WO2022166848A1 - 一种利用酶级联反应合成(1r,2r)-ampp的方法 - Google Patents
一种利用酶级联反应合成(1r,2r)-ampp的方法 Download PDFInfo
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- WO2022166848A1 WO2022166848A1 PCT/CN2022/074774 CN2022074774W WO2022166848A1 WO 2022166848 A1 WO2022166848 A1 WO 2022166848A1 CN 2022074774 W CN2022074774 W CN 2022074774W WO 2022166848 A1 WO2022166848 A1 WO 2022166848A1
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- Prior art keywords
- transaminase
- ampp
- transketolase
- ata117
- formula
- Prior art date
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Definitions
- the present invention is required to be submitted to the China Patent Office on February 5, 2021, the application number is 202110162637.0, and the title of the invention is "A method for synthesizing (1R, 2R)-AMPP using an enzymatic cascade reaction” and submitted on May 24, 2021
- the priority of the Chinese Patent Office, application number 202110567238.2, and the invention title is "A method for synthesizing (1R,2R)-AMPP using an enzymatic cascade reaction", the entire contents of which are incorporated herein by reference.
- the invention relates to the technical field of biocatalysis, in particular to a method for synthesizing (1R, 2R)-AMPP by utilizing an enzyme cascade reaction.
- Florfenicol is a new type of veterinary chloramphenicol antibacterial drug successfully developed by Schering-Plo ⁇ gh Animal Health in the United States in 1988. Its chemical name is 2,2-dichloro-N-[(1R,2R) -1-Fluoromethyl-2-hydroxy-2-[4-(methylsulfonyl)phenyl]ethyl]acetamide, first marketed in Japan, named Nuflor, has broad-spectrum antibacterial activity and can be used to treat cattle , bacterial respiratory diseases in pigs and chickens. The antibacterial mechanism of florfenicol is similar to that of chloramphenicol and thiamphenicol.
- acetyltransferases produced in thiamphenicol and chloramphenicol-resistant bacteria can acylate -OH at the ⁇ -methyl position, thereby losing their pharmacological activity.
- florfenicol replaces -OH at the ⁇ -methyl position with -F to avoid acetylation reaction, so it still has a strong inhibitory effect on chloramphenicol and thiamphenicol-resistant bacteria.
- florfenicol is structurally more CH 3 -SO 2 group replaces NO 2 group, avoids aplastic anemia, and has less residue in animals, gradually replaces chloramphenicol and thiamphenicol, and is widely used in the prevention and treatment of animal infectious diseases .
- the research group improved the activity of this threonine aldolase through directed evolution, and introduced an acetaldehyde elimination system to increase the production of L-threo-p-methylsulfosulfonylphenylserine.
- Escherichia coli transketolase is a ubiquitous thiamine pyrophosphate-dependent enzyme. It links the non-oxidative pentose phosphate pathway and the tricarboxylic acid cycle. Escherichia coli transketolase catalyzes a reversible transketone reaction that transfers the two-carbon unit of a ketol donor to an aldehyde acceptor to form chiral dihydroxyketones. In the transketone reaction, ⁇ -hydroxypyruvate is often used as the ketol donor because ⁇ -hydroxypyruvate is transketoneized to generate volatile carbon dioxide, making the reaction irreversible. In addition to catalyzing aliphatic aldehydes, E.
- coli transketolase was engineered to catalyze aromatic aldehydes.
- Helen C. Hailes' research group reported the site-directed mutation of D469 and F434 on E. coli transketolase derived from E. coli, and realized the catalysis of p-benzaldehyde and m-hydroxybenzaldehyde, but the catalytic efficiency was low, and the conversion rate was ⁇ 10%.
- the hydroxyketone products catalyzed by its mutants are mainly R configuration (ee ⁇ 82%).
- Transaminases are divided into two categories, alpha-transaminases and omega-transaminases.
- Alpha-aminotransferases include subgroups I, III, and IV, and catalyze transamination reactions dependent on the substrate ketone alpha-carboxylic acid.
- the ⁇ -aminotransferases belong to the type II subgroup, and are widely used in the asymmetric transamination reactions of a variety of aliphatic and aromatic ketones to generate stereospecific amines.
- ATA117 is one of the widely used ⁇ -aminotransferases, which can produce stereospecific (R)amines.
- ⁇ -aminotransferases In addition to producing stereospecific amines, ⁇ -aminotransferases also have certain enantioselectivity for ortho chiral centers.
- Wolfgang Kroutil's group reported that R-4-phenyl-2-pyrrolidone was obtained by kinetic resolution of ⁇ -transaminase ATA117 on a racemic aldehyde substrate.
- ATA117 has enantioselectivity to the chiral center at the ortho position of the aldehyde, and the ee of its transamination product can reach 68% by optimizing the cosolvent and pH.
- the ⁇ -transaminase derived from Cytomonas can obtain a highly stereoselective R-Brivaracetam chiral intermediate through one-step kinetic resolution, and the ee can reach 92%.
- Wolfgang Kroutil's research group made several amino acid site mutations based on the ⁇ -transaminase and ArRmut11 mutants derived from Fusarium graminearum respectively.
- its ee can reach 76% and 80% respectively.
- Iván Lavandera's group reported the kinetic resolution of a series of commercial aminotransferases and Bacillus megaterium-derived aminotransferases and their mutants on racemic ⁇ -alkyl- ⁇ -ketoamide substrates.
- This type of substrate has the highest catalytic efficiency and stereoselectivity, and its diastereoselectivity can reach 96%.
- the transaminase derived from Bacillus megaterium and its mutants can only transform part of the substrate, and its diastereoselectivity is low.
- most of the literature reports can obtain high stereoselectivity for the ortho-chiral center of aldehyde substrates by screening ⁇ -aminotransferases from different species, while screening commercial aminotransferases can obtain high stereoselectivity for ketone substrates such as ⁇ -alkane. High stereoselectivity for the ortho-chiral center of yl- ⁇ -ketoamides.
- the enantioselectivity of ⁇ -transaminase to chiral aromatic hydroxyketones has not been reported in the literature.
- the technical problem to be solved by the present invention is how to utilize enzyme-catalyzed synthesis of (1R,2R)-AMPP and its derivatives with high stereoselectivity.
- a method for synthesizing (1R, 2R)-phenylserinol derivatives by enzymatic cascade reaction comprising the following steps:
- Step 1 using the benzaldehyde derivative as a substrate and the Escherichia coli transketolase mutant as a catalyst to obtain a compound shown in formula 2, the Escherichia coli transketolase having an amino acid sequence shown in SEQ ID NO: 1, has the nucleotide sequence shown in SEQ ID NO:2;
- Step 2 using the compound shown in formula 2 as a substrate and a mutant of transaminase ATA117 as a catalyst to obtain (1R, 2R)-AMPP and its derivatives, the transaminase ATA117 having an amino acid sequence as shown in SEQ ID NO: 3, has the nucleotide sequence shown in SEQ ID NO:4;
- step 2 The method according to [1], wherein in step 1, using a benzaldehyde derivative as a substrate, adding lithium hydroxypyruvate, thiamine pyrophosphate, MgCl 2 and Escherichia coli transketolase mutant to react
- the compound represented by the formula 2 is obtained.
- step 2 the compound represented by formula 2 is used as a substrate, and D-alanine (or D-glycine, D-valine) is continuously added Acid, D-Leucine, D-Isoleucine, D-Methionine, D-Proline, D-Tryptophan, D-Serine, D-Tyrosine, D-Cysteine , D-phenylalanine, D-asparagine, D-glutamine, D-threonine, D-aspartic acid, D-glutamic acid, D-lysine, D-arginine , D-histidine or isopropylamine), pyridoxal phosphate, aminotransferase ATA117 mutants continue to react to obtain (1R,2R)-AMPP and its derivatives shown in formula 3.
- D-alanine or D-glycine, D-valine
- step 1 10 mM benzaldehyde derivative and 30 mM hydroxypyruvate are added to 50 ⁇ l of Tris-HCl buffer containing 100 mM Lithium, 4.8 mM thiamine pyrophosphate, 18 mM MgCl 2 , 60 ⁇ M or 100 ⁇ M Escherichia coli transketolase mutant were reacted at 25° C. for 1-3 h to obtain the compound shown in formula 2.
- step 2 The method according to any one of [1] to [4], wherein, in step 2, the reaction system is expanded to 100 ⁇ l, 100 mM Tris-HCl buffer, 200 mM D-alanine (or D -Glycine, D-Valine, D-Leucine, D-Isoleucine, D-Methionine, D-Proline, D-Tryptophan, D-Serine, D-Tyrosine , D-cysteine, D-phenylalanine, D-asparagine, D-glutamine, D-threonine, D-aspartic acid, D-glutamic acid, D-lysine acid, D-arginine, D-histidine or isopropylamine), 2mM pyridoxal phosphate, 50 ⁇ M transaminase ATA117 mutant, react at 25°C for 3-6h to obtain (1R,2R)-AMPP and its derivatives thing.
- D-alanine or D-
- the amino acid mutation site is H26Y site or any one of the following combined mutation sites: H26Y+F434G, H26Y+F434A, H26Y+F434L, H26Y+F434I, H26Y+F434V, H26Y+F434P, H26Y+F434M, H26Y+F434W, H26Y+F434S, H26Y+F434Q, H26Y+F434T, H26Y+F434C, H26Y+F434N, H26Y+F434Y, H26Y+F434D, H26Y+F434E, H26Y+F434K, H26Y+F434R, H26Y+ F434Y+L466F, H
- the transaminase ATA117 mutant has a sequence in which the amino acid mutation of the sequence shown in SEQ ID NO: 3 occurs, and the amino acid mutation occurs in the sequence shown in SEQ ID NO:3.
- the site of amino acid mutation is any of the following mutations: V69F, V69G, V69A, V69L, V69I, V69P, V69M, V69W, V69S, V69Q, V69T, V69C, V69N, V69Y, V69D, V69E, V69K, V69R, V69H, V69A+F122G, V69A+F122A, V69A+F122L, V69A+F122I, V69A+F122V, V69A+F122P, V69A+F122M, V69A+F122W, V69A+F122S, V69A+F122Q, V69A+F122T, V69A+ F122N, V69A+F122Y, V69A+F122D, V69A+F122E, V69A+F122K, V69A+F122R, V69A+F122H, V69A+F122C+I157F, V69A+F122C+
- step 1 using p-methylsulfonylbenzaldehyde as a substrate and transketolase EcTK1_YYH as a catalyst, the compound shown in formula 5 is obtained, and the transketolase EcTK1_YYH has the nucleotide shown in SEQ ID NO:5 sequence;
- step 2 the compound shown in formula 5 is used as a substrate, and the transaminase ATA117_ACHH is used as a catalyst to obtain (1R, 2R)-AMPP, and the transaminase ATA117_ACHH has a nucleotide sequence as shown in SEQ ID NO: 6;
- step 1 using p-methylsulfonylbenzaldehyde as a substrate, adding lithium hydroxypyruvate, thiamine pyrophosphate, MgCl 2 and transketolase EcTK1_YYH to react to obtain The compound represented by the formula 5.
- step 2 the compound shown in formula 5 is used as the substrate, and D-alanine, pyridoxal phosphate, transaminase ATA117_ACHH, NADH, lactate dehydrogenase, glucose and glucose dehydrogenase are continuously added to continue the reaction to obtain the obtained described (1R,2R)-AMPP.
- step 1 10 mM p-methylsulfonylbenzaldehyde, 30 mM lithium hydroxypyruvate, 4.8 mM thiamine are added to 50 ⁇ l of Tris-HCl buffer containing 100 mM Pyrophosphate, 18mM MgCl 2 , 60 ⁇ M transketolase EcTK1_YYH, react at 25° C. for 1 h to obtain the compound shown in formula 5.
- step 2 100 mM Tris-HCl buffer, 200 mM D-alanine, 2 mM pyridoxal phosphate, 50 ⁇ M transaminase ATA117_ACHH, 10 mM NADH, 90 U/ml lactate dehydrogenase, 200 mM NADH are added to the system in step 1 Glucose, 30U/ml glucose dehydrogenase, expand the reaction system to 100 ⁇ l, and react at 25°C for 3h to obtain (1R,2R)-AMPP.
- the present invention provides a method for synthesizing (1R, 2R)-benzeneserinol derivatives by using an enzyme cascade reaction, which comprises the following steps:
- Step 1 Using the benzaldehyde derivative (1 in the formula) and hydroxypyruvate as the substrate, and the Escherichia coli transketolase mutant as the catalyst, the (R)-hydroxyketone intermediate (2 in the formula) is obtained.
