7.5 Activity of the immobilized catalyst in an aqueous medium The ability to convert sucrose and glucose to kojibiose was used to determine the activity of the cross-linked heterogeneous catalyst (with and without leaching treatment) (Scheme 7). A reaction mixture (1 mL) containing 1 M sucrose and 1 M glucose in deionized water was added to the immobilized catalyst. The mixture was incubated (1200 rpm, 55 °C), and after 3 h, a sample (10 μL) of the supernatant was taken to determine the productivity of the catalyst. The sample was diluted with deionized water (190 μL), and 100 mM glycerol (internal standard) in acetonitrile:deionized water (85:15) (200 μL) was added. The mixture was quenched by placing the Eppendorf tube on ice (2 min), and then centrifuged for 2 min (15000 rpm, 21 °C) for debris separation. The supernatant was transferred to a plastic inlet vial and analyzed by HPLC-CAD to determine the conversion of sucrose and glucose to kojibiose (Table 22).

Chemical formula

[0342] Scheme 10. Glycosyl transfer of sucrose (1 M) and glucose (1 M) catalyzed by immobilized SucP2 for kojibiose production

[0343] Table 22. Performance of immobilized SucP2 on propylamine, copolymer propyl + propyl NH-N-aminoethyl, propyl-3N-(4,5-dihydroimidazole), phenethyl-methyl-NH-N-aminoethyl functionalized controlled pore silica (Q30), and subsequent cross-linking treatments with and without GDE, DMS, BS3, GA, and with and without exposure to the leaching protocol. Productivity is based on the activity of the immobilized catalyst for the glycosyl transfer of sucrose and glucose to kojibiose as a function of time and support volume.

Table 25-1

Table 25-2

[0344] Table 23. Crosslinking reagents and possible covalent bonds formed from the crosslinking step [Table 26]

[0345] Example 8 Screening of pore-controlled silica with two coatings 8.1. Synthesis of pore-controlled silica with two different silane coatings As described above (Example section 1.2), a pore-controlled silica support Cariact Q30 or Ecovyst E30 with two different coatings was synthesized. The total volume of silane in the reaction mixture was fixed at 13% (15 mL silane: 100 mL toluene). A group of silanes used to coat the silica support with two coatings and the silane ratios are listed in Table 24.

[0346] Table 24 [Table 27]

[0347] 8.2 Metal deposition and immobilization In the metal deposition step for the immobilization of CalB-N-His, a 0.93 mM metal stock solution was added to the support (see Table 2). For SucP2 and the transaminase reaction, a 0.77 mM zinc stock solution was added to each support well. As described in the previous section, CalB-N-His, TbSADH, SucP2 (section 4.1.1) were immobilized without using crosslinking (results are shown in Tables 10 - 13).

[0348] 8.3 Immobilization of amyloglucosidase on pore-controlled silica containing two coatings Before immobilization, the support was pretreated with 200 mM NaP pH6 (1 mL / 10 mg support) (1 h, 20 rpm, 25 °C) to pH-stabilize the material. The buffer was removed and the material was washed with deionized water (2 × 1 mL, 5 min, 20 rpm, 25 °C). An enzyme solution containing 16.7% enzyme was prepared by diluting a commercially available enzyme stock (supplied by Novozymes) with deionized water. 1 mL of the prepared enzyme solution was added to each support (10 mg) and incubated for 17 h (20 rpm, 25 °C). The supernatant was saved for immobilization yield analysis and the immobilized catalyst was washed with deionized water (2 × 1 mL, 20 rpm, 5 min, 25 °C).

[0349] 8.4 Determination of immobilization yield To an Eppendorf containing either the supernatant (15 μL) from the immobilization reaction or the stock enzyme solution (15 μL), maltose solution (985 μL, 250 mM) was added. These mixtures were incubated for 30 min (1000 rpm, 30 °C). The reaction was then thermally quenched at 95 °C for 5 min. The samples were then diluted 8-fold (75 μL of the reaction + 225 μL of 40 mM ribose + 300 μL of ACN) and analyzed by HPLC. The immobilization yield was determined from the conversion of maltose to glucose.

[0350] 8.5 Activity of the immobilized catalyst in aqueous medium To each Eppendorf containing the washed immobilized catalyst, maltose solution (1 mL, 250 mM) was added. The mixtures were then incubated for 30 min (30 °C, 1200 rpm). The reaction was then thermally quenched at 95 °C for 5 min. The samples were then diluted 8-fold (75 μL of the reaction + 225 μL of 40 mM ribose + 300 μL of ACN) and analyzed by HPLC.

