CN117467733B - High chiral purity sitagliptin and method for preparing same by using immobilized transaminase - Google Patents
High chiral purity sitagliptin and method for preparing same by using immobilized transaminase Download PDFInfo
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- CN117467733B CN117467733B CN202311820581.9A CN202311820581A CN117467733B CN 117467733 B CN117467733 B CN 117467733B CN 202311820581 A CN202311820581 A CN 202311820581A CN 117467733 B CN117467733 B CN 117467733B
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- China
- Prior art keywords
- phosphate
- immobilized
- immobilized transaminase
- transaminase
- sitagliptin
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- 102000003929 Transaminases Human genes 0.000 title claims abstract description 130
- 108090000340 Transaminases Proteins 0.000 title claims abstract description 130
- 229960004034 sitagliptin Drugs 0.000 title claims abstract description 97
- MFFMDFFZMYYVKS-SECBINFHSA-N sitagliptin Chemical compound C([C@H](CC(=O)N1CC=2N(C(=NN=2)C(F)(F)F)CC1)N)C1=CC(F)=C(F)C=C1F MFFMDFFZMYYVKS-SECBINFHSA-N 0.000 title claims abstract description 89
- 238000000034 method Methods 0.000 title claims abstract description 75
- NGVDGCNFYWLIFO-UHFFFAOYSA-N pyridoxal 5'-phosphate Chemical compound CC1=NC=C(COP(O)(O)=O)C(C=O)=C1O NGVDGCNFYWLIFO-UHFFFAOYSA-N 0.000 claims abstract description 136
- 238000006243 chemical reaction Methods 0.000 claims abstract description 83
- -1 diketone compound Chemical class 0.000 claims abstract description 75
- 239000011589 pyridoxal 5'-phosphate Substances 0.000 claims abstract description 68
- 235000007682 pyridoxal 5'-phosphate Nutrition 0.000 claims abstract description 68
- 229960001327 pyridoxal phosphate Drugs 0.000 claims abstract description 68
- 239000012074 organic phase Substances 0.000 claims abstract description 41
- 239000002245 particle Substances 0.000 claims abstract description 36
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims abstract description 34
- 235000019796 monopotassium phosphate Nutrition 0.000 claims abstract description 34
- 238000001914 filtration Methods 0.000 claims abstract description 29
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims abstract description 26
- 239000000706 filtrate Substances 0.000 claims abstract description 25
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims abstract description 13
- 238000004519 manufacturing process Methods 0.000 claims abstract description 13
- 238000001035 drying Methods 0.000 claims abstract description 10
- 239000002244 precipitate Substances 0.000 claims abstract description 8
- 239000000463 material Substances 0.000 claims description 68
- VDZOOKBUILJEDG-UHFFFAOYSA-M tetrabutylammonium hydroxide Chemical compound [OH-].CCCC[N+](CCCC)(CCCC)CCCC VDZOOKBUILJEDG-UHFFFAOYSA-M 0.000 claims description 66
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 64
- 238000003756 stirring Methods 0.000 claims description 59
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- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 36
- 239000000243 solution Substances 0.000 claims description 33
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 32
- 239000001110 calcium chloride Substances 0.000 claims description 32
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 32
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 32
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 32
- 235000019797 dipotassium phosphate Nutrition 0.000 claims description 32
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 32
- 239000007788 liquid Substances 0.000 claims description 29
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 claims description 28
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- 239000007787 solid Substances 0.000 claims description 25
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- ISYORFGKSZLPNW-UHFFFAOYSA-N propan-2-ylazanium;chloride Chemical compound [Cl-].CC(C)[NH3+] ISYORFGKSZLPNW-UHFFFAOYSA-N 0.000 claims description 24
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- 238000004064 recycling Methods 0.000 abstract 1
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- RADKZDMFGJYCBB-UHFFFAOYSA-N pyridoxal hydrochloride Natural products CC1=NC=C(CO)C(C=O)=C1O RADKZDMFGJYCBB-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
- C12P41/006—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by reactions involving C-N bonds, e.g. nitriles, amides, hydantoins, carbamates, lactames, transamination reactions, or keto group formation from racemic mixtures
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
- C12N11/082—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
- C12N11/084—Polymers containing vinyl alcohol units
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1096—Transferases (2.) transferring nitrogenous groups (2.6)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
- C12P17/182—Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y206/00—Transferases transferring nitrogenous groups (2.6)
- C12Y206/01—Transaminases (2.6.1)
Abstract
The present invention relates to high chiral purity sitagliptin and a process for its preparation using immobilized transaminase. Wherein the process for preparing sitagliptin phosphate monohydrate comprises the steps of: reacting a diketone compound in a solvent in the presence of pyridoxal phosphate and isopropylamine and immobilized transaminase, wherein reaction filter residues are used as immobilized transaminase to be used in the next batch; and (3) dropwise adding phosphoric acid into the organic phase of the filtrate, filtering, taking the precipitate, and drying to obtain sitagliptin phosphate monohydrate. The immobilized transaminase comprises an R-transaminase solution, potassium dihydrogen phosphate, pyridoxal phosphate, and enzyme-immobilized particles. The immobilized transaminase can be recycled and sleeved for the next batch after being used by 1-50. The invention also relates to an immobilized transaminase for preparing sitagliptin phosphate monohydrate and a method for preparing the immobilized transaminase. The immobilized enzyme has high recycling rate, extremely low production cost, and high optical purity and normalized purity of the conversion product.
Description
Technical Field
The invention belongs to the technical field of biological medicines, relates to a method for converting a diketone compound into an amino ketone compound through biocatalysis by using immobilized transaminase, in particular to high-chiral-purity sitagliptin and a method for preparing the high-chiral-purity sitagliptin through biocatalysis by using immobilized transaminase.
Background
Sitagliptin (also commonly referred to as Sitagliptin) as a dipeptidyl peptidase-4 (DPP-4) inhibitor has the formula C 16 H 15 F 6 N 5 O, molecular weight 407.3, CAS registry number 486460-32-6. Sitagliptin is the first 10 th 2006 dipeptidyl peptidase-IV (DDP-4) inhibitor approved by the United states Food and Drug Administration (FDA) for the treatment of type II diabetes, which is defined by Merck&Co., inc. under the trade name JANUVIA ® . Clinical studies have shown that sitagliptin as a single drug for treatment of type II diabetics can significantly reduce glycosylated hemoglobin (HbA 1C) levels. When used in combination with metformin or TZDs, has remarkable adjuvant therapeutic effects and can address three main defects of type II diabetes: insulin resistance, beta cell dysfunction, and alpha cell dysfunction.
Clinically pharmaceutically acceptable sitagliptin is commonly used as its phosphate monohydrate (Sitagliptin phosphate monohydrate), the chinese name of the pharmaceutically acceptable sitagliptin is: 7- [ (3R) -3-amino-1-oxo-4- (2, 4, 5-trifluorophenyl) butyl]-5,6,7, 8-tetrahydro-3- (trifluoromethyl) -1,2, 4-triazole [4,3-a ]]Pyrazine phosphate (1:1) monohydrate, english chemical name: 7- [ (3R) -3-amino-1-oxo-4- (2, 4, 5-trifluorophenyl) butyl]-5,6,7,8-tetrahydro-3-(trifluoromethyl)-1,2,4-triazolo[4,3-a]pyrazine phosphate (1:1) monohydrate, molecular weight 523.32, CAS registry number 654671-77-9, having the chemical formula:
。
the invention refers toThe term "sitagliptin" refers generally to the free base form of formula C 16 H 15 F 6 N 5 Of course, in a particular context, the term may also refer to sitagliptin of formula C 16 H 15 F 6 N 5 O•H 3 PO 4 Or sitagliptin phosphate of formula C 16 H 15 F 6 N 5 O•H 3 PO 4 •H 2 Sitagliptin phosphate monohydrate of O. In addition, the term "sitagliptin phosphate" as referred to herein generally refers to a compound of formula C 16 H 15 F 6 N 5 O•H 3 PO 4 •H 2 The sitagliptin phosphate monohydrate of O may of course also be referred to in the particular context as sitagliptin phosphate of the formula c16h15f6n5o.h3po4 (anhydrate, CAS accession number 654671-78-0).
Sitagliptin was previously disclosed in WO03004498 and US6699871 (2003 and 2004, merck) &Co.); the enzyme inhibition profile of sitagliptin is disclosed inD. Kim et al., J. Med. Chem. 48, 141(2005)The method comprises the steps of carrying out a first treatment on the surface of the The improved preparation process is disclosed in K.B. Hansenet al., Org. Process Res. Dev.9, 634 (2005); clinical pharmacokinetics and pharmacodynamics are described in G.A. Hermanet al., Clin. Pharmacol. Ther.78, 675 (2005); for a complete review see c.f. Deacon,Curr. Opin. Invest. Drugs 6, 419-426(2005)。
the prior art discloses some methods for preparing sitagliptin. For example, the process disclosed in US6699871 is outlined in scheme 1, which involves the preparation of the intermediates (3R) -3- [ N- (tert-butoxycarbonyl) amino ] -4- (2, 4, 5-trifluorophenyl) butanoic acid (07) and 3-trifluoromethyl-5, 6,7, 8-tetrahydro- [1,2,4] triazolo [4,3-a ] pyrazine hydrochloride (11), followed by their combination to obtain Boc-protected sitagliptin base (12), which is deprotected using methanol hydrochloride to obtain sitagliptin hydrochloride (13).
