Biocatalyst for organic synthesis

Abstract

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

Description

Technical Field

[0001] The present invention relates to a biocatalyst for organic synthesis, wherein an enzyme is immobilized on a 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] Enzyme acts as a biocatalyst in the metabolism of all living cells. Therefore, enzyme can convert organic molecules into different molecules. Due to the specific three-dimensional structure of each specific enzyme, only some organic molecules will interact with the active site of the enzyme and transform in this way. Therefore, enzyme is usually a highly selective catalyst, and it is very attractive to use enzyme as a catalyst in synthetic organic chemistry for this reason. However, since enzyme is a biomolecule evolved for the cell environment, they are usually not suitable for other environments. When used in an organic solvent, enzyme tends to gather and often precipitate or unfold (i.e. denaturation). Therefore, it is attractive to immobilize enzymes on solid supports and use them as catalysts in this immobilized state, because this can improve the stability of enzymes, allow enzymes to be used for the reaction conditions that they cannot tolerate usually, and also help to separate and recover materials from reaction mixtures. Immobilization also makes it possible to use an immobilized enzyme that is much higher than the free enzyme in the solution in concentration.

[0003] The anticipated industrial-scale synthesis process using biocatalysts requires that the enzyme be immobilized on a suitable support and packaged in a fixed bed reactor suitable for operation in a continuous flow mode. This poses additional challenges to the support material used in the immobilization in order to withstand the pressure and achieve sufficient flow rates. Immobilization of enzymes on solid supports has previously been achieved using different techniques and different solid supports.

[0004] PCT / SE2015 / 050108 discloses a biocatalyst having an enzyme immobilized to controlled porosity glass beads or porous organic beads as a support material by metal chelation. Although controlled porosity glass works well in terms of activity, the support material is expensive and fragile.

[0005] Nagy et al. (ChemCatChem 2018, 10, 3490–3499) disclosed the covalent immobilization of acid phosphatase onto silica nanoparticles for biocatalytic applications. Due to the high back pressure, the biocatalytic nanoparticles were mixed with larger particles of inert silica for continuous flow applications.

[0006] JP2002176974 discloses R-cyanocyanase immobilized on a porous clay-based sintered support or a porous silica-based support by physical adsorption. The support was not surface modified. It is worth noting that 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 controlled pore size, which is used as a carrier for enzyme immobilization. The surface is modified with aminopropyl groups, and glucosaminidase is coupled to the surface using glutaraldehyde.

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

[0009] Nagy et al. (ChemSusChem 2021, pages 1-10 and supporting information pages 1-12) disclosed the covalent immobilization of lipase B (CaLB) from Antarctic Saccharomyces cerevisiae on various functionalized silica nanoparticles.

[0010] It was therefore an object of the present invention to provide improved and / or alternative means and methods for performing biocatalysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 : The data from Table 20 are plotted as conversion versus time on stream, demonstrating the excellent stability of the immobilized sucrose phosphorylase biocatalyst over time under industrial conditions.

[0012] Figure 2 : Activity and immobilization rate of SucP2 immobilized on various types of carriers. It is noteworthy that Cariact Q15, which has a pore size below 20 nm, performs poorly compared to Q30 and Q50. The performance of the support material according to the present invention is much better than the prior art support materials LCAA-CPG, SBA15 and MCM48.

[0013] Figure 3 : Flowchart for optimizing biocatalyst manufacturing.

[0014] definition

[0015] The term multi-step synthesis refers to a reaction in which a starting material is converted into a product by at least one intermediate. As used herein, the term "step" or "multiple steps" refers to one or more individual steps that form the overall step of a multi-step synthesis together. Therefore, the "step" of a multi-step synthesis can refer to the conversion of a starting material to a first intermediate, the conversion of an intermediate to a next intermediate, or the conversion of a last intermediate to a product. Multi-step synthesis can be a linear synthesis, in which a single starting material is converted into a final product in a linear series of continuous steps, or multi-step synthesis can be a convergent synthesis, in which two or more starting materials are converted into a single final product in a series of convergent steps. Each individual enzyme catalyzed step of synthesis is catalyzed by at least one enzyme immobilized on a support material. Any simultaneous reaction (e.g., involving a cofactor or coenzyme regeneration reaction) catalyzed by an immobilized enzyme but not directly involving the conversion of a starting material or any intermediate to a product is not considered as a "step" in the multi-step synthesis herein.

[0016] As used herein in the context of the biocatalysts of the invention, the term surface refers to the entire surface of the controlled porosity silica (CPS) support material and includes the external surface and the pore surface. Due to the nature of CPS, most of the surface area exists in the pores.

[0017] The term sequence identity expressed as a percentage is defined as a value determined by comparing two optimally aligned sequences within a comparison window, wherein for optimal alignment of the two sequences, a portion of the sequence in the comparison window may include additions or deletions (i.e., gaps) compared to a reference sequence (which does not include additions or deletions). The percentage is calculated by determining the number of positions of identical amino acid residues occurring in the two sequences to produce the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 10 to produce the percentage of sequence identity. Unless otherwise indicated, the comparison window is the entire length of the sequence referred to. In this context, the optimal alignment is an alignment generated by the BLASTP algorithm implemented online by the National Center for Biotechnology Information (see NCBI Manual, Second Edition [https: / / www.ncbi.nlm.nih.gov / books / NBK143764 / ]) with the following input parameters: word length = 3, matrix = BLOSUM62, gap cost = 11, gap extension cost = 1.

[0018] As used herein, the term "about" refers to a value or parameter herein, including (and describing) embodiments for the value or parameter itself. For example, a description involving "about 20" includes a description of "20". A numerical range includes the numbers defining the range. In general, the term "about" refers to the indicated value of a variable and the values ​​of all variables within the experimental error of the indicated value (e.g., within a 95% confidence interval of the mean value) or within 10% of the indicated value, whichever is greater.

[0019] The term continuous flow reactor refers to a reactor designed to operate as follows: one or more reactants are fed substantially continuously into the reactor in the form of a feed stream, thereby producing a substantially continuous product stream from the reactor, containing at least one reaction product. In operation, at least a portion of the product stream (preferably the entire product stream) is collected and can optionally be subjected to further processing steps, such as a purification step or a second reactor. Any portion of the product stream that is not collected can be fed back to the reactor as part of the feed stream. Summary of the invention

[0020] The present invention relates to the following items. The subject matter disclosed in the following items should be regarded as disclosed in the same manner as the subject matter is disclosed in the patent claims.

[0021] 1. A biocatalyst for organic synthesis, comprising:

[0022] a. controlled porosity silica (CPS) as a support material having a pore diameter of about 20 to about 100 nm;

[0023] wherein the support material comprises an amino-functionalized surface; and

[0024] b. One or more catalytically active enzymes immobilized to the amino-functionalized surface via a covalent linker comprising a bond selected from the group consisting of amino, amide, and iminoamide.

[0025] 2. A biocatalyst for organic synthesis, comprising:

[0026] a. controlled porosity silica (CPS) as a support material having a pore diameter of about 20 to about 100 nm;

[0027] wherein the support material comprises an amino-functionalized surface; and

[0028] b. One or more catalytically active enzymes immobilized on the surface via non-covalent interactions.

[0029] 3. A biocatalyst for organic synthesis, comprising:

[0030] a. controlled porosity silica (CPS) as a support material having a pore diameter of about 20 to about 100 nm;

[0031] wherein the support material surface comprises at least two different coatings, at least one of the coatings providing an amino-functionalized surface; and

[0032] b. One or more catalytically active enzymes immobilized to said amino-functionalized surface, optionally said immobilization is via a covalent linkage comprising a bond selected from the group consisting of amino, amide and iminoamide.

[0033] 4. A biocatalyst for organic synthesis, comprising:

[0034] a. controlled porosity silica (CPS) as a support material having a pore diameter of about 20 to about 100 nm;

[0035] wherein the support material comprises an amino-functionalized surface; and

[0036] b. One or more catalytically active enzymes immobilized to the amino-functionalized surface, optionally via a covalent linker comprising a bond selected from amino, amide and iminoamide, wherein the enzyme is selected from list (A), preferably from list (B).

[0037] 5. A biocatalyst according to claim 1, 3 or 4, wherein the covalent linker, when present, comprises a bond selected from amino and amide.

[0038] 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.

[0039] 7. A biocatalyst according to any of the preceding items, wherein the support material has a thickness of about 50 m 2 / g to about 200m 2 / g of surface area.

[0040] 8. The biocatalyst according to any of the preceding items, wherein the support material has a pore volume of about 0.5 mL / g to about 2.0 mL / g.

[0041] 9. A biocatalyst according to any of the preceding items, wherein the support material has a pore diameter of about 20 to about 60 nm and a pore diameter of about 50 nm. 2 / g to about 200m 2 / g of surface area.

[0042] 10. The biocatalyst according to any of the preceding items, wherein the support material has a pore diameter of about 20 to about 60 nm, a pore diameter of about 50 nm, a pore diameter of about 100 nm, a pore diameter of about 20 nm, a pore diameter of about 300 nm, a pore diameter of about 500 nm, a pore diameter of about 200 nm, a pore diameter of about 300 nm, a pore diameter of about2 / g to about 200m 2 / g surface area and a pore volume of about 0.5 mL / g to about 2.0 mL / g.

[0043] 11. The biocatalyst according to any one of items 1, 2, 3 or their dependencies, wherein the enzyme is selected from list (A), preferably from list (B).

[0044] 12. The biocatalyst according to any one of items 1 to 9, wherein the support material has a pore diameter of about 20 to about 60 nm, a pore diameter of about 50 nm, a pore diameter of about 100 nm, a pore diameter of about 200 nm, a pore diameter of about 500 nm, a pore diameter of about 200 nm, a pore diameter of about 300 nm, a pore diameter of about 1000 nm, a pore diameter of about 2000 nm, a pore diameter of about 30 ... 2 / g to about 200m 2 / g of surface area and a pore volume of about 0.5 mL / g to about 2.0 mL / g, and wherein the enzyme is selected from list (A), preferably from list (B).

[0045] 13. The biocatalyst according to any of the preceding items, wherein the surface of the support material comprises at least two different coatings, at least one of the coatings providing amino functionalization.

[0046] 14. The biocatalyst according to item 13, wherein the relative amounts of the two coatings are in the range of 9:1 to 1:9 by weight, preferably 5:1 to 1:5, more preferably 2:1 to 1:2, most preferably 1:1.

[0047] 15. The biocatalyst according to any one of items 13-14, wherein one of the coatings not providing amino functionalization provides a hydrophobic coating.

[0048] 16. The biocatalyst of item 15, wherein the hydrophobic coating comprises alkyl moieties and / or aromatic moieties.

[0049] 17. The biocatalyst according to claim 16, wherein the hydrophobic coating comprises C 1-12 Alkyl group, preferably C 1-6 Alkyl group.

[0050] 18. The biocatalyst according to any of the preceding items, wherein the immobilized enzyme is covalently linked intermolecularly via a linker comprising a bond selected from the group consisting of amino, amide, ester, ether, thioester, iminoamide, iminothioamide, thioether and thioamide.

[0051] 19. The biocatalyst according to any one of items 3, 4 or its dependencies, wherein the enzyme is immobilized to the surface via a covalent linker comprising a bond selected from the group consisting of amino, amide and iminoamide.

[0052] 20. A biocatalyst according to any one of items 1, 3, 4 or their dependencies, wherein the enzyme is immobilized to the surface via a covalent linker comprising a bond selected from amino, amide and iminoamide, and wherein prior to forming the bond, the enzyme has been immobilized to the surface via non-covalent interactions.

[0053] 21. The biocatalyst according to item 20, wherein prior to forming said bond, said enzyme has been immobilized to said surface via an interaction mediated by a chelated metal ion.

[0054] 22. The biocatalyst according to any one of items 2, 3, 4 or its dependencies, wherein the enzyme is immobilized to the surface via an interaction mediated by a chelated metal ion.

[0055] 23. A biocatalyst according to item 21 or 22, wherein the chelated metal ion is selected from Ni 2+ , Cu 2+ Mg 2+ , Fe 3+ and Zn 2+ .

[0056] 24. The biocatalyst according to item 23, wherein the metal ion is Zn 2 + ions.

[0057] 25. The biocatalyst of any preceding clause, wherein the covalent linker between the surface and the enzyme, when present, does not comprise an imine bond.

[0058] 26. The biocatalyst according to any of the preceding clauses, wherein when present, the intermolecular covalent linker does not comprise an imine bond.

[0059] 27. The biocatalyst of any preceding clause, wherein the covalent linker, when present, comprises a bridge comprising 3 to 20 atoms comprising any combination of C, H, N and O.

[0060] 28. A biocatalyst according to any of the preceding clauses, wherein the covalent 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, imidates, carbonates, acylisoureas, carbodiimides, maleimides, haloacetyl groups, thiosulfonates, isocyanates and vinyl sulfones.

[0061] 29. The biocatalyst according to any of the preceding clauses, wherein the amino-functionalized surface comprises a structure of formula (I), (II) or (III):

[0062]

[0063] in

[0064] R 1 C 1-6 alkanediyl; and wherein R 1 Provides covalent attachment to the silica surface (vertical line on the left);

[0065] X is a bond (i.e., X is absent) or a benzene ring;

[0066] R 2 C 1-6 Alkanediyl;

[0067] R 3 is W;

[0068] R 4 Is W or selected from the following group: 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 aminocarbonyl, wherein each amino group is substituted by W; and wherein the phenyl group is optionally substituted by one or more substituents C 1-4 Alkyl substitution;

[0069] or R 3 and R 4 are combined together with the nitrogen atom to which they are attached to form a 5- to 7-membered heterocyclic ring which may contain one or more unsaturated bonds and may contain one or more additional heteroatoms, to which is optionally attached a covalent linker comprising a bond selected from the group consisting of amino, amide and iminoamide, wherein the catalytically active enzyme is also attached to the linker;

[0070] or R 3 and R 4 Together they form = CR 5 R 6 , where R 5 and R 6 Each independently is C 1-4 alkyl;

[0071] Each R 7 Independently for C 1-6 alkanediyl; and wherein R 7 Provides covalent attachment to the silica surface (vertical line on the left);

[0072] R8 C 1-3 Alkanediyl;

[0073] Each R 9 is independently W or selected from C 1-4 Alkyl and hydroxy-C 1-4 A group consisting of alkyl groups;

[0074] Each R 10 Independently for C 1-6 alkanediyl; and wherein R 10 Provides covalent attachment to the silica surface (vertical line on the left);

[0075] R 11 Is W or selected from the following group: 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 aminocarbonyl, wherein each amino group is substituted by W; and wherein the phenyl group is optionally substituted by one or more substituents C 1-4 Alkyl substitution; and

[0076] Each W is independently hydrogen or a covalent linker comprising a bond selected from amino, amide, and iminoamide, wherein the catalytically active enzyme is also attached to the linker.

[0077] 30. The biocatalyst according to any of the preceding items, wherein the amino-functionalized surface comprises a combination of a structure of formula (I) as defined above and a structure of formula (IV):

[0078] Where R 12 Selected from C 1-12 Alkyl and amino-C 1-6 alkyl.

[0079] 31. The biocatalyst according to any of the preceding items, wherein the amino functional groups in the surface are not part of a heterocycle.

[0080] 32. The biocatalyst according to any of the preceding items, wherein the surface does not contain any functional groups other than amino groups that can chelate metal ions.

[0081] 33. The biocatalyst according to any of the preceding items, wherein the surface comprises amino-functionalized aliphatic moieties.

[0082] 34. A biocatalyst according to any of the preceding clauses, wherein the amino-functionalized surface comprises any of the following structures, wherein each W is hydrogen or a covalent linker comprising a bond selected from amino, amide and iminoamide, wherein a catalytically active enzyme is also attached to the linker:

[0083]

[0084] 35. The biocatalyst according to any of the preceding clauses, wherein the amino-functionalized surface comprises structure (I'):

[0085]

[0086] in

[0087] 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, and wherein the phenyl group is optionally substituted by one or more substituents C 1-4 Alkyl substituted; and

[0088] W is hydrogen or a covalent linker comprising a bond selected from the group consisting of amino, amide, and iminoamide, wherein the catalytically active enzyme is also attached to the linker;

[0089] Or wherein the amino-functionalized surface comprises structure (I"):

[0090]

[0091] in

[0092] R 4 is W or is selected from the group consisting of 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 wherein the phenyl group is optionally substituted by one or more substituents C 1-4 Alkyl substituted; and

[0093] W is hydrogen or a covalent linker comprising a bond selected from amino, amide and iminoamide, wherein the catalytically active enzyme is also attached to the linker.

[0094] 36. A biocatalyst according to item 34, wherein the amino-functionalized surface comprises structure I', wherein R 4is W or is selected from the group consisting of phenyl, benzyl, amino-C 2-6 Alkyl, N-(phenyl)amino-C 2-6 Alkyl and N-(benzyl)amino-C 2-6 alkyl; or the amino-functionalized surface comprises structure I", wherein R 4 It's W.

[0095] 37. A biocatalyst according to any of the preceding clauses, wherein the amino-functionalized surface comprises any of the following structures, wherein each W is hydrogen or a covalent linker comprising a bond selected from amino, amide and iminoamide, wherein a catalytically active enzyme is also attached to the linker:

[0096]

[0097] 38. A biocatalyst according to any of the preceding clauses, wherein the amino-functionalized surface comprises any of the following structures, wherein each W is hydrogen or a covalent linker comprising a bond selected from amino, amide and iminoamide, wherein a catalytically active enzyme is also attached to the linker: (1)

[0099] (2)

[0101] (3)

[0103] (4)

[0105]

[0106] 39. A biocatalyst according to any of the preceding items, wherein the immobilized enzyme is selected from: Bifidobacterium adolescentis sucrose phosphorylase (SucP), Antarctic Saccharomyces cerevisiae lipase (CalB), Thermoanaerobacterium buchneri secondary alcohol dehydrogenase (TbSADH), amine transaminase (ATA), Mesentericola glycosyltransferase (GT), Thermomyces lanuginosus lipase (TLL) and Aspergillus niger amyloglucosidase.

[0107] 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 compared to the state before immobilization.

[0108] 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 compared to the state before immobilization.

[0109] 42. The biocatalyst according to any of the preceding items, wherein at least two different enzymes are immobilized on the support material.

[0110] 43. The biocatalyst of item 42, wherein each of the at least two immobilized enzymes is capable of catalyzing a different step of a multi-step organic synthesis.

[0111] 44. A biocatalyst according to claim 43, wherein the multi-step synthesis comprises at least 3 intermediates.

[0112] 45. The biocatalyst according to any of the preceding items, wherein at least one immobilized enzyme catalyzes the regeneration of a cofactor.

[0113] 46. ​​A biocatalyst according to any one of items 1 to 45, wherein the lipase (preferably CalB) comprising a metal chelate tag such as a His tag is activated by chelating Zn 2+ Ion-mediated interactions are immobilized to surfaces containing amino-functionalized aliphatic moieties.

[0114] 47. A biocatalyst according to any one of items 1 to 45, wherein the TbSADH comprising a metal chelate tag such as a His tag is chelated by Zn 2+ Ion-mediated interactions are immobilized to the surface.

[0115] 48. A biocatalyst according to any one of items 1-45, wherein ATA comprising a metal chelate tag such as a His tag is immobilized to a surface comprising structure (1), (2), (3) or (4) via an interaction mediated by the chelated metal ion.

[0116] 49. The biocatalyst according to any one of items 1 to 45, wherein SucP comprising a metal chelating tag such as a His tag is immobilized to a surface comprising structure (2).

[0117] 50. The biocatalyst according to any one of items 1-45, wherein a TLL not comprising a metal chelating tag such as a His tag is immobilized to the surface.

[0118] 51. The biocatalyst according to any one of items 1 to 45, wherein a glucosyltransferase comprising a metal chelate tag such as a His tag is immobilized to a surface comprising structure (1).

[0119] 52. The biocatalyst according to any one of items 1 to 45, wherein the lipase CalB is immobilized to a surface comprising structure (1) or (4), preferably non-covalently immobilized, wherein the CalB most preferably does not comprise a metal chelating tag such as a His tag.

[0120] 53. A biocatalyst according to any one of items 1-45, wherein an amyloglucosidase, preferably not comprising a metal chelate tag such as a His tag, is immobilized on a surface functionalized with structure (1), (2), (3) or (4), and optionally has a different second functionalization, which can be structure (1) or an alkyl group such as propyl or octyl.

[0121] 54. Use of a biocatalyst according to any of the preceding items for the synthesis of organic compounds in a reactor.

[0122] 55. The use according to item 54, wherein the reactor is a batch reactor or a continuous flow reactor.