Chemical formula

[0351] Scheme 10. Hydrolysis of maltose (250 mM) catalyzed by immobilized amyloglucosidase.

[0352] 8.6 Immobilization of Transaminase (ATA) onto Pore-Controlled Silica Containing Two Coatings In 20 mM NaP, pH 8 containing 0.3 mM pyridoxal 5'-phosphate, the lyophilized powder (200 mg) of amine transaminase TA633 (EC 2.6.1.62, supplied by Prozomix) was rehydrated to obtain a cell-free extract (CFE) containing 20 mg / mL (lyophilized powder / total volume of solution). The cell-free extract (500 μL) was transferred to an Eppendorf containing 10 mg of pore-controlled silica containing two coatings newly deposited with zinc (1000 ppm), and incubated for 2 hours (20 rpm, 25 °C). Subsequently, the supernatant was removed, and the immobilized catalyst was washed with 20 mM NaP buffer, pH 8 (2 × 1 mL), and used immediately after removing the remaining buffer from the Eppendorf.

[0353] 8.7 Determination of Immobilization Yield Cell-free extracts and supernatants (250 μL) from immobilization were added to 250 μL of reaction mixture (20 mM NaP pH 8, 10% DMSO (5% final concentration), 100 mM phenoxy-2-propanone (50 mM final concentration), and 500 mM isopropylamine (250 mM final concentration)) and incubated for 2 h (600 rpm, 35 °C). After 2 h, 5 M NaOH (150 μL) and EtOAc (500 μL) were added, and the mixture was incubated for an additional 5 min at room temperature (20 rpm), followed by phase separation on the bench for 5 min. Then, the organic phase (200 μL) was transferred to a new Eppendorf containing sodium sulfate. Next, 40 μL of the extracted reaction was transferred to a new Eppendorf containing acetic anhydride (130 μL). Then, 1-methylimidazole (40 μL) was added, and the mixture was incubated at room temperature for 30 min. Deionized water (230 μL) was added and mixed, followed by the addition of EtOAc (460 μL) containing 40 mM dodecane. This mixture was incubated for 5 min (25 °C, 1200 rpm). After phase separation (5 min), 200 μL of the organic phase was transferred to a plate containing sodium sulfate. Then, the extracted samples were analyzed by gas chromatography. The immobilization yield was calculated based on the residual enzyme activity after immobilization compared to the cell-free extracts. All measurements were performed in duplicate and shown as the average of individual samples.

[0354] 8.8 Activity of Immobilized Catalysts in Aqueous Media The activity of the immobilized catalyst was determined by its ability to convert phenoxy-2-propanone to the corresponding amine (Scheme 11). A reaction mixture (20 mM NaP pH 8, DMSO (5% v / v), phenoxy-2-propanone (50 mM), isopropylamine (250 mM)) was prepared and added to the immobilized and washed catalyst (10 mg) (1 mL). The reaction was incubated overnight (25 °C, 1200 rpm). Then, 500 μL of the supernatant was transferred to a new Eppendorf, 5 M NaOH (150 μL) and EtOAc (500 μL) were added, and the mixture was incubated for 5 minutes (25 °C, 1200 rpm). Next, 40 μL of the organic phase was transferred to a new Eppendorf containing acetic anhydride (130 μL) and 1-methylimidazole (40 μL). The mixture was incubated at room temperature for 30 minutes. Deionized water (230 μL) was added and mixed, followed by the addition of EtOAc (460 μL) containing 40 mM dodecane. This mixture was incubated for 5 minutes (25 °C, 1200 rpm). After phase separation (5 minutes), 200 μL of the organic phase was transferred to a plate containing sodium sulfate. The extracted samples were then analyzed by gas chromatography. All measurements were performed in duplicate and shown as the average of individual samples.

Chemical formula

[0355] Scheme 11. Aminotransfer between 1-phenoxypropan-2-one (50 mM) and isopropylamine (250 mM) catalyzed by immobilized ATA.