WO2004/085661 discloses a process for the synthesis of sitagliptin free base (01) in scheme 2, which involves the synthesis of sitagliptin free base from Meldrum's adduct (19) and 3-trifluoromethyl-5, 6,7, 8-tetrahydro [1,2,4]]Triazolo [4,3-a ]]Pyrazine hydrochloride synthesisKeto amide intermediate (14). Intermediate (14) is further treated with (S) -phenylglycine amide in isopropanol and acetic acid to give amide (15) by reaction over catalyst PtO 2 The amide (15) is further converted to an amide (16) by asymmetric hydrogenation in the presence of 90psi and 22 ℃ for 24 hours. Intermediate (16) is converted to sitagliptin free base (01) by a debenzylation reaction carried out in the presence of palladium hydroxide on carbon at 40psi and 50 ℃.
Scheme 3 of US2006/194977 describes the further conversion of intermediate (14) prepared from the reaction of Mitsubishi acid adduct (19) and 3-trifluoromethyl-5, 6,7, 8-tetrahydro [1,2,4] triazolo [4,3-a ] pyrazine hydrochloride (11) to enamine (20) by using ammonium acetate in methanol. The enamine (20) was further converted to enantiomerically enriched sitagliptin free base (01) by asymmetric reduction using a Josiphos catalyst and chloro (1, 5-cyclooctadiene) rhodium (I) dimer. The asymmetric synthesis involves hydrogenating enamine (20) at 200psi and 50 ℃ for 13 hours to obtain enantiomerically enriched sitagliptin free base (01).
WO2004/087650 discloses a synthesis of sitagliptin free base (01), which scheme 4 describes the synthesis of methyl 4- (2, 4, 5-trifluorophenyl) acetoacetate (21) by refluxing the michaelis adduct (19)/methanol, subjecting the formed β -ketoester (21) to asymmetric hydrogenation using ruthenium dichloride and (S) -BINAP to obtain (3S) -4- (2, 4, 5-trifluorophenyl) -3-hydroxybutyric acid (23), which is further converted to oxetane (oxazetidine) (25) via hydroxy intermediate (24), which oxetane (25) is converted to (3R) -3- [ (benzyloxy) amino ] -4- (2, 4, 5-trifluorophenyl) butanoic acid (26), which compound is further converted to enantiomerically enriched sitagliptin free base (01) and sitagliptin dihydrogen phosphate (02).
With the continuous development of biotechnology, enzyme immobilization technology has become an important means in industrial production. The immobilized enzyme has the advantages of high stability, reusability, easy separation and the like, and is widely applied to the fields of medicine synthesis, biofuel production and the like. Wherein, the immobilized aminotransferase has important application value in the aspects of synthesizing medicines such as sitagliptin and the like. Immobilized transaminase is a technique of immobilizing transaminase on a carrier, so that the enzyme can maintain high activity and stability under specific conditions. The process for synthesizing sitagliptin by using immobilized transaminase is an emerging biopharmaceutical synthesis method, and has potential advantages in the aspects of reducing environmental protection pressure, reducing production cost and the like.
There are some prior art methods for biocatalytically converting a precursor diketone compound of sitagliptin (which diketone compound may also be referred to in the art as a substrate or a reaction substrate or a catalytic substrate or an enzyme catalytic substrate, etc.) into an aminoketone compound, sitagliptin, by using an immobilized transaminase, the specific reaction process is as follows:
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chinese patent application No. 201710008432.0 (CN 106834376a, macrosource) discloses a method and apparatus for enzymatic synthesis of sitagliptin, comprising the steps of: culturing transaminase bacteria cells with catalytic activity, crushing the transaminase bacteria cells, suspending in water, and centrifuging to obtain crude enzyme solution; preparing a buffer solvent containing an osmotic pressure stabilizer by adopting the osmotic pressure stabilizer and a sodium chloride solution, adding sodium alginate into the buffer solvent to obtain a sodium alginate solution, taking a crude enzyme solution with the same volume as the sodium alginate solution, uniformly mixing the taken crude enzyme solution with the sodium alginate solution to obtain a mixed solution, dropwise adding the mixed solution into a calcium chloride solution, simultaneously adding pyridoxal phosphate into the calcium chloride solution to obtain a blend solution, and stirring the blend solution for 3-48 hours to obtain a calcium chloride solution containing spheroidized immobilized enzyme; adding diatomite into the calcium chloride solution containing the spheroidizing immobilized enzyme, controlling the pH of the system to be 7.5-9.0, obtaining a mixed system, extracting water in the mixed system, and controlling the water content in the mixed system to be not more than 40%; dissolving a substrate in an aqueous solution of dimethyl sulfoxide, and adding isopropylamine hydrochloride to obtain a buffer solution for reaction; the mixed system is placed in a constant temperature reaction container, and the buffer solution for reaction is filled in the constant temperature reaction container in a circulating flow mode and reacts with the mixed system to obtain sitagliptin. The method is simple to operate, high in conversion efficiency, environment-friendly and low in cost.
Chinese patent application No. 202210330110.9 (CN 114657170B, langkun) discloses a preparation method of high-stability immobilized enzyme, wherein amino resin LXTE-700S is added into crude enzyme liquid prepared by a conventional method for filtering, then boric acid buffer solution and pyridoxal phosphate are added for oscillation reaction; finally adding a long agent Cheng Jiaolian and a short agent Cheng Jiaolian, and carrying out suction filtration after oscillation reaction to obtain the high-stability immobilized enzyme; the epoxy cross-linking agent is a mixture of long Cheng Jiaolian agent polyethylene glycol diglycidyl ether and short-range cross-linking agent glycerol triglycidyl ether, and the invention also discloses a test for preparing sitagliptin by using the immobilized transaminase to perform enzyme catalytic conversion on a diketone compound. The preparation method of the immobilized enzyme provided by the invention is simple to operate, can obviously prolong the storage and service life of the immobilized enzyme, and has good application prospect in the enzyme catalysis industry.
Chinese patent application No. 202310455056.5 (CN 116694697A, langkun) discloses a preparation method of sitagliptin phosphate monohydrate, which takes diketone (CAS: 764667-65-4) as a raw material and reacts with isopropylamine to generate sitagliptin through ammonia conversion under the catalysis of immobilized enzyme; then dilute phosphoric acid is added to form salt, crystallization and purification are carried out, thus obtaining sitagliptin phosphate monohydrate; the method comprises the following specific steps: adding diketone into solvent I (methanol, ethanol and/or isopropanol), sequentially adding purified water, immobilized enzyme and isopropylamine, reacting at 40-50deg.C, filtering, and concentrating the filtrate at 50-60deg.C to obtain concentrate; dissolving the concentrate in dilute hydrochloric acid, adding solvent II (ethyl acetate, isopropyl acetate and/or dichloromethane), layering the solution, taking water phase, adding solvent III (ethyl acetate, isopropyl acetate and/or dichloromethane), and adding alkali liquor to adjust pH value to 11; concentrating the organic phase at 50-60deg.C to dry to obtain sitagliptin; dissolving sitagliptin in a solvent IV (mixed solution of methanol, ethanol and/or isopropanol and water in a ratio of 3:1), adding a phosphoric acid solution, dissolving a product in a reaction at 72-80 ℃, cooling to 62-66 ℃, and adding sitagliptin phosphate monohydrate as a seed crystal; then reacting for 3 hours at 60-65 ℃, then cooling to 20-25 ℃ and reacting for 2 hours at the temperature of heat preservation; then dropwise adding a solvent I into the reaction system, cooling to 0-10 ℃ and reacting for 2h at a constant temperature; and then carrying out suction filtration, leaching a filter cake by using a solvent I, and then drying the filter cake under reduced pressure at 40-45 ℃ to obtain the sitagliptin phosphate monohydrate. The method is simple to operate, mild in condition and suitable for large-scale industrial production.
However, the prior art remains deficient with respect to methods for biocatalytically synthesizing sitagliptin from diketone compounds via immobilized transaminase, there remains a need in the art for new methods for biocatalytically synthesizing sitagliptin from diketone compounds via immobilized transaminase, and such methods are expected to exhibit one or more beneficial effects.
Disclosure of Invention
The object of the present invention is to provide a process for biocatalytically synthesizing sitagliptin from a diketone compound via an immobilized transaminase, which still has drawbacks, and a new process for biocatalytically synthesizing sitagliptin from a diketone compound via an immobilized transaminase is still expected in the art, and which processes are expected to exhibit one or more advantageous effects.
To this end, the first aspect of the invention provides an immobilized transaminase comprising the following components: r-aminotransferase liquid, monopotassium phosphate, dipotassium phosphate, calcium chloride, pyridoxal phosphate, tetrabutylammonium hydroxide, sepiolite particles and polyvinyl alcohol.