[0123] 56. The use according to item 54, wherein the reactor is a continuous stirred tank reactor (CSTR), a slurry bubble column or a fixed bed reactor.

[0124] 57. The use according to item 55, wherein the reactor is a continuous flow fixed bed reactor.

[0125] 58. Use according to any one of items 54-57, wherein the synthesis is carried out under aqueous conditions.

[0126] 59. The use according to any one of items 54-57, wherein the synthesis is carried out in an organic solvent.

[0127] 60. The use according to any one of items 54-57, wherein the synthesis is carried out in a pure substrate.

[0128] 61. The use according to any one of items 54-60, wherein the synthesis is a multi-step synthesis.

[0129] 62. The use according to claim 61, wherein the multi-step synthesis comprises at least 3 intermediates.

[0130] 63. The use according to item 61 or 62, wherein the multi-step synthesis is a linear synthesis.

[0131] 64. A method for synthesizing an organic compound, comprising:

[0132] a. Providing a biocatalyst according to any one of items 1-53, which is placed in a reactor;

[0133] b. feeding a precursor of an organic compound to a flow reactor, whereby the immobilized enzyme catalyzes one or more reactions leading to the synthesis of the organic compound; and

[0134] c. Recovery of organic compounds.

[0135] 65. The use according to item 54, wherein the reactor is a batch reactor or a continuous flow reactor.

[0136] 66. The use according to item 65, wherein the reactor is a continuous stirred tank reactor (CSTR), a slurry bubble column or a fixed bed reactor.

[0137] 67. The use according to item 65, wherein the reactor is a continuous flow fixed bed reactor.

[0138] 68. A method according to any one of items 64-67, wherein the one or more reactions are carried out under aqueous conditions.

[0139] 69. A method according to any one of items 64-67, wherein the one or more reactions are carried out in an organic solvent.

[0140] 70. A method according to any one of items 64-67, wherein the one or more reactions are carried out in a pure substrate.

[0141] 71. A method according to any one of items 64-70, wherein the synthesis is a multi-step synthesis comprising at least two reaction steps.

[0142] 72. A method according to claim 71, wherein the multi-step synthesis comprises at least 3 intermediates.

[0143] 73. A method according to item 71 or 72, wherein the multi-step synthesis is a linear synthesis.

[0144] 74. A method for producing a biocatalyst according to any one of items 1 to 53, comprising:

[0145] a. providing one or more catalytically active enzymes;

[0146] b. providing controlled porosity silica (CPS) as a support material, wherein the pore diameter is between about 20 and about 100 nm, the support material comprising an amino-functionalized surface; and

[0147] c. Immobilizing one or more enzymes on the support material.

[0148] 75. The method according to item 74, further comprising:

[0149] Selecting a bifunctional cross-linking reagent that is capable of generating amino, amide or thioamide bonds when reacting with the enzyme and / or the surface, and

[0150] The bifunctional reagent is used to covalently cross-link the enzyme.

[0151] 76. A method according to claim 75, wherein the bifunctional cross-linking reagent comprises at least two reactive groups independently selected from epoxides, esters, anhydrides, N-hydroxysuccinimide esters, imidoesters, carbonates, acylisoureas, carbodiimides, maleimides, haloacetyl groups, thiosulfonates, isocyanates and vinyl sulfones.

[0152] 77. The method of claim 75, wherein the bifunctional cross-linking agent is defined by the formula:

[0153] wherein Y and Z are independently reactive groups selected from epoxides, esters, anhydrides, imidoesters, N-hydroxysuccinimide esters, carbonates, acylisoureas, carbodiimides, maleimides, haloacetyl groups, thiosulfonates, vinyl sulfones, and isocyanates, and

[0154] L represents a linker between reactive groups containing 3 to 20 atoms, including any combination of C, H, N and O.

[0155] 78. A method according to item 77, wherein Y and Z are each independently an epoxide, an N-hydroxysuccinimide ester or an imidate, wherein preferably Y=Z.

[0156] 79. A method according to item 77, wherein the bifunctional reagent is a diepoxide reagent, a bifunctional sulfonated N-hydroxysuccinimide ester or a bifunctional imidate.

[0157] 80. The method of clause 77, wherein the reagent is a diepoxide defined by the formula:

[0158] wherein L represents a linker containing 3 to 20 atoms, wherein the atoms include any combination of C, H, N and O.

[0159] 81. A method according to claim 77, wherein the reagent is GDE, NPE, PDE, BS3 or DMS.

[0160] 82. A method according to claim 77, wherein the bifunctional reagent is a bifunctional sulfonated N-hydroxysuccinimide ester reagent or a bifunctional imidate.

[0161] 83. A method according to claim 82, wherein the reagent is bis(sulfosuccinimidyl) suberate (BS3) or dimethyl suberate (DMS).

[0162] 84. A method according to any one of items 75-83, wherein the bifunctional reagent is contacted with a support material prior to immobilizing the enzyme.

[0163] 85. A method according to any one of items 75-84, wherein the bifunctional reagent is contacted with the support material after immobilizing the enzyme.

[0164] 86. A method according to any one of item 75 or its dependencies, wherein the enzyme is immobilized to the surface via an interaction mediated by a chelated metal ion, preferably, the metal ion is Ni 2+ , Cu 2+ Mg 2+ , Fe 3+ or Zn 2+ , most preferably Zn 2+ .

[0165] 87. A method according to any one of item 75 or its dependencies, wherein at least one enzyme comprises a polyhistidine tag.

[0166] 88. A method according to any one of item 75 or its dependencies, wherein the enzyme is selected from list (A), more preferably from list (B).

[0167] 89. A method according to any one of item 75 or its dependencies, wherein at least one enzyme is provided in a cell lysate.

[0168] 90. A method according to any one of clause 75 or its dependencies, wherein at least one enzyme is provided as a formulation comprising at least 70% enzyme by weight of total protein in the formulation. DETAILED DESCRIPTION

[0169] The present invention is based on the unexpected discovery that amino-functionalized controlled porosity silica (CPS) is widely used as a support material for immobilizing enzymes for synthesis and provides better results than commercially available support materials (Examples 1-4, 6). Amino-functionalization of CPS supports allows for the immobilization of multiple different types of enzymes with high enzyme loading, high activity retention, high stability, and is suitable for both His-tagged and non-His-tagged enzymes. The industrial applicability of amino-functionalized CPS supports has been further demonstrated in a continuous flow setup (Examples 5, Figure 1 ).

[0170] In Example 7, various cross-linking reagents were tested using SucP2 immobilized on an amino-functionalized CPS as a model, demonstrating the superiority of cross-linking reagents that generate bonds selected from amino, amide, and iminoamide compared to glutaraldehyde that generates imine bonds.

[0171] The utility of using two different coatings on CPS for enzyme immobilization was explored in Example 8, which provides the ability to tailor the functional groups on the surface.

[0172] The results in Example 9 compare different sized CPS Q30 particles (75-150 μm, 280-500 μm and 1.18-2.36 mM) and show that larger species are also feasible, allowing work in certain applications requiring higher flow rates or three-phase reactions.

[0173] Example 10 compares 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.

[0174] Additional benefit is that the cost of CPS is much lower than other supports, such as controlled porosity glass (CPG). Compared with silica nanoparticles, CPS provides useful flow characteristics in packed bed reactors, such as low back pressure at high flow rates. Compared with non-porous silica particles of larger size, CPS surface area is much higher, allowing enzyme immobilization yield (calculated by the weight of support material) to significantly improve. In addition, the CPS support material used in the present invention has been specially designed to be used as a catalyst support, wherein mechanical properties such as crushing strength, wear and abrasion resistance are the characteristics of the material, which means that the catalyst can be used in other reactor types, other reactor types such as batch or continuous stirred tank reactor or bubble column (bubble column) reactor. However, despite having these attractive features for the support of catalyst, silica surface is not often used for enzyme catalysts, because known silica surface can cause enzyme inactivation, and silica support therefore cannot achieve high enzyme loading so far on the support surface with high activity level retention.

[0175] The amino-functionalized CPS support materials are chemically stable, which means that they can be used in a wide range of reaction conditions, such as reactions in aqueous solution, reactions in organic solvents, and reactions in the absence of solvents, such as pure reaction substrates or liquid molten solids.

[0176] In general, a specific balance of properties such as surface area, pore volume, pore diameter, amino functionalization, chemical stability, and mechanical strength combined with low cost provide excellent materials for biocatalytic applications.

[0177] A further feature of the amino-functionalized CPS material is the ability to facilitate the formation of a covalent linkage between the immobilized enzyme and the amino-functionalized coating on the CPS support material. The presence of a covalent linkage can be particularly advantageous for reaction media containing components that would otherwise leach the enzyme from the amino-functionalized support material. Furthermore, the covalent linkage can be installed without any loss of enzyme activity. This is an important advantage compared to support materials that do not contain an amino-functionalized surface coating.

[0178] Biocatalysts for organic synthesis

[0179] In a first aspect, the present invention relates to a biocatalyst for organic synthesis comprising:

[0180] a. controlled porosity silica (CPS) as a support material having a pore diameter of about 20 to about 100 nm;

[0181] wherein the support material comprises an amino-functionalized surface; and

[0182] b. one or more catalytically active enzymes immobilized to the amino-functionalized surface via a covalent linker comprising a bond selected from the group consisting of amino, amide, ester, ether, iminoamide, iminothioamide, thioether, thioester and thioamide.

[0183] In a second aspect, the present invention relates to a biocatalyst for organic synthesis comprising:

[0184] a. controlled porosity silica (CPS) as a support material having a pore diameter of about 20 to about 100 nm;

[0185] wherein the support material comprises an amino-functionalized surface; and

[0186] b. One or more catalytically active enzymes immobilized on the surface via non-covalent interactions.

[0187] In a third aspect, the present invention relates to a biocatalyst for organic synthesis comprising:

[0188] a. controlled porosity silica (CPS) as a support material having a pore diameter of about 20 to about 100 nm;

[0189] wherein the support material surface comprises at least two different coatings, at least one of the coatings providing an amino-functionalized surface; and

[0190] b. One or more catalytically active enzymes immobilized to said amino-functionalized surface, optionally via a covalent linker comprising a bond selected from the group consisting of amino, amide, ester, ether, thioether, iminoamide, iminothioamide, thioester and thioamide.

[0191] In a fourth aspect, the present invention relates to a biocatalyst for organic synthesis comprising:

[0192] a. controlled porosity silica (CPS) as a support material having a pore diameter of about 20 to about 100 nm;

[0193] wherein the support material comprises an amino-functionalized surface; and

[0194] b. one or more catalytically active enzymes immobilized to said amino-functionalized surface, optionally via a covalent linker comprising a bond selected from the group consisting of amino, amide, ester, ether, thioether, iminoamide, iminothioamide, thioester and thioamide,

[0195] Wherein the enzyme is selected from list (A), preferably from list (B) disclosed below.

[0196] Preferably, the covalent linker in the first, third and fourth aspects, when present, comprises a bond selected from amino, amide, ester, ether, thioether, thioester and thioamide.

[0197] More preferably, the covalent linker in the first, third and fourth aspects, when present, comprises a bond selected from amino, amide, thioether, thioester and thioamide. Even more preferably, the covalent linker in the first, third and fourth aspects, when present, comprises a bond selected from amino, amide or thioamide. Still more preferably, the covalent linker in the first, third and fourth aspects, when present, comprises a bond selected from amino, amide and iminoamide.

[0198] Most preferably, the covalent linker for immobilizing the enzyme to the surface in the first, third and fourth aspects, when present, comprises a bond selected from amino and amide.

[0199] Without being bound by theory, the covalent linker between the amino-functionalized surface and the enzyme contains (i) a bond formed in the reaction between the amino-functionalized surface and a functional group on the cross-linking reagent used to form the covalent linker (ii) a bond formed in the reaction between the reactive functional group in the enzyme and the functional group on the cross-linking reagent, and (iii) optionally, any spacer moieties present between the functional groups of the cross-linking reagent, preferably containing 3-20 atoms, the atoms comprising any combination of C, N, H and O. The bond referred to as (i) preferably comprises a bond selected from amino, amide and iminoamide. The bond referred to as (ii) may comprise a bond selected from amino, amide, ester, ether, thioether, iminoamide, iminothioamide, thioester and thioamide.

[0200] Optionally, the immobilized enzyme can be covalently linked intermolecularly by a linker comprising a bond selected from amino, amide, ester, ether, thioester, iminoamide, iminothioamide, thioether and thioamide. These bonds are formed in the reaction between the reactive functional groups in the enzyme and the functional groups on the cross-linking agent. The intermolecular linker can optionally comprise a spacer moiety present between the functional groups of the cross-linking agent, which preferably contains 3-20 atoms comprising any combination of C, N, H and O.

[0201] The amino-functionalized surface comprises primary, secondary or tertiary amines.

[0202] Details and preferred features of the biocatalyst are described below.

[0203] Support material

[0204] The support material according to the first aspect of the present invention is controlled porosity silica (CPS), which is available in a variety of pore diameter ranges. The surface of untreated silica (SiO2) is covered with hydroxyl groups (-OH), while the support material of the present invention is amino-functionalized, as explained in more detail below.

[0205] The ideal controlled pore silica material of the present invention has been specially designed to be used as a catalyst support, wherein mechanical properties such as crushing strength, wear and abrasion resistance are essential features of the material as a catalyst support. Chemically and thermally stable controlled pore silica materials also provide precisely controlled pore diameters and pore diameter distributions. Particle size and particle size ranges can be customized and optimized for different industrial reactor types. For example, they can be deployed in batches or continuously stirred tank reactors (CSTRs) as slurry catalysts or slurry bubbling towers or fixed bed reactors or their known variations. Compared with known fragile and easily abraded (abrasion) or worn (attrition) controlled pore glass materials when stirred in batch reactors or CSTRs, this is an advantage.

[0206] The right pore diameter is a key feature of the support. Pores of the right size provide a favorable microenvironment without being complicated by steric hindrance. The pore structure results in low solution flow resistance and facilitates mass transfer of reactants and products throughout the material. The rigid, non-cage structure of CPS provides a strong, incompressible medium suitable for high-throughput reactor designs and linear scale-up at high flow rates. The material exhibits limited swelling in solvents and is chemically and dimensional stable in most organic media and aqueous environments with a pH below 10.

[0207] The physical properties of controlled porosity silica make it suitable for immobilizing enzymes on the surface of a carrier with high enzyme loading. Generally speaking, the controlled pore silica materials suitable for use in the present invention have a pore diameter of about 20 nm to about 100 nm, preferably about 20 to about 60 nm, and more preferably about 30 to about 50 nm. Preferably, the surface area is about 50 m 2 / g to about 200m 2 / g (preferably greater than about 60m 2 / g, such as greater than about 70m 2 / g). Also preferably, the pore volume is from 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 from about 0.8 mL / g to about 1.5 mL / g). These properties allow high loadings of enzyme to be immobilized on the surface of the support. Preferably, 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 200m 2 Even more preferably, the support material has a pore diameter of about 20 to about 60 nm, about 50 nm 2 / g to about 200m 2 / g surface area and about 0.5mL / g to about 2.0mL / g pore volume. Most preferably, the support material has a pore diameter of 20 to 60nm, a pore diameter of 50m 2 / g to 200m 2 The ranges disclosed herein include the endpoints.

[0208] The enzyme loading capacity exhibited by CPS supports is inversely related to their pore diameter. Thus, CPS supports with large pore diameters cannot load as much protein as CPS supports with smaller pore diameters, primarily due to the inverse relationship between pore diameter and surface area (i.e., lower surface area available for immobilizing enzymes).

[0209] The preferred particle size depends on the type of reactor deployed for use of the immobilized biocatalyst. For example, smaller particle sizes, such as 75-150 μm are well suited for deployment in batch or continuous stirred tank reactors, where the immobilized biocatalyst is operated as a catalyst slurry in the reactor. For deployment in fixed bed reactors, the preferred particle size depends on the chemical reaction and the conditions within the reactor. For liquid phase fixed bed reactions, smaller particles, such as 75-150 μm, are preferred, while for certain other liquid phase reactions and reactions where gas and liquid are deployed together in a fixed bed reactor, larger particle sizes may be deployed, for example, particle sizes greater than 200 μm, and preferably greater than 500 μm.

[0210] Certain types of preferred porous silica materials are commercially available. For example, CARiACT TM Controlled pore silicas of the Fuji CARiACT Q30 and Q-50 classes are available from Fuji Silysia Chemical Ltd (https: / / www.fujisilysia.com / products / cariact / ). These inorganic porous silicas can be produced with varying surface areas and pore diameters. Surprisingly, these materials have been found to be advantageous for immobilizing high enzyme loading enzymes onto the support surface. The properties of Fuji CARiACT Q30 and Q-50 make these materials particularly advantageous. Fuji CARiACT Q-20C, Q30C, and Q40C materials are also suitable. According to the manufacturer, the particles have the following properties:

[0211]

[0212] Another suitable commercially available material is Ecovyst E30. This particle has the following properties:

[0213]

[0214] The controlled porosity silica of the present invention can be prepared according to the method described in JPH0930809A and JP2020001936. Both methods describe the details of the production method, and these production methods provide enough control to change the properties of silica to produce the controlled porosity silica with necessary properties (such as previously specified surface area, pore volume, pore diameter and particle size) balance. In addition, these production methods produce controlled porosity silica with necessary mechanical strength to realize the features of the present invention. Generally speaking, sodium silicate and inorganic acid (such as sulfuric acid) two kinds of raw materials are used together in a solution process to generate monomer silicic acid. Monomer silicic acid polymerizes to generate primary silica particles, called silica sol. Then these primary particles gather together to form a three-dimensional porous structure. The reaction conditions that can make the primary particles grow and the conditions of the dried product are used to control and change physical properties, such as surface area, pore diameter and pore volume.

[0215] Finally, it should be understood that CPS in this context is different from silica mesocellular foams (MCF) known in the literature. MCF has an ordered 3D cage structure with spherical cavities having a diameter of 20-40 nm, interconnected by pores of about 10 nm in size, which are smaller than the size required for immobilizing enzymes. In contrast, CPS is not a cage structure. In the context of MCF materials, the term "pore size" is often used in the literature to define the diameter of the cage, and "pore window" is used to define the cage entrance diameter (or pore diameter), where the window size is usually smaller than the pore size. Due to the cage structure and the smaller window size, the surface area (m 2 / g) is usually significantly higher than the surface area of ​​CPS (over 200m 2 Currently, MCF is not commercially available on an industrial scale and is expensive for industrial applications, so it is not a practical alternative to CPS.

[0216] Controlled pore silica is also different from hollow microsphere silica described in the literature. WO2013 / 078551 describes hollow microsphere silica as microcapsules, which comprise a silica shell having a thickness of about 50 nm to about 500 μm, the shell enclosing a diameter of about 0.1 μm to about 1500 μm and a density of about 0.001 g / cm 3 To about 1.0g / cm 3 The hollow capsules of these materials are described in WO2013 / 078551. Figure 2-5 is clearly shown. These materials are core / shell / functional surface type reservoirs or microcapsules, comprising a core (gaseous or hollow) surrounded by a shell (usually solid), the shell being mainly composed of one or more silica-based materials, and covered with a functional surface having an affinity or adhesion to the matrix of a plastic or composite material or rubber or textile. Microcapsules are designed to be introduced into plastics, composite materials, rubber and textile products at their processing stage. Gaseous or hollow microcapsules are dispersed in plastics, composite materials, rubber and textile products as a density-reducing additive in whole or in part to reduce the density of the final product. Although the hollow sphere microcapsules of WO2013 / 078551 are designed to reduce density additives, which can provide material benefits for the formulated products, the controlled pore silica material of the present invention is specially designed to be used as a catalyst support, wherein mechanical properties such as crushing strength, wear and abrasion resistance are essential features of the material as a catalyst. This low-density hollow sphere support with inherent low crushing strength cannot provide such characteristics. The controlled pore silica of the present invention is not hollow and comprises a three-dimensional silica structure comprising precisely controlled pore diameters and distribution of pore diameters throughout the solid, which is well suited for the synthesis of efficient and robust biocatalysts.

[0217] Immobilized enzyme

[0218] In principle, any enzyme of interest can be immobilized. Preferably, the enzyme is an esterase, a transferase, an oxidoreductase, a hydrolase, a ligase, an isomerase or a lyase.

[0219] The immobilized enzyme may be selected from the enzymes on list (A) consisting of entries 1 to 62 in Table A below and enzymatically active sequence variants thereof, preferably the variants have similar enzymatic activity, and most preferably have substantially the same enzymatic activity.