- step 2 the compound (2 in the formula) and the ammonia donor are used as substrates, and the transaminase ATA117 mutant is used as a catalyst to obtain a (1R,2R)-benzeneserinol derivative (3 in the formula).
- the aldehyde substrate is p-methylsulfone benzaldehyde, p-fluorobenzaldehyde, p-chlorobenzaldehyde, p-bromobenzaldehyde, p-methylbenzaldehyde, p-nitrobenzaldehyde, and benzaldehyde .
- p-methanesulfonyl benzaldehyde p-methylsulfonyl benzaldehyde, and p-methylsulfonyl benzaldehyde have the same meaning and refer to "p-methanesulfonyl benzaldehyde”.
- the amino donor is D-alanine, D-glycine, D-valine, D-leucine, D-isoleucine, D-methionine, D-Proline, D-Tryptophan, D-Serine, D-Tyrosine, D-Cysteine, D-Phenylalanine, D-Asparagine, D-Glutamine, D- Threonine, D-Aspartic Acid, D-Glutamic Acid, D-Lysine, D-Arginine, D-Histidine and Isopropylamine.
- the Escherichia coli transketolase mutant has a sequence in which amino acid mutation occurs in the sequence shown in SEQ ID NO: 1, and the amino acid mutation site is H26Y site or any one of the following combined mutation sites: H26Y+F434G, H26Y+F434A, H26Y+F434L, H26Y+F434I, H26Y+F434V, H26Y+F434P, H26Y+F434M, H26Y+F434W, H26Y+F434S, H26Y+F434Q, H26Y+F434T, H26Y+F434C, H26Y+F434N F434Y, H26Y+F434D, H26Y+F434E, H26Y+F434K, H26Y+F434R, H26Y+F434H, H26Y+F434Y+L466F, H26Y+F434Y+L466G, H26Y+F
- the transaminase mutant has the sequence of amino acid mutation in the sequence shown in SEQ ID NO: 3, and the amino acid mutation site is any one of the following combined mutation sites: V69F, V69G, V69A, V69L, V69I, V69P, V69M, V69W, V69S, V69Q, V69T, V69C, V69N, V69Y, V69D, V69E, V69K, V69R, V69H, V69A+F122G, V69A+F122A, V69A+F122L, V69A+F122I, V69A+F122P, V69A+F V69A+F122M, V69A+F122W, V69A+F122S, V69A+F122Q, V69A+F122T, V69A+F122C, V69A+F122N, V69A+F122Y, V69A+F122D, V69A+F122E, V69A+F122K, V69A
- a method for synthesizing (1R,2R)-AMPP and derivatives thereof using an enzymatic cascade reaction comprising the steps of:
- Step 1 using the benzaldehyde derivative as a substrate and the Escherichia coli transketolase mutant as a catalyst to obtain a compound shown in formula 2, the Escherichia coli transketolase having an amino acid sequence shown in SEQ ID NO: 1, has the nucleotide sequence shown in SEQ ID NO:2;
- Step 2 using the compound shown in formula 2 as a substrate and a mutant of transaminase ATA117 as a catalyst to obtain (1R, 2R)-AMPP and its derivatives, the transaminase ATA117 having an amino acid sequence as shown in SEQ ID NO: 3, Has the nucleotide sequence shown in SEQ ID NO:4.
- step 1 using a benzaldehyde derivative as a substrate, adding lithium hydroxypyruvate, thiamine pyrophosphate, MgCl 2 and a mutant of Escherichia coli transketolase to obtain the compound represented by the formula 2.
- step 2 the compound shown in formula 2 is used as a substrate, and D-alanine, D-glycine, D-valine, D-leucine, D-isoleucine, D- Methionine, D-Proline, D-Tryptophan, D-Serine, D-Tyrosine, D-Cysteine, D-Phenylalanine, D-Asparagine, D-Glutamine Aminoamide, D-threonine, D-aspartic acid, D-glutamic acid, D-lysine, D-arginine, D-histidine or isopropylamine, pyridoxal phosphate, transaminase ATA117
- the mutant continues to react to obtain (1R,2R)-AMPP and its derivatives shown in formula 3.
- step 1 10 mM benzaldehyde derivatives, 30 mM lithium hydroxypyruvate, 4.8 mM thiamine pyrophosphate, 18 mM MgCl 2 , 60 ⁇ M or 100 ⁇ M Escherichia coli were added to 50 ⁇ l of Tris-HCl buffer containing 100 mM.
- the ketolase mutant was reacted at 25 °C for 1-3 h to obtain the compound shown in formula 2.
- step 2 to the reaction system of step 1, add 100mM Tris-HCl buffer, 200mM D-alanine, D-glycine, D-valine, D-leucine, D-isoleucine Acid, D-Methionine, D-Proline, D-Tryptophan, D-Serine, D-Tyrosine, D-Cysteine, D-Phenylalanine, D-Asparagine , D-glutamine, D-threonine, D-aspartic acid, D-glutamic acid, D-lysine, D-arginine, D-histidine or isopropylamine, 2mM pyridine phosphate Doxal, 50 ⁇ M transaminase ATA117 mutant, expand the reaction system to 100 ⁇ l, and react at 25°C for 3-6 h to obtain (1R,2R)-AMPP and its derivatives.
- 100mM Tris-HCl buffer 200mM D-alanine, D-g
- the Escherichia coli transketolase mutant described in step 1 has a sequence of amino acid mutation in the sequence shown in SEQ ID NO: 1, and the amino acid mutation that occurs is H26Y or any one of the following combined mutations: H26Y+F434G, H26Y +F434A, H26Y+F434L, H26Y+F434I, H26Y+F434V, H26Y+F434P, H26Y+F434M, H26Y+F434W, H26Y+F434S, H26Y+F434Q, H26Y+F434T, H26Y+F434C, H26Y+F434N , H26Y+F434D, H26Y+F434E, H26Y+F434K, H26Y+F434R, H26Y+F434H, H26Y+F434Y+L466F, H26Y+F434Y+L466G, H26Y+F
- the step 2 transaminase ATA117 mutant has a sequence of amino acid mutation in the sequence shown in SEQ ID NO: 3, and the amino acid mutation that occurs is any of the following mutations: V69F, V69G, V69A, V69L, V69I, V69P , V69M, V69W, V69S, V69Q, V69T, V69C, V69N, V69Y, V69D, V69E, V69K, V69R, V69H, V69A+F122G, V69A+F122A, V69A+F122L, V69A+F122I, V69A+F122P, V69A , V69A+F122M, V69A+F122W, V69A+F122S, V69A+F122Q, V69A+F122T, V69A+F122C, V69A+F122N, V69A+F122Y, V69A+F122D, V69A+F122E, V69A+
- the pH of the Tris-HCl buffer is 7.5.
- pET28a as a carrier and E.coli BL21 as a host to express the sequence shown in SEQ ID NO: 1 with the above amino acid mutation, and purifying to obtain the Escherichia coli transketolase mutant.
- the production method of the transaminase uses pRSFDuet as a carrier and E.coli BL21 as a host to express the sequence shown in SEQ ID NO: 3 with the above amino acid mutation, and purify to obtain the transaminase ATA117 mutant.
- the host was cultured in LB medium, and when the OD600 in the culture medium reached 0.6-0.8, 0.2 mM IPTG was added to induce expression.
- the cells were collected; the cells were resuspended in a nickel column binding buffer, sonicated, and the supernatant was collected and subjected to Ni ion affinity chromatography.
- the present invention provides a method for synthesizing (1R, 2R)-AMPP and its derivatives by enzymatic cascade reaction, using cheap benzaldehyde derivatives as raw materials, and catalyzing the synthesis of high stereoselectivity by enzymatic cascade reaction.
- Sexual (1R,2R)-AMPP and its derivatives are indispensable for synthesizing (1R, 2R)-AMPP and its derivatives.
- the present invention provides a method for synthesizing (1R,2R)-AMPP using an enzymatic cascade reaction, comprising the steps of:
- transketolase EcTK1_YYH (that is, amino acid mutation H26Y+F434Y+L466H based on SEQ ID NO: 1) is used as a catalyst to obtain the compound shown in formula 5, and the transketone is obtained.
- the enzyme EcTK1_YYH has the nucleotide sequence shown in SEQ ID NO:5;
- transaminase ATA117_ACHH (that is, amino acid mutation V69A+F122C+I157H+F225H based on SEQ ID NO: 3) is a catalyst to obtain (1R, 2R)-AMPP, the transaminase ATA117_ACHH has a nucleotide sequence as shown in SEQ ID NO: 6;
- step 1 using p-methylsulfonylbenzaldehyde as a substrate, adding lithium hydroxypyruvate, thiamine pyrophosphate, MgCl 2 and transketolase EcTK1_YYH to react to obtain the compound represented by the formula 5.
- step 2 using the compound shown in formula 5 as a substrate, continue to add D-alanine, pyridoxal phosphate, transaminase ATA117_ACHH, NADH, lactate dehydrogenase, glucose and glucose dehydrogenase to continue the reaction to obtain the obtained result. described (1R,2R)-AMPP.
- step 1 10 mM p-methylsulfonylbenzaldehyde, 30 mM lithium hydroxypyruvate, 4.8 mM thiamine pyrophosphate, 18 mM MgCl 2 , 60 ⁇ M transketolase were added to 50 ⁇ l of Tris-HCl buffer containing 100 mM.
- EcTK1_YYH react at 25°C for 1 h to obtain the compound represented by formula 5.
- step 2 to the reaction system of step 1, add 100mM Tris-HCl buffer, 200mM D-alanine, 2mM pyridoxal phosphate, 50 ⁇ M transaminase ATA117_ACHH, 10mM NADH, 90U/ml lactate dehydrogenase, 200mM glucose, 30U/ml glucose dehydrogenase, expand the reaction system to 100 ⁇ l, and react at 25°C for 3h to obtain (1R,2R)-AMPP.
- the pH of the Tris-HCl buffer is 7.5.
- transaminase using pRSFDuet as a carrier and E.coli BL21 as a host to express the nucleotide sequence shown in SEQ ID NO: 6, and purify to obtain the transaminase ATA117_ACHH.
- the host was cultured in LB medium, and when the OD600 in the culture medium reached 0.6-0.8, 0.2 mM IPTG was added to induce expression.
- the cells were collected; the cells were resuspended in a nickel column binding buffer, sonicated, and the supernatant was collected and subjected to Ni ion affinity chromatography.
- the present invention provides a method for synthesizing (1R, 2R)-AMPP by enzymatic cascade reaction, using cheap p-methylsulfonylbenzaldehyde as raw material, and catalyzing the synthesis of stereospecific AMPP through enzymatic cascade reaction.
- (1R,2R)-AMPP in 76% yield, product stereoselectivity 96% de, >99% ee.
- Fig. 1 is the liquid phase diagram of the reaction product under C18 column condition with p-methanesulfonyl benzaldehyde as substrate;
- Fig. 2 is the liquid phase diagram of the threo-type reaction product under chiral liquid phase condition with p-methanesulfonyl benzaldehyde as substrate;
- Fig. 3 is the hydrogen spectrum of reaction product (1R, 2R)-AMPP
- Fig. 4 is the carbon spectrum of reaction product (1R, 2R)-AMPP
- Fig. 5 is the liquid phase diagram of the reaction product with benzaldehyde as a substrate under C18 column conditions, namely the high performance liquid chromatography identification of the enzyme cascade catalyzed benzaldehyde reaction product; wherein, A is the HPLC of the product of the enzyme cascade reaction Chromatography; B is the standard of (1R, 2R)-phenylserinol; C is the mixture of Thresh and erythro products.
- Fig. 6 is the liquid phase diagram of the reaction product taking p-tolualdehyde as substrate under C18 column condition, namely the high performance liquid chromatography identification of enzyme cascade catalyzed p-tolualdehyde reaction product; wherein, A is the enzyme level HPLC chromatogram of the combined reaction product; B is the standard of (1R,2R)-p-methylphenylserinol; C is the standard of the mixture of Thresh and erythro products.
- the invention provides a method for synthesizing (1R, 2R)-AMPP and derivatives thereof.