An immobilized transaminase according to the first aspect of the invention comprises the following proportions of components: comprises 100g of solid enzyme powder, 0.08-0.12 mol of monopotassium phosphate, 0.1-0.14 mol of dipotassium phosphate, 0.16-0.24 mol of calcium chloride, 12-18 g of pyridoxal phosphate, 1.6-2.4 g of tetrabutylammonium hydroxide, 400-600 g of sepiolite particles and 4-6 g of polyvinyl alcohol.
The immobilized transaminase according to the first aspect of the invention, wherein the sepiolite particles are particles of diameter 1-3mm obtained by calcination of sepiolite at 650-750 ℃.
The immobilized transaminase according to the first aspect of the invention, wherein the polyvinyl alcohol is polyvinyl alcohol of type PVA 17-88.
An immobilized transaminase according to the first aspect of the invention comprises the following proportions of components: comprises 100g of solid enzyme powder, 0.1mol of monopotassium phosphate, 0.12mol of dipotassium phosphate, 0.2mol of calcium chloride, 15g of pyridoxal phosphate, 2g of tetrabutylammonium hydroxide, 500g of sepiolite particles and 5g of polyvinyl alcohol.
An immobilized transaminase according to the first aspect of the invention comprises the following proportions of components: comprises 10g of solid enzyme powder, 0.012mol of monopotassium phosphate, 0.010mol of dipotassium phosphate, 0.024mol of calcium chloride, 1.2g of pyridoxal phosphate, 0.24g of tetrabutylammonium hydroxide, 40g of sepiolite and 4g of polyvinyl alcohol.
An immobilized transaminase according to the first aspect of the invention comprises the following proportions of components: comprises 10g of solid enzyme powder, 0.008mol of monopotassium phosphate, 0.014mol of dipotassium phosphate, 0.016mol of calcium chloride, 1.8g of pyridoxal phosphate, 0.16g of tetrabutylammonium hydroxide, 60g of sepiolite and 6g of polyvinyl alcohol.
The immobilized transaminase according to the first aspect of the invention is prepared according to a process comprising the following steps:
(i) Adding monopotassium phosphate, dipotassium phosphate, calcium chloride, pyridoxal phosphate and tetrabutylammonium hydroxide into the R-aminotransferase liquid at the temperature of 5-10 ℃ and stirring for 2 hours to dissolve;
(ii) Then adding the mixed solution into sepiolite particles, sealing a container for containing the solid-liquid mixed material, mixing the materials for 1 hour by turning the container over, and standing for 24 hours;
(iii) Turning over the container to mix the materials for 30min, adding 5% polyvinyl alcohol solution into the solid-liquid mixture, and continuing turning over the container to mix the materials for 1 h;
(iv) And (5) draining the water by centrifugation until the water content is less than 5%, thus obtaining the immobilized transaminase.
Further, the second aspect of the present invention provides a process for preparing sitagliptin phosphate monohydrate, comprising the steps of:
(1) Adding 0.1mol of diketone compound into a reaction bottle, adding 0.5L of methyl tertiary butyl ether, stirring to dissolve, then adding 8-12 mmol of pyridoxal phosphate, 0.4-0.6 mol of isopropylamine hydrochloride, and carrying out hot filtration, wherein the immobilized transaminase is 40-60 g which is recovered and used for the first time or after the method is used for multiple times, stirring at 55-75 ℃ to carry out reflux reaction on materials for 12-18 hours;
(2) Centrifuging the filter residue to dryness, and taking the filter residue as recovered immobilized transaminase to be used in the next batch; the filtrate was cooled to room temperature and washed with water to give an organic phase containing the aminoketone compound;
(3) Dropwise adding 0.12-0.15 mol of phosphoric acid into the organic phase under stirring, continuously stirring for 4 hours at the temperature of 5-10 ℃, filtering, washing the precipitate with ethanol and isopropanol in sequence, and drying to obtain sitagliptin phosphate monohydrate.
The method according to the second aspect of the invention, wherein the immobilized transaminase comprises the following components: r-aminotransferase liquid, monopotassium phosphate, dipotassium phosphate, calcium chloride, pyridoxal phosphate, tetrabutylammonium hydroxide, sepiolite particles and polyvinyl alcohol.
The method according to the second aspect of the invention, wherein the immobilized transaminase comprises the following proportions of components: comprises 100g of solid enzyme powder, 0.08-0.12 mol of monopotassium phosphate, 0.1-0.14 mol of dipotassium phosphate, 0.16-0.24 mol of calcium chloride, 12-18 g of pyridoxal phosphate, 1.6-2.4 g of tetrabutylammonium hydroxide, 400-600 g of sepiolite particles and 4-6 g of polyvinyl alcohol.
The method according to the second aspect of the present invention, wherein the sepiolite particles in the immobilized transaminase are particles having a diameter of 1 to 3mm obtained by calcining sepiolite at 650 to 750 ℃.
The method according to the second aspect of the present invention, wherein the polyvinyl alcohol in the immobilized transaminase is polyvinyl alcohol of type PVA 17-88.
The method according to the second aspect of the invention, wherein the immobilized transaminase comprises the following proportions of components: comprises 100g of solid enzyme powder, 0.1mol of monopotassium phosphate, 0.12mol of dipotassium phosphate, 0.2mol of calcium chloride, 15g of pyridoxal phosphate, 2g of tetrabutylammonium hydroxide, 500g of sepiolite particles and 5g of polyvinyl alcohol.
The method according to the second aspect of the invention, wherein the immobilized transaminase comprises the following proportions of components: comprises 10g of solid enzyme powder, 0.012mol of monopotassium phosphate, 0.010mol of dipotassium phosphate, 0.024mol of calcium chloride, 1.2g of pyridoxal phosphate, 0.24g of tetrabutylammonium hydroxide, 40g of sepiolite and 4g of polyvinyl alcohol.
The method according to the second aspect of the invention, wherein the immobilized transaminase comprises the following proportions of components: comprises 10g of solid enzyme powder, 0.008mol of monopotassium phosphate, 0.014mol of dipotassium phosphate, 0.016mol of calcium chloride, 1.8g of pyridoxal phosphate, 0.16g of tetrabutylammonium hydroxide, 60g of sepiolite and 6g of polyvinyl alcohol.
The method according to the second aspect of the invention, wherein the immobilized transaminase is prepared according to a method comprising the following steps:
(i) Adding monopotassium phosphate, dipotassium phosphate, calcium chloride, pyridoxal phosphate and tetrabutylammonium hydroxide into the R-aminotransferase liquid at the temperature of 5-10 ℃ and stirring for 2 hours to dissolve;
(ii) Then adding the mixed solution into sepiolite particles, sealing a container for containing the solid-liquid mixed material, mixing the materials for 1 hour by turning the container over, and standing for 24 hours;
(iii) Turning over the container to mix the materials for 30min, adding 5% polyvinyl alcohol solution into the solid-liquid mixture, and continuing turning over the container to mix the materials for 1 h;
(iv) And (5) draining the water by centrifugation until the water content is less than 5%, thus obtaining the immobilized transaminase.
The method according to the second aspect of the present invention, wherein the recovered immobilized transaminase after the multiple uses means the recovered immobilized transaminase after 1 to 50 uses.
The method according to the second aspect of the present invention, wherein the recovered immobilized transaminase after the multiple uses means the recovered immobilized transaminase after 1 to 30 uses.
The method according to the second aspect of the invention comprises the steps of:
(1) Adding 0.1mol of diketone compound into a reaction bottle, adding 0.5L of methyl tertiary butyl ether, stirring to dissolve, then adding 10mmol of pyridoxal phosphate, 0.5mol of isopropylamine hydrochloride, and 50g of immobilized transaminase which is used for the first time or recovered after the method is used for a plurality of times, stirring at 65 ℃ to make the materials reflux-react for 15 hours, and filtering while the materials are hot;
(2) Centrifuging the filter residue to dryness, and taking the filter residue as recovered immobilized transaminase to be used in the next batch; the filtrate was cooled to room temperature and washed with water to give an organic phase containing the aminoketone compound;
(3) Dropwise adding 0.13mol of phosphoric acid into the organic phase under stirring, continuously stirring for 4 hours at the temperature of 5-10 ℃, filtering, washing the precipitate with ethanol and isopropanol in sequence, and drying to obtain sitagliptin phosphate monohydrate.
The method according to the second aspect of the invention comprises the steps of:
(1) Adding 0.1mol of diketone compound into a reaction bottle, adding 0.5L of methyl tertiary butyl ether, stirring to dissolve, then adding 8mmol of pyridoxal phosphate, 0.6mol of isopropylamine hydrochloride, and 40g of immobilized transaminase which is used for the first time or recovered after the method is used for multiple times, stirring at 55 ℃ to make the materials reflux-react for 18 hours, and filtering while the materials are hot;
(2) Centrifuging the filter residue to dryness, and taking the filter residue as recovered immobilized transaminase to be used in the next batch; the filtrate was cooled to room temperature and washed with water to give an organic phase containing the aminoketone compound;
(3) Dropwise adding 0.12mol of phosphoric acid into the organic phase under stirring, continuously stirring for 4 hours at the temperature of 5-10 ℃, filtering, washing the precipitate with ethanol and isopropanol in sequence, and drying to obtain sitagliptin phosphate monohydrate.