[0220] As is well known, the amino acid sequence of enzyme can be greatly changed and can not significantly affect enzymatic activity.For example, artificial sequence such as His tag can be added to realize metal chelation.In other cases, residues unimportant to enzymatic activity can be modified to improve characteristics (such as stability or production efficiency) or alternative sequences are only provided to bypass patent protection.In some cases, enzyme sequence can be shortened to produce more compact enzyme by omitting the enzyme part unimportant to enzymatic activity.Therefore, the enzymatic activity sequence variant of the enzyme identified in Table A can also be used and therefore be included in the list (A).

[0221] Table A: Preferred immobilized enzymes

[0222]

[0223]

[0224]

[0225]

[0226] Reference 1: Global Substance Registration System Ver 3.0.3UNII record A370TYK9KO

[0227] Reference 2: Cuetos A et al. (2016) Angew Chem Int Ed 2016,55:3144-3147

[0228] The immobilized enzyme is preferably selected from List (B), which consists of: Thermomyces lanuginosus lipase (TLL) (TLL, #1 in Table A), Saccharomyces antarctica lipase (CalB, #2 in Table A), Bifidobacterium adolescentis sucrose phosphorylase (SucP, #3 in Table A), Thermoanaerobacter buchneri secondary alcohol dehydrogenase (TbSADH, #4 in Table A), Arthrobacter species transaminase (ATA, #60 in Table A), Mesentericum glycosyltransferase (GT, #18 in Table A), amyloglucosidase from Aspergillus niger (#61 in Table A) and transaminase (Arthrobacter species) (#62 in Table A); and their enzyme-active sequence variants, preferably the variants have similar enzyme activity, most preferably have substantially the same enzyme activity.

[0229] The enzyme activities relevant in the context of Tables (A) and (B) are indicated in Table A by the "EC Number" column, which refers to the Enzyme Commission's numerical classification scheme for enzymes according to the reaction they catalyze.

[0230] The Uniprot id column in Table (A) refers to entries in the public Uniprot database located at www.uniprot.org as of December 1, 2021. Preferably, each selected enzyme of List (A) or List (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 of the database entry identified by "Uniprot id" referred to in Table A.

[0231] In some embodiments, the enzyme comprises a metal affinity tag, such as a polyhistidine tag (a His tag of 2-8 consecutive histidines, preferably 6), 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.

[0232] Amino-functionalized surfaces

[0233] The amino-functionalized surface may comprise a structure of formula (I), (II) or (III):

[0234]

[0235] in

[0236] R 1 C 1-6 alkanediyl; and wherein R 1 Provides covalent attachment to the silica surface (vertical line on the left);

[0237] X is a bond (i.e., X is absent) or a benzene ring;

[0238] R 2 C 1-6 Alkanediyl;

[0239] R 3 is W;

[0240] R 4 Is W or selected from the following group: 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 aminocarbonyl; and wherein the phenyl group is optionally substituted by one or more substituents C 1-4 Alkyl substitution;

[0241] or R 3 and R 4 are combined together with the nitrogen atom to which they are attached to form a 5- to 7-membered heterocyclic ring, which may contain one or more unsaturated bonds and may contain one or more additional heteroatoms, to which is optionally attached a covalent linker comprising a bond selected from the group consisting of amino, amide and iminoamide, wherein the catalytically active enzyme is also attached to the linker;

[0242] or R 3 and R 4 Together they form = CR 5 R 6 , where R 5 and R 6 Each independently is C 1-4 alkyl;

[0243] Each R 7 Independently for C 1-6 alkanediyl; and wherein R 7 Provides covalent attachment to the silica surface (vertical line on the left);

[0244] R 8 C 1-3 Alkanediyl;

[0245] Each R 9 is independently W or selected from C 1-4 Alkyl and hydroxy-C 1-4 A group consisting of alkyl groups;

[0246] Each R 10 Independently for C 1-6 alkanediyl; and wherein R 10 Provides covalent attachment to the silica surface (vertical line on the left);

[0247] R 11 Is W or selected from the following group: 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 aminocarbonyl; and wherein the phenyl group is optionally substituted by one or more substituents C 1-4 Alkyl substitution; and

[0248] Each W is independently hydrogen or a covalent linker comprising a bond selected from amino, amide, and iminoamide, wherein the catalytically active enzyme is also attached to the linker.

[0249] In some embodiments, the amino-functionalized surface comprises a combination of a structure of formula (I) as defined above and a structure of formula (IV):

[0250] Where R 12 Selected from C 1-12 Alkyl and amino-C 1-6 alkyl.

[0251] Preferably, the amino functional groups in the surface are not part of an aromatic heterocycle, more preferably not any heterocycle.

[0252] Preferably, the surface does not contain any functional groups other than amino groups that could chelate metal ions.

[0253] More preferably, the surface comprises amino-functionalized aliphatic moieties.

[0254] In some cases, it is beneficial to have at least two different coatings on the CPS material, at least one of which provides amino functionalization. For certain enzymes, the presence of two different coatings can provide a better balance of properties suitable for the certain enzymes, resulting in a more effective biocatalyst. For example, a certain enzyme may prefer a coating that is naturally more hydrophobic, and may also benefit from the presence of amino groups and additional metal ions. This situation can be accommodated by the presence of at least two coatings, one coating providing amino functionalization and the other providing a hydrophobic coating. The hydrophobic coating may contain alkyl moieties and / or aromatic moieties. Preferably, the hydrophobic coating contains C 1-12 Alkyl, preferably C 1-6 Alkyl and / or phenyl.

[0255] For example, one coating may contain fatty chains or phenyl groups, providing a hydrophobic environment, and a second coating may contain amino functionalized groups, which may further accommodate metal ions. The relative proportions of the two coatings on the surface may be controlled and optimized to maximize the efficiency of the biocatalyst. The relative amounts of the two coatings by weight may range from 9:1 to 1:9, preferably from 5:1 to 1:5, more preferably from 2:1 to 1:2, and most preferably from 1:1.

[0256] In addition, the presence of two coatings can favor situations where covalent bonding is preferred between the enzyme and the surface of the CPS material. The presence of two coatings can tailor a surface with two coatings that can a) provide enzyme-preferred chemical groups, such as hydrophobic groups such as aliphatic chains or phenyl groups, and b) provide amino-functionalized groups to achieve covalent bonding between the enzyme and the CPS surface.

[0257] As used herein, the term “C 1-6 "Alkanediyl" refers to a straight-chain or branched divalent group having 1 to 6 carbon atoms. 1-6 Examples of the alkanediyl group include methanediyl, ethylenediyl, 1,3-propylenediyl and 2,2-dimethyl-1,4-butanediyl.

[0258] As used herein, the term “C 1-10 "Alkyl" refers to a straight or branched chain alkyl group having 1 to 10 carbon atoms, and the term "C 1-4"Alkyl" refers to a straight or branched chain alkyl group having 1 to 4 carbon atoms. 1-4 Examples of the alkyl group include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl.

[0259] As used herein, the term “C 5-7 "Cycloalkyl" refers to a monocyclic saturated hydrocarbon ring having 5 to 7 carbon atoms. 5-7 Examples of cycloalkyl groups include cyclopentyl, cyclohexyl and cycloheptyl.

[0260] As used herein, the term "hydroxy-C 1-4 "Alkyl" refers to a C 1-4 Alkyl group.

[0261] As used herein, the term "phenyl-C 1-6 "Alkyl" refers to a C 1-6 Alkyl group.

[0262] As used herein, the term "amino-C 2-8 "Alkyl" refers to a C 2-8 Alkyl group.

[0263] The term "amino" refers to primary, secondary, and tertiary amines.

[0264] The term "aminocarbonyl" refers to the group -C(=O)NH2.

[0265] As used herein, the term "N-(phenyl)amino-C 2-8 Alkyl" and "N-(phenyl-C 1-6 -alkyl)amino-C 2-8 "Alkyl" refers to amino-C 2-8 Alkyl groups in which one of the hydrogen atoms on the amino group is replaced by a phenyl or phenyl-C 1-6 Alkyl group replacement.

[0266] Preferably, wherein the amino-functionalized surface comprises any of the following structures, wherein each W is hydrogen or a covalent linker comprising a bond selected from amino, amide and iminoamide, wherein the catalytically active enzyme is also attached to the linker:

[0267]

[0268] In some embodiments, the amino-functionalized surface comprises structure (I'):

[0269]

[0270] in

[0271] 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, and wherein the phenyl group is optionally substituted by one or more substituents C 1-4 Alkyl substituted; and

[0272] W is hydrogen or a covalent linker comprising a bond selected from the group consisting of amino, amide, and iminoamide, wherein the catalytically active enzyme is also attached to the linker;

[0273] Or wherein the amino-functionalized surface comprises structure (I"):

[0274]

[0275] in

[0276] R 4 is W or is selected from the group consisting of 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 wherein the phenyl group is optionally substituted by one or more substituents C 1-4 Alkyl substitution; and

[0277] W is hydrogen or a covalent linker comprising a bond selected from amino, amide and iminoamide wherein the catalytically active enzyme is also attached to the linker.

[0278] In certain preferred embodiments, the amino-functionalized surface comprises structure I', wherein R 4 is W or is selected from the group consisting of phenyl, benzyl, amino-C 26 Alkyl, N-(phenyl)amino-C 26 Alkyl and N-(benzyl)amino-C 26 Alkyl; or

[0279] The amino-functionalized surface comprises structure I", where R 4 It's W.

[0280] More preferably, wherein the amino-functionalized surface comprises any of the following structures, wherein each W is hydrogen or a covalent linker comprising a bond selected from amino, amide and iminoamide, wherein the catalytically active enzyme is also attached to the linker:

[0281]

[0282] Most preferably, wherein the amino-functionalized surface comprises any of the following structures, wherein each W is hydrogen or a covalent linker comprising a bond selected from amino, amide and iminoamide, wherein the catalytically active enzyme is also attached to the linker:

[0283] (1) Propylamine

[0284]

[0285] (2) Propyl NH-N-aminoethyl

[0286]

[0287] (3) Propyl-NH-N-aminohexyl (LCAA)

[0288]

[0289] (4) Phenethyl-methyl-NH-N-aminoethyl

[0290]

[0291] For the avoidance of any doubt, it should be understood that the above names of structures (1)-(4) are derived from the case where W=H, but the structure still also encompasses the case where W is a covalent linker comprising a bond selected from amino, amide and iminoamide, wherein the catalytically active enzyme is also attached to the linker.

[0292] Enzyme immobilization

[0293] In preferred embodiments of the third and fourth aspects, the enzyme is immobilized to the surface via a covalent linker comprising a bond selected from amino, amide and iminoamide, but non-covalent immobilization is also an option.

[0294] In preferred embodiments of the first, third and fourth aspects, the enzyme is immobilized to the surface via a covalent linker comprising a bond selected from amino, amide and iminoamide, and prior to forming the bond, the enzyme has been immobilized to the surface via non-covalent interactions, preferably via interactions mediated by chelated metal ions. Without being bound by theory, immobilization after non-covalent binding may allow for more uniform crosslinking between the CPS support surface and the enzyme, thereby improving retention of activity. In other embodiments, initial immobilization is via a covalent linker, optionally followed by additional intermolecular crosslinking.

[0295] In preferred embodiments of the second, third and fourth aspects, the enzyme is non-covalently immobilized to the surface via interactions mediated by chelated metal ions.

[0296] The above-mentioned chelated metal ion can be selected from Ni 2+, Cu 2+ Mg 2+ , Fe 3+ or Zn 2+ Preferably, the metal ion is Zn 2+ Ion. This Zn 2+ Ion-mediated interactions provide excellent results for certain enzymes such as CalB and TbSADH (see Example 2).

[0297] In certain embodiments of the first, second, third or fourth aspect, the immobilized enzyme is covalently linked between molecules via a linker comprising a bond selected from the group consisting of amino, amide, ester, ether, thioether, iminoamide, iminothioamide, thioester and thioamide.

[0298] Preferably, the covalent linker between the surface and the enzyme, when present, does not comprise an imine bond, which may be susceptible to cleavage by hydrolysis. Preferably, the intermolecular covalent linker, when present, does not comprise an imine bond. Preferably, the covalent linker, when present, comprises a bridge comprising 3 to 20 atoms, said atoms comprising any combination of C, H, N and O.

[0299] In some embodiments, the enzyme comprises a metal affinity tag, such as a polyhistidine tag (a His tag of 2-8 consecutive histidines, preferably 6), 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 many industrially relevant enzymes can be immobilized by metal chelation even without affinity tags due to the inherent metal chelation properties of the enzyme. However, compared with physical adsorption, the advantage of immobilization by metal chelation using affinity tags is selective enzyme binding, and in addition, the spatial orientation of the immobilized enzyme may be more uniform compared to the case where immobilization occurs, for example, by non-specific physical adsorption. Affinity tags can also improve the activity after immobilization by driving the immobilization to a controlled position, thereby allowing better and / or more consistent access to the active site. On the other hand, initial immobilization by a covalent linker can also be applied because it can provide a simplified manufacturing process because the step of non-covalent immobilization can be omitted.

[0300] Covalent cross-linking (between different enzyme molecules and / or between enzyme molecules and amino groups of amino-functionalized surfaces) has certain advantages. Cross-linking allows for better retention of enzyme activity under industrial flow conditions and the possibility of recovering and reusing the catalyst in batch operations.

[0301] Suitable cross-linking reagents for forming covalent cross-links are disclosed below.

[0302] Cross-linking reagents

[0303] Various covalent cross-linking strategies applied to amino functional groups and enzymes are known in the field of protein chemistry. See, for example, the Cross-linking Technology Handbook published by Thermo Scientific, which is incorporated herein by reference, https: / / tools.thermofisher.com / content / sfs / brochures / 1602163-Crosslinking-Reagents-Handbook.pdf.

[0304] An effective cross-linking reagent for connecting the enzyme to the amino-functionalized support surface will require at least two (preferably no more than two) reactive groups separated by a linker. An effective cross-linking reagent needs to contain at least one first reactive group that can react with the amino functional group attached to the CPS surface to form a stable covalent bond. In addition, the cross-linking reagent needs to contain at least one second reactive group that can react with the functional group present on the enzyme structure to form a stable covalent bond, thereby creating a covalent connection between the enzyme and the support surface. Alternatively, the reagent can contain at least two reactive groups, each of which can react with the functional group present on the enzyme structure to form a stable covalent bond, thereby creating an intermolecular covalent connection between the enzyme molecules.

[0305] The functional group that is easy to cross-link on enzyme comprises amino group, carboxylic acid group, thiol (sulfhydryl) and hydroxyl group.By appropriately selecting cross-linking reagent, these functional groups can easily form covalent bond, such as amino, amide, ester, ether, thioether, iminoamide, iminothioamide, thioester and thioamide.As a specific example, in the case of epoxide cross-linking reagent, the functional group on enzyme can react with epoxy group to produce covalent bond, such as amino, ester, ether and thioether bond.For example, in the case of diepoxide cross-linking reagent, one of epoxy group can react with the amino functional group on amino functionalized controlled pore silica carrier surface to form amino covalent bond.The second epoxy group can react with the functional group on the amino acid residue on enzyme.For example, the amino functional group from lysine amino acid residue can react with epoxy group to form amino covalent bond.Alternatively, the thiol functional group from cysteine ​​amino acid residue can react with epoxy group to form thioether covalent bond. In yet further embodiments, the carboxylic acid functional group from a glutamic acid amino acid residue can react with an epoxy group to form an ester covalent bond, or can react with a hydroxyl group from a serine amino acid residue to form an ether covalent bond.

[0306] When desired, cross-linking reagents can similarly be used to form intermolecular linkers between immobilized enzymes.

[0307] The cross-linking reagent may be homobifunctional or heterobifunctional, meaning 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, said atoms comprising any combination of C, H, N and O.

[0308] An effective cross-linking reagent should create a covalent bond between the enzyme and the support surface and retain enzyme activity upon cross-linking. Retention of enzyme activity upon cross-linking is an important desired feature of a cross-linking reagent, which can be modulated by appropriate choice of reactive groups, linker groups, and linker length.

[0309] The preferred reactive group selected is selected based on its ability to react with amine, hydroxyl, carboxylate or sulfhydryl to create a stable covalent linkage, which is linked by amino, amide, ether, ester, iminoamide, iminothioamide, thioether, thioester or thioamide. The cross-linking reagent capable of cross-linking with the functional group of the enzyme can contain at least two reactive groups independently selected from epoxides, esters, anhydrides, N-hydroxysuccinimide esters, iminoesters, carbonates, acylisoureas, carbodiimides, maleimides, haloacetyl groups, thiosulfonates, isocyanates and vinyl sulfones. Reactive groups such as carbodiimides can be assisted in forming covalent linkages by reacting with, for example, a carboxylate group, which itself is then displaced by a second reactive group to form a covalent bond such as an amide bond.

[0310] Preferred cross-linking agents are defined by the formula:

[0311]

[0312] Wherein Y and Z are independently selected from the reactive groups of epoxide, ester, anhydride, imidoester, N-hydroxysuccinimide ester, carbonate, acylisourea, carbodiimide, maleimide, haloacetyl, thiosulfonate, vinyl sulfone and isocyanate. Preferably, Y and Z are each independently epoxide, N-hydroxysuccinimide ester or imidoester.

[0313] L represents a linker between reactive groups containing 3 to 20 atoms, including any combination of C, H, N and O.

[0314] More preferred cross-linking reagents are homobifunctional, wherein Y = Z, and contain a reactive group selected from epoxides, esters, anhydrides, N-hydroxysuccinimide esters, imidates, carbonates, acylisoureas, maleimides, haloacetyl groups, and vinyl sulfones, most preferably epoxides, N-hydroxysuccinimide esters, or imidates.

[0315] Even more preferably, the cross-linking agent is a diepoxide agent, still more preferably a diepoxide defined by the formula:

[0316]

[0317] Wherein L represents a linker between epoxy groups containing 3 to 20 atoms, wherein the atoms include any combination of C, H, N and O.

[0318] Most preferably, the diepoxide reagent is glycerol diglycidyl ether (GDE, which may be 1,3- or 1,2-substituted glycerol diglycidyl ether, or a mixture of the two isomers), neopentylglycerol diglycidyl ether (NPE), poly(tetraethylene oxide) diglycidyl ether (PDE), 1,6-hexanediol diglycidyl ether (HDDE) or glycerol triglycidyl ether (GTGE).

[0319] Alternative preferred cross-linking agents comprise difunctional sulfonated N-hydroxysuccinimide esters and difunctional imidates, such as bis(sulfosuccinimidyl) suberate (BS3) or dimethyl suberate (DMS).

[0320] A known alternative cross-linking agent is glutaraldehyde, but it results in the formation of imine bonds and is therefore not covered by the present invention. Glutaraldehyde is a particularly reactive cross-linking agent and can result in significant inactivation of the enzyme. The formation of imine bonds is also reversible and therefore the connection is not very stable.

[0321] Preferred properties of biocatalysts

[0322] In preferred embodiments of the first aspect, the support material has a pore diameter of about 20 to about 60 nm, a pore diameter of about 50 nm, and a pore diameter of about 100 nm. 2 / g to about 200m 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), or even more preferably from list (B).

[0323] In preferred embodiments of the second aspect, the support material has a pore diameter of about 20 to about 60 nm, a pore diameter of about 50 nm, and a pore diameter of about 100 nm. 2 / g to about 200m 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), or even more preferably from list (B).

[0324] In preferred embodiments of the third aspect, the support material has a pore diameter of about 20 to about 60 nm, a pore diameter of about 50 nm, and a pore diameter of about 100 nm. 2 / g to about 200m 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), or even more preferably from list (B).

[0325] In some embodiments, the immobilized enzyme retains at least 20% of its activity after immobilization, preferably at least 30%; more preferably at least 40%, even more preferably at least 50%, still more preferably at least 60%, still more preferably at least 70%, still even more preferably at least 80%, and most preferably at least 90% compared to the state before immobilization. In rare cases, immobilization can even enhance the activity, resulting in an apparent retention of more than 100%. In some embodiments, the immobilized enzyme retains 20%-100% of its activity after immobilization, preferably 30%-100%; more preferably 40%-100%, even more preferably 50%-100%, still more preferably 60%-100%, still more preferably 70%-100%, still even more preferably 80%-100%, and most preferably 90%-100%.