[0356] Table 25. Amyloglucosidase activity against CPS containing two coatings

Table 28

[0357] Table 26. SucP2 activity against CPS containing two coatings

Table 29

[0358] Table 27. CalB activity for CPS containing two coatings

Table 30

[0359] Table 28. TbSADH activity for CPS containing two coatings

Table 31

[0360] Table 29. ATA activity for CPS containing two coatings

Table 32

[0361] Example 9. Formulation of heterogeneous biocatalysts using pore-controlled silica with large particle sizes 9.1 Isolation of pore-controlled silica with a particle size range of 280 - 500 μm Using a Haver EML 200 Premium sieve apparatus, isolation of CPS Q30 particles with a size range of 280 - 500 μm was achieved by sieving 75 - 500 μm CPS Q30 (200 g, supplied by Fuji) through a 280 μm mesh filter. After several cycles (amplitude 2, 1 minute), the isolated material (>280 μm) was removed and stored for further use.

[0362] 9.2 Synthesis of amino-functionalized pore-controlled silica with two particle size ranges (1: 280 - 500 μm, and 2: 1.18 - 2.36 mm) Following the aforementioned protocol (section 1.2), the synthesis of amino-functionalized CPS particles with diameters of 280 - 500 μm and 1.18 - 2.36 mm was generated.

[0363] 9.3 Immobilization of SucP2 onto Amino-Functionalized 280 - 500 μm and 1.18 - 2.36 mm CPS Q30 Particles The lyophilized powder of SucP2 (500 mg) was rehydrated in 150 mM MOPS buffer, pH 7 (50 mL) to obtain a cell-free extract concentration of 10 mg / mL (lyophilized powder / buffer solution). The prepared cell-free extract was resuspended on a tilt / roller mixer for 10 minutes (60 rpm, 21 °C), and then centrifuged for 10 minutes (8000 rpm, 21 °C). After centrifugation, 1 mL of the cell-free extract was transferred to a 1.5 mL Eppendorf tube containing 10 mg of the support (CPS Q30 functionalized with propylamine). The Zn from a solution of propylamine-functionalized Q30 at a final concentration of 0.105 g / L was 2+ subjected to deposition. The sample was incubated for 2 hours (20 rpm, 21 °C), and after immobilization, the supernatant was removed. The immobilized support was washed with 150 mM MOPS buffer, pH 7 (2 × 1 mL).

[0364] 9.4 Determination of Immobilization Yield An aliquot of the initial enzyme solution or an aliquot of the supernatant from immobilization (100 μL) was mixed with a reaction mixture (400 μL) containing 1 M sucrose and 1 M glucose in deionized water and incubated for 1 hour (600 rpm, 50 °C). Then, the reaction was quenched thermally at 95 °C for 5 minutes. Next, 50 μL of the sample was added to 450 μL of 30 mM ribose (internal standard), followed by the addition of 500 μL of ACN. The sample was then analyzed by HPLC to determine the conversion of sucrose and glucose to kojibiose. The immobilization yield was calculated by determining the percentage of enzyme activity remaining in the supernatant after immobilization compared to the enzyme activity in the cell-free extract.

[0365] 9.5 Crosslinking Treatment A crosslinking solution of glycerol diglycidyl ether (GDE) was prepared in a mixture of ethanol and 150 mM Tris, pH 9 (4% GDE, 20% EtOH). Then, the crosslinking solution was added to the immobilized catalyst (1 mL / 100 mg sample) and incubated for 24 h (20 rpm, 25 °C). Then, the crosslinked catalyst was washed with 150 mM Tris, pH 9 (2 × 1 mL, 1 min, 20 rpm, 25 °C). All samples were performed in duplicate.

[0366] 9.6 Leaching treatment As a protocol for determining the efficiency of crosslinking, a leaching test was performed on the catalyst. Samples were subjected to leaching treatment, and samples from the same conditions were kept separately and used as controls to enable the determination of the productivity of the immobilized catalyst with and without using the leaching protocol. 0.5 M sodium phosphate buffer, pH 7 (1 mL) was added to the immobilized catalyst and leaching was carried out by incubating on a tube rotator for 2 h (800 rpm, 50 °C). After the leaching protocol, the catalyst was washed with deionized water (3 × 1 mL). According to the control test, treating the immobilized catalyst with 0.5 M sodium phosphate is an effective method for leaching enzymes that have not undergone crosslinking from the immobilized catalyst.