The method according to the second aspect of the invention comprises the steps of:
(1) Adding 0.1mol of diketone compound into a reaction bottle, adding 0.5L of methyl tertiary butyl ether, stirring to dissolve, then adding 12mmol of pyridoxal phosphate, 0.4mol of isopropylamine hydrochloride, and 60g of immobilized transaminase which is used for the first time or recovered after the method is used for a plurality of times, stirring at 75 ℃ to make the materials reflux and react for 12 hours, and filtering while the materials are hot;
(2) Centrifuging the filter residue to dryness, and taking the filter residue as recovered immobilized transaminase to be used in the next batch; the filtrate was cooled to room temperature and washed with water to give an organic phase containing the aminoketone compound;
(3) Dropwise adding 0.15mol of phosphoric acid into the organic phase under stirring, continuously stirring for 4 hours at the temperature of 5-10 ℃, filtering, washing the precipitate with ethanol and isopropanol in sequence, and drying to obtain sitagliptin phosphate monohydrate.
Further, a third aspect of the present invention provides a process for preparing an immobilized transaminase comprising the following components: r-transaminase solution, monopotassium phosphate, dipotassium phosphate, calcium chloride, pyridoxal phosphate, tetrabutylammonium hydroxide, sepiolite particles and polyvinyl alcohol, the method comprises the following steps:
(i) Adding monopotassium phosphate, dipotassium phosphate, calcium chloride, pyridoxal phosphate and tetrabutylammonium hydroxide into the R-aminotransferase liquid at the temperature of 5-10 ℃ and stirring for 2 hours to dissolve;
(ii) Then adding the mixed solution into sepiolite particles, sealing a container for containing the solid-liquid mixed material, mixing the materials for 1 hour by turning the container over, and standing for 24 hours;
(iii) Turning over the container to mix the materials for 30min, adding 5% polyvinyl alcohol solution into the solid-liquid mixture, and continuing turning over the container to mix the materials for 1 h;
(iv) And (5) draining the water by centrifugation until the water content is less than 5%, thus obtaining the immobilized transaminase.
The method according to the third aspect of the invention, wherein the immobilized transaminase comprises the following proportions of components: comprises 100g of solid enzyme powder, 0.08-0.12 mol of monopotassium phosphate, 0.1-0.14 mol of dipotassium phosphate, 0.16-0.24 mol of calcium chloride, 12-18 g of pyridoxal phosphate, 1.6-2.4 g of tetrabutylammonium hydroxide, 400-600 g of sepiolite particles and 4-6 g of polyvinyl alcohol.
The method according to the third aspect of the present invention, wherein the sepiolite particles are particles having a diameter of 1 to 3mm obtained by calcining sepiolite at 650 to 750 ℃.
The method according to the third aspect of the present invention, wherein the polyvinyl alcohol is polyvinyl alcohol of type PVA 17-88.
The method according to the third aspect of the invention, wherein the immobilized transaminase comprises the following proportions of components: comprises 100g of solid enzyme powder, 0.1mol of monopotassium phosphate, 0.12mol of dipotassium phosphate, 0.2mol of calcium chloride, 15g of pyridoxal phosphate, 2g of tetrabutylammonium hydroxide, 500g of sepiolite particles and 5g of polyvinyl alcohol.
The method according to the third aspect of the invention, wherein the immobilized transaminase comprises the following proportions of components: comprises 10g of solid enzyme powder, 0.012mol of monopotassium phosphate, 0.010mol of dipotassium phosphate, 0.024mol of calcium chloride, 1.2g of pyridoxal phosphate, 0.24g of tetrabutylammonium hydroxide, 40g of sepiolite and 4g of polyvinyl alcohol.
The method according to the third aspect of the invention, wherein the immobilized transaminase comprises the following proportions of components: comprises 10g of solid enzyme powder, 0.008mol of monopotassium phosphate, 0.014mol of dipotassium phosphate, 0.016mol of calcium chloride, 1.8g of pyridoxal phosphate, 0.16g of tetrabutylammonium hydroxide, 60g of sepiolite and 6g of polyvinyl alcohol.
Any of the embodiments of any of the aspects of the invention may be combined with any of the other embodiments, provided that such combination does not create a conflict. The present invention is further described below.
In the present invention, when% is referred to, weight/volume percentage is the case of solid solutes and liquid solvents, and weight/weight percentage is the case of solids distributed in solids, unless otherwise specified.
Sitagliptin is used clinically as a classical dipeptidyl peptidase 4 (DPP-4) inhibitor for improving glycemic control in type 2 diabetics by increasing the level of active incretin in type 2 diabetics. The incretin insulinotropic hormone, including glucagon-like polypeptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), is released throughout the gut and increases in levels after a meal. The incretin insulinotropic hormone is part of the endogenous system involved in physiological regulation of glucose homeostasis. GLP-1 and GIP can increase pancreatic β cell synthesis and release insulin through intracellular signaling pathways involving adenosine cyclophosphate when blood glucose concentrations are normal or elevated. GLP-1 or DPP-4 inhibitor treatment can improve pancreatic beta cell responsiveness to glucose and promote insulin biosynthesis and release in an animal model of type 2 diabetes. As insulin levels increase, the uptake of glucose by tissues increases. In addition, GLP-1 can inhibit pancreatic alpha cells from secreting glucagon. A decrease in glucagon concentration and an increase in insulin levels may decrease hepatic glucose production, thereby lowering blood glucose levels. The effects of GLP-1 and GIP are glucose dependent, and GLP-1 does not promote insulin release nor inhibit glucagon secretion when blood glucose levels are low. GLP-1 and GIP have enhanced effects in promoting insulin release when glucose levels are above normal concentrations. In addition, GLP-1 does not impair the body's normal glucagon-releasing response to hypoglycemia. GLP-1 and GIP activities are limited by the DPP-4 enzyme, which can rapidly hydrolyze the incretin to produce inactive products. Sitagliptin prevents DPP-4 from hydrolyzing the incretin, thereby increasing the plasma concentration of GLP-1 and GIP in active form. By increasing the level of active incretin, sitagliptin can increase insulin release and decrease glucagon levels in a glucose dependent manner. For type 2 diabetics with hyperglycemia, the above-described changes in insulin and glucagon levels can reduce glycosylated hemoglobin A1c (HbA 1 c) and lower fasting and postprandial blood glucose levels. The glucose-dependent mechanism of action of sitagliptin is different from that of sulfonylurea drugs, which can increase insulin secretion even at low glucose levels, resulting in hypoglycemia in type 2 diabetics and normal subjects. Sitagliptin is an effective and highly selective DPP-4 enzyme inhibitor, and does not inhibit DPP-8 or DPP-9 closely related to DPP-4 at therapeutic concentration [ Xu Hui ], and the like, the clinical application of sitagliptin has been recently researched and developed, jining medical college, 2014]. Studies of the pharmacokinetic profile of sitagliptin have been widely conducted in healthy subjects and type 2 diabetics. After oral administration of 100mg dose to healthy subjects, sitagliptin is rapidly absorbed and plasma drug concentration reaches a peak (median Tmax) 1 to 4 hours after administration. The blood AUC of sitagliptin increases in proportion to the dose. After single dose oral administration of 100mg in healthy volunteers, the mean blood AUC of sitagliptin was 8.52 μm·hr, cmax was 950nM, and apparent terminal half-life (t 1/2) was 12.4 hours. The plasma AUC at steady state reached with sitagliptin 100mg was increased by about 14% compared to the initial administration. The individual self and individual sitagliptin AUC have smaller coefficients of variation (5.8% and 15.1%). The pharmacokinetic index of sitagliptin in healthy subjects and type 2 diabetics is substantially similar.
It is well known to those skilled in the art that biological enzymes have been widely accepted due to their specific advantages of high catalytic efficiency, high specific selectivity, mild reaction conditions, less three-waste emission, etc., and the practical effects in the pharmaceutical industry are continuously expanding, especially in process substitution, realization of greener pharmaceutical processes. The biological enzymes used for industrialization comprise carbonyl reductase, alcohol dehydrogenase, transaminase, lipase, acylase, nitrilase and the like, when the biological enzymes are used for catalyzing and synthesizing small molecular compounds, compared with the original chemical conversion or resolution process, after the biological enzyme process is adopted, the total reaction yield is obviously improved, the reaction time is greatly shortened, the productivity is obviously improved, the use of chemical solvents is greatly reduced, the use of noble metal catalysts is avoided, the emission of three wastes is reduced, and the national environment-friendly concept is met. The above-mentioned biological enzymes are readily available commercially, and for example, in the present invention, unless otherwise specified, the biological enzymes used are obtained from R-aminotransferase solutions (solid-state enzyme concentration 10%, percentage of target protein in SDS-PAGE: 75% or more, and effective protein content in target protein: 85% or more) obtained from Jiangsu Mei Biotech Co., ltd.
In addition, sitagliptin precursor diketone compounds (CAS: 764667-65-4) can be synthesized with reference to the relevant literature or are readily available directly from the market (e.g., hubei Nanoz technologies, anqing Qijacka, etc., or can be readily obtained from the Gede chemical industry network).