[0326] In some embodiments, compared to the state before immobilization, after 20 hours of immobilization under continuous flow conditions, the immobilized enzyme retains at least 20%, preferably at least 30% of its activity; more preferably at least 40%, even more preferably at least 50%, still more preferably at least 60%, still more preferably at least 70%, still even more preferably at least 80%, and most preferably at least 90%. In some embodiments, compared to the state before immobilization, after 20 hours of immobilization under continuous flow conditions, the immobilized enzyme retains 20%-100%, preferably 30%-100%; more preferably 40%-100%, even more preferably 50%-100%, still more preferably 60%-100%, still more preferably 70%-100%, still even more preferably 80%-100%, and most preferably 90%-100%. In some embodiments, compared to the state before immobilization, after 70 hours of immobilization under continuous flow conditions, the immobilized enzyme retains at least 20% of its activity, preferably at least 30%; more preferably at least 40%, even more preferably at least 50%, still more preferably at least 60%, still more preferably at least 70%, still even more preferably at least 80%, and most preferably at least 90%. In some embodiments, compared to the state before immobilization, after 70 hours of immobilization under continuous flow conditions, the immobilized enzyme retains 20%-100%, preferably 30%-100%; more preferably 40%-100%, even more preferably 50%-100%, still more preferably 60%-100%, still more preferably 70%-100%, still even more preferably 80%-100%, and most preferably 90%-100%.

[0327] In some embodiments, compared to the state before immobilization, after 200 hours of immobilization under continuous flow conditions, the immobilized enzyme retains at least 20%, preferably at least 30% of its activity; more preferably at least 40%, even more preferably at least 50%, still more preferably at least 60%, still more preferably at least 70%, still even more preferably at least 80%, and most preferably at least 90%. In some embodiments, compared to the state before immobilization, after 200 hours of immobilization under continuous flow conditions, the immobilized enzyme retains 20%-100%, preferably 30%-100%; more preferably 40%-100%, even more preferably 50%-100%, still more preferably 60%-100%, still more preferably 70%-100%, still even more preferably 80%-100%, and most preferably 90%-100%.

[0328] In some embodiments, compared to the state before immobilization, after 500 hours of immobilization under continuous flow conditions, the immobilized enzyme retains at least 20%, preferably at least 30% of its activity; more preferably at least 40%, even more preferably at least 50%, still more preferably at least 60%, still more preferably at least 70%, still even more preferably at least 80%, and most preferably at least 90%. In some embodiments, compared to the state before immobilization, after 500 hours of immobilization under continuous flow conditions, the immobilized enzyme retains 20%-100%, preferably 30%-100%; more preferably 40%-100%, even more preferably 50%-100%, still more preferably 60%-100%, still more preferably 70%-100%, further more preferably 80%-100%, and most preferably 90%-100%.

[0329] In the above preferred embodiments, the continuous flow conditions can be carried out in aqueous solution, organic solvent or using neat substrate.

[0330] In the above preferred embodiments, the continuous flow is performed in an aqueous solution having a pH of 4.0 to 9.0, or more preferably 6.0 to 7.5.

[0331] In the above preferred embodiments, the continuous flow conditions may comprise a temperature of 4°C to 100°C or more preferably 30°C to 70°C.

[0332] It should be noted that certain steps may also facilitate reaction steps that are performed at lower conversions, such as at least 20%, or preferably at least 40%, particularly when the product of interest is of high value.

[0333] In some embodiments, at least two different enzymes are immobilized on a support material. More preferably, at least two immobilized enzymes are capable of catalyzing the different steps of a multi-step organic synthesis. Multi-step synthesis can include at least 3 intermediates, such as 3, 4, 5, 6, 7, 8, 9 or 10 intermediates. In some embodiments, multi-step synthesis includes 3 to 10 steps, such as 3 to 5 steps, such as 4 to 6 steps, such as 5 to 7 steps, such as 6 to 8 steps; or such as 4 to 10 steps, such as 5 to 10 steps, such as 6 to 10 steps, such as 7 to 10 steps, for example 8 to 10 steps, for example 9 to 10 steps.

[0334] In certain embodiments, at least one immobilized enzyme catalyzes the regeneration of a cofactor.

[0335] The most preferred embodiment of the biocatalyst

[0336] In a preferred embodiment of the second, third or fourth aspect of the invention, the enzyme is a lipase (preferably CalB) comprising a metal chelate tag such as a His tag, wherein the enzyme is activated by the chelated Zn 2+ Ion-mediated interactions are immobilized to surfaces containing amino-functionalized aliphatic moieties.

[0337] In another preferred embodiment of the second, third or fourth aspect, the enzyme is TbSADH comprising a metal chelate tag such as a His tag, wherein the enzyme is activated by the chelated Zn 2+ Ion-mediated interactions are immobilized to the surface.

[0338] In a further preferred embodiment of the second, third or fourth aspect, the enzyme is ATA comprising a metal chelate tag such as a His tag, wherein the enzyme is immobilized to a surface comprising structure (1), (2), (3) or (4) via an interaction mediated by the chelated metal ion.

[0339] In yet another preferred embodiment of the second, third or fourth aspect, the enzyme is SucP comprising a metal chelate tag such as a His tag, wherein the enzyme is immobilized to a surface comprising structure (1).

[0340] In another preferred embodiment of the second, third or fourth aspect, the enzyme is TLL which preferably does not comprise a metal chelating tag such as a His tag, wherein the enzyme is immobilized to a surface comprising structure (1) or (4).

[0341] 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, wherein the enzyme is immobilized to a surface comprising structure (1).

[0342] In another preferred embodiment of the first, second, third or fourth aspect, the lipase CalB is immobilized to a surface comprising structure (1) or (4), preferably non-covalently immobilized, wherein said CalB most preferably does not comprise a metal chelating tag such as a His tag.

[0343] In yet another preferred embodiment of the second, third or fourth aspect, the enzyme is CalB (preferably not comprising a metal chelating tag such as a His tag), wherein the enzyme is immobilized to a surface comprising structure (4).

[0344] 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). It is preferably immobilized on a surface functionalized with structure (1), (2), (3) or (4), optionally with a different second functionalization. The second functionalization may be structure (1) or an alkyl group such as propyl or octyl.

[0345] Uses of biocatalysts

[0346] In a fifth aspect, the present invention provides use of a biocatalyst according to the first, second, third or fourth aspect in a reactor for synthesizing an organic compound.

[0347] The reactor can be any industrial reactor type. The reactor can be a batch reactor or a continuous flow reactor. The reactor includes a continuous stirred tank reactor (CSTR), a slurry bubble column or a fixed bed reactor or known variations thereof. A continuous flow fixed bed reactor is preferred.

[0348] 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 a pure substrate. The latter is particularly preferred for lipase. The biocatalyst can also be used for three-phase reactions, such as those reactions that require liquid solutions and gaseous reagents to exist, such as oxidation using oxygen or air as reactants. In this case, the particle size is preferably adjusted for operation in a fixed bed reactor, such as a trickle bed or CSTR, or a bubble tower, another reactor form suitable for specific three-phase conditions.

[0349] The synthesis of the fifth aspect may be a multi-step synthesis, preferably comprising at least 3 intermediates, such as 3, 4, 5, 6, 7, 8, 9 or 10 intermediates. The multi-step synthesis is preferably a linear synthesis.

[0350] Synthesis methods using biocatalysts

[0351] In a sixth aspect, the present invention provides a method for synthesizing an organic compound, comprising:

[0352] a. Providing a biocatalyst according to the first, second, third or fourth aspect, which is placed in a reactor;

[0353] b. feeding a precursor of an organic compound to a reactor, whereby the immobilized enzyme catalyzes one or more reactions leading to the synthesis of the organic compound; and

[0354] c. Recovery of organic compounds.

[0355] The reactor can be any industrial reactor type. The reactor can be a batch reactor or a continuous flow reactor. The reactor includes a continuous stirred tank reactor (CSTR), a slurry bubble column or a fixed bed reactor or known variations thereof. A continuous flow fixed bed reactor is preferred.

[0356] The one or more reactions can be carried out under aqueous conditions, in an organic solvent or in a pure substrate. The latter is particularly preferred for lipase. The method can also relate to a three-phase reaction, such as those reactions that require a liquid solution and a gaseous reagent to exist, such as the oxidation using oxygen or air as a reactant. In this case, the particle size is preferably adjusted for operation in a fixed bed reactor, such as a trickle bed or a CSTR, or a bubbling tower, another reactor form that is suitable for concrete three-phase conditions.

[0357] The synthesis of the third aspect may be a multi-step synthesis, preferably comprising at least 3 intermediates, such as 3, 4, 5, 6, 7, 8, 9 or 10 intermediates. The multi-step synthesis is preferably a linear synthesis.

[0358] Method for producing a biocatalyst

[0359] In a seventh aspect, the present invention provides a method of making a biocatalyst according to the first, second, third or fourth aspect, comprising:

[0360] a. providing one or more catalytically active enzymes;

[0361] b. providing a controlled porosity silica (CPS) as defined in the first, second, third or fourth aspect as a support material, wherein the pore diameter is between about 20 and about 100 nm, the support material comprising an amino-functionalized surface; and

[0362] c. Immobilizing one or more enzymes on the support material.

[0363] In certain embodiments, the method of the seventh aspect further comprises selecting a bifunctional cross-linking reagent that is capable of producing an amino, amide, ester, ether, thioether, iminoamide, iminothioamide, thioester or thioamide bond (preferably amino, amide or thioamide, more preferably amino, amide (amido) or iminoamide (imidoamido), most preferably amino or amide) when reacting with the enzyme and the amino-functionalized surface, and using the bifunctional reagent to covalently cross-link the enzyme.

[0364] Suitable bifunctional cross-linking reagents for use in the method of the seventh aspect have been discussed above in the context of the first to fourth aspects.

[0365] The bifunctional reagent may be contacted with the amino-functionalized CPS before immobilizing the enzyme, after immobilizing the enzyme, or both before and after immobilization. Whether the bifunctional cross-linking reagent is added before and / or after the enzyme immobilization step depends on aspects and embodiments of the present invention. For example, the bifunctional cross-linking reagent may be added to the amino-functionalized support before enzyme immobilization, whereby one of the reactive groups of the cross-linking reagent will react with the amino-functionalized surface of the support material. For example, where the cross-linking reagent is a diepoxide, it is expected that one of the epoxy groups will react with the surface of the amino-functionalized CPS, while the second epoxy group is then able to react with an amino acid present in the enzyme, thereby directly immobilizing the enzyme in a covalent manner.

[0366] When the bifunctional cross-linking reagent is added prior to immobilizing the enzyme, the bifunctional cross-linking reagent may be added in molar excess, molar equivalent, or submolar equivalent relative to the molar number of amino groups on the CPS surface. In addition, in certain embodiments, the bifunctional cross-linking reagent may be added after initial non-covalent immobilization in anticipation that the first reactive group will react with the amino-functionalized CPS surface, while the second reactive group may subsequently react with the amino acid present in the enzyme.

[0367] In addition, in certain embodiments, a cross-linking agent (double cross-linking) can be added before and after immobilization. The cross-linking agent can also react between two enzyme structures in a covalently linked manner. The inventors were surprised to find that the use of described difunctional (preferably diepoxide, difunctional sulfonated N-hydroxysuccinimide ester or difunctional imidate) cross-linking agents is likely to retain the activity of the enzyme fully when cross-linked. The retention of activity during cross-linking is applied to many enzyme classes, thereby providing a general scheme for preparing stable immobilized biocatalysts. This is contrary to reports in the literature, where loss of activity is a universal feature of cross-linking.

[0368] The inventors have also found that when immobilization and cross-linking are attempted on CPS surfaces that do not have amino functionalization, a significant loss of activity is evident. It is possible to immobilize enzymes on CPS surfaces that do not have amino functionalization, but the resulting immobilized biocatalysts have only limited stability, and therefore CPS that do not have amino functionalized surfaces do not represent a widely useful support material for immobilizing synthetic enzymes. See Example 4.3, which illustrates that biocatalysts produced using CPS surfaces that do not have amino functionalization cannot tolerate the presence of cross-linking agents without significant loss of activity. Therefore, compared to the case of immobilization, for example, by non-specific physical adsorption on surfaces without a customized coating, the invention described herein provides significant advantages.

[0369] Examples 4.1-4.4 illustrate complete retention of activity in the presence of covalent attachment. In such embodiments, the immobilized biocatalyst may have additional stability in the presence of the reaction components. The additional stability may manifest itself in a longer stability period when operated under continuous flow conditions, such as those operating in a fixed bed reactor. Examples 5.2-5.3 illustrate this longer stability period in a fixed bed reactor compared to an immobilized enzyme in the absence of a covalent attachment.

[0370] The enzyme can be immobilized to the surface through an interaction mediated by a chelated metal ion, preferably Ni 2+ , Cu 2+ Mg 2+ , Fe 3+ or Zn 2+ , most preferably Zn 2+ .

[0371] The enzyme may be selected from list (A), more preferably from list (B). The enzyme may optionally comprise a polyhistidine tag.

[0372] At least one enzyme may be provided in a cell lysate for the immobilization step. Alternatively, the enzyme may be provided in a purified or isolated form.

[0373] The at least one enzyme may be provided as a formulation comprising at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95%, such as 70%-100%, 80%-100%, 90%-100% or 95%-100% enzyme by weight of the total protein in the formulation.

[0374] The present disclosure also provides methods for optimizing biocatalysts 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).

[0375] Secondly, two, three, four, five or more different controlled porosity silica (CPS) support materials are provided that meet the criteria provided in the section entitled Support Materials above. In short, the pore diameter is between about 20 and about 100 nm, and the support material comprises an amino-functionalized surface. Preferably, the support material is as defined above for the first, second, third or fourth aspect of the present invention. The support material can be amino-functionalized with, for example, propylamine, propyl NH-N-aminoethyl, phenethyl-methyl-NH-N-aminoethyl or propyl-NH-N-aminohexyl. The support material can comprise an immobilized metal chelating group. In order to identify a suitable support material, one or more enzymes are immobilized on a support material, and the activity of the immobilized enzymes between (different) support materials is compared (200). Various immobilization conditions can be tested. Initial conditions can include the absence and presence of chelated metal ions, such as Ni 2+ , Cu 2+ Mg 2+ , Fe 3+ and Zn 2+ The immobilization conditions may include, for example, a buffer with 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.

[0376] Optionally, further immobilization conditions can be screened to maximize the immobilized enzyme activity by performing immobilization at at least two different values ​​of at least one parameter of the optimal initial conditions: initial enzyme concentration, metal ion concentration, buffer type, pH, salt concentration, additives, time and temperature, and then comparing the immobilized enzyme activity between the (different) immobilization conditions and selecting the best conditions for optimizing immobilization (300).

[0377] As described above, covalent cross-linking can be used for certain applications. Optionally, cross-linking conditions can be screened to maximize the activity and / or stability of the immobilized enzyme by testing at least two values ​​of at least one of the following parameters: type of cross-linking reagent, amount of cross-linking reagent, concentration of cross-linking reagent, composition of cross-linking solution, cross-linking time, and cross-linking temperature. The cross-linking reagent meets the criteria provided in the above-mentioned section entitled cross-linking reagent. The cross-linking reagent type can include at least one diepoxide, at least one difunctional sulfonated N-hydroxysuccinimide ester, and / or at least one difunctional imidate. Typically, the activity is compared with a non-cross-linked immobilized enzyme. The cross-linking reagent concentration can be 0.1-3% (v / v), the cross-linking temperature is 0-25°C, the cross-linking time is 2-24 hours, and / or the cross-linking solution can have a pH of 6-8. The immobilized enzyme activity can be compared between the cross-linking conditions, and then the optimal cross-linking conditions (400) are selected.

[0378] Optionally, one or more of the above steps (200)-(400) can be repeated with additional variations to further optimize the results (500). The method can result in at least one immobilized enzyme retaining at least 20% (such as 20%-100%, preferably 50%, or 50%-100%) of its activity after immobilization compared to the state before immobilization, preferably after 20 hours of immobilization under continuous flow conditions, compared to the state before immobilization. The optimized biocatalyst is then scaled up and manufactured as needed (600).

[0379] General Statements Relating to the Present Disclosure

[0380] The term "comprising (or including)" should be interpreted as including (or including) but not limited to. All references are incorporated herein by reference. The arrangement of the present disclosure into sections with headings and subheadings is merely to improve readability and should not be interpreted as limiting in any way. In particular, the division does not in any way exclude or limit the combination of features under different headings and subheadings with each of the others.

[0381] Example

[0382] The following examples are not to be considered limiting. For more information on experimental details, the skilled reader is referred to the separate section entitled "Materials and Methods".

[0383] Example 1 Support Preparation

[0384] 1.1 Support

[0385] All controlled pore glass (CPG) and controlled pore silica (CPS) amino-functionalized supports used for immobilization were synthesized in-house. CPG cores (particle size 70-125 μm, pore size 50 nm, surface area 60 m 2 ) was purchased from Schott AG. CPS core (Cariact Q30: particle size 70-150 μm, pore size 30 nm, surface area 99 m 2 , and CariactQ50: particle size 70-150μm, pore size 50nm, surface area 80m 2 ) was purchased from Fuji Silysia Chemical. Polymer beads used for comparison with CPG and CPS were purchased from commercial sources. ReliZyme EA403 / S (ethylamine functionalized on methacrylate cross-linked copolymer) and ReliZyme HA403 / S (hexylamine functionalized on methacrylate cross-linked copolymer) were purchased from Biokal. 66 free base (polyamine groups on styrene-divinylbenzene matrix) was purchased from Sigma-Aldrich.

[0386] 1.2 Synthesis of amino-functionalized supports

[0387] Controlled pore core (CPG or CPS) (5g) is transferred to a jacketed glass reactor with an overhead stirrer and toluene (100mL) is added. The core is amino-functionalized (Table 1) with four different silanes. Each silane (15mL) is added to the reactor and incubated for 22 hours (250-290rpm, 80°C). The solid is filtered under vacuum, washed with toluene (2×200mL), then washed with 99.7% EtOH (2×200mL). After the washing step, a glass filter funnel is used to filter the solid under vacuum for 60min. The amino-functionalized support is transferred to a crystallizing dish and vacuum dried overnight at 120°C.

[0388] Table 1. Amino-functional silanes for controlled pore glass and silica (CPG and CPS).

[0389]

[0390] 1.3 Chelating agent attachment

[0391] 2', 4'-dihydroxyacetophenone (500mg, 3.3mmol) is added to a 250mL conical flask containing methanol (16mL). Amino functionalized support (2g) is added to the mixture and incubated for 2 hours (150rpm, 25°C). Sodium borohydride (600mg, 15.9mmol) is slowly added to the flask and incubated for another 2 hours (150rpm, 25°C). The slurry containing the support is washed stepwise as follows: the support slurry is transferred to a glass filter and washed with deionized water (40mL) under vacuum. Deionized water (10mL) is added and allowed to drip without vacuum. Deionized water (40mL) is added and vacuum is applied. Deionized water (10mL) is added and allowed to drip without vacuum. Afterwards, the pH of each support is evaluated, and if the pH is higher than 7, deionized water (45mL) is further added and vacuum is applied. After washing with deionized water to the desired pH 7, the support was washed with ethanol (50 mL) under vacuum, followed by acetone (25 mL) under vacuum. The support with attached chelator was dried in a desiccator under vacuum overnight.

[0392] 1.4 Metal stripping from EziG support

[0393] CPG carrier LCAA CPG (Fe 3+ )、HybCPG VBC(Fe 3+) and HybCPG copo(Fe 3+ ) (see WO2015 / 115993) was also used as a comparative example of the immobilization technology described in the present invention. In order to evaluate the effect of different metals on the CPG carrier, the chelated Fe 3+ The CPG material (5g) is transferred to a 100mL Duran bottle and 2M HCl (50mL) is added. The flask is placed on a tilting / drum mixer for 35min (70rpm, 21°C). The program repeats 3 other cycles, and between each cycle, HCl is removed, fresh 2M HCl (50mL) is added and incubated for 35min (70rpm, 21°C). Solid is filtered on a glass filter funnel under vacuum and washed with deionized water (500mL), then washed with 25mM (300mL) sodium phosphate (NaP) of pH 7.3, other deionized water (300mL) and acetone (50mL). The support stripped is dried overnight in a desiccator under vacuum.

[0394] 1.5 Metal Deposition

[0395] Each immobilized support (100mg) is weighed and put into 15mL Falcon pipe, and deionized water (2mL) is added.This mixture is used as support slurry, fills 6 holes on plate at most, and prepares mixture according to the required support of measuring each time.The slurry containing support (200 μ L) is transferred in each hole in 96-hole deep-well plates, to reach the support of about 10mg in every hole.Prepare two kinds of stock solutions (FeCl3, ZnCl2, NiCl2, MgCl2, CuCl2, concentration is 1.55 and 0.26mM) of every kind of metal salt, for depositing metal ion on amino-functionalized support.For CalB immobilization, the metal stock solution of 1.55mM (300 μ L) is added in the support, to reach the ultimate concentration (table 2) of 0.93mM in hole. For TbSADH immobilization, 0.26 mM (300 μL) of metal stock solution was added to the support to reach a final concentration of 0.15 mM in the well (Table 2). The plate containing the support and metal solution was incubated for 30 min (1300 rpm, 21 ° C). The metal solution was then removed and the support was washed with deionized water (4x1 mL), followed by washing with buffer solution or deionized water (1x1 mL) (Table 2). For comparison purposes, the preparation without metal was also included in the reaction screening of each enzyme.