- the benzaldehyde derivative is used as a substrate, and a transketolase reaction is carried out under the action of a mutant of Escherichia coli transketolase to obtain the formula 2
- the reaction product shown, and then the compound shown in formula 2 is used as the substrate, and the transamination reaction is carried out under the action of the transaminase ATA117 mutant, and the amino donor is D-alanine, D-glycine, D-valine, D-Leucine, D-Isoleucine, D-Methionine, D-Proline, D-Tryptophan, D-Serine, D-Tyrosine, D-Cysteine, D -Phenylalanine, D-Asparagine, D-Glutamine, D-Threonine, D-Aspartic Acid, D-Glutamic Acid, D-Lysine,
- the nucleotide sequence in which the above-mentioned amino acid mutation occurs in the sequence shown in SEQ ID NO: 2 is introduced into the vector pET28a, and into the host E.coli BL21; then on the LB solid medium, pick the Escherichia coli E.coli BL21 containing the recombinant plasmid on the LB solid medium
- a single colony was inoculated into 40ml LB liquid medium (containing 50 ⁇ g/ml kanamycin antibiotic), and cultured overnight at 37°C and 220rpm; 10ml of bacterial culture was transferred to a 2L shake flask containing 500ml liquid LB medium, and two cells were inoculated. bottle, continue to culture at 37°C, 220rpm until OD600 reaches 0.6-0.8, add 0.2mM IPTG for induction, and induce culture at 30°C, 200rpm for 5h;
- the crushed mixture was centrifuged at 12000rpm for 30min-1h (for example, 30min or 1h), the supernatant was filtered through a 0.22 ⁇ m filter; then, the filtered sample was loaded and pre-equilibrated with a nickel column binding buffer.
- 2 ml of nickel filler wash the impurity protein with 20 column volumes of 50 mM imidazole elution buffer, and then elute the target protein with 5 ml of 250 mM imidazole elution buffer.
- Measure the protein concentration of each tube combine several tubes of protein solution with higher concentration, dilute or concentrate to 2.5ml, and load the sample into the desalting column equilibrated with glycerol buffer. After the protein solution is drained, add 3.5ml of glycerol buffer to elute protein to obtain the pure protein of Escherichia coli transketolase mutant.
- the molar extinction coefficient of the enzyme was predicted by the software Vector NTI.
- the nucleotide sequence with the above amino acid mutation in the sequence shown in SEQ ID NO: 4 is introduced into the vector pRSFDuet, and into the host E.coli BL21; then on the LB solid medium, pick the Escherichia coli E.coli containing the recombinant plasmid on the LB solid medium
- a single colony of BL21 was inoculated into 40ml LB liquid medium (containing 50 ⁇ g/ml kanamycin antibiotic), and cultured overnight at 37°C and 220rpm; 10ml of bacterial culture was transferred to a 2L shake flask containing 500ml liquid LB medium, inoculated Two bottles, continue to culture at 37°C, 220rpm until OD600 reaches 0.6-0.8, add 0.2mM IPTG for induction, and induce culture at 20°C, 200rpm for 15h.
- the crushed mixture was centrifuged at 12,000 rpm for 30min-1h (for example, 30min or 1h), and the supernatant was filtered through a 0.22 ⁇ m filter; then, the filtered sample was loaded on a nickel-column binding buffer that had been pre-equilibrated with buffer.
- the yellow color is due to the binding of the transaminase to the cofactor PLP.
- the conversion rate is 0-40%*, the conversion rate is 40-60%**, the conversion rate is 60-80%**, and the conversion rate is >80%**.
- Embodiment 5 The following is a specific description of Embodiment 5:
- the synthetic method of (1R,2R)-AMPP uses p-methylsulfonylbenzaldehyde as a substrate, and carries out a keto group transfer reaction under the action of transketolase EcTK1_YYH to obtain the reaction product shown in formula 5, followed by formula 5
- the compound shown is the substrate, and the transamination reaction is carried out under the action of transaminase ATA117_ACHH to synthesize (1R,2R)-1,3-dihydroxy-2-amino-1-p-methylsulfonyl phenylpropane ((1R,2R) -AMPP), wherein, in order to accelerate the consumption of pyruvate, NADH and lactate dehydrogenase are added to make the reaction proceed towards the synthesis of (1R,2R)-AMPP.
- the nucleotide sequence shown in SEQ ID NO: 5 was introduced into the vector pET28a, and into the host E.coli BL21; then on the LB solid medium, the single colony of E. coli E.coli BL21 containing the recombinant plasmid was picked and inoculated To 40ml of LB liquid medium (containing 50 ⁇ g/ml kanamycin antibiotic), cultivate overnight at 37°C and 220rpm; transfer 10ml of bacterial culture to a 2L shake flask containing 500ml of liquid LB medium, inoculate two bottles, and continue in Cultivate at 37°C and 220rpm until OD600 reaches 0.6-0.8, add 0.2mM IPTG for induction, and induce and culture at 30°C and 200rpm for 5h;
- the crushed mixture was centrifuged at 12,000 rpm for 1 h, and the supernatant was filtered through a 0.22 ⁇ m membrane filter; then, the filtered sample was loaded into 2 ml of nickel filler that had been pre-equilibrated with the nickel column binding buffer, using 20 times A column volume of 50 mM imidazole elution buffer was used to wash the impurity protein, and then 5 ml of 250 mM imidazole elution buffer was used to elute the target protein. Measure the protein concentration of each tube, combine several tubes of protein solution with higher concentration, dilute or concentrate to 2.5ml, and load the sample into the desalting column equilibrated with glycerol buffer.
- transketolase EcTK1_YYH After the protein solution is drained, add 3.5ml of glycerol buffer to elute Protein, obtains transketolase EcTK1_YYH, it has nucleotide sequence as shown in SEQ ID NO:5, amino acid sequence as shown in SEQ ID NO:7.
- the molar extinction coefficient of the enzyme was predicted by the software Vector NTI.
- the nucleotide sequence shown in SEQ ID NO: 6 was introduced into the vector pRSFDuet, and into the host E.coli BL21; then on the LB solid medium, the single colony of Escherichia coli E.coli BL21 containing the recombinant plasmid was picked and inoculated To 40ml of LB liquid medium (containing 50 ⁇ g/ml kanamycin antibiotic), cultivate overnight at 37°C and 220rpm; transfer 10ml of bacterial culture to a 2L shake flask containing 500ml of liquid LB medium, inoculate two bottles, and continue in Cultivate at 37°C and 220rpm until OD600 reaches 0.6-0.8, add 0.2mM IPTG for induction, and induce culture at 20°C and 200rpm for 15h.
- LB liquid medium containing 50 ⁇ g/ml kanamycin antibiotic
- the crushed mixture was centrifuged at 12,000 rpm for 1 h, and the supernatant was filtered through a 0.22 ⁇ m membrane filter; then, the filtered sample was loaded onto 2 ml of nickel filler that had been pre-equilibrated with a nickel column binding buffer, using 10 times
- the impurity protein was washed with a column volume of 25mM imidazole elution buffer, and then the target protein was eluted with 5ml of 250mM imidazole elution buffer.
- the yellow color is due to the binding of the transaminase to the cofactor PLP.
- ATA117_ACHH which has the nucleotide sequence shown in SEQ ID NO:6 and the amino acid sequence shown in SEQ ID NO:8.
- the present invention further detects the reaction product on an Agilent 1200 liquid chromatograph, and the specific detection method includes: using a C18 column (4.6 ⁇ 150 mm, particle size 3 ⁇ m), column temperature 30° C., 224 nm, 0.5 ml/min, phase A : H 2 O (10 mM KH 2 PO 4 , pH 8.5), phase B: acetonitrile, chromatographic conditions are shown in Table 3:
- Fig. 1 is the liquid phase diagram of the reaction product of embodiment 5 using p-methanesulfonyl benzaldehyde as a substrate under C18 column conditions
- the top is (erythro)-p-methanesulfonyl phenylserine
- the liquid phase diagram of alcohol and (threo)-p-methylsulfosulfonyl phenylserinol standard in this example, it is (erythro)-1,3-dihydroxy-2-amino-1-p-methylsulfone phenylpropane and (threo)-1,3-dihydroxy-2-amino-1-p-methylsulfonyl phenylpropane standard
- the middle is (1R,2R)-p-methylsulfonyl phenylserinol (Specifically the standard product of (1R,2R)-1,3-dihydroxy-2-amino-1-p-methylsulfonyl phenyl
- FIG. 2 is the liquid phase diagram of (threo)-p-methanesulfonyl phenylserinol product under chiral liquid column conditions, in Fig. 2, the top is the erythro+threo standard control, the second row is the threo standard The third row is (1R,2R)-p-methanesulfonyl phenylserinol ((1R,2R)-AMPP) standard control, the fourth row is the last row (threo)-p-methanesulfonic acid Sulfonylphenylserinol ((threo)-AMPP) products, as shown in Figure 2, (1R,2R)- and (1S,2S)-p-methanesulfonylphenylserinol ((1R, The retention times of 2R)- and (1S, 2S)-AMPP) are 5.6 and 9.4 min, respectively.
- ee [(RR-SS)/(RR+SS)] ⁇ 100%
- the ee value is greater than 99%, where : RR and SS represent (1R,2R)- and (1S,2S)-p-methylsulfosulfonylphenylserinol ((1R,2R)- and (1S,2S)-1,3-dihydroxy-2 - amino-1-p-methylsulfonyl phenylpropane).
- Fig. 3 is the hydrogen spectrum of the reaction product (1R, 2R)-AMPP
- Fig. 4 is the carbon spectrum of the reaction product (1R, 2R)-AMPP.
- the (1R,2R)-AMPP is synthesized, and combined with the de value and the ee value, the (1R,2R)-AMPP prepared by the method provided by the present invention has high stereoselectivity.