[0367] 9.7 Activity of the immobilized catalyst in an aqueous medium The activity (with and without leaching treatment) of the heterogeneous catalyst after crosslinking was determined by its ability to convert sucrose and glucose to kojibiose (Scheme 7). A reaction mixture (1 mL) containing 1 M sucrose and 1 M glucose in deionized water was added to the immobilized catalyst. The mixture was incubated for 0.5 h (1200 rpm, 55 °C). Then, the reaction was thermally quenched at 95 °C for 5 min. Then, 50 μL of the sample was added to 450 μL of 30 mM ribose (internal standard), followed by the addition of 500 μL of ACN. Then, the sample was analyzed by HPLC to determine the conversion of sucrose and glucose to kojibiose.

[0368] Summary of the immobilization yields and productivities of propylamine-functionalized mesoporous control silica with different particle sizes, including both uncrosslinked and crosslinked samples, with and without treatment by leaching conditions.

Table 33

[0369] 9.8 Synthesis of amino-functionalized mesoporous control silicas Q30 and Q50 with different particle size ranges.

[0370] According to the aforementioned protocol, propylamine-functionalized CPS Q50 and CPS Q30 with three different particle size ranges were prepared.

[0371] 9.9 Metal deposition and enzyme immobilization The procedure for metal deposition was carried out as described above. The lyophilized powder of SucP2 (2.4 g) was rehydrated in 150 mM MOPS, pH 7 to obtain a cell-free extract (CFE) containing 120 mg / mL (lyophilized powder / total volume of solution). Cell-free extract (1000 μL) was transferred to Eppendorf tubes containing 100 mg of amino-functionalized CPS Q30 support and amino-functionalized CPS Q50 support with freshly deposited zinc (1000 ppm), and incubated for 24 h (20 rpm, 25 °C). Subsequently, the supernatant was removed, and the immobilized catalyst was washed with 150 mM MOPS, pH 7 (2 × 1 mL), and used immediately after removing the remaining buffer in the Eppendorf tube.

[0372] 9.10 Determination of immobilization yield An aliquot of the initial enzyme solution or an aliquot of the supernatant from immobilization (100 μL) was mixed with a reaction mixture (400 μL) containing 1 M sucrose and 1 M glucose in deionized water and incubated for 1 h (600 rpm, 50 °C). The reaction was then thermally quenched at 95 °C for 5 min. Then, 50 μL of the sample was added to 450 μL of 30 mM ribose (internal standard), followed by the addition of 500 μL of ACN. The sample was then analyzed by HPLC to determine the conversion of sucrose and glucose to kojibiose. The immobilization yield was calculated by determining the percentage of enzyme activity remaining in the supernatant after immobilization compared to the enzyme activity in the cell-free extract.

[0373] 9.11 Activity of the Immobilized Catalyst in Aqueous Media The activity of the heterogeneous catalyst was determined by its ability to convert sucrose and glucose to kojibiose (Scheme 7). A reaction mixture (1 mL) containing 1 M sucrose and 1 M glucose in deionized water was added to the immobilized catalyst. The mixture was incubated for 0.5 h (1200 rpm, 55 °C). The reaction was then thermally quenched at 95 °C for 5 min. Then, 50 μL of the sample was added to 450 μL of 30 mM ribose (internal standard), followed by the addition of 500 μL of ACN. The sample was then analyzed by HPLC to determine the conversion of sucrose and glucose to kojibiose.

[0374] Table 31. Summary of the immobilization yield and productivity of propylamine-functionalized pore-controlled silica with different particle sizes and different pore diameters.

Table 34

[0375] Example 10 Amino-Functionalized Pore-Controlled Silica Prepared Using Various Methods Two different pore control silicas, Q30 (supplied by Fuji) and Ecovyst E30 CPS (supplied by Ecovyst), were functionalized with propylamine using three different application methods, namely, slurry coating, spray coating, and initial wetting coating. Slurry coating is a method in which amino-functionalization of the CPS support is carried out by suspending the CPS support in a solution containing an appropriate silane. Initial coating is a method in which amino-functionalization of the CPS support is carried out by applying a controlled volume of solution containing an appropriate silane, where the controlled volume exactly matches the pore volume of the CPS support. Spray coating is a process in which a solution of an appropriate silane is converted into droplets via a nozzle and then sprayed onto the CPS support to enable a uniform distribution of the coating.

[0376] Any of the methods described herein can produce immobilized supports with similar performance.