[0396] Table 2. Metal deposition conditions on amino-functionalized supports for different enzymes.

[0397]

[0398] 1.6 gram zinc deposition

[0399] Immobilized support (1g) is weighed and put into 100mL Duran bottle and deionized water (10.4mL).Preparation is used for the ZnCl2 stock solution of metal ion deposition, and adds in the support according to required final metal concentration.Bottle is placed on tilting / roller mixer for 1 hour (20rpm, 21 ℃).The slurry containing support is transferred to glass filter column and washed with deionized water (60mL), then washed with acetone (30mL) under vacuum.The support is transferred in 15mL Falcon pipe and vacuum dried in desiccator overnight.This material is used in embodiment 4.1 to 4.5 and 5.1 to 5.3.

[0400] 1.7 Enzymes

[0401] Lipase from Antarctic pseudomyces B (CalB) with an N-terminal His tag; alcohol dehydrogenase from Thermoanaerobacterium buchneri (TbSADH) with an N-terminal His tag; wild-type sucrose phosphorylase from Bifidobacterium adolescentis (SucP1) with an N-terminal His tag, sucrose phosphorylase variant from Bifidobacterium adolescentis with an N-terminal His tag (SucP2, see WO 2016 / 075219 and J. Agric. Food Chem. 2017, 65, 6030-6041) and transglycosylation from Leuconostoc mesenteroides with an N-terminal His tag were produced in-house using known culture procedures. Lipases from Candida species and lipases from Aspergillus oryzae were purchased from Sigma-Aldrich. TL 100L and TL IM was purchased from Novozymes.

[0402] 1.8 Analysis methods

[0403] Chiral analysis was performed on an Agilent 6890NGC 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 with dimethylpolysiloxane), using H2 (2 mL / min flow rate) as the carrier gas.

[0404] GC-FID method for reactions using lipase: Injector temperature: 220°C; Detector temperature: 250°C; Injection volume: 1 μL and Split ratio: 20:1. Oven temperature program: Start at 115°C, hold time 9 min; Total run time: 9 min. Retention times: Dodecane 4.3 min; (R)-1-phenylethyl acetate 5.4 min; (R)-1-phenylethanol 7.5 min; (S)-1-phenylethanol 8.2 min.

[0405] 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: Start at 100°C, hold time 2 min; Increase at a rate of 15°C / min to 195°C, hold time 2 min; Total run time: 11 min. Retention times: phenoxy-2-propanone 5.7 min; (S)-1-phenoxy-2-propanol 6.3 min; (R)-1-phenoxy-2-propanol 6.4 min.

[0406] HPLC-RID method for reactions using SucP1: Oven temperature: 30°C; Detector temperature: 30°C; Injection volume: 10 μL; Flow rate: 0.8 mL / min; Total run time: 12 min. Retention times: phosphorylated sugars 3.8 min; sucrose 4.5 min; glucose 5.4 min; fructose 5.9 min.

[0407] High performance liquid chromatography for reactions using SucP2 was performed in isocratic mode using acetonitrile:methanol:water (75:10:15) as the mobile phase, 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 x 250 mm; stationary phase: polymer-based containing chemically bonded amino and hydrophilic groups).

[0408] HPLC-CAD method for reactions using SucP2: Oven temperature: 50°C; Injection volume: 10 μL; Flow rate: 0.8 mL / min; Total run time: 14 min. Retention times: fructose 6.1 min; glucose 7.2 min; sucrose 9.4 min; kojibiose 11.6 min.

[0409] The HPLC-MS / MS instrument was used in isocratic mode with acetonitrile at pH 4.5 (pH adjusted with formic acid) and 50 mM ammonium acetate (75:25) as the mobile phase, using a Rigol L3500 UV-Vis detector and Merck -HILIC 3.5μm, High performance liquid chromatography for reactions using glycosyltransferases was performed using a Rigol L3400 HPLC system using 100 μM HPLC-MS / MS (size: 4.6 mm x 150 mm; stationary phase: polyetheretherketone).

[0410] HPLC-UV-Vis method for reactions using glycosyltransferases: Oven temperature: 40°C; Injection volume: 2 μL; Flow rate: 0.75 mL / min; Total run time: 6 min. Retention times: hydroquinone 1.4 min; α-arbutin 2.0 min.

[0411] Example 2 Reaction Screening Using Amino-Functionalized Supports

[0412] 2.1 Immobilization of CalB

[0413] 2.1.1 Immobilization

[0414] By centrifugal 60min (11000rpm, 4 ℃), then use 0.45 μ m PVDF filter to filter, make the cell-free extract of 20mM MOPS (50mL) of pH 7.5 containing CalB-N-His clarify.Use 0.1M NaOH the pH of the cell-free extract of clarification is adjusted to 7.5.Cell-free extract (500 μ L) is transferred in the 96-well deep well plates containing the support of fresh deposited metal, and hatched 2 hours (1200rpm, 25 ℃).Remove supernatant, and wash immobilized support with 250mM MOPS (2x1 mL) of pH 7.5.Catalyst is vacuum dried overnight in desiccator.

[0415] 2.1.2 Determination of immobilization yield

[0416] The cell-free extract and the supernatant from the immobilization were diluted 400-fold with 20 mM MOPS at pH 7.5. Aliquots (30 μL) of the diluted enzyme solution and the supernatant were mixed with a reaction mixture (170 μL) containing 20 mM MOPS at pH 7.5 and 3 mM 4-nitrophenyl acetate in acetonitrile. The absorbance (A) at 410 nm was measured every 10 s using a plate reader. 410 ) was continued for 3 min to measure the formation of 4-nitrophenol. The reaction rates (A) from each supernatant and fresh enzyme solution from immobilization were extracted from the linear regression of the data points. 410 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 and the results are presented as the average of each sample.

[0417]

[0418] Protocol 1. Hydrolysis of 4-nitrophenylacetate (3 mM) by lipase for immobilization yield determination.

[0419] 2.1.3 Activity of immobilized catalysts in organic solvents

[0420] The activity of the heterogeneous catalyst is determined by its ability to convert (±)-1-phenylethanol and vinyl acetate into (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 min (1200 rpm, 25°C). After stopping the mixing and allowing the catalyst to settle, an aliquot of the supernatant (10 μL) 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 and the results are presented as the average of each sample.

[0421]

[0422] Scheme 2. Transesterification of (±)-1-phenylethanol (2 M) with vinyl acetate (1.2 M) catalyzed by immobilized CalB.

[0423] Table 3. Performance of immobilized CalB on amino-functionalized supports chelated with various metals. Immobilization yields are based on the activity of the supernatant after immobilization towards the hydrolysis of 4-nitrophenylacetate, and conversions are based on the activity of the immobilized catalyst towards the transesterification of (±)-1-phenylethanol with vinyl acetate. The CPS used in Table 3 is CPS Q30.

[0424]

[0425]

[0426]

[0427]

[0428]

[0429]

[0430] 2.2 Immobilization of TbSADH-N-His

[0431] 2.2.1 Immobilization

[0432] TbSADH-N-His lyophilized powder (300 mg) was rehydrated in deionized water (8.61 mL) and diluted with 50 mM MOPS, 150 mM NaCl (21.39 mL) at pH 7 to obtain a cell-free extract containing 10 mg / mL (total volume of lyophilized powder / solution). The cell-free extract (500 μL) was transferred to a 96-well deep well plate containing a freshly deposited metal support and incubated for 2 hours (1200 rpm, 25 ° C). The supernatant was removed and the immobilized support was washed with 20 mM MOPS (2x1 mL) at pH 7.5 and used directly after removing the remaining buffer in the well.

[0433] 2.2.2 Determination of immobilization yield

[0434] The cell-free extract and the supernatant from the immobilization were diluted 8-fold with 50 mM MOPS, 150 mM NaCl, pH 7. Aliquots (20 μL) of the diluted enzyme solution and the supernatant were mixed with 50 mM MOPS, 1 mM NADP, pH 8. + The reaction mixture (180 μL) of 100 mM 2-propanol was mixed. The absorbance at 340 nm (A) was measured every 10 s using a plate reader. 340 ) was continued for 3 min to measure the formation of NADPH (Scheme 3). The reaction rates (A) from each supernatant and fresh enzyme solution from immobilization were extracted from the linear regression of the data points. 340 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 4). All measurements were performed in duplicate and the results are presented as the average of each sample.

[0435]

[0436] Scheme 3. 2-Propanol (100 mM) oxidation and NADPH formation by TbSADH for immobilization yield determination.

[0437] 2.2.3 Activity of immobilized catalyst in aqueous media

[0438] The activity of the heterogeneous catalyst is determined by its ability to convert phenoxy-2-propanone to 1-phenoxy-2-propanol in the presence of NADPH as a hydride donor. + Regeneration to NADPH (Scheme 4). +The reaction mixture (1 mL) of 2-propanol pH 7.5 in 10% (v / v) 20 mM MOPS was added to the immobilized catalyst, and the mixture was incubated for 1 h (1500 rpm, 30 ° C). After stopping mixing and allowing the catalyst to settle, an aliquot of supernatant (500 μL) was added to EtOAc (500 μL), and the reaction was extracted for 5 min (1500 rpm, 25 ° C). The 96-well deep well plate was left to stand for 5 min to promote phase separation. The organic layer (200 μL) was transferred to another plate and dried with Na2SO4. An aliquot (100 μL) of the dried organic layer was added to EtOAc (900 μL) and analyzed by GC-FID to determine the conversion of phenoxy-2-acetone (Table 4). All measurements were performed in duplicate, and the results were expressed as the average value of each sample.

[0439]

[0440] Scheme 4. Reduction of phenoxy-2-propanone (20 mM) catalyzed by immobilized TbSADH using 2-propanol (10% v / v) for cofactor regeneration.

[0441] 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 for the oxidation of 2-propanol, and the conversion is based on the activity of the immobilized catalyst for the reduction of phenoxy-2-propanone. The CPS used in Table 4 is CPS Q30.

[0442]

[0443]

[0444]

[0445]

[0446] 2.4 Immobilization of non-His-tagged enzymes: lipase from Candida species

[0447] 2.4.1 Immobilization

[0448] Commercial lipase solution from Candida species was diluted four times in 20 mM MOPS at pH 7.5. Enzyme solution (500 μL) was transferred to a 96-well deep well plate containing a support of freshly deposited metal and incubated for 2 hours (1200 rpm, 25 ° C). The supernatant was removed and the immobilized support was washed with 20 mM MOPS (2x1 mL) at pH 7.5. The catalyst was vacuum dried in a desiccator overnight.

[0449] 2.4.2 Determination of immobilization yield

[0450] The enzyme solution and the supernatant from the immobilization were diluted 400 times with 20 mM MOPS at 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 at pH 7.5 and 3 mM 4-nitrophenyl acetate in acetonitrile. The absorbance (A) at 410 nm was measured every 10 s using a plate reader. 410 ) was continued for 3 min to measure the formation of 4-nitrophenol (Scheme 1). The reaction rates (A) from each supernatant and fresh enzyme solution from immobilization were extracted from the linear regression of the data points. 410 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 and the results are presented as the average of each sample.

[0451] 2.4.3 Activity of immobilized catalysts in organic solvents

[0452] The activity of the heterogeneous catalyst is determined by its ability to convert (±)-1-phenylethanol and vinyl acetate into (R)-phenylethyl acetate (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 solution was added to the immobilized catalyst and the mixture was incubated for 3 h (1200 rpm, 25 ° C). After stopping mixing and allowing the catalyst to settle, an aliquot of the supernatant (10 μL) 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 and the results were expressed as the mean value of each sample. For comparison purposes, a support without amino functionalization was also included in the reaction screening of the enzyme.

[0453]

[0454] Scheme 5. Transesterification of (±)-1-phenylethanol (2 M) with vinyl acetate (1.2 M) catalyzed by immobilized lipase from Candida sp.

[0455] Table 5. Performance of immobilized lipase from Candida species on amino-functionalized supports chelated with various metals. Immobilization yields are based on the activity of the supernatant after immobilization towards the hydrolysis of 4-nitrophenylacetate, and conversions are based on the activity of the immobilized catalyst towards the transesterification of (±)-1-phenylethanol with vinyl acetate. The CPS used in Table 6 is CPS Q30.

[0456]

[0457]

[0458]

[0459] 2.5 Immobilization of the non-His-tagged enzyme, lipase from Aspergillus oryzae.

[0460] 2.5.1 Immobilization

[0461] The commercial lipase solution from Aspergillus oryzae was diluted twice in deionized water, and the enzyme solution (500 μL) was transferred to a 96-well deep well plate containing a freshly deposited metal support and incubated for 2 hours (1200 rpm, 25°C). The supernatant was removed, and the immobilized support was washed with a 50 mM HEPES, 50 mM ammonium acetate, 10 mM CaCl2 solution (2x1 mL) at pH 7, and then washed with a 50 mM HEPES, 50 mM ammonium acetate, 10 mM CaCl2, 130 mM sucrose, pH 7 solution (1x1 mL). The catalyst was dried in a desiccator under vacuum overnight.

[0462] 2.5.2 Determination of immobilization yield

[0463] The enzyme solution and the supernatant from the immobilization were diluted 10-fold with deionized water. The absorbance (A) of aliquots (200 μL) of the diluted enzyme solution and the supernatant was measured at 280 nm using a plate reader. 280 The immobilization yield was calculated by determining the absorbance in the supernatant after immobilization (related to the amount of protein present in solution) compared to the absorbance in the initial enzyme solution (Table 6).

[0464] 2.5.3 Activity of immobilized catalysts in organic solvents

[0465] The activity of the heterogeneous catalyst is determined by its ability to convert (±)-1-phenylethanol and vinyl acetate into (R)-phenylethyl acetate (Scheme 6). A reaction mixture (1 mL) containing 2 M (±)-1-phenylethanol, 1.2 M vinyl acetate and 1% (v / v) dodecane (internal standard) in MTBE solution was added to the immobilized catalyst and the mixture was incubated for 2 h (1200 rpm, 25 ° C). After stopping mixing and allowing the catalyst to settle, an aliquot of the supernatant (10 μL) 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 and the results were expressed as the mean value of each sample. For comparison purposes, a support without amino functionalization was also included in the reaction screening of the enzyme.

[0466]

[0467] Scheme 6. Transesterification of (±)-1-phenylethanol (2 M) with vinyl acetate (1.2 M) catalyzed by immobilized lipase from Aspergillus oryzae.

[0468] Table 6. Performance of immobilized lipase from Aspergillus oryzae on amino-functionalized supports chelated with various metals. 280 ), and the conversion is based on the activity of the immobilized catalyst towards the transesterification of (±)-1-phenylethanol with vinyl acetate. The CPS used in Table 7 is CPS Q30.

[0469]

[0470]

[0471]

[0472] 2.6 Non-His-tagged enzymes TL 100L——Fixation.

[0473] 2.6.1 Immobilization of TLL on CPS Propylamine

[0474] A lipase containing Thermomyces lanuginosus (TLL) was prepared by diluting the commercial enzyme (60% v / v) in deionized water (pH 6.7 of the final solution). TL 100L solution, for immobilization. The diluted enzyme (250mL) is transferred to a 500mL Duran bottle containing 50g of a support (CPS Q30 or Q50 functionalized with propylamine). The sample is incubated on a tilting / roller mixer for 1.5 hours (70rpm, 21°C). After immobilization, the supernatant is removed, and the immobilized support is washed with 50mM HEPES, 50mM ammonium acetate, 10mM CaCl2, pH 7 solution (1x250 mL), and then washed with 50mM HEPES, 50mM ammonium acetate, 10mM CaCl2, 130mM sucrose, pH 7 solution (1x250 mL). For each washing step, the immobilized catalyst is mixed with the washing solution on a tilting / roller mixer for 3min (70rpm, 21°C). The catalyst was vacuum filtered to remove most of the wash solution (6 min), transferred to a 3 L glass flask and dried on a rotary evaporator for 115-120 min (40 mbar, 40 °C).

[0475] 2.6.2 Immobilization of TLL on CPS Phenethyl-Methyl-NH-N-Aminoethyl

[0476] A lipase containing Thermomyces lanuginosus (TLL) was prepared by diluting the commercial enzyme (60% v / v) in deionized water (pH 6.7 of the final solution). TL 100L solution, for immobilization.The enzyme (100mL) of dilution is transferred to the 250mL Duran bottle containing 20g support (with the CPS Q50 functionalized with phenethyl-methyl-NH-N-aminoethyl).Sample is incubated 1.5 hours (70rpm, 21 ℃) on tilting / roller mixer.After immobilization, remove supernatant, and wash immobilized support with multiple solutions, to find the most active immobilized formulation 50mM HEPES, 50mM ammonium acetate, 9mM CaCl , pH 7 solutions (3x100 mL).For each washing step, immobilized catalyst is mixed with washing solution on tilting / roller mixer for 3min (70rpm, 21 ℃).Vacuum filtration catalyst is transferred in the crystallization dish and vacuum dried in desiccator for 26 hours to remove most of washing solution (45min).

[0477] 2.6.3 Determination of immobilization yield

[0478] The enzyme solution and the supernatant from the immobilization were diluted 50-fold and 10-fold with deionized water. The absorbance (A) of aliquots (200 μL) of the diluted enzyme solution and the supernatant was measured at 280 nm using a plate reader. 280 The immobilization yield was calculated by determining the absorbance in the supernatant after immobilization (related to the amount of protein present in solution) compared to the absorbance in the initial enzyme solution (Table 7).

[0479] 2.6.4 Activity of immobilized catalysts in organic solvents

[0480] The activity of the heterogeneous catalyst was determined by its ability to convert (±)-1-phenylethanol and vinyl acetate into (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 min (1500 rpm, 35 ° C). After stopping the mixing and allowing the catalyst to settle, an aliquot of the supernatant (10 μL) 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 and the results are expressed as the average of each sample. The activity of the immobilized catalyst was compared with a commercial immobilized catalyst from Novozymes TL IM were compared (Table 7).

[0481] Table 7. Immobilization Performance of TL 100L on amino-functionalized supports. The immobilization yield is based on the absorbance of the supernatant after immobilization (A 280 ), and the conversion is based on the activity of the immobilized catalyst towards the transesterification of (±)-1-phenylethanol with vinyl acetate.

[0482]

[0483] 2.6.5 Activity of immobilized catalysts for interesterification of blended soybean oil

[0484] The activity of immobilized catalyst was evaluated for the transesterification of the blended soybean oil consisting of 60% refined and bleached soybean oil and 40% fully hydrogenated soybean oil supplied by Bunge (BG F41120-000SOY SHTG) and the product with lower melting end temperature. Melting soybean blended oil (500 μ L) was added in the 2mL Eppendorf tube containing immobilized TLL (15 mg), and incubated for 30 min (1500 rpm, 70 ℃). After stopping mixing and allowing catalyst sedimentation, the aliquots (350 μ L) of oil were transferred to the 2mL Eppendorf tube, and placed in ice-cold water, fat was solidified, and then stored in a refrigerator (-20 ℃) ​​for at least 1 hour and analyzed. The melting end range of the sampling reaction was analyzed using a Melting Point System MP55 (Mettler Toledo) with a capillary (Hirschmann, L 75 mm, ID 0.95 mm, OD 1.35 mm) between 34°C and 67°C with a heating rate of 2°C / min, and the melting end temperature was recorded as the temperature when the intensity signal (transmittance) tended to be stable and no further increase in intensity was detected (Table 8). The melting end range of the initial soybean blended oil was 64.7°C, and the lower the melting end range, the higher the degree of transesterification, thereby showing a higher enzyme activity.

[0485] Table 8. Immobilization TL 100L is available on amino-functionalized supports and Performance on TL IM. The efficiency of the catalyst in the transesterification of soybean oil blends depends on the melting end range of the soybean oil blends after the reaction.

[0486]

[0487] Example 3 Screening of different silanes for functionalization of controlled pore silica

[0488] 3.1 Synthesis of various amino-functionalized supports using controlled pore silica

[0489] The synthesis of different amino-functionalized supports using controlled pore silica was performed as described previously. A panel of structurally different aminosilanes was used, as shown in Table 9.

[0490] Table 9. Amino-functionalized silanes for controlled pore silica.

[0491]

[0492] *Oligomerized co-hydrolyzate of N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (CAS 1760-24-3) and n-propyltrimethoxysilane (CAS 1067-25-0), purchased from Gelest (product code SIA0591.3).