In the present invention, the carrier used for immobilizing transaminase is sepiolite (sepiolite) or a processed product thereof, and sepiolite isA natural mineral containing a water-containing magnesium silicate salt having a chemical structure of (Si 12 )(Mg 8 )O 30 (OH) 4 (OH 2 ) 4 ·8H 2 O, sepiolite ore is widely distributed in China, for example, hunan Xiangtan, hebei Yixian county, jiangxi Leping, henan Neixiang and the like are typically distributed. The preferred support is sepiolite calcined at 650-750 ℃ to obtain particles of 1-3mm diameter, referred to herein as sepiolite calcined particles or sepiolite particles. The sepiolite particles used in example 1 of the present invention are extremely low cost, for example, less than one tenth of the LXTE-700S resin used in the prior art.
Pyridoxal phosphate (PLP) is a derivative of vitamin B6, an active form of vitamin B6 involved in various metabolic reactions of proteins, fats and carbohydrates. It is a coenzyme for transaminase and decarboxylase in amino acid metabolism, and can promote glutamic acid decarboxylation and increase the generation of gamma-aminobutyric acid. Pyridoxal phosphate plays a role as a coenzyme in all transamination reactions and in decarboxylation and deamination reactions of some amino acids. Schiff base bonds (internal aldimines) are formed between the aldehyde group of pyridoxal phosphate and the epsilon-amino group of a specific lysine group in aminotransferase. The alpha-amino group in the amino acid substrate replaces the epsilon-amino group of the active site lysine residue. The resulting external aldimine becomes deprotonated to form a quinoid intermediate which in turn accepts a proton at a different position to form a ketimine. The ketimine is hydrolyzed to regenerate the amino group of the complex enzyme. In addition, pyridoxal phosphate is also a cofactor for certain transaminases with unusual sugars (e.g., peroxoglycoamine, deoxysugar amine) as substrates. In these reactions, pyridoxal phosphate reacts with glutamic acid, and the α -amino group of glutamic acid is transferred to pyridoxal phosphate to produce pyridoxamine phosphate (PMP). Pyridoxamine phosphate then transfers its nitrogen to the sugar, forming an amino sugar. The biochemical processes involved in pyridoxal phosphate are also: beta-elimination reactions, such as serine dehydratases and GDP-4-keto-6-deoxymannose-3-dehydratases (ColDs), are involved in the completed reaction; condensation reaction in heme synthesis; a reaction of dopa to dopamine conversion; reaction of excitatory transmitter glutamate with conversion of inhibitory transmitter gamma-aminobutyric acid; histidine (decarboxylation) to histamine conversion reactions, and the like. Pyridoxal phosphate for use in the present invention is readily available from commercial sources.
It has been unexpectedly found that by using the immobilized transaminase of example 1 of the present invention, it exhibits many advantageous effects such as high reuse rate of immobilized enzyme, extremely low production cost of immobilized enzyme, and high optical purity of converted product, and high normalized purity of final product when biocatalytic conversion of diketone compound to ammonone compound is performed in example 2.
Drawings
Fig. 1: typical HPLC profile of an ammonione compound.
Fig. 2: typical HPLC profile of precursor diketo compounds.
Fig. 3: typical mixture chromatograms of R-sitagliptin and S-sitagliptin are shown in the figure, wherein S-sit is S-sitagliptin and R-sit is R-sitagliptin.
Fig. 4: representative chromatograms of R-sitagliptin phosphate monohydrate test samples.
Detailed Description
The present invention will be further described by the following examples, however, the scope of the present invention is not limited to the following examples. Those skilled in the art will appreciate that various changes and modifications can be made to the invention without departing from the spirit and scope thereof. The present invention generally and/or specifically describes the materials used in the test as well as the test methods. Although many materials and methods of operation are known in the art for accomplishing the objectives of the present invention, the present invention will be described in as much detail herein.
In the following experiments of the invention, the feeding amount of each material can be proportionally enlarged or reduced according to specific requirements.
Test example 1: HPLC method for determining content of diketone compound and ammonone compound
The test method can be used for determining the content of the raw material diketone compound and also can be used for determining the content of the ammonione compound or the phosphate or the hydrate thereof.
Chromatographic conditions and system suitability test: chromatographic column (INERTSIL CN-3,5 mu m, 4.6X1250 mm) with cyano silane bonded silica gel as filler, phosphate buffer (0.01 mol/L potassium dihydrogen phosphate solution, pH value adjusted to 2.0) with phosphoric acid) -acetonitrile (17:3) as mobile phase, column temperature of 30 ℃ and detection wavelength of 225nm;
preparation of a control solution: weighing a proper amount of sitagliptin phosphate reference substance or diketone compound reference substance, precisely weighing, dissolving and diluting with a mobile phase to prepare a solution containing 80 mug of sitagliptin or 80 mug of diketone compound in each 1m < 1 >, and shaking uniformly to obtain the sitagliptin phosphate reference substance or diketone compound;
preparation of test solution: taking a proper amount of a test sample (a diketone compound, an ammonifloric acid compound or a sitagliptin phosphate monohydrate), precisely weighing, dissolving and diluting with a mobile phase to prepare a solution containing 80 mug of sitagliptin or 80 mug of the diketone compound in each 1m < 1 >, and shaking uniformly to obtain the sitagliptin-phosphate;
Assay: and respectively injecting 20 mu l of the sample solution and 20 mu l of the reference solution into a liquid chromatograph, recording the chromatograms, and calculating the content of sitagliptin in the sample according to an external standard method and using peak areas (the conversion factor of sitagliptin and sitagliptin phosphate monohydrate is 0.7783). The measurement of the aminoketone compound is calculated and converted by using a sitagliptin reference solution.
Typical HPLC of the aminoketone compound is shown in FIG. 1 and typical HPLC of the precursor diketo compound is shown in FIG. 2, as determined using the method of this test example.
Test example 2: determination of optical purity of sitagliptin phosphate monohydrate by HPLC method
The assay method can be used to determine the optical purity of sitagliptin phosphate monohydrate.
Chromatographic column: chirapak AD-H column (5 mu m, 4.6X1250 mm), column temperature of 30 ℃,
mobile phase: a solution of 0.2% diethylamine in heptane-acetonitrile (3:7) at a flow rate of 0.8ml/min,
detection wavelength: 268nm and 20 μl of sample injection amount;
weighing 5mg of S-sitagliptin reference substance and 5mg of R-sitagliptin reference substance (purchased from Sigma-Aldrich), respectively adding 10ml of mobile phase to dissolve to prepare stock solutions, taking 2ml of each stock solution, adding 2ml of mobile phase to dilute, respectively injecting into a liquid chromatograph, recording a chromatogram, wherein the S-sitagliptin retention time is about 6.9min and the R-sitagliptin retention time is about 8.2min; mixing the two stock solutions at a ratio of 1:1 to obtain a mixed solution, injecting into a liquid chromatograph, recording a chromatogram, and typically, the chromatogram of the mixture is shown in fig. 3; taking 5mg of sitagliptin phosphate monohydrate test sample, adding 20ml of mobile phase to obtain solution, injecting into a liquid chromatograph, recording chromatogram, calculating optical purity by using area normalization method, wherein typical test sample chromatogram is shown in figure 4; from the results of fig. 3 and 4, it can be seen that the two isomers are well separated (degree of separation is greater than 3).
Example 1: preparation of immobilized transaminase
(1) Adding 0.1mol of monopotassium phosphate, 0.12mol of dipotassium phosphate, 0.2mol of calcium chloride, 15g of pyridoxal phosphate and 2g of tetrabutylammonium hydroxide into R-aminotransferase solution (1L, 10 percent, corresponding to 100g containing solid enzyme powder) at a temperature of 5-10 ℃ and stirring for 2 hours to dissolve;
(2) Then adding the mixed solution into 500g of sepiolite particles (particles with the diameter of 1-3mm obtained by calcining sepiolite at 650-750 ℃), sealing a container for containing the solid-liquid mixed material, mixing the materials for 1 hour by turning the container, and standing for 24 hours;
(3) Turning over the container to mix the materials for 30min, adding 100ml of 5% polyvinyl alcohol (PVA 17-88) solution into the solid-liquid mixture, and continuing turning over the container to mix the materials for 1 h;
(4) The immobilized transaminase 652g (1 g solid enzyme powder is equivalent to each 6.52g immobilized transaminase) is obtained by centrifuging and draining until the water content is less than 5%, and the immobilized transaminase is refrigerated at 4 ℃ for standby.