[0377] 10.1 Immobilization of TLL onto CPS propylamine A solution of Lipozyme® TL 100L containing Thermomyces lanuginosa lipase (TLL) for immobilization was prepared by diluting a commercially available enzyme (60% v / v) with deionized water (final solution at pH 6.7). Diluted enzyme (50 mL) was transferred to a 100 mL Duran bottle containing 10 g of support (propylamine-functionalized CPS Q30 supplied by Fuji and a second CPS sample supplied by Ecovyst). The sample was incubated on a tilt / roller mixer for 1.5 h (70 rpm, 21 °C). After immobilization, the supernatant was removed and the immobilized support was washed with 50 mM HEPES, 50 mM ammonium acetate, 10 mM CaCl 2 2, in a pH 7 solution (1 × 50 mL), followed by 50 mM HEPES, 50 mM ammonium acetate, 10 mM CaCl 2、It was washed with 60 mM sucrose, pH 7 solution (1×50 mL). For each washing step, the immobilized catalyst was mixed with the washing solution on a tilt / roller mixer for 3 minutes (70 rpm, 21 °C). The catalyst was filtered under vacuum to remove most of the washing solution (6 minutes), transferred to a 2 L glass flask, and dried using a rotary evaporator for 55 - 70 minutes (40 mbar, 40 °C).

[0378] 10.2 Activity of immobilized TLL catalyst for transesterification of blended soybean oil To obtain a product with a low final melting temperature, the activity of the immobilized catalyst was evaluated for the transesterification of a soybean oil blend composed of 60% refined and bleached soybean oil and 40% fully hydrogenated soybean oil supplied by Bunge (BG F41120 - 000 SOY SHTG). The melted soybean oil blend (500 μL) was added to a 2 mL Eppendorf tube containing immobilized TLL (10 mg) and incubated for 30 minutes (1500 rpm, 70 °C). After stopping the mixing and allowing the catalyst to settle, an aliquot of the oil (350 μL) was transferred to a 2 mL Eppendorf tube, placed in ice-cold water to solidify the fat, and subsequently stored in a freezer (-20 °C) for at least 1 hour before analysis. The end point of the melting range of the sampled reaction was analyzed using a Melting Point System MP55 (Mettler Toledo) equipped with a capillary (Hirschmann, L 75 mm, I.D 0.95 mm, O.D. 1.35 mm) using a heating rate of 2 °C / min from 34 °C to 67 °C, and the end point of the melting temperature was recorded as the temperature at which the intensity signal (transmittance) reached a plateau and no further increase in intensity was detected (Table 32). The end point of the melting range of the initial soybean oil blend was 64.7 °C, and the lower end of the melting range indicates a high degree of the transesterification reaction that occurred, thereby indicating high enzyme activity.

[0379] Table 32. Performance of immobilized Lipozyme® TL 100L on amino-functionalized pore-controlled silica supports prepared using various methods. The efficiency of the catalyst in the transesterification of blended soybean oil is based on the end point of the melting range of the soybean oil blend after the reaction.

Table 35

Claims

**Claim 1** a. Porosity-controlled silica (CPS) as a support material having a pore diameter of about 20 to about 100 nm, and wherein the support material includes an amino-functionalized surface, b. One or more catalytically active enzymes immobilized on the amino-functionalized surface by a covalent linker including a bond selected from amino, amide, and imidoamide, and A biocatalyst for organic synthesis comprising the same. **Claim 2** a. Porosity-controlled silica (CPS) as a support material having a pore diameter of about 20 to about 100 nm, and wherein the support material includes an amino-functionalized surface, b. One or more catalytically active enzymes immobilized on the surface via non-covalent interactions, and A biocatalyst for organic synthesis comprising the same. **Claim 3** a. Porosity-controlled silica (CPS) as a support material having a pore diameter of about 20 to about 100 nm, and wherein the support material surface includes at least two different coatings, at least one of which provides an amino-functionalized surface, b. Optionally, one or more catalytically active enzymes immobilized on the surface by a covalent linker including a bond selected from amino, amide, and imidoamide, and A biocatalyst for organic synthesis comprising the same. **Claim 4** a. Porosity-controlled silica (CPS) as a support material having a pore diameter of about 20 to about 100 nm, and wherein the support material includes an amino-functionalized surface, b. Optionally, one or more catalytically active enzymes immobilized on the amino-functionalized surface by a covalent linker including a bond selected from amino, amide, and imidoamide, and wherein The enzyme is selected from List (A), preferably from List (B), A biocatalyst for organic synthesis. **Claim 5** The biocatalyst according to claim 1, 3 or 4, wherein the covalent linker, when present, includes a bond selected from the group consisting of amino and amide. **Claim 6** The support material has a pore diameter of about 20 to about 60 nm and a surface area of about 50 m ２ / g to about 200 m ２ / g, and the biocatalyst according to any one of claims 1 to 5. **Claim 7** The support material has a pore diameter of about 20 to about 60 nm, a surface area of about 50 m ２ / g to about 200 m ２ / g, and a pore volume of about 0.5 mL / g to about 2.0 mL / g. The biocatalyst according to any one of claims 1 to 6. **Claim 8** The biocatalyst according to claim 1, 2, 3 or any claim dependent thereon, wherein the enzyme is selected from List (A). **Claim 9** The biocatalyst according to any one of claims 1 to 8, wherein the enzyme is selected from List (B). **Claim 10** The biocatalyst according to any one of claims 1 to 9, wherein the support material surface includes at least two different coatings, at least one of which provides amino-functionalization. **Claim 11** The biocatalyst according to claim 10, wherein one of the coatings that does not provide amino functionalization provides a hydrophobic coating.