[0493] 3.2 Metal deposition and immobilization

[0494] The metal deposition procedures used to screen different supports were performed as described previously. This set of experiments evaluated the Zn2 + As the case of metal presence and absence. For CalB-N-His immobilization, 1.55mM (300 μL) of metal stock solution was added to the support to reach a final concentration of 0.93mM in the well (Table 2). For lipase and TbSADH immobilization from Candida species, 0.26mM (300 μL) of metal stock solution was added to the support to reach a final concentration of 0.16mM in the well (Table 2). As described in the previous section, CalB-N-His, lipase from Candida species and TbSADH were immobilized (the results are presented in Tables 10-13). For comparison purposes, CPSs without amino functionalization were also included in the reaction screening of this set of experiments.

[0495] Table 10. Performance of immobilized CalB-N-His on amino-functionalized controlled porous silica. Immobilization yield is based on the activity of the supernatant after immobilization towards the hydrolysis of 4-nitrophenylacetate, and conversion is based on the activity of the immobilized catalyst towards the transesterification of (±)-1-phenylethanol with vinyl acetate. The CPS used in Table 11 is CPS Q30.

[0496]

[0497] Table 11. Performance of immobilized lipase from Candida species on amino-functionalized controlled porous silica. Immobilization yield is based on the activity of the supernatant after immobilization towards the hydrolysis of 4-nitrophenylacetate, and conversion is based on the activity of the immobilized catalyst towards the transesterification of (±)-1-phenylethanol with vinyl acetate. The CPS used in Table 12 is CPS Q30.

[0498]

[0499]

[0500] Table 12. Performance of immobilized TbSADH-N-His on amino-functionalized controlled porous silica. The immobilization yield is based on the activity of the supernatant after immobilization for the oxidation of 2-propanol, and the conversion is based on the activity of the immobilized catalyst for the reduction of phenoxy-2-propanone. The CPS used in Table 13 is CPS Q30.

[0501]

[0502] Example 4 Cross-linking of enzymes to amino-functionalized CPS supports

[0503] 4.1 Cross-linking of SucP2 immobilized on amino-functionalized controlled pore silica

[0504] 4.1.1 Immobilization

[0505] SucP2 lyophilized powder (760 mg) was rehydrated in 150 mM MOPS buffer (19 mL) at pH 7 to obtain a cell-free extract at a concentration of 40 mg / mL (lyophilized powder / buffer solution). The prepared cell-free extract was resuspended on a tilt / roll 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 20 mg support (CPS Q30 functionalized with propylamine and Zn from a solution with a final concentration of 0.15 mM) 2+ The immobilized support was washed with 150 mM MOPS buffer (2 x 1 mL) at pH 7.

[0506] 4.1.2 Cross-linking

[0507] The cross-linking reagents used were glutaraldehyde (GA) and bis-epoxides neopentylglycol diglycidyl ether (NPE), glycerol diglycidyl ether (GDE) and polyethylene glycol diglycidyl ether (PDE). A solution of each cross-linking reagent 2% (v / v) and ethanol 20% (v / v) was prepared in 150mM MOPS buffer at pH 7 and added (1 mL) to the immobilized catalyst. A control catalyst (non-cross-linked) was prepared by adding 150mM MOPS buffer at pH 7 (1 mL) to immobilized SucP2. Both the control and cross-linked catalysts were incubated overnight on a tube rotator (15 rpm, 2°C). The buffer / cross-linking solution was removed and the catalyst was washed with 150mM MOPS buffer at pH 7 (3x1 mL). The cross-linking treatment under different conditions was performed in duplicate.

[0508] 4.1.3 Activity of immobilized catalysts in aqueous media

[0509] The activity of the heterogeneous catalyst after cross-linking (with and without leaching treatment) was determined by the ability to convert sucrose and glucose into 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 (1200 rpm, 55°C) and supernatant samples (10 μL) were collected after 1 hour, 2 hours, 3 hours and 4 hours to determine the catalyst productivity. 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 Eppendorf tube was placed on ice (2 min) to quench the mixture and then centrifuged for 2 min (15000 rpm, 21°C) to separate the fragments. 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).

[0510]

[0511] Scheme 7. Transglycosylation of sucrose (1 M) and glucose (1 M) to produce kojibiose catalyzed by immobilized SucP2.

[0512] Table 13. Performance of immobilized SucP2 on propylamine functionalized controlled porous silica (Q30) followed by crosslinking. Productivity is based on the activity of the immobilized catalyst for transglycosylation of sucrose and glucose to kojibiose as a function of time and amount of support. Productivity retention is based on productivity retention of the catalyst after crosslinking compared to the uncrosslinked control catalyst.

[0513]

[0514] 4.2 Effect of pH on the cross-linking of immobilized SucP2

[0515] 4.2.1 Immobilization

[0516] SucP2 lyophilized powder (840 mg) was rehydrated in 150 mM MOPS buffer (pH 7) (10 mL) to obtain a cell-free extract at a concentration of 40 mg / mL (lyophilized powder / buffer solution). The prepared cell-free extract was resuspended on a tilt / roll 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 20 mg support (CPS Q30 functionalized with propylamine and Zn from a solution with a final concentration of 0.15 mM) 2+ The immobilized support was washed with 150 mM MOPS buffer (2 x 1 mL) at pH 7.

[0517] 4.2.2 Cross-linking treatment

[0518] Diepoxides used as cross-linking agents are glycerol diglycidyl ether (GDE) and polyethylene glycol diglycidyl ether (PDE). A solution of each diepoxide 2% (v / v) and ethanol 20% (v / v) was prepared in 150mM MOPS buffer at pH 7 and pH 8 and 150mM Tris / HCl buffer at pH 9 and added (1 mL) to the immobilized catalyst. A control catalyst (non-cross-linked) was prepared by adding 150mM MOPS buffer at pH 7 and pH 8 and 150mM Tris / HCl buffer at pH 9 (1 mL) to immobilized SucP2. Both control and cross-linked catalysts were incubated overnight on a tube rotator (15 rpm, 25 ° C). Buffer / cross-linking solution was removed and the catalyst was washed with 150mM MOPS buffer at pH 7 (2x1 mL). Cross-linking treatments under different conditions were performed in duplicate.

[0519] 4.2.3 Leaching treatment

[0520] The catalyst was subjected to leaching test as a scheme to determine the efficiency of cross-linking. A sample from each different cross-linking condition (different cross-linking reagents and pH) was subjected to leaching treatment, while the sample from the same condition was kept separate and used as a control, so as to be able to determine the productivity of the immobilized catalyst with and without the leaching scheme. Leaching was performed by adding 0.5M sodium phosphate buffer (1mL) of pH 7 to the immobilized catalyst and incubating on a tube rotator for 2 hours (15rpm, 21°C). After the leaching scheme, the catalyst was washed with 150mM MOPS buffer (2x1 mL) of pH 7. According to control studies, treating the immobilized catalyst with 0.5M sodium phosphate is an effective method for leaching enzymes that have not been cross-linked from the immobilized catalyst.

[0521] 4.2.4 Activity of immobilized catalysts in aqueous media

[0522] The activity of the heterogeneous catalyst after cross-linking (with and without leaching treatment) was determined by the ability to convert sucrose and glucose into 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 supernatant samples (10 μL) were collected after 1 hour, 2 hours, 3 hours and 4 hours to determine the catalyst productivity. 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 Eppendorf tube was placed on ice (2 min) to quench the mixture, and then centrifuged for 2 min (15000 rpm, 21 ° C) to separate the fragments. 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).

[0523] Table 14. Performance of immobilized SucP2 on propylamine functionalized controlled porous silica (Q30) followed by cross-linking treatment at different pH. Productivity is based on the activity of the immobilized catalyst for transglycosylation of sucrose and glucose to kojibiose as a function of time and amount of support. Productivity retention is based on the productivity retention of the catalyst after leaching treatment compared to the sample without leaching treatment.

[0524]

[0525] 4.3 Cross-linking treatment of immobilized SucP2 with and without amino functionalization of CPS supports

[0526] 4.3.1 Immobilization

[0527] SucP2 lyophilized powder was rehydrated in 150 mM MOPS buffer (10 mL) at pH 7 to obtain a cell-free extract at a concentration of 120 mg / mL (lyophilized powder / buffered solution). The prepared cell-free extract was resuspended on a tilt / roll mixer for 10 min (70 rpm, 21° C.) and then centrifuged for 10 min (8000 rpm, 21° C.). After centrifugation and separation of cell debris, the cell-free extract (1 mL) was added to 50 mg of a support (CPS Q30 functionalized with propylamine and Zn from a solution with a final concentration of 0.15 mM). 2+ Deposition of either non-functionalized CPS Q30 and Zn from a solution with a final concentration of 0.15 mM 2+ The enzyme was immobilized on the support on a tilt / roll mixer (70 rpm, 21° C.) for 1 hour, and the supernatant was removed after immobilization. The immobilized support was washed with 150 mM MOPS buffer, pH 7 (2×1 mL) and used as a wet catalyst for crosslinking.

[0528] 4.3.2 Cross-linking treatment

[0529] The cross-linking solution was prepared using glycerol diglycidyl ether (GDE) 2% (v / v) and ethanol 20% (v / v) in 150 mM Tris / HCl buffer at pH 9. The cross-linking solution (1 mL) was added to the immobilized catalyst and incubated overnight on a tilt / roll mixer (70 rpm, 21° C.). The cross-linking solution was removed and the catalyst was washed with 150 mM MOPS buffer at pH 7 (2×1 mL).

[0530] 4.3.3 Activity of immobilized catalysts in aqueous media

[0531] The activity of heterogeneous catalysts (cross-linked or non-cross-linked) was determined by the ability to convert sucrose and glucose into 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 out after 30 min, 50 min, 70 min and 90 min to determine the catalyst productivity. The aliquots taken out were diluted in deionized water (95 μL) containing 13.5 mM ribose (internal standard), and acetonitrile (200 μL) was then added. The Eppendorf tube was placed on ice (2 min) to quench the mixture, and then centrifuged for 2 min (15000 rpm, 21 ° C) to separate the solids. 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).

[0532] Table 15. Performance of immobilized SucP2 on propylamine functionalized or non-functionalized controlled porous silica (Q30). Productivity is based on the activity of the immobilized catalyst for transglycosylation of sucrose and glucose to kojibiose as a function of time and amount of support. Productivity retention is based on the productivity retention of the catalyst after crosslinking treatment compared to the sample without crosslinking treatment.

[0533]

[0534] 4.4 Modification of CPS-Q30 propylamine by diepoxide

[0535] In addition to the stability of enzymes immobilized on CPS-Q30 by cross-linking treatment with diepoxides, modification of amino-functionalized CPS with diepoxides prior to enzyme immobilization was demonstrated. Diepoxide support modification was performed using glycerol diglycidyl ether (GDE). CPS-Q30 propylamine in a 2 mL Eppendorf tube was wetted with deionized water (1 mL) and Zn from a solution with a final concentration of 0.15 mM (10 mg) was added prior to adding the diepoxide solution. 2+ Deposition. GDE 15% (v / v) and ethanol 20% (v / v) in 150 mM Tris / HCl buffer, pH 9 (500 μL) were added to the support and incubated overnight (1200 rpm, 30° C.). The diepoxide solution was removed and the catalyst was washed with deionized water (2×1 mL) followed by 25 mM sodium phosphate buffer, 150 mM NaCl, pH 8 (1×1 mL).

[0536] 4.4.1 Immobilization

[0537] Glycosyltransferase lyophilized powder was prepared in 25 mM sodium phosphate buffer, 150 mM NaCl, pH 8 to obtain a cell-free extract with a concentration of 14 mg / mL (lyophilized powder / buffer solution). The prepared cell-free extract (1 mL) was transferred to a 10 mg support (CPS-Q30 propylamine and Zn from a solution with a final concentration of 0.15 mM) 2+ Deposition and GDE modified CPS-Q30 propylamine as described previously and Zn from a solution with a final concentration of 0.15 mM 2+ The immobilized support was washed with 50 mM MOPS buffer (3 x 1 mL) at pH 7.2.

[0538] 4.4.2 Determination of immobilization yield

[0539] The initial enzyme solution or the aliquot (250 μ L) from the immobilized supernatant was mixed with a reaction mixture containing 0.67 M sucrose, 0.13 M hydroquinone, 0.013 M ascorbic acid in a 50 mM MOPS buffer (750 μ L) at pH 7.2, and incubated for 1 hour (600 rpm, 30 ° C). The aliquot (25 μ L) of the mixture was diluted in deionized water (55 μ L), and then acetonitrile (100 μ L) was added. The formation of β-arbutin was measured by HPLC UV-Vis (Scheme 8). The immobilization yield (Table 16) was calculated by determining the percentage of the enzyme activity remaining in the supernatant after immobilization compared with the enzyme activity in the cell-free extract.

[0540] 4.4.3 Cross-linking and double cross-linking treatment

[0541] A solution of GDE 6% (v / v) and ethanol 20% (v / v) was prepared in 25 mM sodium phosphate buffer at pH 7 and added (1 mL) to the propylamine + Zn immobilized on CPS-Q30. 2+ On (cross-linked) and CPS-Q30 propylamine + Zn 2+ GDE modified (double cross-linked) catalyst. Control catalyst (non-cross-linked) was prepared by adding 25mM sodium phosphate buffer (1mL) of pH 7 to immobilized glycosyltransferase. Both control and cross-linked catalysts were incubated overnight on a tube rotator (15rpm, 25°C). Buffer / cross-linking solution was removed and the catalyst was washed with 50mM MOPS buffer (3x1 mL) of pH 7.2. Cross-linking treatments under different conditions were performed in duplicate, and a portion of the repeated treatments including non-cross-linked catalysts was leached.

[0542] 4.4.4 Leaching treatment

[0543] For each different condition (for CPS CPS-Q30 propylamine + Zn 2+ or GDE modified CPS-Q30 propylamine + Zn 2+ A sample of the immobilized catalyst (with or without cross-linking treatment) was subjected to the leaching protocol, while a sample from the same condition was separated and used as a control to be able to determine the productivity of the immobilized catalyst with and without the leaching protocol. The leaching protocol was performed by adding 0.5M sodium phosphate buffer (1 mL) at pH 7 to the immobilized catalyst and incubating on a tube rotator for 2 hours (20 rpm, 21°C). After leaching, the supernatant was removed and retained to evaluate the enzyme leached from the catalyst. The leached support was washed with 50mM MOPS buffer (3x1 mL) at pH 7.2.

[0544] 4.4.5 Determination of leached protein

[0545] The supernatant of the leached solution was tested for protein leached from the support using the bathocuproine disulfonate (BCS) assay. Bovine serum albumin (BSA) protein standards and reagents for protein determination were purchased from ThermoFisher as a commercial kit. The BSA solution and leached solution incubated samples for the standard protein curve were prepared according to the instructions provided with the BCS kit. The absorbance at 562 nm (A) was measured using a plate reader. 562 ) protein concentration. The amount of leached protein expressed in mg / mL is based on A 562 With BSA [mg / ml] The BSA calibration curve constructed by linear interpolation of the linear region of the graph was extrapolated (y = 1.6285x + 0.1335, R2 = 99.8%).

[0546] 4.4.6 Activity of immobilized catalyst

[0547] The activity of the heterogeneous catalyst was determined by its ability to convert sucrose and hydroquinone into α-arbutin and fructose (Scheme 8) in the presence of ascorbic acid as an antioxidant. A reaction mixture (1 mL) containing 0.5 M sucrose, 0.1 M hydroquinone, 0.01 M ascorbic acid in a 50 mM MTBE solution at pH 7.2 was added to the immobilized catalyst and the mixture was incubated for 1 h (1300 rpm, 30 ° C). After stopping the mixing and allowing the catalyst to settle, an aliquot (25 μL) of the supernatant was diluted in deionized water (55 μL) and 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.

[0548]

[0549] Scheme 8. Transglycosylation reactions catalyzed by glycosyltransferases used to evaluate the immobilization yields on the test supports and the performance of different resulting immobilized catalysts.

[0550] Table 16. Performance of immobilized glycosyltransferases on propylamine functionalized controlled pore silica (Q30) with and without diepoxide modification followed by cross-linking treatment. Productivity is based on the activity of the immobilized catalyst in the production of α-arbutin. Productivity retention is based on the relative productivity of the treated (support modification / x-cross-linking) catalyst compared to the catalyst without any treatment (control).

[0551]

[0552] Example 5 Catalyst stability under continuous flow conditions

[0553] 5.1 Stability of CalB immobilized on amino-functionalized controlled pore silica under continuous flow in organic solvents

[0554] 5.1.1 Immobilization

[0555] The cell-free extract of 20 mM MOPS (2 x 40 mL) containing CalB-N-His at pH 7.5 was clarified by centrifugation for 60 min (11000 rpm, 4°C) and then filtered 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 140 mM solution containing 700 mg of support (CPS Q30 functionalized with propylamine and Zn from a solution with a final concentration of 140 mM). 2+ The enzyme was immobilized on a support for 3 hours on a tilt / drum mixer (70 rpm, 25° C.). The supernatant was removed and the immobilized support was transferred to a glass filter and washed with 250 mM MOPS (3 x 20 mL) at pH 7.5. The catalyst was transferred to a 15 mL Falcon tube and vacuum dried overnight in a desiccator.

[0556] 5.1.2 Continuous flow operation and sample analysis

[0557] The catalyst of drying (300mg) is loaded in Uniqsis column reactor (1cm OD). The reaction mixture of feed stream is prepared with MTBE (1L) solution of 2M (±)-1-phenylethanol, 1.2M vinyl acetate and 1% (v / v) dodecane. The reactor of charging is assembled in Uniqsis FlowSyn system. The reaction mixture is continuously fed to reactor (0.61mL / min, 30 ℃) and samples are collected regularly. The sample (10 μ L) from the outlet is diluted in EtOAc (990 μ L) in a glass bottle, and analyzed by GC-FID, to determine the conversion of (±)-1-phenylethanol, the result is shown in Table 17, showing the stable operation of continuous flow exceeding 73 hours.

[0558] Table 17. Immobilization in a solution containing 140 mM Zn 2+ Flow stability of CPS-Q30 CalB in propylamine. Conversion is based on the activity of the immobilized catalyst for transesterification of (±)-1-phenylethanol with vinyl acetate.

[0559]

[0560] 5.2 Stability of SucP1 in continuous flow with and without cross-linking

[0561] 5.2.1 Immobilization

[0562] Purified SucP1 was diluted in 50 mM MOPS buffer at pH 8 to obtain an enzyme solution with a concentration of 0.6 mg / mL (enzyme / buffer solution). The prepared enzyme solution (10 mL) was transferred to a 300 mg support (CPSQ30 functionalized with propylamine and Zn from a solution with a final concentration of 0.15 mM). 2+ The immobilized support was washed with 50 mM MOPS buffer (2 x 10 mL) at pH 8. The procedure was repeated twice, and one of the immobilized catalysts was cross-linked.

[0563] 5.2.2 Cross-linking

[0564] The cross-linking solution was prepared using neopentylglycerol diglycidyl ether (NPE) 2% (v / v) and ethanol 20% (v / v) in 50 mM MOPS buffer at pH 8. The cross-linking solution (30 mL) was added to the immobilized catalyst and incubated overnight on a tilt / roll mixer (60 rpm, 21° C.). The solution was removed and the catalyst was washed with 50 mM MOPS buffer at pH 8 (2×10 mL).

[0565] 5.2.3 Continuous flow operation and sample analysis

[0566] After immobilization or cross-linking treatment, wet catalyst (300mg) is directly loaded into Uniqsis column reactor (1cm OD). In 100mM sodium phosphate buffer (1L) of pH 7.8, 2M sucrose is used to prepare the reaction mixture of feed stream. According to the reactor feed needs, multiple batches of reaction mixtures are prepared. The reactor of loading is assembled in Uniqsis FlowSyn system. Reactor (0.2mL / min, 25°C) is continuously fed to the reactor and samples are collected regularly. Samples (50μL) from the outlet are quenched with 1MHCl (50μL), diluted with deionized water (100μL), and centrifuged for 2min (15000rpm, 21°C) to separate fragments. Supernatant is transferred to a plastic inlet vial and analyzed by HPLC-RID to determine the conversion (Scheme 9) (Table 18) of phosphate to glucose 1-phosphate (G1P).

[0567]

[0568] Scheme 9. Conversion of sucrose (1 M) into G1P and fructose catalyzed by immobilized SucP1.

[0569] Table 18. Comparison of flow stability of immobilized SucP1 with and without cross-linking treatment. Conversion is based on the activity of the immobilized catalyst in the run for the production of G1P.

[0570]

[0571] *For the non-crosslinked catalyst, after 91.5 h the conversion dropped to a level below the reliable detection limit of the HPLC method.