- Figure 5 is a liquid phase diagram of the reaction product of Example 9 using benzaldehyde as a substrate under C18 column conditions, in Figure 5, the upper part is the reaction product obtained in Example 9, and the middle is (1R, 2R)-p-methanesulfonic acid
- Fig. 6 is the liquid phase diagram of the reaction product of Example 13 using p-methylbenzaldehyde as a substrate under C18 column conditions
- the upper part is the reaction product obtained in Example 13
- the middle is (erythro)-para Methylphenylserinol
- the bottom is (threo type)-p-methylphenylserinol standard product
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Abstract
提供了一种利用酶级联反应合成(1R,2R)-AMPP及其衍生物的方法，包括如下步骤：步骤1、以苯甲醛衍生物为底物，大肠杆菌转酮酶突变体为催化剂，合成式2所示的化合物；步骤2、以式2所示的化合物为底物，转氨酶ATA117突变体为催化剂，合成(1R,2R)-AMPP及其衍生物。
Description
本发明要求于2021年2月5日提交中国专利局、申请号为202110162637.0、发明名称为“一种利用酶级联反应合成(1R,2R)-AMPP的方法”和2021年5月24日提交中国专利局、申请号为202110567238.2、发明名称为“一种利用酶级联反应合成(1R,2R)-AMPP的方法”的中国专利申请的优先权，其全部内容通过引用结合在本发明中。
本发明涉及生物催化技术领域，尤其涉及一种利用酶级联反应合成(1R,2R)-AMPP的方法。
氟苯尼考，是美国Schering-Ploμgh Animal Health公司于1988年研发成功的一种新型兽用氯霉素类抗菌药，其化学名称为2,2-二氯-N-[(1R,2R)-1-氟甲基-2-羟基-2-[4-(甲磺酰基)苯基]乙基]乙酰胺，最早于日本上市，名为Nuflor，具有广谱的抗菌活性，可用于治疗牛、猪和鸡的细菌性呼吸系统疾病。氟苯尼考的抗菌机理与氯霉素、甲砜霉素类似，通过抑制细菌70S核糖体，与50S亚基结合，抑制肽酰基转移酶，从而抑制肽链的延伸，干扰细菌蛋白质的合成，从而达到抑菌的作用。通常，甲砜霉素和氯霉素耐药菌中产生的乙酰转移酶能够使α-甲基位的-OH发生酰基化反应，从而失去药理活性。而氟苯尼考通过-F取代α-甲基位的-OH，免于乙酰化反应，所以对氯霉素、甲砜霉素的耐药菌仍有较强的抑制作用。由于氯霉素、甲砜霉素的使用引起再生障碍性贫血等不良反应，许多国家已经禁止该类抗菌药在动物治疗中使用，比起氯霉素而言，氟苯尼考在结构上以CH
3-SO
2基团取代了NO
2基团，避免了再生障碍性贫血症，且在动物体内残留量少，逐渐替代氯霉素和甲砜霉素，广泛应用于动物感染性疾病的防治。
2018年，陈绍兴课题组利用酮还原酶介导的动态动力学拆分，以芳香α-氨基-β-酮酯为底物，合成了立体专一性的对甲磺砜基苯丝氨酸酯，但是其反应前体商业不可获取，需要额外的三步化学反应来获得。2019年林娟课题组报道了一种假单胞菌属来源的L-苏氨酸转醛缩酶，该酶具备较高的非对映选择性，在优化的反应条件下，该酶催化生成的L-苏式-对甲砜基苯丝氨酸的非对映选择性为94.5％。2020年该课题组通过定向进化，改善了这个苏氨酸醛缩酶的活性，并引入乙醛消除系统，提升了L-苏式-对甲磺砜基苯丝氨酸的产量。
除了以上方法合成氟苯尼考手性中间体，还有文献报道通过L-苏氨酸醛缩酶催化合成对甲磺砜基苯丝氨酸。2007年，Herfried Griengl课题组利用L-苏氨酸醛缩酶催化芳香醛和甘氨酸合成了一系列手性苯基丝氨酸衍生物，但是其非对映选择性不高。为了增强L-苏氨酸醛缩酶的工业应用，浙江大学吴坚平课题组通过酶进化成功地提高了L-苏氨酸醛缩酶的立体选择性。其突变体Y31H/N305R催化产生L-苏式-对甲磺砜基苯丝氨酸的转化率为87.2％，de为93.1％。次年，该课题组进一步提高了L-苏氨酸醛缩酶的Cβ选择性，所获突变体RS1(Y8H/Y31H/I143R/N305R)催化合成的L-苏式-对甲磺砜基苯丝氨酸，de值为99.5％，转化率为73.2％。
L-苏式-对甲磺砜基苯丝氨酸转化成最终的氟苯尼考需要先经过两步化学反应(酯化和还原)转成(1R,2R)-2-氨基-1-(4-(甲磺酰)苯基)丙烷-1,3-二醇((1R,2R)-AMPP)(也称为(1R,2R)-1,3-二羟基-2-氨基-1-对甲砜基苯丙烷)，其化学结构式如式4所示。为了节省反应步骤，我们设计了(1R,2R)-AMPP的合成路线。我们设计以商业易获取的对甲磺砜苯甲醛为起始原料，经过转酮和转氨反应合成(1R,2R)-AMPP。
大肠杆菌转酮酶是一种广泛存在的焦磷酸硫胺素依赖的酶。它连接了非氧化的磷酸戊糖途径和三羧酸循环。大肠杆菌转酮酶催化可逆的转酮反应，将酮醇供体的二碳单位转移到醛受体中，形成手性的二羟酮类化合物。在转酮反应中，β-羟基丙酮酸通常用作酮醇供体，因为β-羟基丙酮酸被转酮后生成挥发性的二氧化碳，从而使反应不可逆。除了催化脂肪醛，大肠杆菌转酮酶被改造后还可以催化芳香醛。2010年Helen C.Hailes课题组报道对大肠杆菌来源的大肠杆菌转酮酶进行了D469和F434的定点突变，实现了对苯甲醛和间羟基苯甲醛的催化，但催化效率较低，转化率≤10％。其突变体催化产生的羟酮产物主要为R构型(ee≤82％)。Paul A.Dalby课题组2015年报道在大肠杆菌TK/D469T的基础上作R520和S385的迭代饱和突变，筛选所得突变体EcTK/D469T/R520Q/S385Y对苯甲醛衍生物包括间羧基苯甲醛，间羟基苯甲醛和对羧基苯甲醛均具有较高的催化活性，拓宽了大肠杆菌转酮酶的底物谱，但其立体选择性未知。Wolf-Dieter Fessner课题组2017年报道通过对嗜热脂肪芽孢杆菌来源的大肠杆菌转酮酶定向进化，提高了大肠杆菌转酮酶对苯基乙醛，苯基丙醛，苯氧基乙醛，苄氧基乙醛的催化效率，产率为60-72％，其羟酮产物具有绝对的S立体选择性(ee＞99％)。另外，定向进化所得的大肠杆菌转酮酶突变体L382N/D470S对苯甲醛底物具有良好的催化活性，但未报道其立体选择性。综上，文献报道通过对大肠杆菌转酮酶的改造提高了大肠杆菌转酮酶对芳香醛的催化活性，其催化芳香醛底物如苯基乙醛，苯基丙醛和苯氧基乙醛产生S立体构型的羟酮产物，而催化苯甲醛的产物构型未知。因此未有文献报道能高效催化芳香醛尤其是苯甲醛衍生物产生R构型羟酮产物的大肠杆菌转酮酶。
转氨酶分为两类，α-转氨酶和ω-转氨酶。α-转氨酶包括I、III和IV型亚族，催化转氨反应时依赖底物酮的α-羧酸。而ω-转氨酶属于II型亚族，不依赖底物酮的α-羧酸，被广泛应用于多种脂肪酮和芳香酮的不对称转氨反应，生成立体专一性的胺。其中ATA117是广泛应用的ω-转氨酶之一，能产生立体专一性的(R)胺。ω-转氨酶除了能产生立体专一性的胺，同时对邻位的手性中心也具有一定的对映选择性。2009年Wolfgang Kroutil课题组报道通过ω-转氨酶ATA117对外消旋醛底物的动力学拆分获得了R-4-苯基-2-吡咯烷酮。ATA117对醛邻位的手性中心存在对映选择性，通过助溶剂和pH的优化，其转氨产物的ee可达68％。2013年Vicente Gotor课题组考察了24种商业S-和R-转氨酶对外消旋的α-烷基-β-酮酯的动力学拆分情况，结果表明其催化效率较高，但非对映选择性较差。2014年Wolfgang Kroutil课题组考察了假单胞菌属、节杆菌属、土曲霉属和生丝单胞菌属来源的ω-转氨酶对外消旋的2-苯基丙醛及对位间位邻位甲基和甲氧基取代的2-苯基丙醛的动力学拆分情况，表征了这些转氨酶对邻位手性中心的立体选择性，其ee(R)＝76-98％。Wolfgang Kroutil课题组在2018年考察了节杆菌属KNK168、土曲霉、禾谷镰孢菌、生丝单胞菌、费氏新萨托菌、巨大芽孢杆菌等来源的ω-转氨酶对外消旋脂肪醛的动力学拆分情况，通过一步转氨反应，获得了高立体选择性的抗癫痫药物布瓦西坦和普瑞巴林。其中，生丝单胞菌来源的ω-转氨酶通过一步动力学拆分可获得高立体选择性的R-布瓦西坦手性中间体，ee可达92％。Wolfgang Kroutil课题组分别在禾谷镰孢菌来源的ω-转氨酶和ArRmut11突变体的基础上做了几个氨基酸位点的突变，所获得的突变体催化外消旋脂肪醛合成了S-普瑞巴林，其ee分别可达76％和80％。2019年Iván Lavandera课题组报道了一系列商业转氨酶和巨大芽孢杆菌来源的转氨酶及其突变体对外消旋的α-烷基-β-酮酰胺底物的动力学拆分情况，其中商业R转氨酶对该类底物的催化效率和立体选择性最高，其非对映选择性de可达96％。而巨大芽孢杆菌来源的转氨酶及其突变体仅可转化部分底物，其非对映选择性较低。综上，文献报道中大多通过筛选不同种属来源的ω-转氨酶，可获得对醛底物邻位手性中心的高立体选择性，而通过筛选商业转氨酶可获得对酮底物如α-烷基-β-酮酰胺的邻位手性中心的高立体选择性。但目前未有文献报道ω-转氨酶对手性芳香羟酮化合物的对映选择性。
发明内容
有鉴于现有技术的上述缺陷，本发明所要解决的技术问题是如何利用酶催化合成高立体选择性的 (1R,2R)-AMPP及其衍生物。
本发明提供了以下技术方案：
[1].一种利用酶级联反应合成(1R,2R)-苯丝氨醇衍生物的方法，其中，包括如下步骤：
步骤1、以苯甲醛衍生物为底物，大肠杆菌转酮酶突变体为催化剂，得到式2所示的化合物，所述大肠杆菌转酮酶具有如SEQ ID NO:1所示的氨基酸序列，具有如SEQ ID NO:2所示的核苷酸序列；
步骤2、以式2所示的化合物为底物，转氨酶ATA117突变体为催化剂，得到(1R,2R)-AMPP及其衍生物，所述转氨酶ATA117具有如SEQ ID NO:3所示氨基酸序列，具有如SEQ ID NO:4所示的核苷酸序列；
[2].根据[1]所述的方法，其中，步骤1中以苯甲醛衍生物为底物，添加羟基丙酮酸锂、硫胺素焦磷酸、MgCl
2以及大肠杆菌转酮酶突变体反应得到所述式2所示的化合物。
[3].根据[1]或[2]所述的方法，其中，步骤2中以式2所示的化合物为底物，继续添加D-丙氨酸(或D-甘氨酸、D-缬氨酸、D-亮氨酸、D-异亮氨酸、D-甲硫氨酸、D-脯氨酸、D-色氨酸、D-丝氨酸、D-酪氨酸、D-半胱氨酸、D-苯丙氨酸、D-天冬酰胺、D-谷氨酰胺、D-苏氨酸、D-天冬氨酸、D-谷氨酸、D-赖氨酸、D-精氨酸、D-组氨酸或异丙胺)、磷酸吡哆醛、转氨酶ATA117突变体继续反应得到式3所示的(1R,2R)-AMPP及其衍生物。
[4].根据[1]～[3]中任一项所述的方法，其中，步骤1中，在50μl，含100mM的Tris-HCl缓冲液中加入10mM苯甲醛衍生物、30mM羟基丙酮酸锂、4.8mM硫胺素焦磷酸、18mM MgCl
2、60μM或100μM大肠杆菌转酮酶突变体，在25℃下反应1-3h得到式2所示的化合物。
[5].根据[1]～[4]中任一项所述的方法，其中，步骤2中，扩充反应体系至100μl，加入100mM Tris-HCl缓冲液、200mM D-丙氨酸(或D-甘氨酸、D-缬氨酸、D-亮氨酸、D-异亮氨酸、D-甲硫氨酸、D-脯氨酸、D-色氨酸、D-丝氨酸、D-酪氨酸、D-半胱氨酸、D-苯丙氨酸、D-天冬酰胺、D-谷氨酰胺、D-苏氨酸、D-天冬氨酸、D-谷氨酸、D-赖氨酸、D-精氨酸、D-组氨酸或异丙胺)、2mM磷酸吡哆醛、50μM转氨酶ATA117突变体，在25℃下反应3-6h，得到(1R,2R)-AMPP及其衍生物。