Example 2: conversion of diketo compounds to aminoketone compounds and preparation of cigliptin phosphate using immobilized transaminase
Tine monohydrate
The overall reaction process is as follows:
the operation steps are as follows:
(1) 40.63g of a diketone compound (0.1 mol, C16H12F6N4O2, mr 406.3) was added to the reaction flask, methyl t-butyl ether was added and 0.5L was stirred to dissolve, followed by adding 2.47g (10 mmol) of pyridoxal phosphate, 47.8g (0.5 mol) of isopropylamine hydrochloride, 50g of the immobilized transaminase obtained in example 1, and the mixture was stirred at 65℃to reflux the reaction for 15 hours, and filtered while it was hot;
(2) Centrifuging the filter residue to dryness, and taking the filter residue as recovered immobilized transaminase to be used in the next batch; the filtrate was cooled to room temperature and washed 3 times with water (1:1) to give an organic phase containing the aminoketone compound;
[ 2ml of this organic phase was taken and determined by HPLC method of test example 1, the main peak of the retention time of the liquid chromatograph was coincident with that of the sitagliptin free base, the typical HPLC diagram of this ammonification compound is shown in FIG. 1, and the typical HPLC diagram of the precursor diketone compound is shown in FIG. 2; calculating the molar conversion rate from the diketone compound to the aminoketone compound to be 98.6% based on the obtained organic phase and the content of the aminoketone compound detected in the organic phase; the organic phase was separated 20mL, washed 3 times with 60mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent, affording a colorless oil whose nuclear magnetic hydrogen profile, as determined, matches that of sitagliptin free base: 1 H NMR(400MHz，CDCl 3 ) δ 1.86(b，2H)，2.61-2.73(m，2H)，2.78-2.95(m，2H)，3.98-4.39(m，5H)，4.95(s，1H)，4.96-5.09(m，1H)，6.89-6.91(m，1H)，6.98-7.03(m，1H)]
(3) 15g of phosphoric acid (85%, 0.13 mol) was added dropwise to the organic phase under stirring, stirring was continued for 4 hours at a temperature of 5 to 10 ℃, filtration was carried out, and the precipitate was washed 2 times with ethanol and isopropanol, respectively, and dried to obtain a white crystalline powder as a sitagliptin phosphate monohydrate (49.77 g,0.0951mol, total molar yield 95.1%. Elemental analysis measurement result: C36.72%, H3.85%, F21.78%, N13.38%, O18.34%, P5.92%, melting point: 215 to 217%, optical rotation [ alpha ] D-74.4 ° (c=1.0, in water), in accordance with the results reported in the literature, the normalized purity of the sitagliptin phosphate monohydrate was 99.83% as measured by the HPLC method of test example 1, and the optical purity of the sitagliptin phosphate monohydrate was 99.67% as measured by the HPLC method of test example 2.
Example 2a: conversion of diketo compounds to aminoketone compounds and preparation of cigliptin phosphate using immobilized transaminase
Tine monohydrate
Reference example 2 was carried out as follows:
(1) 40.63g of a diketone compound (0.1 mol) was added to a reaction flask, methyl t-butyl ether (0.5L) was added and stirred to dissolve, followed by 1.98g (8 mmol) of pyridoxal phosphate, 57.4g (0.6 mol) of isopropylamine hydrochloride, 40g of the immobilized transaminase obtained in example 1, and stirred at 55℃to reflux the material for 18 hours, and filtered while it is still hot;
(2) Centrifuging the filter residue to dryness, and taking the filter residue as recovered immobilized transaminase to be used in the next batch; the filtrate was cooled to room temperature and washed 3 times with water (1:1) to give an organic phase containing the aminoketone compound (yield of the aminoketone compound was measured to give a molar conversion of diketone to aminoketone of 98.2%);
(3) Dropwise adding 0.12mol of phosphoric acid (85%) into an organic phase under stirring, continuously stirring for 4 hours at a temperature of 5-10 ℃, filtering, sequentially washing ethanol and isopropanol for 2 times respectively, and drying to obtain white crystalline powder, namely sitagliptin phosphate monohydrate (94.6 mmol, total molar yield of 94.6%. Melting point of a product: 215-216 degrees, optical rotation [ alpha ] D-74.5 degrees (c=1.0 in water), normalized purity of 99.89%, and optical purity of 99.55%.
Example 2b: conversion of diketo compounds to aminoketone compounds and preparation of cigliptin phosphate using immobilized transaminase
Tine monohydrate
Reference example 2 was carried out as follows:
(1) 40.63g of a diketone compound (0.1 mol) was added to a reaction flask, methyl t-butyl ether (0.5L) was added and stirred to dissolve, followed by addition of 2.96g (12 mmol) of pyridoxal phosphate, 38.3g (0.4 mol) of isopropylamine hydrochloride, 60g of the immobilized transaminase obtained in example 1, stirring at 75℃and allowing the material to reflux for 12 hours, and filtering while it is still hot;
(2) Centrifuging the filter residue to dryness, and taking the filter residue as recovered immobilized transaminase to be used in the next batch; the filtrate was cooled to room temperature and washed 3 times with water (1:1) to give an organic phase containing the aminoketone compound (yield of the aminoketone compound was measured to give a molar conversion of diketone to aminoketone of 98.8%);
(3) Dropwise adding 0.15mol phosphoric acid (85%) into an organic phase under stirring, continuously stirring for 4 hours at a temperature of 5-10 ℃, filtering, sequentially washing ethanol and isopropanol for 2 times respectively, and drying to obtain white crystalline powder, namely sitagliptin phosphate monohydrate (95.6 mmol, total molar yield of 95.6%. Melting point of the product: 216-217 DEG optical rotation [ alpha ] D-74.1 DEG (c=1.0 in water), wherein the normalized purity is 99.78%, and the optical purity is 99.72%.
From the results of example 2 and examples 2a and 2b according to the present invention, the immobilized transaminase of the present invention has high catalytic efficiency, and the obtained product has excellent purity and relatively high yield; in addition, the amounts of enzyme used in the three examples were slightly different but there was no significant difference in the transamination efficiency, indicating that the amount of immobilized transaminase used was sufficient.
Example 3: examine the reusability of the immobilized transaminase
(1) Use of immobilized transaminase 1: adding 0.01mol of diketone compound into a reaction bottle, adding 50ml of methyl tertiary butyl ether, stirring to dissolve, then adding 1mmol of pyridoxal phosphate, 0.05mol of isopropylamine hydrochloride and 5g of immobilized transaminase obtained in example 1, stirring at 65 ℃ to reflux and react the materials for 15 hours, and filtering while the materials are hot; centrifuging the filter residue (immobilized transaminase) to dryness; the filtrate was cooled to room temperature and washed 3 times with water (1:1) to give an organic phase containing the aminoketone compound (molar conversion from diketone to aminoketone was measured and calculated);
(2) Use of immobilized transaminase 2: adding 0.01mol of diketone compound into a reaction bottle, adding 50ml of methyl tertiary butyl ether, stirring to dissolve, then adding 1mmol of pyridoxal phosphate, 0.05mol of isopropylamine hydrochloride and 5g of filter residue (immobilized transaminase) in the previous step, stirring at 65 ℃ to reflux and react the materials for 15 hours, and filtering while the materials are hot; centrifuging the filter residue until the filter residue is dry; the filtrate was cooled to room temperature and washed 3 times with water (1:1) to give an organic phase containing the aminoketone compound (molar conversion from diketone to aminoketone was measured and calculated); [ in this step, an immobilized transaminase in the form of a residue sufficient for this step can be obtained by increasing the total amount of the above-mentioned steps, and the amount of the immobilized transaminase in the form of a residue in the subsequent step is also so operated as to ensure that the test can be performed ]
(3) 3 rd use of immobilized transaminase: adding 0.01mol of diketone compound into a reaction bottle, adding 50ml of methyl tertiary butyl ether, stirring to dissolve, then adding 1mmol of pyridoxal phosphate, 0.05mol of isopropylamine hydrochloride and 5g of filter residue (immobilized transaminase) in the previous step, stirring at 65 ℃ to reflux and react the materials for 15 hours, and filtering while the materials are hot; centrifuging the filter residue until the filter residue is dry; the filtrate was cooled to room temperature and washed 3 times with water (1:1) to give an organic phase containing the aminoketone compound (molar conversion from diketone to aminoketone was measured and calculated);
(4) Immobilized transaminase was used 4 times: adding 0.01mol of diketone compound into a reaction bottle, adding 50ml of methyl tertiary butyl ether, stirring to dissolve, then adding 1mmol of pyridoxal phosphate, 0.05mol of isopropylamine hydrochloride and 5g of filter residue (immobilized transaminase) in the previous step, stirring at 65 ℃ to reflux and react the materials for 15 hours, and filtering while the materials are hot; centrifuging the filter residue until the filter residue is dry; the filtrate was cooled to room temperature and washed 3 times with water (1:1) to give an organic phase containing the aminoketone compound (molar conversion from diketone to aminoketone was measured and calculated);
and so on to continue the execution of the reaction,
(n) nth use of immobilized transaminase: adding 0.01mol of diketone compound into a reaction bottle, adding 50ml of methyl tertiary butyl ether, stirring to dissolve, then adding 1mmol of pyridoxal phosphate, 0.05mol of isopropylamine hydrochloride and 5g of filter residue (immobilized transaminase) in the previous step, stirring at 65 ℃ to reflux and react the materials for 15 hours, and filtering while the materials are hot; centrifuging the filter residue until the filter residue is dry; the filtrate was cooled to room temperature and washed 3 times with water (1:1) to give an organic phase containing the aminoketone compound (molar conversion from diketo aminoketone was measured and calculated).