[0572] 5.3 Stability of SucP2 in continuous flow with and without cross-linking treatment

[0573] 5.3.1 Preparation and continuous flow operation of non-crosslinked catalyst

[0574] 5.3.1.1 Immobilization

[0575] SucP2 lyophilized powder was rehydrated in 150 mM MOPS buffer (50 mL) at pH 7 to obtain a cell-free extract at a concentration of 40 mg / mL (lyophilized powder / buffer solution). The prepared cell-free extract was resuspended on a tilt / roll mixer for 10 min (70 rpm, 21° C.) and then centrifuged for 10 min (8000 rpm, 21° C.). After centrifugation and separation of cell debris, the cell-free extract was transferred to a 3 g support (CPS-Q30 functionalized with propylamine and 0.15 mM Zn 2+ The enzyme was immobilized on the support for 1 hour on a tilt / roll mixer (70 rpm, 21° C.), and the supernatant was removed after immobilization. The immobilized support was washed with 150 mM MOPS buffer, pH 7 (3 x 45 mL) and used as a wet catalyst for flow operation.

[0576] 5.3.1.2 Continuous flow operation and sample analysis

[0577] The wetted catalyst (4.5 g) was loaded into a Uniqsis column reactor (1 cm OD). The reaction mixture of the feed stream was prepared with 1M sucrose, 1M glucose in deionized water (1 L). Multiple batches of reaction mixtures were prepared according to the reactor feed needs. The reactor of the charge was assembled in the Uniqsis FlowSyn system. The initial feed reaction mixture (0.125 mL / min, 25 ° C) was until the product could be detected from the reactor outlet. Afterwards, the temperature was adjusted to 55 ° C and kept flowing at 0.125 mL / min. Samples were collected regularly, and productivity was calculated in kg kojibiose / kg support * hour. The sample (5 μL) from the outlet was diluted in deionized water (95 μL), and 100 mM glycerol (internal standard) in acetonitrile: deionized water (85:15) (100 μL) was added. The Eppendorf tube was placed on ice (2 min) to quench the mixture, and then centrifuged for 2 min (15000 rpm, 21 ° C) to separate the fragments. 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).

[0578] 5.3.2 Preparation of cross-linking catalyst and continuous flow operation

[0579] 5.3.2.1 Immobilization

[0580] SucP2 lyophilized powder was rehydrated in 150 mM MOPS buffer (2 x 50 mL) at pH 7 to obtain a cell-free extract at a concentration of 40 mg / mL (lyophilized powder / buffer solution). The prepared cell-free extract was resuspended on a tilt / roll mixer for 10 min (70 rpm, 21 ° C) and then centrifuged for 10 min (8000 rpm, 21 ° C). After centrifugation and separation of cell debris, the two test tubes containing the cell-free extract were combined into a 10.5 g support (CPS functionalized with propylamine and 0.15 mM Zn 2+ The enzyme was immobilized on the support for 1 hour on a tilt / roll mixer (70 rpm, 21° C.), and the supernatant was removed after immobilization. The immobilized support was washed with 150 mM MOPS buffer (3×90 mL) at pH 7.

[0581] 5.3.2.2 Cross-linking

[0582] The cross-linking solution was prepared in 150 mM Tris / HCl buffer at pH 9 using glycerol diglycidyl ether (GDE) 2% (v / v) and ethanol 20% (v / v). The cross-linking solution (100 mL) was added to the immobilized catalyst and incubated overnight on a tilt / roll mixer (70 rpm, 21° C.). The solution was removed and the catalyst was washed with 150 mM MOPS buffer at pH 7 (1 x 90 mL) and 150 mM MOPS buffer at pH 7, 250 mM sucrose (1 x 90 mL). The catalyst was vacuum filtered to remove excess buffer and then vacuum dried in a desiccator overnight.

[0583] 5.3.3 Continuous flow operation and sample analysis

[0584] The dried catalyst (2.6 g) was loaded into two Uniqsis column reactors (1 cm OD) connected in series. The reaction mixture of the feed stream was prepared with 1M sucrose and 1M glucose in deionized water (1 L). Multiple batches were prepared according to the reactor feed needs. The reactor of the charge was assembled in the Uniqsis FlowSyn system. The initial feed reaction mixture (0.125 mL / min, 25 ° C) was until the product could be detected from the reactor outlet. Afterwards, the temperature was adjusted to 55 ° C and kept flowing at 0.125 mL / min. Samples were collected regularly, and productivity was calculated in kg kojibiose / kg support * hour. The sample (5 μL) from the outlet was diluted in deionized water (95 μL), and 100 mM glycerol (internal standard) in acetonitrile: deionized water (85:15) (100 μL) was added. The Eppendorf tube was placed on ice (2 min) to quench the mixture, and then centrifuged for 2 min (15000 rpm, 21 ° C) to separate the fragments. 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).

[0585] Table 19. Comparison of flow stability of immobilized SucP2 with and without crosslinking treatment. When the catalyst was loaded in the form of a wet formulation, the flow productivity is expressed as the yield of kojibiose as a function of the amount of immobilized SucP2 per hour in the run [kg 曲二糖 / kg 支持物 *Hour].

[0586]

[0587] 5.4 Extended continuous flow runs using cross-linked SucP2 catalyst

[0588] 5.4.1 Zinc deposition

[0589] CPS Q30 functionalized with propylamine (5 g) was weighed into a 50 mL Falcon tube and deionized water (46.45 mL) was added. 36.7 mM ZnCl2 stock solution (1.05 mL) was added and the tube was placed on a tilt / roll mixer for 1 hour (70 rpm, 21°C). The support was washed with deionized water (3x50 mL) and 150 mM MOPS buffer at pH 7 (1x50 mL). The support was incubated with ZnCl2 for 1 h. 2+ Used directly for immobilization after the deposition procedure.

[0590] 5.4.2 Immobilization

[0591] SucP2 lyophilized powder (6 g) was rehydrated in 150 mM MOPS buffer (50 mL) at pH 7 to obtain a cell-free extract at a concentration of 120 mg / mL (lyophilized powder / buffer solution). The prepared cell-free extract was resuspended on a tilt / roll mixer for 10 min (70 rpm, 21° C.) and then centrifuged for 15 min (8000 rpm, 21° C.). After centrifugation and separation of cell debris, all cell-free extract preparations were added to a 1% flask containing fresh Zn. 2+ The enzyme was immobilized on the support for 90 min on a tube rotator (20 rpm, 21° C.), and the supernatant was removed after immobilization. The immobilized support was washed with 150 mM MOPS buffer (2×50 mL) at pH 7.

[0592] 5.4.3 Cross-linking

[0593] The cross-linking solution was prepared using glycerol diglycidyl ether (GDE) 4% (v / v) and ethanol 20% (v / v) in 150mM Tris / HCl buffer at pH 9. The cross-linking solution (100 mL) was added to the immobilized catalyst and incubated overnight on a tube rotator (20 rpm, 21° C.). The solution was removed and washed with 150mM MOPS buffer at pH 7 (1×10 mL) and 400mM sucrose, 50mg / mL maltodextrin in 150mM MOPS buffer at pH 7 (1×90 mL). The catalyst was vacuum filtered to remove excess buffer and then vacuum dried in a desiccator for 6 hours.

[0594] 5.4.4 Continuous flow operation and sample analysis

[0595] The dried catalyst (2 g) was loaded into two Uniqsis column reactors (1 cm OD). The reaction mixture of the feed stream was prepared with 1M sucrose, 1M glucose in deionized water (1 L). Multiple batches were prepared according to the reactor feed needs. The reactor of the charge was assembled in the Uniqsis FlowSyn system. The reaction mixture was initially added (0.25 mL / min, 45 ° C), then the flow was reduced and maintained at 0.1 mL / min. Samples were collected regularly, and productivity was calculated as kg kojibiose / kg support*hour. The sample (5 μL) from the outlet was diluted in deionized water (95 μL) containing 13.5 mM ribose (internal standard), and acetonitrile (100 μL) was added. The Eppendorf tube was placed on ice (2 min) to quench the mixture, and then centrifuged for 2 min (15000 rpm, 21 ° C) to separate the fragments. 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 20).

[0596] Table 20. Run times of immobilized SucP2 after cross-linking. Productivity is based on the yield of kojibiose as a function of the amount of immobilized SucP2 catalyst per hour on the support.

[0597]

[0598]

[0599] Example 6 Comparison of CPS support with other supports

[0600] The reaction for converting sucrose and glucose to kojibiose shown in Scheme 7 was used to evaluate the activity of SucP2 enzymes immobilized on the controlled pore silica supports of the present invention (Q30 and Q50, provided by Fuji) and compared with the activity of SucP2 enzymes immobilized on other commercial porous silica supports and controlled pore glass supports. The following commercial silica supports were used: 1) MCM 48 (- provided by Sigma Aldrich), a mesoporous silica with an ordered cage structure, similar in properties to the mesoporous honeycomb foam (MCF) structure previously described in this disclosure, SBA-15 (provided by Sigma Aldrich), a mesoporous silica containing ordered hexagonal pores, and Q15, a controlled pore silica containing pores with a pore diameter of 15 nm (provided by Fuji, pore volume 1.00 ml / g, surface area 200 m 2 / g). In addition, the comparative study also included amino-functionalized controlled pore glass material carrier (LCAA CPG (Zn 2+Each silica support was first treated with (3-aminopropyl)trimethoxysilane as described in Section 1.2 to produce an amino-functionalized silica support-, and then further treated with Zn as described in Section 1.5. 2+ Processing to produce Zn 2+ SucP2 was immobilized onto porous silica and LCAA CPG (Zn 2+ ) without cross-linking. 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 The results of the comparative study are presented and clearly demonstrate the superior performance of the controlled pore silica of the present invention.

[0601] Example 7 Cross-linking of SucP2 immobilized on amino-functionalized controlled pore silica

[0602] 7.1. Immobilization

[0603] SucP2 lyophilized powder (500 mg) was rehydrated in 150 mM MOPS buffer (50 mL) at pH 7 to obtain a cell-free extract at a concentration of 10 mg / mL (lyophilized powder / buffer solution). The prepared cell-free extract was resuspended on a tilt / roll 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 10 mg support (CPS Q30 functionalized with propylamine, copolymer propyl + propyl NH-N-aminoethyl, propyl-3N-(4,5-dihydroimidazole), phenethyl-methyl-NH-N-aminoethyl and Zn from a solution with a final concentration of 0.105 g / mL. 2+ The samples were incubated for 2 hours (20 rpm, 21° C.) and the supernatant was removed after immobilization. The immobilized support was washed with 150 mM MOPS buffer at pH 7 (2×1 mL).

[0604] 7.2 Determination of immobilization yield

[0605] The initial enzyme solution or aliquots (100 μL) of the supernatant from the immobilization were mixed with a reaction mixture containing 1M sucrose, 1M glucose in deionized water (400 μL) and incubated for 2 hours (600 rpm, 55°C). An aliquot (10 μL) of the mixture was taken out and diluted in deionized water (190 μL), and 100 mM glycerol (internal standard) in acetonitrile: deionized water (85:15) (200 μL) was added. The Eppendorf tube was placed on ice (2 min) to quench the mixture, and then centrifuged for 2 min (15000 rpm, 21°C) to separate the fragments. 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 (21) 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.

[0606] 7.3 Cross-linking

[0607] The cross-linking reagent used is glutaraldehyde (GA), dimethyl suberimidate (DMS), bis(sulfosuccinimidyl) suberate (BS3) and glycerol diglycidyl ether (GDE) (see Table 23). According to Table 21, a solution of each cross-linking reagent is prepared in 150mM MOPS buffer, and 0.5mL is subsequently added to the immobilized catalyst. The control catalyst (non-cross-linked) is prepared by adding 150mM MOPS buffer (0.5mL) of pH 7 to immobilized SucP2. Both the control and cross-linked catalysts are incubated overnight on a tube rotator (20rpm, 21°C). The buffer / cross-linking solution is removed, and the catalyst treated with DMS and BS3 is washed once with 1mL20mM TRIS of pH 7, and then all catalysts are washed (2x1mL) with 150mM MOPS buffer of pH 7. The cross-linking treatment under different conditions is carried out in duplicate.

[0608] Table 21. Solutions used for cross-linking on immobilized SucP2

[0609]

[0610] 7.4 Leaching treatment

[0611] The catalyst was subjected to leaching test as a scheme to determine the efficiency of cross-linking. A sample from each different cross-linking condition (different cross-linking reagent) was subjected to leaching treatment, while samples from the same condition were separated and used as controls to be able to determine the productivity of the immobilized catalyst with and without the leaching scheme. Leaching was performed by adding 0.5M sodium phosphate buffer (1mL) at pH 7 to the immobilized catalyst and incubating on a tube rotator for 2 hours (20rpm, 21°C). After the leaching scheme, the catalyst was washed with 150mM MOPS buffer (2x1 mL) at pH 7. According to control studies, treating the immobilized catalyst with 0.5M sodium phosphate is an effective method for leaching enzymes that have not been cross-linked from the immobilized catalyst.

[0612] 7.5 Activity of immobilized catalysts in aqueous media

[0613] The activity of the heterogeneous catalyst after cross-linking (with and without leaching treatment) was determined by the ability to convert sucrose and glucose into 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 (1200 rpm, 55°C) and a supernatant sample (10 μL) was collected after 3 hours to determine the catalyst productivity. 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 Eppendorf tube was placed on ice (2 min) to quench the mixture and then centrifuged for 2 min (15000 rpm, 21°C) to separate the fragments. 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).

[0614]

[0615] Scheme 10. Transglycosylation of sucrose (1 M) and glucose (1 M) to produce kojibiose catalyzed by immobilized SucP2.

[0616] Table 22. Performance of SucP2 immobilized on propylamine, copolymer propyl + propyl NH-N-aminoethyl, propyl-3N-(4,5-dihydroimidazole), phenethyl-methyl-NH-N-aminoethyl functionalized controlled porous silica (Q30) followed by crosslinking with and without GDE, DMS, BS3, GA and with and without leaching protocol. Productivity is based on the activity of the immobilized catalyst for transglycosylation of sucrose and glucose to kojibiose as a function of time and amount of support.

[0617]

[0618]

[0619]

[0620] Table 23. Cross-linking reagents and possible covalent bond types formed from the cross-linking step

[0621]

[0622] Example 8 Screening of controlled pore silica with two coatings

[0623] 8.1. Synthesis of controlled-pore silica with two different silane coatings

[0624] The synthesis of controlled pore silica supports Cariact Q30 or Ecovyst E30 with 2 different coatings was performed as described previously (Examples Section 1.2). The total volume of silane in the reaction mixture was fixed at 13% (15 mL silane: 100 mL toluene). The silane groups and silane ratios used to coat the silica supports with the 2 coatings are described in Table 24.

[0625] Table 24.

[0626]

[0627] 8.2 Metal Deposition and Immobilization

[0628] For the metal deposition step of immobilized CalB-N-His, 0.93 mM of the metal stock solution was added to the support (see Table 2). For the SucP2 and transaminase reactions, 0.77 mM of the zinc stock solution was added to each support well. Immobilization of CalB-N-His, TbSADH, SucP2 (Section 4.1.1) was performed as described in the previous section without crosslinking (results are shown in Tables 10-13).

[0629] 8.3 Immobilization of amyloglucosidase on controlled pore silica containing two coatings

[0630] Before immobilization, the support was pretreated (1 hour, 20rpm, 25°C) with 200mM NaP pH 6 (1mL / 10mg support) to stabilize the pH of the material. Remove the buffer and wash the material with deionized water (2x1 mL, 5min, 20rpm, 25°C). Prepare an enzyme solution containing 16.7% enzyme by diluting a commercial enzyme storage solution (provided by Novozymes) with deionized water. 1mL of the prepared enzyme solution was added to each support (10mg) and incubated for 17 hours (20rpm, 25°C). Save the supernatant for immobilization yield analysis, and wash the immobilized catalyst with deionized water (2x1 mL, 20rpm, 5min, 25°C).

[0631] 8.4 Determination of immobilization yield

[0632] Maltose solution (985 μ L, 250 mM) is added to the Eppendorf containing immobilized reaction supernatant (15 μ L) or enzyme stock solution (15 μ L). These mixtures are incubated for 30 min (1000 rpm, 30 ° C). Then the reaction is thermally quenched at 95 ° C for 5 min. Then the sample is diluted 8 times (75 μ L reaction solution + 225 μ L 40 mM ribose + 300 μ L ACN) and analyzed by HPLC. Immobilized yield is determined by the conversion of maltose to glucose.

[0633] 8.5 Activity of Immobilized Catalysts in Aqueous Media

[0634] Maltose solution (1mL, 250mM) was added to each Eppendorf containing washed immobilized catalyst. The mixture was then incubated for 30min (30°C, 1200rpm). The reaction was then thermally quenched at 95°C for 5min. The sample was then diluted 8 times (75μL reaction solution + 225μL 40mM ribose + 300μL ACN) and analyzed by HPLC.

[0635]

[0636] Scheme 10. Hydrolysis of maltose (250 mM) catalyzed by immobilized amyloglucosidase.

[0637] 8.6 Immobilization of transaminase (ATA) on controlled pore silica containing two coatings

[0638] Lyophilized powder of amine transaminase TA633 (EC 2.6.1.62, provided by Prozomix) (200 mg) was rehydrated in 20 mM NaP at pH 8 containing 0.3 mM pyridoxal 5'-phosphate to obtain a cell-free extract (CFE) containing 20 mg / mL (lyophilized powder / total volume solution). The cell-free extract (500 μL) was transferred to Eppendorfs containing 10 mg of controlled pore silica, which contained 2 coatings that had been freshly deposited with zinc (1000 ppm) and incubated for 2 hours (20 rpm, 25° C.). The supernatant was then removed and the immobilized catalyst was washed with 20 mM NaP buffer (2x1 mL) at pH 8 and used directly after removing the remaining buffer from the Eppendorfs.

[0639] 8.7 Determination of Immobilization Yield

[0640] The cell-free extract and the supernatant from the immobilization (250 μL) were added to 250 μL of the reaction mixture (20 mM NaP at pH 8, 10% DMSO (final concentration 5%), 100 mM phenoxy-2-propanone (final concentration 50 mM), and 500 mM isopropylamine (final concentration 250 mM) and incubated for 2 hours (600 rpm, 35°C). After 2 hours, 5 M NaOH (150 μL) and EtOAc (500 μL), and incubate for another 5 min at room temperature (20 rpm), then perform phase separation for 5 min on the bench. Then the organic phase (200 μL) is transferred to a new Eppendorf containing sodium sulfate. Then the reaction solution extracted by 40 μL is transferred to a new Eppendorf containing acetic anhydride (130 μL). Then 1-methylimidazole (40 μL) is added, and the mixture is incubated at room temperature for 30 min. Deionized water (230 μL) is added and mixed, and then EtOAc (460 μL) containing 40 mM dodecane is added. The mixture is incubated for 5 min (25 ° C, 1200 rpm). After phase separation (5 min), 200 μL of organic phase is transferred to a plate containing sodium sulfate. Then the extracted sample is analyzed by gas chromatography. The immobilization yield is calculated according to the enzyme activity remaining after immobilization than the enzyme activity of the cell-free extract. All measurements are performed in duplicate and are represented by the average value of each sample.

[0641] 8.8 Activity of Immobilized Catalysts in Aqueous Media

[0642] The activity of the immobilized catalyst is determined by its ability to convert phenoxy 2-acetone into the corresponding amine (Scheme 11). A reaction mixture (20 mM NaP, DMSO (5% v / v), phenoxy-2-acetone (50 mM), isopropylamine (250 mM) at pH 8) was prepared and added (1 mL) to the immobilized and washed catalyst (10 mg). The reactants were incubated overnight (25 ° C, 1200 rpm). Subsequently, 500 μL of supernatant was transferred to a new Eppendorf, 5 M NaOH (150 μL) and EtOAc (500 μL) were added, and incubated for 5 min (25 ° C, 1200 rpm). Then 40 μL of 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 min. Deionized water (230 μL) was added and mixed, followed by addition of EtOAc (460 μL) containing 40 mM dodecane. The 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. The extracted samples were then analyzed by gas chromatography. All measurements were performed in duplicate and are represented by the average value of each sample.

[0643]

[0644] Scheme 11. Transamination between 1-phenoxypropan-2-one (50 mM) and isopropylamine (250 mM) catalyzed by immobilized ATA.