[6].根据[1]～[5]中任一项所述的方法，其中，步骤1中所述大肠杆菌转酮酶突变体具有SEQ ID NO:1所示序列发生氨基酸突变的序列，所述发生氨基酸突变的位点为H26Y位点或如下任一种组合突变位点：H26Y+F434G、H26Y+F434A、H26Y+F434L、H26Y+F434I、H26Y+F434V、H26Y+F434P、H26Y+F434M、H26Y+F434W、H26Y+F434S、H26Y+F434Q、H26Y+F434T、H26Y+F434C、H26Y+F434N、H26Y+F434Y、H26Y+F434D、H26Y+F434E、H26Y+F434K、H26Y+F434R、H26Y+F434H、H26Y+F434Y+L466F、H26Y+F434Y+L466G、H26Y+F434Y+L466A、H26Y+F434Y+L466I、H26Y+F434Y+L466V、H26Y+F434Y+L466P、H26Y+F434Y+L466M、H26Y+F434Y+L466W、H26Y+F434Y+L466S、H26Y+F434Y+L466Q、H26Y+F434Y+L466T、H26Y+F434Y+L466C、H26Y+F434Y+L466N、H26Y+F434Y+L466Y、H26Y+F434Y+L466D、H26Y+F434Y+L466E、H26Y+F434Y+L466K、H26Y+F434Y+L466R、H26Y+F434Y+L466H、H26Y+F434Y+L466H+H261F、H26Y+F434Y+L466H+H261G、H26Y+F434Y+L466H+H261A、H26Y+F434Y+L466H+H261L、H26Y+F434Y+L466H+H261I、H26Y+F434Y+L466H+H261V、H26Y+F434Y+L466H+H261P、H26Y+F434Y+L466H+H261M、H26Y+F434Y+L466H+H261W、H26Y+F434Y+L466H+H261S、 H26Y+F434Y+L466H+H261Q、H26Y+F434Y+L466H+H261T、H26Y+F434Y+L466H+H261C、H26Y+F434Y+L466H+H261N、H26Y+F434Y+L466H+H261Y、H26Y+F434Y+L466H+H261D、H26Y+F434Y+L466H+H261E、H26Y+F434Y+L466H+H261K、H26Y+F434Y+L466H+H261R、H26Y+F434Y+L466H+H461F、H26Y+F434Y+L466H+H461G、H26Y+F434Y+L466H+H461A、H26Y+F434Y+L466H+H461L、H26Y+F434Y+L466H+H461I、H26Y+F434Y+L466H+H461V、H26Y+F434Y+L466H+H461P、H26Y+F434Y+L466H+H461M、H26Y+F434Y+L466H+H461W、H26Y+F434Y+L466H+H461S、H26Y+F434Y+L466H+H461Q、H26Y+F434Y+L466H+H461T、H26Y+F434Y+L466H+H461C、H26Y+F434Y+L466H+H461N、H26Y+F434Y+L466H+H461Y、H26Y+F434Y+L466H+H461D、H26Y+F434Y+L466H+H461E、H26Y+F434Y+L466H+H461K、H26Y+F434Y+L466H+H461R。
[7].根据[1]～[6]中任一项所述的方法，其中，所述步骤2中转氨酶ATA117突变体具有SEQ ID NO:3所示序列发生氨基酸突变的序列，所述发生氨基酸突变的位点为如下任一种突变：V69F、V69G、V69A、V69L、V69I、V69P、V69M、V69W、V69S、V69Q、V69T、V69C、V69N、V69Y、V69D、V69E、V69K、V69R、V69H、V69A+F122G、V69A+F122A、V69A+F122L、V69A+F122I、V69A+F122V、V69A+F122P、V69A+F122M、V69A+F122W、V69A+F122S、V69A+F122Q、V69A+F122T、V69A+F122C、V69A+F122N、V69A+F122Y、V69A+F122D、V69A+F122E、V69A+F122K、V69A+F122R、V69A+F122H、V69A+F122C+I157F、V69A+F122C+I157G、V69A+F122C+I157A、V69A+F122C+I157L、V69A+F122C+I157I、V69A+F122C+I157V、V69A+F122C+I157P、V69A+F122C+I157M、V69A+F122C+I157W、V69A+F122C+I157S、V69A+F122C+I157Q、V69A+F122C+I157T、V69A+F122C+I157C、V69A+F122C+I157N、V69A+F122C+I157Y、V69A+F122C+I157D、V69A+F122C+I157E、V69A+F122C+I157K、V69A+F122C+I157R、V69A+F122C+I157H、V69A+F122C+I157H+F225G、V69A+F122C+I157H+F225A、V69A+F122C+I157H+F225L、V69A+F122C+I157H+F225I、V69A+F122C+I157H+F225V、V69A+F122C+I157H+F225P、V69A+F122C+I157H+F225M、V69A+F122C+I157H+F225W、V69A+F122C+I157H+F225S、V69A+F122C+I157H+F225Q、V69A+F122C+I157H+F225T、V69A+F122C+I157H+F225C、V69A+F122C+I157H+F225N、V69A+F122C+I157H+F225Y、V69A+F122C+I157H+F225D、V69A+F122C+I157H+F225E、V69A+F122C+I157H+F225K、V69A+F122C+I157H+F225R、V69A+F122C+I157H+F225H。
[8].根据[1]～[7]中任一项所述的方法，其中，步骤1和步骤2中，所述Tris-HCl缓冲液的pH为7.5。
[9].根据[1]～[8]中任一项所述的方法，其中，以pET28a为载体、E.coli BL21为宿主表达SEQ ID NO:1所示序列发生上述氨基酸突变的序列，纯化得到所述大肠杆菌转酮酶突变体。
[10].根据[1]～[9]中任一项所述的方法，其中，所述转氨酶的生产方法，以pRSFDuet为载体、E.coli BL21为宿主表达SEQ ID NO:3所示序列发生上述氨基酸突变的序列，纯化得到所述转氨酶ATA117突变体。
[11].根据[1]～[10]中任一项所述的方法，其中，将所述宿主在LB培养基中进行培养，当培养液中OD600达到0.6-0.8时加入0.2mM IPTG诱导表达。
[12].根据[1]～[11]中任一项所述的方法，其中，培养结束后，收集细胞；将细胞重悬于镍柱结合缓冲液中，进行超声处理，收集上清液，并进行Ni离子亲和层析。
[13].根据[1]～[12]中任一项所述的方法，其为一种利用酶级联反应合成(1R,2R)-AMPP的方法，其中，包括如下步骤：
在步骤1中、以对甲砜基苯甲醛为底物，转酮酶EcTK1_YYH为催化剂，得到式5所示的化合物，所述转酮酶EcTK1_YYH具有如SEQ ID NO:5所示的核苷酸序列；
在步骤2中、以式5所示的化合物为底物，转氨酶ATA117_ACHH为催化剂，得到(1R,2R)-AMPP，所述转氨酶ATA117_ACHH具有如SEQ ID NO:6所示核苷酸序列；
[14].如[13]所述的方法，其中，步骤1中以对甲砜基苯甲醛为底物，添加羟基丙酮酸锂、硫胺素焦磷酸、MgCl
2以及转酮酶EcTK1_YYH反应得到所述式5所示的化合物。
优选地，步骤2中以式5所示的化合物为底物，继续添加D-丙氨酸、磷酸吡哆醛、转氨酶ATA117_ACHH、NADH、乳酸脱氢酶、葡萄糖以及葡萄糖脱氢酶继续反应得到所述(1R,2R)-AMPP。
[15].如[13]所述的方法，其中，步骤1中，在50μl，含100mM的Tris-HCl缓冲液中加入10mM对甲砜基苯甲醛、30mM羟基丙酮酸锂、4.8mM硫胺素焦磷酸、18mM MgCl
2、60μM转酮酶EcTK1_YYH，在25℃下反应1h得到式5所示的化合物。
优选地，步骤2中，向步骤1的体系中加入100mM Tris-HCl缓冲液、200mM D-丙氨酸、2mM磷酸吡哆醛、50μM转氨酶ATA117_ACHH、10mM NADH、90U/ml乳酸脱氢酶、200mM葡萄糖、30U/ml葡萄糖脱氢酶，扩充反应体系至100μl，在25℃下反应3h，得到(1R,2R)-AMPP。
进一步地，为实现上述目的，本发明提供了一种利用酶级联反应合成(1R,2R)-苯丝氨醇衍生物的方法，其中，包括如下步骤：
步骤1、以苯甲醛衍生物(式中1)和羟基丙酮酸为底物，大肠杆菌转酮酶突变体为催化剂，得到(R)-羟酮中间体(式中2)。
步骤2、以化合物(式中2)和氨供体为底物，转氨酶ATA117突变体为催化剂，得到(1R,2R)-苯丝氨醇衍生物(式中3)。
在本发明的一些具体实施方案中，醛底物为对甲磺砜苯甲醛、对氟苯甲醛、对氯苯甲醛、对溴苯甲醛、对甲基苯甲醛、对硝基苯甲醛和苯甲醛。
在本发明中，对甲磺砜苯甲醛、对甲磺砜基苯甲醛、对甲砜基苯甲醛具有相同的含义，指“对甲磺酰基苯甲醛”。
在本发明的一些具体实施方案中，氨基供体为D-丙氨酸、D-甘氨酸、D-缬氨酸、D-亮氨酸、D-异亮氨酸、D-甲硫氨酸、D-脯氨酸、D-色氨酸、D-丝氨酸、D-酪氨酸、D-半胱氨酸、D-苯丙氨酸、D-天冬酰胺、D-谷氨酰胺、D-苏氨酸、D-天冬氨酸、D-谷氨酸、D-赖氨酸、D-精氨酸、D-组氨酸和异丙胺。
所述大肠杆菌转酮酶突变体具有SEQ ID NO:1所示序列发生氨基酸突变的序列，所述发生氨基酸突变的位点为H26Y位点或如下任一种组合突变位点：H26Y+F434G、H26Y+F434A、H26Y+F434L、H26Y+F434I、H26Y+F434V、H26Y+F434P、H26Y+F434M、H26Y+F434W、H26Y+F434S、H26Y+F434Q、H26Y+F434T、H26Y+F434C、H26Y+F434N、H26Y+F434Y、H26Y+F434D、H26Y+F434E、H26Y+F434K、H26Y+F434R、H26Y+F434H、H26Y+F434Y+L466F、H26Y+F434Y+L466G、H26Y+F434Y+L466A、H26Y+F434Y+L466I、H26Y+F434Y+L466V、H26Y+F434Y+L466P、H26Y+F434Y+L466M、H26Y+F434Y+L466W、 H26Y+F434Y+L466S、H26Y+F434Y+L466Q、H26Y+F434Y+L466T、H26Y+F434Y+L466C、H26Y+F434Y+L466N、H26Y+F434Y+L466Y、H26Y+F434Y+L466D、H26Y+F434Y+L466E、H26Y+F434Y+L466K、H26Y+F434Y+L466R、H26Y+F434Y+L466H、H26Y+F434Y+L466H+H261F、H26Y+F434Y+L466H+H261G、H26Y+F434Y+L466H+H261A、H26Y+F434Y+L466H+H261L、H26Y+F434Y+L466H+H261I、H26Y+F434Y+L466H+H261V、H26Y+F434Y+L466H+H261P、H26Y+F434Y+L466H+H261M、H26Y+F434Y+L466H+H261W、H26Y+F434Y+L466H+H261S、H26Y+F434Y+L466H+H261Q、H26Y+F434Y+L466H+H261T、H26Y+F434Y+L466H+H261C、H26Y+F434Y+L466H+H261N、H26Y+F434Y+L466H+H261Y、H26Y+F434Y+L466H+H261D、H26Y+F434Y+L466H+H261E、H26Y+F434Y+L466H+H261K、H26Y+F434Y+L466H+H261R、H26Y+F434Y+L466H+H461F、H26Y+F434Y+L466H+H461G、H26Y+F434Y+L466H+H461A、H26Y+F434Y+L466H+H461L、H26Y+F434Y+L466H+H461I、H26Y+F434Y+L466H+H461V、H26Y+F434Y+L466H+H461P、H26Y+F434Y+L466H+H461M、H26Y+F434Y+L466H+H461W、H26Y+F434Y+L466H+H461S、H26Y+F434Y+L466H+H461Q、H26Y+F434Y+L466H+H461T、H26Y+F434Y+L466H+H461C、H26Y+F434Y+L466H+H461N、H26Y+F434Y+L466H+H461Y、H26Y+F434Y+L466H+H461D、H26Y+F434Y+L466H+H461E、H26Y+F434Y+L466H+H461K、H26Y+F434Y+L466H+H461R。