12. The biocatalyst according to claim 11, wherein the hydrophobic coating contains an alkyl moiety and / or an aromatic moiety.

13. The hydrophobic coating is C １－１２ an alkyl group, preferably C １－６ an alkyl group, the biocatalyst according to claim 12.

14. The biocatalyst according to any one of claims 1 to 13, wherein the immobilized enzyme is covalently bonded intermolecularly by a linker containing a bond selected from amino, amide, ester, ether, thioester, thioether, imidoamide, imidothioamide and thioamide.

15. The biocatalyst according to claim 3, 4 or any claim dependent thereon, wherein the enzyme is immobilized on the surface by a covalent linker containing a bond selected from amino, amide and imidoamide, preferably amino or amide.

16. The enzyme is immobilized on the surface by a covalent linker containing a bond selected from amino, amide and imidoamide (preferably amino or amide), before the formation of the bond, the enzyme was immobilized on the surface via non-covalent interactions, The biocatalyst according to claim 1, 3, 4 or any claim dependent thereon.

17. The biocatalyst according to claim 16, wherein before the formation of the bond, the enzyme was immobilized on the surface via an interaction mediated by a chelating metal ion.

18. The biocatalyst according to claim 2, 3, 4 or any claim dependent thereon, wherein the enzyme is immobilized on the surface via an interaction mediated by a chelating metal ion.

19. The chelated metal ion is Ni ２＋ , Cu ２＋ , Mg ２＋ , Fe ３＋ and Zn ２＋ The biocatalyst according to claim 17 or 18, selected from

20. wherein the metal ion is Zn ２＋ The biocatalyst according to claim 19, wherein the ion is an ion of Zn

21. The biocatalyst according to any one of claims 1 to 20, wherein when present, the covalent linker between the surface and the enzyme does not contain an imine bond.

22. The biocatalyst according to any one of claims 1 to 21, wherein when present, the intermolecular covalent linker does not contain an imine bond.

23. The biocatalyst according to any one of claims 1 to 22, wherein when present, the covalent linker contains a bridge containing 3 to 20 atoms in any combination of C, H, N and O.

24. The biocatalyst according to any one of claims 1 to 23, wherein the co - binding linker, when present, is formed using a bifunctional reagent preferably containing at least two reactive groups independently selected from epoxides, esters, anhydrides, N - hydroxysuccinimide esters, imido esters, carbonates, acyl isoureas, carbodiimides, maleimides, haloacetyls, thiosulfonates, isocyanates and vinyl sulfones.