[0645] Table 25. Amyloglucosidase activity on CPS containing 2 coatings

[0646]

[0647] Table 26. SucP2 activity on CPS containing 2 coatings

[0648]

[0649]

[0650] Table 27. CalB activity on CPS containing 2 coatings

[0651]

[0652] Table 28. TbSADH activity on CPS containing 2 coatings

[0653]

[0654]

[0655] **Signal interference hinders measurement

[0656] Table 29. ATA activity on CPS containing 2 coatings

[0657]

[0658] Example 9 Preparation of heterogeneous biocatalyst using controlled pore silica with larger particle size

[0659] 9.1 Separation of controlled pore silica with a particle size range of 280–500 μm

[0660] Separation of 280–500 μm CPS Q30 particles was achieved by sieving 75–500 μm CPS Q30 (200 g, supplied by Fuji) through a 280 μm mesh filter using a Haver EML 200 Premium sieving instrument. After several cycles (amplitude 2, 1 min), the separated material (>280 μm) was removed and saved for further use.

[0661] 9.2 Synthesis of amino-functionalized controlled-pore silica with two particle size ranges (1: 280-500 μm, and 2: 1.18-2.36 mm)

[0662] Amino-functionalized CPS particles with synthetic diameters of 280-500 μm and 1.18-2.36 mm were produced according to a previously described protocol (Section 1.2).

[0663] 9.3 Immobilization of SucP2 on Amino-Functionalized 280–500 μm and 1.18–2.36 mm CPS Q30 Particles

[0664] SucP2 lyophilized powder (500 mg) was rehydrated in 150 mM MOPS buffer (50 mL) at pH 7 to obtain a cell-free extract at a concentration of 10 mg / mL (lyophilized powder / buffer solution). The prepared cell-free extract was resuspended on a tilt / roll 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 10 mg of support (CPS Q30 functionalized with propylamine). The propylamine functionalized Q30 was subjected to Zn from a solution with a final concentration of 0.105 g / L. 2+ The samples were incubated for 2 hours (20 rpm, 21° C.) and the supernatant was removed after immobilization. The immobilized support was washed with 150 mM MOPS buffer, pH 7 (2×1 mL).

[0665] 9.4 Determination of Immobilization Yield

[0666] The initial enzyme solution or aliquots (100 μL) of the supernatant from the immobilization were mixed with a reaction mixture containing 1M sucrose, 1M glucose in deionized water (400 μL) and incubated for 1 hour (600rpm, 50°C). The reaction was then thermally quenched at 95°C for 5min. The 50 μL sample was then added to 450 μL 30mM ribose (internal standard) and 500 μL ACN was then added. 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 the enzyme activity remaining in the supernatant after immobilization compared to the enzyme activity in the cell-free extract.

[0667] 9.5 Cross-linking

[0668] A cross-linking solution of glycerol diglycidyl ether (GDE) was prepared in a mixture of ethanol and 150 mM Tris at pH 9 (4% GDE, 20% EtOH). The cross-linking solution was then added to the immobilized catalyst (1 mL / 100 mg sample) and incubated for 24 hours (20 rpm, 25° C.). The cross-linked catalyst was then washed with 150 mM Tris, pH 9 (2x1 mL, 1 min, 20 rpm, 25° C.). Samples were run in duplicate.

[0669] 9.6 Leaching treatment

[0670] The catalyst was subjected to leaching test as a protocol to determine the efficiency of cross-linking. The samples were subjected to leaching treatment while samples from the same conditions were separated and used as controls to be able to determine the productivity of the immobilized catalyst with and without the leaching protocol. Leaching was performed by adding 0.5M sodium phosphate buffer (1 mL) at pH 7 to the immobilized catalyst and incubating on a tube rotator for 2 hours (800 rpm, 50°C). After the leaching protocol, the catalyst was washed with deionized water (3x1 mL). According to control studies, treating the immobilized catalyst with 0.5M sodium phosphate is an effective method to leach enzymes that have not been cross-linked from the immobilized catalyst.

[0671] 9.7 Activity of Immobilized Catalysts in Aqueous Media

[0672] The activity of the heterogeneous catalyst after cross-linking (with and without leaching treatment) was determined by the ability to convert sucrose and glucose into 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 hours (1200 rpm, 55°C). The reaction was then thermally quenched at 95°C for 5 min. 50 μL of the sample was then added to 450 μL of 30 mM ribose (internal standard) followed by 500 μL of ACN. The samples were then analyzed by HPLC to determine the conversion of sucrose and glucose to kojibiose.

[0673] Table 30. Summary of immobilization yields and productivity of propylamine functionalized controlled pore silica with different particle sizes, including both non-crosslinked and crosslinked samples with and without leaching conditions.

[0674]

[0675] 9.8 Synthesis of amino-functionalized controlled pore silicas Q30 and Q50 with different particle size ranges.

[0676] Propylamine functionalized CPS Q50 and CPS Q30 with 3 different particle size ranges were prepared according to a previously described protocol.

[0677] 9.9 Metal Deposition and Enzyme Immobilization

[0678] The procedure of metal deposition was carried out as described above. SucP2 lyophilized powder (2.4 g) was rehydrated in 150 mM MOPS at pH 7 to obtain a cell-free extract (CFE) containing 120 mg / mL (total volume of lyophilized powder / solution). The cell-free extract (1000 μL) was transferred to an Eppendorf containing 100 mg amino-functionalized-CPS Q30 and Q50 supports, which had been freshly deposited with zinc (1000 ppm) and incubated for 24 hours (20 rpm, 25 ° C). The supernatant was subsequently removed and the immobilized catalyst was washed with 150 mM MOPS (2x1 mL) at pH 7 and used directly after removing the remaining buffer in the Eppendorfs.

[0679] 9.10 Determination of Immobilization Yield

[0680] An aliquot (100 μL) of the initial enzyme solution or the supernatant from immobilization was mixed with a reaction mixture containing 1 M sucrose, 1 M glucose in deionized water (400 μL) and incubated for 1 hour (600 rpm, 50°C). The reaction was then heat quenched at 95°C for 5 min. 50 μL of the sample was then added to 450 μL of 30 mM ribose (internal standard) followed by 500 μL of ACN. The samples were 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. 9.11 Activity of immobilized catalysts in aqueous media

[0681] The activity of the heterogeneous catalysts (Scheme 7) was determined by their ability to convert sucrose and glucose into kojibiose. 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. 50 μL of the sample was then added to 450 μL of 30 mM ribose (internal standard) followed by 500 μL of ACN. The samples were then analyzed by HPLC to determine the conversion of sucrose and glucose into kojibiose.

[0682] Table 31. Summary of immobilization yields and productivity of propylamine functionalized controlled pore silica with different particle sizes and different pore diameters.

[0683]

[0684]

[0685] Example 10 Amino-functionalized controlled pore silica prepared using different methods

[0686] Two different controlled pore 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 incipient wetness coating. Slurry coating is a method in which amino-functionalization of a CPS support is performed by suspending the CPS support in a solution containing an appropriate silane. Incipient coating is a method in which amino-functionalization of a CPS support is performed by applying a controlled volume of a solution containing an appropriate silane, wherein the controlled volume closely matches the pore volume of the CPS support. Spray coating is a process in which an appropriate silane solution is converted into droplets through a nozzle and then sprayed onto the CPS support to evenly distribute the coating.

[0687] Any of the methods described herein will produce immobilization supports with similar properties.

[0688] 10.1 Immobilization of TLL on CPS Propylamine

[0689] A lipase containing Thermomyces lanuginosus (TLL) was prepared by diluting the commercial enzyme (60% v / v) in deionized water (pH 6.7 of the final solution). TL 100L solution for immobilization. The diluted enzyme (50mL) is transferred to a 100mL Duran bottle containing 10g of a support (propylamine functionalized CPS Q30 provided by Fuji and a second CPS sample provided by Ecovyst). The sample is incubated on a tilting / roller mixer for 1.5 hours (70rpm, 21°C). After immobilization, the supernatant is removed, and the immobilized support is washed with 50mM HEPES, 50mM ammonium acetate, 10mM CaCl2, pH 7 solution (1x50 mL), followed by washing with 50mM HEPES, 50mM ammonium acetate, 10mM CaCl2, 60mM sucrose, pH 7 solution (1x50 mL). For each washing step, the immobilized catalyst is mixed with the washing solution on a tilting / roller mixer for 3min (70rpm, 21°C). The catalyst was vacuum filtered to remove most of the wash solution (6 min), transferred to a 2 L glass flask and dried on a rotary evaporator for 55-70 min (40 mbar, 40 °C).

[0690] 10.2 Activity of Immobilized TLL Catalyst for Transesterification of Blended Soybean Oil

[0691] The activity of immobilized catalyst was evaluated for the transesterification of the blended soybean oil composed of 60% refined and bleached soybean oil and 40% fully hydrogenated soybean oil supplied by Bunge (BG F41120-000SOY SHTG) and produced with a product with a lower melting end temperature. Melted soybean blended oil (500 μ L) was added to the 2 mL Eppendorf tube containing immobilized TLL (10 mg), and incubated for 30 min (1500 rpm, 70 ° C). After stopping mixing and allowing the catalyst to settle, the aliquots (350 μ L) of oil were transferred to the 2 mL Eppendorf tube, and placed in ice-cold water, fat was solidified, and then stored in a refrigerator (-20 ° C) for at least 1 hour and analyzed. The melting end range of the sampling reaction was analyzed using a Melting Point System MP55 (Mettler Toledo) with a capillary (Hirschmann, L 75mm, ID 0.95mm, OD 1.35mm) at a heating rate of 2°C / min between 34°C and 67°C, and the melting end temperature was recorded as the temperature when the intensity signal (transmittance) tended to be stable and no further increase in intensity was detected (Table 32). The melting end range of the initial soybean blended oil was 64.7°C, and the lower the melting end range, the higher the degree of transesterification occurred, thereby showing a higher enzyme activity.

[0692] Table 32. Immobilization of 2-amino-functionalized controlled pore silica supports prepared using different methods. Performance of TL 100L. The efficiency of the catalyst in the transesterification of blended soybean oil depends on the melting end range of the soybean blended oil after the reaction.

[0693]

Claims

1. A biocatalyst for organic synthesis, comprising: a. controlled porosity 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; and b. One or more catalytically active enzymes immobilized to the amino-functionalized surface via a covalent linker comprising a bond selected from the group consisting of amino, amide, and iminoamide.

2. A biocatalyst for organic synthesis, comprising: a. controlled porosity 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; and b. One or more catalytically active enzymes immobilized to the surface via non-covalent interactions.

3. A biocatalyst for organic synthesis, comprising: a. controlled porosity 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 the coatings providing an amino-functionalized surface; and b. One or more catalytically active enzymes immobilized to said surface, optionally via a covalent linker comprising a bond selected from the group consisting of amino, amide and iminoamide.

4. A biocatalyst for organic synthesis, comprising: a. controlled porosity 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; and b. one or more catalytically active enzymes immobilized to said amino-functionalized surface, optionally via a covalent linker comprising a bond selected from the group consisting of amino, amide and iminoamide, Wherein the enzyme is selected from list (A), preferably from list (B).

5. A biocatalyst according to claim 1, 3 or 4, wherein the covalent linker, when present, comprises a bond selected from amino and amide.

6. The biocatalyst according to any one of the preceding claims, wherein the support material has a pore diameter of about 20 to about 60 nm and a pore diameter of about 50 nm. 2 / g to about 200m 2 / g of surface area.

7. The biocatalyst according to any one of the preceding claims, wherein the support material has a pore diameter of about 20 to about 60 nm, a pore diameter of about 50 nm, a pore diameter of about 100 nm, a pore diameter of about 20 nm, a pore diameter of about 300 nm, a pore diameter of about 500 nm, a pore diameter of about 200 nm, a pore diameter of about 300 nm, a pore diameter of about 2 / g to about 200m 2 / g surface area and a pore volume of about 0.5 mL / g to about 2.0 mL / g.

8. The biocatalyst according to any one of claims 1, 2, 3 or claims dependent therefrom, wherein the enzyme is selected from list (A).

9. The biocatalyst according to any one of the preceding claims, wherein the enzyme is selected from list (B).

10. The biocatalyst according to any one of the preceding claims, wherein the surface of the support material comprises at least two different coatings, at least one of the coatings providing the amino functionalization.

11. The biocatalyst according to claim 10, wherein one of the coatings not providing amino functionalization provides a hydrophobic coating.

12. The biocatalyst of claim 11, wherein the hydrophobic coating comprises alkyl moieties and / or aromatic moieties.

13. The biocatalyst of claim 12, wherein the hydrophobic coating comprises C 1-12 Alkyl group, preferably C 1-6 Alkyl group.

14. The biocatalyst according to any one of the preceding claims, wherein the immobilized enzyme is covalently linked between molecules via a linker comprising a bond selected from the group consisting of amino, amide, ester, ether, thioester, thioether, iminoamide, iminothioamide and thioamide.

15. The biocatalyst according to any one of claims 3, 4 or dependent therefrom, wherein the enzyme is immobilized to the surface via a covalent linker comprising a bond selected from the group consisting of amino, amide and iminoamide, preferably amino or amide.

16. The biocatalyst according to any one of claims 1, 3, 4 or claims dependent therefrom, wherein the enzyme is immobilized to the surface via a covalent linker comprising a bond selected from the group consisting of amino, amide and iminoamide (preferably amino or amide), and Therein, prior to forming said bond, said enzyme has been immobilized to said surface via non-covalent interactions.

17. The biocatalyst according to claim 16, wherein prior to forming the bonds, the enzyme has been immobilized to the surface via an interaction mediated by chelated metal ions.

18. The biocatalyst of any one of claims 2, 3, 4 or claims dependent therefrom, wherein the enzyme is immobilized to the surface via an interaction mediated by chelated metal ions.

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

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

21. The biocatalyst of any preceding claim, wherein the covalent linker between the surface and the enzyme, when present, does not comprise an imine bond.

22. A biocatalyst according to any preceding claim, wherein when present, the intermolecular covalent linker does not comprise an imine bond.

23. The biocatalyst of any preceding claim, wherein the covalent linker, when present, comprises a bridge comprising 3 to 20 atoms comprising any combination of C, H, N and O.

24. A biocatalyst according to any preceding claim, wherein the covalent 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, imidates, carbonates, acylisoureas, carbodiimides, maleimides, haloacetyl groups, thiosulfonates, isocyanates and vinyl sulfones.

25. The biocatalyst of any preceding claim, wherein the amino-functionalized surface comprises a structure of formula (I), (II) or (III): in R 1 C 1-6 alkanediyl; and wherein R 1 Provides covalent attachment to the silica surface (vertical line on the left); X is a bond (i.e., X is absent) or a benzene ring; R 2 C 1-6 Alkanediyl; R 3 is W; R 4 Is W or selected from the following group: 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 aminocarbonyl, wherein each amino group is substituted by W; and wherein the phenyl group is optionally substituted by one or more substituents C 1-4 Alkyl substitution; or R 3 and R 4 are combined together with the nitrogen atom to which they are attached to form a 5- to 7-membered heterocyclic ring, which may contain one or more unsaturated bonds and may contain one or more additional heteroatoms, to which is optionally attached a covalent linker comprising a bond selected from the group consisting of amino, amide and iminoamide, wherein the catalytically active enzyme is also attached to the linker; or R 3 and R 4 Together they form = CR 5 R 6 , where R 5 and R 6 Each independently is C 1-4 alkyl; Each R 7 Independently for C 1-6 alkanediyl; and wherein R 7 Provides covalent attachment to the silica surface (vertical line on the left); R 8 C 1-3 Alkanediyl; Each R 9 is independently W or selected from C 1-4 Alkyl and hydroxy-C 1-4 A group consisting of alkyl groups; Each R 10 Independently for C 1-6 alkanediyl; and wherein R 10 Provides covalent attachment to the silica surface (vertical line on the left); R 11 Is W or selected from the following group: 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 aminocarbonyl, wherein each amino group is substituted by W; and wherein the phenyl group is optionally substituted by one or more substituents C 1-4 Alkyl substitution; and Each W is independently hydrogen or a covalent linker comprising a bond selected from amino, amide, and iminoamide, wherein the catalytically active enzyme is also attached to the linker.

26. A biocatalyst according to any one of the preceding claims, wherein the amino-functionalized surface comprises a combination of a structure of formula (I) as defined above and a structure of formula (IV): Where R 12 Selected from C 1-12 Alkyl and amino-C 1-6 alkyl.

27. The biocatalyst according to any one of the preceding claims, wherein the amino functional groups in the surface are not part of a heterocycle.

28. The biocatalyst according to any one of the preceding claims, wherein the surface does not contain any functional groups other than amino groups that can chelate metal ions.

29. The biocatalyst of any preceding claim, wherein the surface comprises amino-functionalized aliphatic moieties.

30. The biocatalyst of any one of the preceding claims, wherein the amino-functionalized surface comprises any of the following structures, wherein each W is hydrogen or a covalent linker comprising a bond selected from amino, amide and iminoamide, wherein a catalytically active enzyme is also attached to the linker:

31. The biocatalyst according to any one of the preceding claims, wherein the amino-functionalized surface comprises structure (I'): in 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, and wherein the phenyl group is optionally substituted by one or more substituents C 1-4 Alkyl substituted; and W is hydrogen or a covalent linker comprising a bond selected from the group consisting of amino, amide, and iminoamide, wherein the catalytically active enzyme is also attached to the linker; Or wherein the amino-functionalized surface comprises structure (I"): in R 4 is W or is selected from the group consisting of 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 wherein the phenyl group is optionally substituted by one or more substituents C 1-4 Alkyl substituted; and W is hydrogen or a covalent linker comprising a bond selected from amino, amide and iminoamide, wherein the catalytically active enzyme is also attached to the linker.

32. The biocatalyst of claim 34, wherein the amino-functionalized surface comprises structure I', wherein R 4 is W or is selected from the group consisting of phenyl, benzyl, amino-C 2-6 Alkyl, N-(phenyl)amino-C 2-6 Alkyl and N-(benzyl)amino-C 2-6 alkyl; or the amino-functionalized surface comprises structure I", wherein R 4 It's W.

33. The biocatalyst according to any one of the preceding claims, wherein the amino-functionalized surface comprises any of the following structures, wherein each W is hydrogen or a covalent linker comprising a bond selected from amino, amide and iminoamide, wherein a catalytically active enzyme is also attached to the linker:

34. The biocatalyst according to any one of the preceding claims, wherein the amino-functionalized surface comprises any of the following structures, wherein each W is hydrogen or a covalent linker comprising a bond selected from amino, amide and iminoamide, wherein a catalytically active enzyme is also attached to the linker: (1) (2) (3) (4) 35. The biocatalyst of any one of the preceding claims, wherein the immobilized enzyme is selected from the group consisting of Bifidobacterium adolescentis sucrose phosphorylase (SucP), Coccidioides antarctica lipase (CalB), Thermoanaerobacterium buchneri secondary alcohol dehydrogenase (TbSADH), amine transaminase (ATA), Leuconostoc mesenteroides glycosyltransferase (GT), Thermomyces lanuginosus lipase (TLL) and Aspergillus niger amyloglucosidase.

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

37. The biocatalyst according to any one of claims 1 to 36, wherein the lipase (preferably CalB) comprising a metal chelate tag such as a His tag is activated by chelating Zn 2+ Ion-mediated interactions are immobilized to surfaces containing amino-functionalized aliphatic moieties.

38. The biocatalyst according to any one of claims 1 to 36, wherein the TbSADH comprising a metal chelating tag such as a His tag is activated by chelating Zn 2+ Ion-mediated interactions are immobilized to the surface.

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

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

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

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

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

44. The biocatalyst of any one of claims 1-36, wherein an amyloglucosidase, preferably not comprising a metal chelating tag such as a His tag, is immobilized on a surface functionalized with structure (1), (2), (3) or (4), and wherein the surface optionally has a different second functionalization.

45. Use of a biocatalyst according to any preceding claim for the synthesis of organic compounds in a reactor.

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

47. A method for synthesizing an organic compound, comprising: a. Providing a biocatalyst according to any one of claims 1-44, which is placed in a reactor; b. feeding a precursor of the organic compound to a flow reactor, whereby the immobilized enzyme catalyzes one or more reactions leading to the synthesis of the organic compound; and c. Recovering the organic compound.

48. The process of claim 47, wherein the reactor is a batch reactor or a continuous flow reactor.

49. The process of claim 48, wherein the reactor is a continuous flow fixed bed reactor.

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

51. The method of claim 50, further comprising: selecting a bifunctional cross-linking reagent capable of generating amino, amide or thioamide bonds when reacting with the enzyme and the surface, and The bifunctional reagent is used to covalently cross-link the enzyme.

52. The method of claim 51, wherein the bifunctional reagent is a diepoxide, an imidate, or an N-hydroxysuccinimide ester.

53. The method of claim 50 or any one of its dependencies, wherein the enzyme is immobilized to the surface via an interaction mediated by a chelated metal ion, preferably Ni 2+ , Cu 2+ Mg 2+ , Fe 3+ or Zn 2+ , most preferably Zn 2+ .

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