所述转氨酶突变体具有SEQ ID NO:3所示序列发生氨基酸突变的序列，所述发生氨基酸突变的位点为如下任一种组合突变位点：V69F、V69G、V69A、V69L、V69I、V69P、V69M、V69W、V69S、V69Q、V69T、V69C、V69N、V69Y、V69D、V69E、V69K、V69R、V69H、V69A+F122G、V69A+F122A、V69A+F122L、V69A+F122I、V69A+F122V、V69A+F122P、V69A+F122M、V69A+F122W、V69A+F122S、V69A+F122Q、V69A+F122T、V69A+F122C、V69A+F122N、V69A+F122Y、V69A+F122D、V69A+F122E、V69A+F122K、V69A+F122R、V69A+F122H、V69A+F122C+I157F、V69A+F122C+I157G、V69A+F122C+I157A、V69A+F122C+I157L、V69A+F122C+I157I、V69A+F122C+I157V、V69A+F122C+I157P、V69A+F122C+I157M、V69A+F122C+I157W、V69A+F122C+I157S、V69A+F122C+I157Q、V69A+F122C+I157T、V69A+F122C+I157C、V69A+F122C+I157N、V69A+F122C+I157Y、V69A+F122C+I157D、V69A+F122C+I157E、V69A+F122C+I157K、V69A+F122C+I157R、V69A+F122C+I157H、V69A+F122C+I157H+F225G、V69A+F122C+I157H+F225A、V69A+F122C+I157H+F225L、V69A+F122C+I157H+F225I、V69A+F122C+I157H+F225V、V69A+F122C+I157H+F225P、V69A+F122C+I157H+F225M、V69A+F122C+I157H+F225W、V69A+F122C+I157H+F225S、V69A+F122C+I157H+F225Q、V69A+F122C+I157H+F225T、V69A+F122C+I157H+F225C、V69A+F122C+I157H+F225N、V69A+F122C+I157H+F225Y、V69A+F122C+I157H+F225D、V69A+F122C+I157H+F225E、V69A+F122C+I157H+F225K、V69A+F122C+I157H+F225R、V69A+F122C+I157H+F225H。
在本发明的一些实施方案中，提供了一种利用酶级联反应合成(1R,2R)-AMPP及其衍生物的方法，包括如下步骤：
步骤1、以苯甲醛衍生物为底物，大肠杆菌转酮酶突变体为催化剂，得到式2所示的化合物，所述大肠杆菌转酮酶具有如SEQ ID NO:1所示的氨基酸序列，具有如SEQ ID NO:2所示的核苷酸序列；
步骤2、以式2所示的化合物为底物，转氨酶ATA117突变体为催化剂，得到(1R,2R)-AMPP及其衍生物，所述转氨酶ATA117具有如SEQ ID NO:3所示氨基酸序列，具有如SEQ ID NO:4所示的核苷酸序列。
进一步地，步骤1中以苯甲醛衍生物为底物，添加羟基丙酮酸锂、硫胺素焦磷酸、MgCl
2以及大肠杆菌转酮酶突变体反应得到所述式2所示的化合物。
进一步地，步骤2中以式2所示的化合物为底物，继续添加D-丙氨酸、D-甘氨酸、D-缬氨酸、D-亮氨酸、D-异亮氨酸、D-甲硫氨酸、D-脯氨酸、D-色氨酸、D-丝氨酸、D-酪氨酸、D-半胱氨酸、D-苯丙氨酸、D-天冬酰胺、D-谷氨酰胺、D-苏氨酸、D-天冬氨酸、D-谷氨酸、D-赖氨酸、D-精氨酸、D-组氨酸或异丙胺、磷酸吡哆醛、转氨酶ATA117突变体继续反应得到式3所示的(1R,2R)-AMPP及其衍生物。
进一步地，步骤1中，在50μl，含100mM的Tris-HCl缓冲液中加入10mM苯甲醛衍生物、30mM羟基丙酮酸锂、4.8mM硫胺素焦磷酸、18mM MgCl
2、60μM或100μM大肠杆菌转酮酶突变体，在25℃下反应1-3h得到式2所示的化合物。
进一步地，步骤2中，向步骤1反应体系中，加入100mM Tris-HCl缓冲液、200mM D-丙氨酸、D-甘氨酸、D-缬氨酸、D-亮氨酸、D-异亮氨酸、D-甲硫氨酸、D-脯氨酸、D-色氨酸、D-丝氨酸、D-酪氨酸、D-半胱氨酸、D-苯丙氨酸、D-天冬酰胺、D-谷氨酰胺、D-苏氨酸、D-天冬氨酸、D-谷氨酸、D-赖氨酸、D-精氨酸、D-组氨酸或异丙胺、2mM磷酸吡哆醛、50μM转氨酶ATA117突变体，扩充反应体系至100μl，在25℃下反应3-6h，得到(1R,2R)-AMPP及其衍生物。
进一步地，步骤1所述大肠杆菌转酮酶突变体具有SEQ ID NO:1所示序列发生氨基酸突变的序列，所述发生的氨基酸突变为H26Y或如下任一种组合突变：H26Y+F434G、H26Y+F434A、H26Y+F434L、H26Y+F434I、H26Y+F434V、H26Y+F434P、H26Y+F434M、H26Y+F434W、H26Y+F434S、H26Y+F434Q、H26Y+F434T、H26Y+F434C、H26Y+F434N、H26Y+F434Y、H26Y+F434D、H26Y+F434E、H26Y+F434K、H26Y+F434R、H26Y+F434H、H26Y+F434Y+L466F、H26Y+F434Y+L466G、H26Y+F434Y+L466A、H26Y+F434Y+L466I、H26Y+F434Y+L466V、H26Y+F434Y+L466P、H26Y+F434Y+L466M、H26Y+F434Y+L466W、H26Y+F434Y+L466S、H26Y+F434Y+L466Q、H26Y+F434Y+L466T、H26Y+F434Y+L466C、H26Y+F434Y+L466N、H26Y+F434Y+L466Y、H26Y+F434Y+L466D、H26Y+F434Y+L466E、H26Y+F434Y+L466K、H26Y+F434Y+L466R、H26Y+F434Y+L466H、H26Y+F434Y+L466H+H261F、H26Y+F434Y+L466H+H261G、H26Y+F434Y+L466H+H261A、H26Y+F434Y+L466H+H261L、H26Y+F434Y+L466H+H261I、H26Y+F434Y+L466H+H261V、H26Y+F434Y+L466H+H261P、H26Y+F434Y+L466H+H261M、H26Y+F434Y+L466H+H261W、H26Y+F434Y+L466H+H261S、H26Y+F434Y+L466H+H261Q、H26Y+F434Y+L466H+H261T、H26Y+F434Y+L466H+H261C、H26Y+F434Y+L466H+H261N、H26Y+F434Y+L466H+H261Y、H26Y+F434Y+L466H+H261D、H26Y+F434Y+L466H+H261E、H26Y+F434Y+L466H+H261K、H26Y+F434Y+L466H+H261R、H26Y+F434Y+L466H+H461F、H26Y+F434Y+L466H+H461G、H26Y+F434Y+L466H+H461A、H26Y+F434Y+L466H+H461L、H26Y+F434Y+L466H+H461I、H26Y+F434Y+L466H+H461V、H26Y+F434Y+L466H+H461P、H26Y+F434Y+L466H+H461M、H26Y+F434Y+L466H+H461W、H26Y+F434Y+L466H+H461S、H26Y+F434Y+L466H+H461Q、H26Y+F434Y+L466H+H461T、H26Y+F434Y+L466H+H461C、H26Y+F434Y+L466H+H461N、H26Y+F434Y+L466H+H461Y、H26Y+F434Y+L466H+H461D、H26Y+F434Y+L466H+H461E、H26Y+F434Y+L466H+H461K、H26Y+F434Y+L466H+H461R。
进一步地，所述步骤2转氨酶ATA117突变体具有SEQ ID NO:3所示序列发生氨基酸突变的序列，所述发生的氨基酸突变为如下任一种突变：V69F、V69G、V69A、V69L、V69I、V69P、V69M、V69W、V69S、V69Q、V69T、V69C、V69N、V69Y、V69D、V69E、V69K、V69R、V69H、V69A+F122G、V69A+F122A、 V69A+F122L、V69A+F122I、V69A+F122V、V69A+F122P、V69A+F122M、V69A+F122W、V69A+F122S、V69A+F122Q、V69A+F122T、V69A+F122C、V69A+F122N、V69A+F122Y、V69A+F122D、V69A+F122E、V69A+F122K、V69A+F122R、V69A+F122H、V69A+F122C+I157F、V69A+F122C+I157G、V69A+F122C+I157A、V69A+F122C+I157L、V69A+F122C+I157I、V69A+F122C+I157V、V69A+F122C+I157P、V69A+F122C+I157M、V69A+F122C+I157W、V69A+F122C+I157S、V69A+F122C+I157Q、V69A+F122C+I157T、V69A+F122C+I157C、V69A+F122C+I157N、V69A+F122C+I157Y、V69A+F122C+I157D、V69A+F122C+I157E、V69A+F122C+I157K、V69A+F122C+I157R、V69A+F122C+I157H、V69A+F122C+I157H+F225G、V69A+F122C+I157H+F225A、V69A+F122C+I157H+F225L、V69A+F122C+I157H+F225I、V69A+F122C+I157H+F225V、V69A+F122C+I157H+F225P、V69A+F122C+I157H+F225M、V69A+F122C+I157H+F225W、V69A+F122C+I157H+F225S、V69A+F122C+I157H+F225Q、V69A+F122C+I157H+F225T、V69A+F122C+I157H+F225C、V69A+F122C+I157H+F225N、V69A+F122C+I157H+F225Y、V69A+F122C+I157H+F225D、V69A+F122C+I157H+F225E、V69A+F122C+I157H+F225K、V69A+F122C+I157H+F225R、V69A+F122C+I157H+F225H。
进一步地，步骤1和步骤2中，所述Tris-HCl缓冲液的pH为7.5。
进一步地，以pET28a为载体、E.coli BL21为宿主表达SEQ ID NO:1所示序列发生上述氨基酸突变的序列，纯化得到所述大肠杆菌转酮酶突变体。
进一步地，所述转氨酶的生产方法，以pRSFDuet为载体、E.coli BL21为宿主表达SEQ ID NO:3所示序列发生上述氨基酸突变的序列，纯化得到所述转氨酶ATA117突变体。
进一步地，将所述宿主在LB培养基中进行培养，当培养液中OD600达到0.6-0.8时加入0.2mM IPTG诱导表达。
进一步地，培养结束后，收集细胞；将细胞重悬于镍柱结合缓冲液中，进行超声处理，收集上清液，并进行Ni离子亲和层析。
综上，本发明提供了一种利用酶级联反应合成(1R,2R)-AMPP及其衍生物的方法，以廉价的苯甲醛衍生物为原料，通过酶级联反应催化合成了高立体选择性的(1R,2R)-AMPP及其衍生物。
在一些具体的实施方案中，本发明提供了一种利用酶级联反应合成(1R,2R)-AMPP的方法，包括如下步骤：
步骤1、以对甲砜基苯甲醛为底物，转酮酶EcTK1_YYH(即基于SEQ ID NO:1发生氨基酸突变H26Y+F434Y+L466H)为催化剂，得到式5所示的化合物，所述转酮酶EcTK1_YYH具有如SEQ ID NO:5所示的核苷酸序列；
步骤2、以式5所示的化合物为底物，转氨酶ATA117_ACHH(即基于SEQ ID NO:3发生氨基酸突变V69A+F122C+I157H+F225H)为催化剂，得到(1R,2R)-AMPP，所述转氨酶ATA117_ACHH具有如SEQ ID NO:6所示核苷酸序列；
进一步地，步骤1中以对甲砜基苯甲醛为底物，添加羟基丙酮酸锂、硫胺素焦磷酸、MgCl
2以及转酮酶EcTK1_YYH反应得到所述式5所示的化合物。
进一步地，步骤2中以式5所示的化合物为底物，继续添加D-丙氨酸、磷酸吡哆醛、转氨酶ATA117_ACHH、NADH、乳酸脱氢酶、葡萄糖以及葡萄糖脱氢酶继续反应得到所述(1R,2R)-AMPP。
进一步地，步骤1中，在50μl，含100mM的Tris-HCl缓冲液中加入10mM对甲砜基苯甲醛、30mM羟基丙酮酸锂、4.8mM硫胺素焦磷酸、18mM MgCl