25. The amino - functionalized surface comprises a structure of formula (I), (II) or (III): 【Chemical 1】 wherein, R １ is C １－６ alkanediyl, and R １ provides a covalent bond to the silica surface (left vertical line), X is a bond (i.e., X is absent) or a phenyl ring, R ２ is C １－６ alkanediyl, and R ３ is W, R ４ is W or C １－１０ alkyl, C ５－７ cycloalkyl, phenyl, phenyl-C １－６ -alkyl, amino-C ２－８ alkyl, N-(phenyl)amino-C ２－８ alkyl, and N-(phenyl-C １－６ -alkyl)amino-C ２－８ alkyl, and is selected from the group consisting of aminocarbonyl, each amino being substituted by W, and phenyl being, in each case, optionally substituted by one or more substituents C １－４ alkyl, or R ３ and R ４ may, together with the nitrogen atom to which they are attached, form a 5- to 7-membered heterocyclic ring containing one or more unsaturated bonds and may contain another additional heteroatom, on which a covalent linker containing a bond selected from amino, amide and imidoamide may be attached, and the catalytically active enzyme may also be attached to said linker, or or R ３ and R ４ is =CR ５ R ６ together form, R ５ and R ６ are each independently, C １－４ alkyl, Each R ７ is independently a C １－６ alkanediyl, and R ７ provides a covalent bond to the silica surface (left vertical line), R ８ is C １－３ alkanediyl, and Each R ９ is independently W or C １－４ alkyl, and hydroxy-C １－４ selected from the group consisting of alkyl Each R １０ is independently a C １－６ alkanediyl, and R １０ provides a covalent bond to the silica surface (left vertical line), R １１ is W or C １－１０ alkyl, C ５－７ cycloalkyl, phenyl, phenyl-C １－６ -alkyl, amino-C ２－８ alkyl, N-(phenyl)amino-C ２－８ alkyl, and N-(phenyl-C １－６ -alkyl)amino-C ２－８ alkyl, and is selected from the group consisting of aminocarbonyl, each amino being substituted by W, and phenyl being, in each case, optionally substituted by one or more substituents C １－４ alkyl each W is independently a covalent linker containing a bond selected from hydrogen or amino, amide and imidoamide, and the catalytically active enzyme is also bound to the linker, the biocatalyst according to any one of claims 1 to 24.

26. The amino - functionalized surface comprises a structure of formula (I) as defined above in combination with a structure of formula (IV): [Chemical Formula 2] In the formula, R １２ is selected from C １－１２ alkyl and amino-C １－６ alkyl, and is the biocatalyst according to any one of claims 1 to 25.

27. The amino - functional group of the surface is not part of a heterocyclic ring, the biocatalyst according to any one of claims 1 to 26.

28. The surface, except for the amino group, does not contain a functional group capable of chelating metal ions, the biocatalyst according to any one of claims 1 to 27.

29. The surface comprises an amino - functionalized aliphatic moiety, the biocatalyst according to any one of claims 1 to 28.

30. The amino - functionalized surface comprises any of the following structures, each W is a covalent linker containing a bond selected from hydrogen or amino, amide and imidoamide, and the catalytically active enzyme is also bound to the linker, the biocatalyst according to any one of claims 1 to 29: [Chemical 3]

31. The amino - functionalized surface comprises structure (I’): [Chemical Formula 4] wherein, R ４ is W or is selected from the group consisting of phenyl, phenyl-C １－６ -alkyl, amino-C ２－８ -alkyl, N-(phenyl)amino-C ２－８ -alkyl, and N-(phenyl-C １－６ -alkyl)amino-C ２－８ -alkyl, wherein phenyl may in each case be substituted by one or more substituents C １－４ -alkyl, W is a covalent linker containing a bond selected from hydrogen or amino, amide and imidoamide, and the catalytically active enzyme is also bound to the linker, or the amino - functionalized surface comprises structure (I”): 【Chemical Formula 5】 wherein, R ４ is W or amino-C ２－８ alkyl, N-(phenyl)amino-C ２－８ alkyl, and N-(phenyl-C １－６ -alkyl)amino-C ２－８ alkyl, and phenyl is optionally substituted in each case by one or more substituents C １－４ alkyl, W is a covalent linker containing a bond selected from hydrogen or amino, amide and imidoamide, and the catalytically active enzyme is also bound to the linker, the biocatalyst according to any one of claims 1 to 30.

32. wherein said amino-functionalized surface is R ４ is W, or phenyl, benzyl, amino-C ２－６ alkyl, N-(phenyl)amino-C ２－６ alkyl, and N-(benzyl)amino-C ２－６ alkyl, or comprises a structure I' selected from the group consisting of The amino-functionalized surface is R ４ The biocatalyst according to claim 34, comprising Structure I" in which is W.

33. The biocatalyst according to any one of claims 1 to 32, wherein the amino-functionalized surface comprises any of the following structures, each W is a covalent linker containing hydrogen or a bond selected from amino, amide and imide amide, and the catalytically active enzyme is also bound to the linker: 【Chemical Formula 6】

34. The biocatalyst according to any one of claims 1 to 33, wherein the amino-functionalized surface comprises any of the following structures, each W is a covalent linker containing hydrogen or a bond selected from amino, amide and imide amide, and the catalytically active enzyme is also bound to the linker: （１） 【Chemical 7】 （２） [Chemical Formula 8] （３） 【Chemical Formula 9】 （４） 【Chemical Formula 10】

35. The biocatalyst according to any one of claims 1 to 34, wherein the immobilized enzyme is selected from Bifidobacterium adolescentis sucrose phosphorylase (SucP), Candida antarctica lipase (CalB), Thermoanaerobacter brockii secondary alcohol dehydrogenase (TbSADH), aminotransaminase (ATA), Leuconostoc mesenteroides glucosyltransferase (GT), Thermomyces lanuginosus lipase (TLL), and Aspergillus niger amyloglucosidase.