The reactions of the steps are sequentially carried out for 20 times, and the conversion rates are respectively as follows: (1) 98.8%, (2) 98.4%, (3) 98.6%, (4) 97.4%, (5) 97.1%, (6) 97.3%, (7) 96.8%, (8) 96.4%, (9) 96.2%, (10) 95.5%, (11) 95.8%, (12) 95.1%, (13) 94.6%, (14) 95.1%, (15) 94.4%, (16) 93.8%, (17) 93.4%, (18) 92.7%, (19) 93.2%, (20) 92.4%. The results show that the molar conversion rate of diketone to aminoketone can still reach more than 92% after the immobilized transaminase obtained in the example 1 is reused for 20 times, which indicates that the immobilized transaminase can be used in large batches.
Since the operation scale of step (3) of example 2 is easily significantly enlarged on an actual production line as compared with the first two steps, it is quite advantageous to perform the salt conversion of step (3) at one time if the organic phase containing the aminoketone compound obtained in a plurality of batches can be mixed and then the organic phase mixture of the plurality of batches is subjected to the following test for this purpose to enlarge the production scale:
mixing 20 batches of the organic phase containing the aminoketone compound obtained by using the immobilized aminotransferase for the 1 st to 20 th times in the embodiment, measuring the content of the diketone compound, dropwise adding 1.3 equivalent phosphoric acid (85%) into the mixed organic phase under stirring, continuously stirring for 4 hours at the temperature of 5-10 ℃, filtering, sequentially washing the precipitate with ethanol and isopropanol for 2 times respectively, and drying to obtain the white crystalline powder which is the sitagliptin phosphate monohydrate. The molar yield of the salt forming step is 96.4% by measurement, and the melting point of the obtained sitagliptin phosphate monohydrate is: 215-216 deg., optical rotation [ alpha ] D-74.6 deg. (c=1.0 in water), normalized purity 99.79%, optical purity 99.82%. This result shows that the method of the invention is particularly suitable for industrial mass production.
Example 4: conversion of diketo compounds to aminoketone compounds using immobilized transaminases
Immobilized transaminase was prepared by the method of reference example 1, and the dosage of each material was changed to: 100ml of R-aminotransferase solution, 0.012mol of monopotassium phosphate, 0.010mol of dipotassium phosphate, 0.024mol of calcium chloride, 1.2g of pyridoxal phosphate, 0.24g of tetrabutylammonium hydroxide, 40g of sepiolite and 8ml of polyvinyl alcohol solution were prepared, and the remaining operation conditions were the same as in example 1 to obtain immobilized aminotransferase designated as immobilized enzyme 4a.
Immobilized transaminase was prepared by the method of reference example 1, and the dosage of each material was changed to: 100ml of R-aminotransferase solution, 0.008mol of monopotassium phosphate, 0.014mol of dipotassium phosphate, 0.016mol of calcium chloride, 1.8g of pyridoxal phosphate, 0.16g of tetrabutylammonium hydroxide, 60g of sepiolite and 12ml of polyvinyl alcohol solution were prepared, and the remaining conditions were the same as in example 1 to obtain immobilized aminotransferase designated as immobilized enzyme 4b.
Diketon 0.01mol was added to the flask, methyl tert-butyl ether 50ml was added and stirred to dissolve, followed by pyridoxal phosphate 1mmol, isopropylamine hydrochloride 0.05mol, 5g immobilized enzyme 4a, stirred at 65℃to reflux the material for 15 hours, filtered while hot, the filtrate cooled to room temperature and washed 3 times with water (1:1) to give an organic phase containing the aminoketone compound (the molar conversion from diketo aminoketone was determined and calculated to be 98.5%).
Diketon 0.01mol was added to the flask, methyl tert-butyl ether 50ml was added and stirred to dissolve, followed by pyridoxal phosphate 1mmol, isopropylamine hydrochloride 0.05mol, 5g immobilized enzyme 4b and stirred at 65℃to reflux the material for 15 hours, filtered while hot, the filtrate cooled to room temperature and washed 3 times with water (1:1) to give an organic phase containing the aminoketone compound (the molar conversion from diketo aminoketone was determined and calculated to be 98.1%). The results of this example show that the immobilized enzyme prepared by properly adjusting the amounts of the respective components also has substantially the same amino group conversion effect as in the example.
Example 5: conversion of diketo compounds to aminoketone compounds using immobilized transaminases
The immobilized transaminase was prepared by the formulation and preparation of reference example 1 except that tetrabutylammonium hydroxide was not added, resulting in immobilized transaminase denoted immobilized enzyme 5a; the immobilized transaminase is prepared by the ingredients and the preparation method of reference example 1, except that polyvinyl alcohol is replaced by sodium alginate with equal concentration and other addition amounts, and the immobilized transaminase is recorded as immobilized enzyme 5b; the ingredients and the preparation method of reference example 1 are used for preparing immobilized transaminase, except that sepiolite is replaced by diatomite with equal addition amount, and immobilized transaminase is recorded as immobilized enzyme 5c; the immobilized transaminase obtained by the method described in the specifications [0045] to [0048] of CN114657170B using LXTE-700S resin was referred to as immobilized enzyme 5d in the case of the R-transaminase solution used in example 1 of the present invention.
Adding 0.01mol of a diketone compound into a reaction bottle, adding 50ml of methyl tertiary butyl ether, stirring to dissolve, then adding 1mmol of pyridoxal phosphate, 0.05mol of isopropylamine hydrochloride and 5g of immobilized enzyme 5a, stirring at 65 ℃ to make the materials reflux-react for 15 hours, filtering while the materials are hot, cooling the filtrate to room temperature, and washing 3 times with water (1:1) to obtain an organic phase containing an aminoketone compound (the molar conversion rate from diketone to aminoketone is measured and calculated to be 68.5%); adding 0.01mol of a diketone compound into a reaction bottle, adding 50ml of methyl tertiary butyl ether, stirring to dissolve, then adding 1mmol of pyridoxal phosphate, 0.05mol of isopropylamine hydrochloride and 5g of immobilized enzyme 5a, stirring at 35 ℃ to make the materials reflux-react for 15 hours, filtering while the materials are hot, cooling the filtrate to room temperature, and washing 3 times with water (1:1) to obtain an organic phase containing an aminoketone compound (the molar conversion rate from diketone to aminoketone is measured and calculated to be 63.8%); diketon 0.01mol was added to the flask, methyl tert-butyl ether 50ml was added and stirred to dissolve, followed by pyridoxal phosphate 1mmol, isopropylamine hydrochloride 0.05mol, 5g immobilized enzyme 5a, stirred at 35℃to reflux the material for 30 hours, filtered while hot, the filtrate cooled to room temperature and washed 3 times with water (1:1) to give an organic phase containing the aminoketone compound (the molar conversion of diketo aminoketone was determined and calculated to be 81.3%).
Adding 0.01mol of a diketone compound into a reaction bottle, adding 50ml of methyl tertiary butyl ether, stirring to dissolve, then adding 1mmol of pyridoxal phosphate, 0.05mol of isopropylamine hydrochloride and 5g of immobilized enzyme 5b, stirring at 65 ℃ to make the materials reflux-react for 15 hours, filtering while the materials are hot, cooling the filtrate to room temperature, and washing 3 times with water (1:1) to obtain an organic phase containing an aminoketone compound (the molar conversion rate from diketone to aminoketone is measured and calculated to be 71.2%); adding 0.01mol of a diketone compound into a reaction bottle, adding 50ml of methyl tertiary butyl ether, stirring to dissolve, then adding 1mmol of pyridoxal phosphate, 0.05mol of isopropylamine hydrochloride and 5g of immobilized enzyme 5b, stirring at 35 ℃ to make the materials reflux-react for 15 hours, filtering while the materials are hot, cooling the filtrate to room temperature, and washing 3 times with water (1:1) to obtain an organic phase containing an aminoketone compound (the molar conversion rate from diketone to aminoketone is measured and calculated to be 74.7%); diketon 0.01mol was added to the flask, methyl tert-butyl ether 50ml was added and stirred to dissolve, followed by pyridoxal phosphate 1mmol, isopropylamine hydrochloride 0.05mol, 5g of immobilized enzyme 5b and stirred at 35℃to reflux the material for 30 hours, filtered while hot, the filtrate cooled to room temperature and washed 3 times with water (1:1) to give an organic phase containing the aminoketone compound (the molar conversion of diketo aminoketone was determined and calculated to be 85.6%). These results suggest that immobilized enzyme 5a and immobilized enzyme 5b are not suitable for use at high temperature, and that the conversion is improved but not as effective as the immobilized enzyme of example 1 when the temperature is lowered and the reaction time is prolonged.
Adding 0.01mol of a diketone compound into a reaction bottle, adding 50ml of methyl tertiary butyl ether, stirring to dissolve, then adding 1mmol of pyridoxal phosphate, 0.05mol of isopropylamine hydrochloride and 5g of immobilized enzyme 5C, stirring at 65 ℃ to make the materials reflux-react for 15 hours, filtering while the materials are hot, cooling the filtrate to room temperature, and washing 3 times with water (1:1) to obtain an organic phase containing an aminoketone compound (the molar conversion rate from diketone to aminoketone is measured and calculated to be 94.7%); in view of the availability of the ketone/ammonia conversion efficiency of immobilized enzyme 5c prepared by using diatomaceous earth, the ginseng of the present invention has the repeated use performance of immobilized enzyme 5c measured according to the method of example 3 herein, and as a result, the conversion rate is reduced to below 70% after 4 continuous repeated use: (1) 95.2%, (2) 91.3%, (3) 86.7%, (4) 64.1%. The above results indicate that the reuse of immobilized enzyme 5c is very poor.