2、60μM转酮酶EcTK1_YYH，在25℃下反应1h得到式5所示的化合物。
进一步地，步骤2中，向步骤1反应体系中，加入100mM Tris-HCl缓冲液、200mM D-丙氨酸、2mM磷酸吡哆醛、50μM转氨酶ATA117_ACHH、10mM NADH、90U/ml乳酸脱氢酶、200mM葡萄糖、30U/ml葡萄糖脱氢酶，扩充反应体系至100μl，在25℃下反应3h，得到(1R,2R)-AMPP。
进一步地，步骤1和步骤2中，所述Tris-HCl缓冲液的pH为7.5。
进一步地，以pET28a为载体、E.coli BL21为宿主表达如SEQ ID NO:5所示的核苷酸序列，纯化得到所述转酮酶EcTK1_YYH。
进一步地，所述转氨酶的生产方法，以pRSFDuet为载体、E.coli BL21为宿主表达如SEQ ID NO:6所示核苷酸序列，纯化得到所述转氨酶ATA117_ACHH。
进一步地，将所述宿主在LB培养基中进行培养，当培养液中OD600达到0.6-0.8时加入0.2mM IPTG诱导表达。
进一步地，培养结束后，收集细胞；将细胞重悬于镍柱结合缓冲液中，进行超声处理，收集上清液，并进行Ni离子亲和层析。
综上，本发明提供了一种利用酶级联反应合成(1R,2R)-AMPP的方法，以廉价的对甲砜基苯甲醛为原料，通过酶级联反应催化合成了立体专一性的(1R,2R)-AMPP，产率为76％，产物的立体选择性为96％de，＞99％ee。
图1为以对甲磺砜基苯甲醛为底物的反应产物在C18柱条件下的液相图；
图2为以对甲磺砜基苯甲醛为底物的苏式反应产物在手性液相条件下的液相图；
图3为反应产物(1R,2R)-AMPP的氢谱；
图4为反应产物(1R,2R)-AMPP的碳谱；
图5为以苯甲醛为底物的反应产物在C18柱条件下的液相图，即酶级联催化苯甲醛反应产物的高效液相色谱鉴定；其中，A为酶级联反应的产物的HPLC色谱；B为(1R,2R)-苯基丝氨醇的标准品；C为苏氏和赤式产物的混合物标品。
图6为以对甲基苯甲醛为底物的反应产物在C18柱条件下的液相图，即酶级联催化对甲基苯甲醛反应产物的高效液相色谱鉴定；其中，A为酶级联反应产物的HPLC色谱；B为(1R,2R)-对甲基苯基丝氨醇的标准品；C为苏氏和赤式产物的混合物标品。
以下参考具体实施方式介绍本发明，使其技术内容更加清楚和便于理解。本发明可以通过许多不同形式的实施例来得以体现，本发明的保护范围并非仅限于文中提到的实施例。
本发明提供了一种(1R,2R)-AMPP及其衍生物的合成方法，以苯甲醛衍生物为底物，在大肠杆菌转酮酶突变体的作用下进行转酮反应，得到式2所示的反应产物，接着以式2所示的化合物为底物，在转氨酶ATA117突变体的作用下进行转氨反应，氨基供体为D-丙氨酸、D-甘氨酸、D-缬氨酸、D-亮氨酸、D-异亮氨酸、D-甲硫氨酸、D-脯氨酸、D-色氨酸、D-丝氨酸、D-酪氨酸、D-半胱氨酸、D-苯丙氨酸、D-天冬酰胺、D-谷氨酰胺、D-苏氨酸、D-天冬氨酸、D-谷氨酸、D-赖氨酸、D-精氨酸、D-组氨酸或异丙胺，合成式3所示的(1R,2R)-AMPP及其衍生物。
所涉及的反应式如下：
以下就该方法进行详细阐述：
步骤1、大肠杆菌转酮酶突变体的表达纯化
将SEQ ID NO:2所示序列发生上述氨基酸突变的核苷酸序列导入载体pET28a中，并导入宿主E.coli BL21；随后在LB固体培养基上挑取含重组质粒的大肠杆菌E.coli BL21单菌落，接种至40ml LB液体培养基(含50μg/ml卡那霉素抗生素)，37℃、220rpm培养过夜；将10ml细菌培养液转移至含500ml液体LB培养基的2L摇瓶中，接种两瓶，继续在37℃、220rpm培养至OD600达到0.6-0.8，加入0.2mM IPTG诱导，于30℃、200rpm下诱导培养5h；
培养结束后，收集1L发酵液，于5000rpm，离心20min收集细胞，将收集得到的细胞重悬于30mL镍柱结合缓冲液中，置于冰水混合物中进行超声破碎，超声破碎条件为工作5s，停顿10s，总计30min。
将经过破碎处理后的混合物，于12000rpm离心30min-1h(例如30min或1h)后，上清液过0.22μm滤膜过滤；随后，将过滤后的样品上样经镍柱结合缓冲液预先平衡过的2ml镍填料，用20倍柱体积的50mM咪唑洗脱缓冲液冲洗杂蛋白，然后用5ml 250mM咪唑洗脱缓冲液洗脱目的蛋白，分管接样，每管500μl。测定每管的蛋白浓度，合并浓度较高的几管蛋白液，稀释或者浓缩至2.5ml，上样经甘油缓冲液平衡过的脱盐柱，蛋白液流干后，加入3.5ml甘油缓冲液洗脱蛋白，得到大肠杆菌转酮酶突变体纯蛋白。
步骤1中，蛋白浓度的测定使用Thermo Scientific Nanodrop 8000型检测器检测280nm处的吸光值E，目标蛋白浓度根据摩尔消光系数换算得到，即蛋白浓度(mg/mL)＝E/摩尔消光系数，重组酶的摩尔消光系数通过软件Vector NTI预测获得。
步骤2、转氨酶ATA117突变体的表达纯化
将如SEQ ID NO:4所示序列发生上述氨基酸突变的核苷酸序列导入载体pRSFDuet中，并导入宿主E.coli BL21；随后在LB固体培养基上挑取含重组质粒的大肠杆菌E.coli BL21单菌落，接种至40ml LB液体培养基(含50μg/ml卡那霉素抗生素)，37℃、220rpm培养过夜；将10ml细菌培养液转移至含500ml液体LB培养基的2L摇瓶中，接种两瓶，继续在37℃、220rpm培养至OD600达到0.6-0.8，加入0.2mM IPTG诱导，于20℃，200rpm下诱导培养15h。
培养结束后，收集1L发酵液，于5000rpm，离心20min收集细胞，将收集得到的细胞重悬于30mL镍柱结合缓冲液中，置于冰水混合物中进行超声破碎，超声破碎条件为工作5s，停顿10s，总计30min。
将经过破碎处理后的混合物，于12000rpm离心30min-1h(例如30min或1h)，上清液过0.22μm滤膜过滤；随后，将过滤后的样品上样经镍柱结合缓冲液预先平衡过的2ml镍填料，用10倍柱体积的50mM咪唑洗脱缓冲液冲洗杂蛋白，然后用5ml 250mM咪唑洗脱缓冲液洗脱目的蛋白，分管接样，每管500μl。由于转氨酶结合了辅因子PLP，因此呈现出黄色。合并黄色较深的几管蛋白液，稀释或者浓缩蛋白液至2.5ml，上样经甘油缓冲液平衡过的层析柱，蛋白液流干后，加入3.5ml甘油缓冲液洗脱蛋白，得到转氨酶突变体纯蛋白。
步骤3、合成(1R,2R)-AMPP及其衍生物
在50μl，含100mM的Tris-HCl buffer(pH 7.5)中加入10mM苯甲醛衍生物、30mM羟基丙酮酸锂(LiHPA，作为转酮供体)、4.8mM硫胺素焦磷酸(TPP，作为转酮反应的辅因子)、18mM MgCl
2(作为转酮反应的金属离子)、100μM或60μM大肠杆菌转酮酶突变体，在25℃下反应1-3h；
加入100mM Tris-HCl buffer(pH 7.5)、200mM D-Ala、或D-甘氨酸、或D-缬氨酸、或D-亮氨酸、或D-异亮氨酸、或D-甲硫氨酸、或D-脯氨酸、或D-色氨酸、或D-丝氨酸、或D-酪氨酸、或D-半胱氨酸、或D-苯丙氨酸、或D-天冬酰胺、或D-谷氨酰胺、或D-苏氨酸、或D-天冬氨酸、或D-谷氨酸、或D-赖氨酸、或D-精氨酸、或D-组氨酸、或异丙胺(作为转氨供体)、2mM磷酸吡哆醛(PLP，作为转氨反应的辅因子)、50μM转氨酶ATA117突变体，扩充反应体系至100μl，在25℃下反应3-6h，得到(1R,2R)-AMPP及其衍生物。
表1 级联反应产物的转化率和立体选择性
转化率在0-40％的*，转化率在40-60％的**，转化率在60-80％的***，转化率在>80％的****。
de在0-40％的*，de在40-70％的**，de在70-90％的***，de在>90％的****。
ee在0-40％的*，ee在40-70％的**，ee在70-90％的***，ee在90-99％的****,ee在>99％的*****。
本发明进一步对反应产物在Agilent 1200型液相色谱仪上进行检测，具体检测方法包括：使用C18柱(4.6×150mM，粒径3μm)，柱温30℃，0.5ml/min，A相：H
2O(10mM KH
2PO
4，pH＝8.5)，B相：乙腈。
表2 液相色谱的色谱条件
以下为对实施例5的具体说明：
(1R,2R)-AMPP的合成方法，以对甲砜基苯甲醛为底物，在转酮酶EcTK1_YYH的作用下进行酮基转移反应，得到式5所示的反应产物，接着以式5所示的化合物为底物，在转氨酶ATA117_ACHH的作用下进行氨基转移反应，合成(1R,2R)-1,3-二羟基-2-氨基-1-对甲砜基苯丙烷((1R,2R)-AMPP)，其中，为了加速消耗丙酮酸，加入NADH和乳酸脱氢酶使反应向着合成(1R,2R)-AMPP的方向进行。
所涉及的反应式如下：
以下就该方法进行详细阐述：
步骤1、转酮酶EcTK1_YYH的表达纯化
将如SEQ ID NO:5所示的核苷酸序列导入载体pET28a中，并导入宿主E.coli BL21；随后在LB固体培养基上挑取含重组质粒的大肠杆菌E.coli BL21单菌落，接种至40ml LB液体培养基(含50μg/ml卡那霉素抗生素)，37℃、220rpm培养过夜；将10ml细菌培养液转移至含500ml液体LB培养基的2L摇瓶中，接种两瓶，继续在37℃、220rpm培养至OD600达到0.6-0.8，加入0.2mM IPTG诱导，于30℃、200rpm下诱导培养5h；
培养结束后，收集1L发酵液，于5000rpm，离心20min收集细胞，将收集得到的细胞重悬于30mL镍柱结合缓冲液中，置于冰水混合物中进行超声破碎，超声破碎条件为工作5s，停顿10s，总计30min。
将经过破碎处理后的混合物，于12000rpm离心1h后，上清液过0.22μm滤膜过滤；随后，将过滤后的样品上样经镍柱结合缓冲液预先平衡过的2ml镍填料，用20倍柱体积的50mM咪唑洗脱缓冲液冲洗杂蛋白，然后用5ml 250mM咪唑洗脱缓冲液洗脱目的蛋白，分管接样，每管500μl。测定每管的蛋白浓度，合并浓度较高的几管蛋白液，稀释或者浓缩至2.5ml，上样经甘油缓冲液平衡过的脱盐柱，蛋白液流干后，加入3.5ml甘油缓冲液洗脱蛋白，得到转酮酶EcTK1_YYH，其具有如SEQ ID NO:5所示的核苷酸序列，如SEQ ID NO:7所示的氨基酸序列。
步骤1中，蛋白浓度的测定使用Thermo Scientific Nanodrop 8000型检测器检测280nm处的吸光值E， 目标蛋白浓度根据摩尔消光系数换算得到，即蛋白浓度(mg/mL)＝E/摩尔消光系数，重组酶的摩尔消光系数通过软件Vector NTI预测获得。
步骤2、转氨酶ATA117_ACHH的表达纯化
将如SEQ ID NO:6所示的核苷酸序列导入载体pRSFDuet中，并导入宿主E.coli BL21；随后在LB固体培养基上挑取含重组质粒的大肠杆菌E.coli BL21单菌落，接种至40ml LB液体培养基(含50μg/ml卡那霉素抗生素)，37℃、220rpm培养过夜；将10ml细菌培养液转移至含500ml液体LB培养基的2L摇瓶中，接种两瓶，继续在37℃、220rpm培养至OD600达到0.6-0.8，加入0.2mM IPTG诱导，于20℃，200rpm下诱导培养15h。
培养结束后，收集1L发酵液，于5000rpm，离心20min收集细胞，将收集得到的细胞重悬于30mL镍柱结合缓冲液中，置于冰水混合物中进行超声破碎，超声破碎条件为工作5s，停顿10s，总计30min。
将经过破碎处理后的混合物，于12000rpm离心1h后，上清液过0.22μm滤膜过滤；随后，将过滤后的样品上样经镍柱结合缓冲液预先平衡过的2ml镍填料，用10倍柱体积的25mM咪唑洗脱缓冲液冲洗杂蛋白，然后用5ml 250mM咪唑洗脱缓冲液洗脱目的蛋白，分管接样，每管500μl。由于转氨酶结合了辅因子PLP，因此呈现出黄色。合并黄色较深的几管蛋白液，稀释或者浓缩蛋白液至2.5ml，上样经甘油缓冲液平衡过的层析柱，蛋白液流干后，加入3.5ml甘油缓冲液洗脱蛋白，得到转氨酶ATA117_ACHH，其具有如SEQ ID NO:6所示的核苷酸序列，如SEQ ID NO:8所示的氨基酸序列。
步骤3、合成(1R,2R)-AMPP
在50μl，含100mM的Tris-HCl buffer(pH 7.5)中加入10mM对甲砜基苯甲醛、30mM羟基丙酮酸锂(LiHPA，作为转酮供体)、4.8mM硫胺素焦磷酸(TPP，作为转酮反应的辅因子)、18mM MgCl
2(作为转酮反应的金属离子)、60μM转酮酶EcTK1_YYH，在25℃下反应1h；
扩充反应体系至100μl，加入100mM Tris-HCl buffer(pH 7.5)、200mM D-Ala(作为转氨供体)、2mM磷酸吡哆醛(PLP，作为转氨反应的辅因子)、50μM转氨酶ATA117_ACHH、10mM NADH(作为丙酮酸脱氢反应的氢供体)、90U/ml乳酸脱氢酶(LDH)、200mM葡萄糖、30U/ml葡萄糖脱氢酶(GDH)，在25℃下反应3h，得到(1R,2R)-AMPP。