36. The biocatalyst according to any one of claims 1 to 35, wherein at least one immobilized enzyme retains at least 50% of its activity after 20 hours under continuous flow conditions compared to its state before immobilization.

37. A lipase (preferably CalB) containing a metal chelate tag such as a His tag is immobilized on a surface containing an amino-functionalized aliphatic moiety via an interaction mediated by chelated Zn ２＋ The biocatalyst according to any one of claims 1 to 36, wherein the biocatalyst is immobilized on a surface containing an amino-functionalized aliphatic moiety via an interaction mediated by chelated Zn ions.

38. TbSADH containing a metal chelate tag such as a His tag is immobilized on the surface through an interaction mediated by chelated Zn ２＋ The biocatalyst according to any one of claims 1 to 36, which is immobilized on the surface through an interaction mediated by chelated Zn ions.

39. The biocatalyst according to any one of claims 1 to 36, wherein ATA containing a metal chelate tag such as a His tag is immobilized on a surface comprising structure (1), (2), (3) or (4) via an interaction mediated by a chelated metal ion.

40. The biocatalyst according to any one of claims 1 to 36, wherein SucP containing a metal chelate tag such as a His tag is immobilized on a surface comprising structure (2).

41. The biocatalyst according to any one of claims 1 to 36, wherein TLL not containing a metal chelate tag such as a His tag is immobilized on the surface.

42. The biocatalyst according to any one of claims 1 to 36, wherein glucosyltransferase containing a metal chelate tag such as a His tag is immobilized on a surface comprising structure (1).

43. The lipase CalB is immobilized on a surface comprising structure (1) or (4), preferably non-covalently, and the CalB most preferably does not contain a metal chelate tag such as a His tag, the biocatalyst according to any one of claims 1 to 36.

44. An amyloglucosidase preferably not containing a metal chelate tag such as a His tag is immobilized on a surface functionalized by structure (1), (2), (3) or (4), the surface optionally having a different second functionalization, the biocatalyst according to any one of claims 1 to 36.

45. Use of the biocatalyst according to any one of claims 1 to 44 in a reactor for the synthesis of organic compounds.

46. The use according to claim 45, wherein the reactor is a continuous flow fixed bed reactor.

47. a. Providing the biocatalyst according to any one of claims 1 to 44 disposed within a reactor; b. Supplying a precursor to an organic compound to the flow reactor, whereby the immobilized enzyme catalyzes the reaction, resulting in the synthesis of the organic compound; and c. Recovering the organic compound, A method for the synthesis of an organic compound comprising.

48. The method according to claim 47, wherein the reactor is a batch reactor or a continuous flow reactor.

49. The method according to claim 48, wherein the reactor is a continuous flow fixed bed reactor.

50. a. Providing one or more catalytically active enzymes; b. Providing a porosity-controlled silica (CPS) as a support material, wherein the pore diameter is from about 20 to about 100 nm, the support material comprising an amino-functionalized surface; and c. Immobilizing the one or more enzymes on the support material, A method for producing the biocatalyst according to any one of claims 1 to 44 comprising.

51. Selecting a bifunctional crosslinking reagent capable of forming an amino bond, an amide bond or a thioamide bond when reacted with the enzyme and the surface; and Using the bifunctional reagent to covalently crosslink the enzyme, The method according to claim 50, further comprising.

52. The method according to claim 51, wherein the bifunctional reagent is a bisepoxide, an imidoester or an N-hydroxysuccinimide ester.

53. The enzyme is immobilized on the surface via an interaction mediated by a chelating metal ion, preferably Ni ２＋ , Cu ２＋ , Mg ２＋ , Fe ３＋ or Zn ２＋ , most preferably Zn ２＋ The method according to claim 50 or any claim dependent thereon, wherein the method is immobilized on the surface via an interaction mediated by a chelating metal ion, preferably Ni, Cu, Mg, Fe or Zn, most preferably Zn

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