Adding 0.01mol of a diketone compound to a reaction flask, adding 50ml of methyl tert-butyl ether, stirring to dissolve, then adding 1mmol of pyridoxal phosphate, 0.05mol of isopropylamine hydrochloride, and 5d of immobilized enzyme (corresponding to the amount of immobilized enzyme 5d obtained from 7.7ml of R-aminotransferase solution, which is comparable to the operating conditions of example 2), stirring at 65 ℃ to reflux the material for 15 hours, filtering while hot, cooling the filtrate to room temperature, and washing 3 times with water (1:1) to obtain an organic phase containing an aminoketone compound (the molar conversion of diketone to aminoketone was measured and calculated to be 97.3%); the results show that the immobilized enzyme prepared by the literature method and the resin has better ketone/ammonia conversion efficiency, but the effect of the immobilized enzyme is not as good as that of the embodiment 1-2, and the cost of the resin is far higher than that of the sepiolite, so that the overall effect is obviously poorer than that of the embodiment 1-2, and the scheme of the embodiment 1-2 is particularly remarkably advantageous when the immobilized enzyme is used in industrial mass production.
The present invention is described in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e., it does not mean that the present invention must be practiced depending on the above detailed methods. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.
Claims (11)
1. A process for preparing sitagliptin phosphate monohydrate comprising the steps of:
(1) Adding 0.1mol of diketone compound into a reaction bottle, adding 0.5L of methyl tertiary butyl ether, stirring to dissolve, then adding 8-12 mmol of pyridoxal phosphate, 0.4-0.6 mol of isopropylamine hydrochloride, and carrying out hot filtration, wherein the immobilized transaminase is 40-60 g which is recovered and used for the first time or after the method is used for multiple times, stirring at 55-75 ℃ to carry out reflux reaction on materials for 12-18 hours; the diketone compound is a compound of the formula:
；
(2) Centrifuging the filter residue to dryness, and taking the filter residue as recovered immobilized transaminase to be used in the next batch; the filtrate was cooled to room temperature and washed with water to give an organic phase containing the aminoketone compound; the aminoketone compound is a compound of the formula:
；
(3) Dropwise adding 0.12-0.15 mol of phosphoric acid into the organic phase under stirring, continuously stirring for 4 hours at the temperature of 5-10 ℃, filtering, washing the precipitate with ethanol and isopropanol in sequence, and drying to obtain sitagliptin phosphate monohydrate;
wherein,
the immobilized transaminase recovered and reused after multiple uses refers to the immobilized transaminase recovered and reused after 1-20 uses;
the immobilized transaminase comprises the following components in proportion: comprises 100g of solid enzyme powder, 0.08-0.12 mol of monopotassium phosphate, 0.1-0.14 mol of dipotassium phosphate, 0.16-0.24 mol of calcium chloride, 12-18 g of pyridoxal phosphate, 1.6-2.4 g of tetrabutylammonium hydroxide, 400-600 g of sepiolite particles and 4-6 g of polyvinyl alcohol;
the immobilized transaminase is prepared by the following steps:
(i) Adding monopotassium phosphate, dipotassium phosphate, calcium chloride, pyridoxal phosphate and tetrabutylammonium hydroxide into the R-aminotransferase liquid at the temperature of 5-10 ℃ and stirring for 2 hours to dissolve;
(ii) Then adding the mixed solution into sepiolite particles, sealing a container for containing the solid-liquid mixed material, mixing the materials for 1 hour by turning the container over, and standing for 24 hours;
(iii) Turning over the container to mix the materials for 30min, adding 5% polyvinyl alcohol solution into the solid-liquid mixture, and continuing turning over the container to mix the materials for 1 h;
(iv) And (5) draining the water by centrifugation until the water content is less than 5%, thus obtaining the immobilized transaminase.
2. The method of claim 1, wherein the sepiolite particles in the immobilized transaminase are particles of 1-3mm in diameter that are obtained by calcination of sepiolite at 650-750 ℃.
3. The method of claim 1, wherein the polyvinyl alcohol in the immobilized transaminase is polyvinyl alcohol model number PVA 17-88.
4. The method of claim 1, wherein the immobilized transaminase comprises the following proportions of components: comprises 100g of solid enzyme powder, 0.1mol of monopotassium phosphate, 0.12mol of dipotassium phosphate, 0.2mol of calcium chloride, 15g of pyridoxal phosphate, 2g of tetrabutylammonium hydroxide, 500g of sepiolite particles and 5g of polyvinyl alcohol.
5. The method of claim 1, wherein the immobilized transaminase comprises the following proportions of components: comprises 10g of solid enzyme powder, 0.012mol of monopotassium phosphate, 0.010mol of dipotassium phosphate, 0.024mol of calcium chloride, 1.2g of pyridoxal phosphate, 0.24g of tetrabutylammonium hydroxide, 40g of sepiolite and 4g of polyvinyl alcohol.
6. The method of claim 1, wherein the immobilized transaminase comprises the following proportions of components: comprises 10g of solid enzyme powder, 0.008mol of monopotassium phosphate, 0.014mol of dipotassium phosphate, 0.016mol of calcium chloride, 1.8g of pyridoxal phosphate, 0.16g of tetrabutylammonium hydroxide, 60g of sepiolite and 6g of polyvinyl alcohol.
7. An immobilized transaminase comprising the following proportions of components: comprises 100g of solid enzyme powder, 0.08-0.12 mol of monopotassium phosphate, 0.1-0.14 mol of dipotassium phosphate, 0.16-0.24 mol of calcium chloride, 12-18 g of pyridoxal phosphate, 1.6-2.4 g of tetrabutylammonium hydroxide, 400-600 g of sepiolite particles and 4-6 g of polyvinyl alcohol; the sepiolite particles are particles with the diameter of 1-3mm obtained by calcining sepiolite at 650-750 ℃, and the polyvinyl alcohol is the polyvinyl alcohol with the model number of PVA 17-88; the immobilized transaminase is prepared by the following steps:
(i) Adding monopotassium phosphate, dipotassium phosphate, calcium chloride, pyridoxal phosphate and tetrabutylammonium hydroxide into the R-aminotransferase liquid at the temperature of 5-10 ℃ and stirring for 2 hours to dissolve;
(ii) Then adding the mixed solution into sepiolite particles, sealing a container for containing the solid-liquid mixed material, mixing the materials for 1 hour by turning the container over, and standing for 24 hours;
(iii) Turning over the container to mix the materials for 30min, adding 5% polyvinyl alcohol solution into the solid-liquid mixture, and continuing turning over the container to mix the materials for 1 h;
(iv) And (5) draining the water by centrifugation until the water content is less than 5%, thus obtaining the immobilized transaminase.
8. The immobilized transaminase of claim 7 comprising the following proportions of components: comprises 100g of solid enzyme powder, 0.1mol of monopotassium phosphate, 0.12mol of dipotassium phosphate, 0.2mol of calcium chloride, 15g of pyridoxal phosphate, 2g of tetrabutylammonium hydroxide, 500g of sepiolite particles and 5g of polyvinyl alcohol.
9. The immobilized transaminase of claim 7 comprising the following proportions of components: comprises 10g of solid enzyme powder, 0.012mol of monopotassium phosphate, 0.010mol of dipotassium phosphate, 0.024mol of calcium chloride, 1.2g of pyridoxal phosphate, 0.24g of tetrabutylammonium hydroxide, 40g of sepiolite and 4g of polyvinyl alcohol.
10. The immobilized transaminase of claim 7 comprising the following proportions of components: comprises 10g of solid enzyme powder, 0.008mol of monopotassium phosphate, 0.014mol of dipotassium phosphate, 0.016mol of calcium chloride, 1.8g of pyridoxal phosphate, 0.16g of tetrabutylammonium hydroxide, 60g of sepiolite and 6g of polyvinyl alcohol.
11. A process for preparing the immobilized transaminase of any one of claims 7 to 10 comprising the following steps:
(i) Adding monopotassium phosphate, dipotassium phosphate, calcium chloride, pyridoxal phosphate and tetrabutylammonium hydroxide into the R-aminotransferase liquid at the temperature of 5-10 ℃ and stirring for 2 hours to dissolve;
(ii) Then adding the mixed solution into sepiolite particles, sealing a container for containing the solid-liquid mixed material, mixing the materials for 1 hour by turning the container over, and standing for 24 hours;
(iii) Turning over the container to mix the materials for 30min, adding 5% polyvinyl alcohol solution into the solid-liquid mixture, and continuing turning over the container to mix the materials for 1 h;
(iv) And (5) draining the water by centrifugation until the water content is less than 5%, thus obtaining the immobilized transaminase.
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