本发明进一步对反应产物在Agilent 1200型液相色谱仪上进行检测，具体检测方法包括：使用C18柱(4.6×150mm，粒径3μm)，柱温30℃，224nm，0.5ml/min，A相：H
2O(10mM KH
2PO
4，pH8.5)，B相：乙腈，色谱条件见表3：
表3 液相色谱的色谱条件
图1为实施例5以对甲磺砜基苯甲醛为底物的反应产物在C18柱条件下的液相图，图1中，上方为(赤式)-对甲磺砜基苯基丝氨醇和(苏式)-对甲磺砜基苯基丝氨醇标品的液相图(本实施例中具体为(赤式)-1，3-二羟基-2-氨基-1-对甲砜基苯丙烷和(苏式)-1，3-二羟基-2-氨基-1-对甲砜基苯丙烷标品)，中间为(1R,2R)-对甲磺砜基苯基丝氨醇(本实施例中具体为(1R,2R)-1,3-二羟基-2-氨基-1-对甲砜基苯丙烷)的标品，最下方为实施例5得到的反应产物，如图1所示，实施例5制备得到的产物中以苏式为主，并根据公式：de＝[(苏 式-赤式)/(苏式+赤式)]×100％计算得到de>90％(96％)，其中：赤式和苏式代表(赤式)-和(苏式)-对甲磺砜基苯基丝氨醇(本实施例中具体为(赤式)-和(苏式)-1，3-二羟基-2-氨基-1-对甲砜基苯丙烷)。
根据液相出峰，收集(苏式)-对甲磺砜基苯基丝氨醇((苏式)-1，3-二羟基-2-氨基-1-对甲砜基苯丙烷)产物，浓缩，进一步用手性液相柱分离，考察产物的对映选择性。对映异构体分析条件为：手性柱IG柱，柱温25℃，0.5ml/min，224nm，色谱条件：纯甲醇(含0.1％二乙胺)，15min。图2为(苏式)-对甲磺砜基苯基丝氨醇产物在手性液相柱条件下的液相图，图2中，最上方为赤式+苏式标品对照，第二行为苏式标品对照，第三行为(1R,2R)-对甲磺砜基苯基丝氨醇((1R,2R)-AMPP)标品对照，第四行即最后一行为(苏式)-对甲磺砜基苯基丝氨醇((苏式)-AMPP)产物，如图2所示，(1R,2R)-和(1S,2S)-对甲磺砜基苯基丝氨醇((1R,2R)-和(1S,2S)-AMPP)的保留时间分别为5.6和9.4min，根据公式ee＝[(RR-SS)/(RR+SS)]×100％计算ee值大于99％，其中：RR和SS表示(1R,2R)-和(1S,2S)-对甲磺砜基苯基丝氨醇((1R,2R)-和(1S,2S)-1,3-二羟基-2-氨基-1-对甲砜基苯丙烷)。
图3为反应产物(1R,2R)-AMPP的氢谱，图4为反应产物(1R,2R)-AMPP的碳谱，根据图3-4和图1-2的标品对照可知，本发明合成了(1R,2R)-AMPP，结合de值和ee值，本发明提供的方法制备得到的(1R,2R)-AMPP的立体选择性性较高。
图5为实施例9以苯甲醛为底物的反应产物在C18柱条件下的液相图，图5中，上方为实施例9得到的反应产物，中间为(1R,2R)-对甲磺砜基苯基丝氨醇的标品，下方为(赤式)-苯基丝氨醇和(苏式)-苯基丝氨醇标品，如图5所示，实施例9制备得到的产物以苏式为主，并根据公式：de＝[(苏式-赤式)/(苏式+赤式)]×100％计算得到de>90％，其中：赤式和苏式代表(赤式)-和(苏式)-苯基丝氨醇。
图6为实施例13以对甲基苯甲醛为底物的反应产物在C18柱条件下的液相图，图6中，上方为实施例13得到的反应产物，中间为(赤式)-对甲基苯基丝氨醇，下方为(苏式)-对甲基苯基丝氨醇标品，如图6所示，实施例13制备得到的产物以苏式为主，并根据公式：de＝[(苏式-赤式)/(苏式+赤式)]×100％计算得到de>90％，其中：赤式和苏式代表(赤式)-和(苏式)-对甲基苯基丝氨醇。
以上详细描述了本发明的较佳具体实施例。应当理解，本领域的普通技术无需创造性劳动就可以根据本发明的构思作出诸多修改和变化。因此，凡本技术领域中技术人员依本发明的构思在现有技术的基础上通过逻辑分析、推理或者有限的实验可以得到的技术方案，皆应在由权利要求书所确定的保护范围内。
大肠杆菌转酮酶的氨基酸序列(SEQ ID NO:1)
大肠杆菌转酮酶的核苷酸序列(SEQ ID NO:2)
转氨酶ATA117的氨基酸序列(SEQ ID NO:3)
转氨酶ATA117的核苷酸序列(SEQ ID NO:4)
转酮酶EcTK1_YYH的核苷酸序列(SEQ ID NO:5)：
转氨酶ATA117_ACHH的核苷酸序列(SEQ ID NO:6)：
转酮酶EcTK1_YYH的氨基酸序列(SEQ ID NO:7)：
转氨酶ATA117_ACHH的氨基酸序列(SEQ ID NO:8)：
Claims (13)
- 一种利用酶级联反应合成(1R,2R)-AMPP的方法，其特征在于，包括如下步骤：步骤1、以对甲砜基苯甲醛为底物，转酮酶EcTK1_YYH为催化剂，得到式5所示的化合物，所述转酮酶EcTK1_YYH具有如SEQ ID NO:5所示的核苷酸序列；步骤2、以式5所示的化合物为底物，转氨酶ATA117_ACHH为催化剂，得到(1R,2R)-AMPP，所述转氨酶ATA117_ACHH具有如SEQ ID NO:6所示核苷酸序列；
- 如权利要求1所述的方法，其特征在于，步骤1中以对甲砜基苯甲醛为底物，添加羟基丙酮酸锂、硫胺素焦磷酸、MgCl 2以及转酮酶EcTK1_YYH反应得到所述式5所示的化合物。
- 如权利要求2所述的方法，其特征在于，步骤2中以式5所示的化合物为底物，继续添加D-丙氨酸、磷酸吡哆醛、转氨酶ATA117_ACHH、NADH、乳酸脱氢酶、葡萄糖以及葡萄糖脱氢酶继续反应得到所述(1R,2R)-AMPP。
- 如权利要求1所述的方法，其特征在于，步骤1中，在50μl，含100mM的Tris-HCl缓冲液中加入10mM对甲砜基苯甲醛、30mM羟基丙酮酸锂、4.8mM硫胺素焦磷酸、18mM MgCl 2、60μM转酮酶EcTK1_YYH，在25℃下反应1h得到式5所示的化合物。
- 如权利要求4所述的方法，其特征在于，步骤2中，向步骤1的反应体系，加入100mM Tris-HCl缓冲液、200mM D-丙氨酸、2mM磷酸吡哆醛、50μM转氨酶ATA117_ACHH、10mM NADH、90U/ml乳酸脱氢酶、200mM葡萄糖、30U/ml葡萄糖脱氢酶，扩充反应体系至100μl，在25℃下反应3h，得到(1R,2R)-AMPP。
- 如权利要求5所述的方法，其特征在于，步骤1和步骤2中，所述Tris-HCl缓冲液的pH为7.5。
- 如权利要求1所述的方法，其特征在于，以pET28a为载体、E.coli BL21为宿主表达如SEQ ID NO:5所示的核苷酸序列，纯化得到所述转酮酶EcTK1_YYH。
- 如权利要求1所述的方法，其特征在于，所述转氨酶的生产方法，以pRSFDuet为载体、E.coli BL21为宿主表达如SEQ ID NO:6所示核苷酸序列，纯化得到所述转氨酶ATA117_ACHH。
- 如权利要求7或8所述的方法，其特征在于，将所述宿主在LB培养基中进行培养，当培养液中OD600达到0.6-0.8时加入0.2mM IPTG诱导表达。
- 如权利要求9所述的方法，其特征在于，培养结束后，收集细胞；将细胞重悬于镍柱结合缓冲液中，进行超声处理，收集上清液，并进行Ni离子亲和层析。
- 一种利用酶级联反应合成(1R,2R)-苯丝氨醇衍生物的方法，其特征在于，包括如下步骤：步骤1、以苯甲醛衍生物1和羟基丙酮酸为底物，大肠杆菌转酮酶突变体为催化剂，得到(R)-羟酮中间体2；步骤2、以化合物2和氨供体为底物，转氨酶ATA117突变体为催化剂，得到(1R,2R)-苯丝氨醇衍生物3；
- 根据权利要求11所述的方法，其中的醛底物为对甲磺砜苯甲醛，对氟苯甲醛，对氯苯甲醛，对溴苯甲醛，对甲基苯甲醛，对硝基苯甲醛和苯甲醛。
- 根据权利要求11或12所述的方法，其中，氨基供体为D-丙氨酸、D-甘氨酸、D-缬氨酸、D-亮氨酸、D-异亮氨酸、D-甲硫氨酸、D-脯氨酸、D-色氨酸、D-丝氨酸、D-酪氨酸、D-半胱氨酸、D-苯丙氨酸、D-天冬酰胺、D-谷氨酰胺、D-苏氨酸、D-天冬氨酸、D-谷氨酸、D-赖氨酸、D-精氨酸、D-组氨酸和异丙胺。
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| MX2023009215A MX2023009215A (es) | 2021-02-05 | 2022-01-28 | Un metodo para sintetizar (1r,2r)-ampp mediante la reaccion en cascada enzimatica. |
| CN202280019986.XA CN116964212A (zh) | 2021-02-05 | 2022-01-28 | 一种利用酶级联反应合成(1r,2r)-ampp的方法 |
| EP22749127.1A EP4289961A1 (en) | 2021-02-05 | 2022-01-28 | Method for synthesizing (1r,2r)-ampp by using enzyme cascade reaction |
| KR1020237029609A KR20230137995A (ko) | 2021-02-05 | 2022-01-28 | 효소 캐스케이드 반응을 이용한 (1r,2r)-ampp 합성 방법 |
| BR112023015722A BR112023015722A2 (pt) | 2021-02-05 | 2022-01-28 | Método para sintetizar (1r, 2r) - ampp usando a reação em cascata de enzimas e método para sintetizar derivados de (1r,2r)-fenilserina usando uma reação em cascata de enzimas |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101265220A (zh) * | 2008-04-30 | 2008-09-17 | 上海立科药物化学有限公司 | 氟苯尼考的合成方法 |
| CN101941927A (zh) * | 2010-09-28 | 2011-01-12 | 湖北美天生物科技有限公司 | 氟苯尼考中间体(1r,2r)-2-氨基-1–(4-(甲砜基)苯基)-1,3-丙二醇的合成方法 |
| CN102442930A (zh) * | 2011-11-02 | 2012-05-09 | 江苏宇翔化工有限公司 | Dl-对甲砜基苯丝氨酸乙酯的制备方法 |
| CN110747181A (zh) * | 2019-11-27 | 2020-02-04 | 江南大学 | 一种ω-转氨酶突变体及其在生产手性芳香胺中的应用 |
| CN111607544A (zh) * | 2019-02-26 | 2020-09-01 | 韩国科学技术院 | 能够产生邻氨基苯甲酸甲酯的重组微生物和使用其产生邻氨基苯甲酸甲酯的方法 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101265220A (zh) * | 2008-04-30 | 2008-09-17 | 上海立科药物化学有限公司 | 氟苯尼考的合成方法 |
| CN101941927A (zh) * | 2010-09-28 | 2011-01-12 | 湖北美天生物科技有限公司 | 氟苯尼考中间体(1r,2r)-2-氨基-1–(4-(甲砜基)苯基)-1,3-丙二醇的合成方法 |
| CN102442930A (zh) * | 2011-11-02 | 2012-05-09 | 江苏宇翔化工有限公司 | Dl-对甲砜基苯丝氨酸乙酯的制备方法 |
| CN111607544A (zh) * | 2019-02-26 | 2020-09-01 | 韩国科学技术院 | 能够产生邻氨基苯甲酸甲酯的重组微生物和使用其产生邻氨基苯甲酸甲酯的方法 |
| CN110747181A (zh) * | 2019-11-27 | 2020-02-04 | 江南大学 | 一种ω-转氨酶突变体及其在生产手性芳香胺中的应用 |
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| BR112023015722A2 (pt) | 2023-11-07 |
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| KR20230137995A (ko) | 2023-10-05 |
| CL2023002312A1 (es) | 2024-04-26 |
| MX2023009215A (es) | 2023-08-22 |
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