WO2023061237A1 - 转氨酶及其用于制备西他列汀或其中间体的用途 - Google Patents

转氨酶及其用于制备西他列汀或其中间体的用途 Download PDF

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WO2023061237A1
WO2023061237A1 PCT/CN2022/122803 CN2022122803W WO2023061237A1 WO 2023061237 A1 WO2023061237 A1 WO 2023061237A1 CN 2022122803 W CN2022122803 W CN 2022122803W WO 2023061237 A1 WO2023061237 A1 WO 2023061237A1
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amino acid
transaminase
acid residue
acid sequence
amino
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PCT/CN2022/122803
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French (fr)
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田振华
王舒
焦琦
徐晓岚
程占冰
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弈柯莱生物科技(上海)股份有限公司
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    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
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    • 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
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    • C12R2001/19—Escherichia coli

Definitions

  • the invention belongs to the field of biotechnology, and specifically relates to a transaminase, the use of the transaminase for preparing sitagliptin or its intermediate, and a sitagliptin or its intermediate (R)-3-amino-1-
  • Diabetes mellitus is a metabolic disease that occurs due to changes in insulin secretion, leading to insulin deficiency and weakened action, or decreased insulin activity, or under the combined influence of the two. It is characterized by high blood sugar and accompanied by protein, sugar and fat metabolism. disorder. Diabetes and its complications are the third most harmful to human health after cardiovascular diseases and tumors, becoming an important disease that endangers human health. Among the four types of diabetes, type II diabetes accounts for more than 90%, and is more common in middle-aged and elderly people over 30 years old. The cause of disease is mainly due to the body's insensitivity to insulin.
  • Sitagliptin phosphate is the first dipeptidylase-IV (DPP-4) inhibitor approved by the FDA in 2006 for the treatment of type II diabetes. It has obvious hypoglycemic effect when used alone or in combination with metformin and pioglitazone, and is safe to take, with good tolerance and few adverse reactions.
  • DPP-4 dipeptidylase-IV
  • WO2019011236A1 reported that 1-morpholine-4-(2,4,5-trifluorophenyl)-1,3-butanedione (also referred to as morpholinedione in the present invention) can be catalyzed by transaminase (R)-3-amino-1-morpholine-4-(2,4,5-trifluorophenyl)-1-butanone, (R)-3-amino-1-morpholine-4- (2,4,5-trifluorophenyl)-1-butanone can further obtain Boc-(R)-3-amino-4-(2,4,5-trifluorophenyl)butyric acid (abbreviated as Listin-Boc butyric acid), sitagliptin-Boc butyric acid is reacted to prepare sitagliptin phosphate again, but the enzyme activity in the reaction also needs to be further improved.
  • the technical problem to be solved by the present invention is to provide a transaminase and its For the preparation of sitagliptin intermediate or purposes of sitagliptin.
  • the transaminase of the present invention is used to catalyze the morpholino substrate to produce the sitagliptin intermediate or sitagliptin, the conversion rate is high and the stability is good, thereby reducing the production cost and being beneficial to industrial production.
  • the first aspect of the present invention provides a transaminase
  • the amino acid sequence of the transaminase comprises one, two or three residues selected from the amino acid sequence as shown in SEQ ID NO:1 compared with Amino acid residue differences at positions:
  • the 150th amino acid residue is A, L, Q, V, W, T, E or C;
  • the 152nd amino acid residue is A, H, I, K, L, M, N, Q, S, T, V or Y;
  • the 155th amino acid residue is A, K, N or R.
  • the transaminase has S150A/L/Q/V/W/T/E/C, C152A/H/I/K/L/M/N /Q/S/T/V/Y and/or Q155A/K/N/R changes.
  • the change does not necessarily need to be mutated on the basis of SEQ ID NO: 1, as long as the transaminase finally realizes that compared with the amino acid sequence shown in SEQ ID NO: 1, it has S150A/L/Q/V/ Amino acid differences in W/T/E/C, C152A/H/I/K/L/M/N/Q/S/T/V/Y and/or Q155A/K/N/Q/R are also subject to Into the protection scope of the present invention.
  • the amino acid sequence of the transaminase may additionally have 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1 -9, 1-10, 1-11, 1-12, 1-13, 1-14, 1-15, 1-16, 1-17, 1-18, 1-19 or 1-20 amino acid residues differences; differences in these residues include substitutions with conservative amino acid residues. Usually these substitutions do not affect the enzymatic activity of the transaminase.
  • the amino acid sequence of the transaminase comprises amino acid residue differences selected from the following two residue positions compared with the amino acid sequence shown in SEQ ID NO: 1: the 150th amino acid residue is A , L, Q, V or W; the 152nd amino acid residue is L, Q, T, A or S; the 155th amino acid residue is A or R.
  • the amino acid sequence of the transaminase comprises amino acid residue differences selected from the following three residue positions compared with the amino acid sequence shown in SEQ ID NO: 1: the 150th amino acid residue is A, V, W, T, E or C; the 152nd amino acid residue is H, I, K, L, M, N, Q, S, T, V or Y; the 155th amino acid residue is A, K, N or R.
  • the amino acid sequence of the transaminase comprises amino acid residue differences selected from the following two residue positions compared with the amino acid sequence shown in SEQ ID NO: 1: the 150th amino acid residue is A, L, V or W; the 152nd amino acid residue is L, T, A or S.
  • the amino acid sequence of the transaminase comprises amino acid residue differences selected from the following two residue positions compared with the amino acid sequence shown in SEQ ID NO: 1: the 150th amino acid residue is Q or V; the 155th amino acid residue is A or R.
  • the amino acid sequence of the transaminase comprises amino acid residue differences selected from the following two residue positions compared with the amino acid sequence shown in SEQ ID NO: 1: the 152nd amino acid residue is L or Q; the 155th amino acid residue is R.
  • the amino acid sequence of the transaminase comprises amino acid residue differences selected from the following group compared with the amino acid sequence shown in SEQ ID NO: 1, as shown in the following table:
  • Group Amino acid residue positions and differences 1 C152L and Q155R 2 C152Q and Q155R 3 S150T, C152L and Q155R 4 S150W and C152T 5 S150A, C152L and Q155R 6 S150E, C152K and Q155N 7 S150W, C152H and Q155R 8 S150A and C152L 9 S150A, C152L and Q155K 10 S150A, C152T and Q155R 11 S150V and Q155A 12 S150C, C152S and Q155K 13 S150Q and Q155R 14 S150T, C152V and Q155R 15 S150V, C152S and Q155A 16 S150L and C152A 17 S150W, C152M and Q155R
  • the nucleotide sequence encoding the transaminase comprises nucleotide differences selected from the following group compared with the nucleotide sequence shown in SEQ ID NO: 2, as shown in the table below:
  • 152CTG indicates that the codon encoding the 152nd amino acid residue is replaced by TGC to CTG
  • 155CGT indicates that the codon encoding the 155th amino acid residue is replaced by CGT
  • 150ACC indicates that the codon encoding the 150th amino acid residue
  • the codon is replaced by ACC from AGT, and the replacement of other amino acid residue codons in this table is expressed in a similar manner.
  • the second aspect of the present invention provides an isolated nucleic acid encoding the transaminase described in the first aspect of the present invention.
  • the third aspect of the present invention provides a recombinant expression vector, which includes the isolated nucleic acid as described in the second aspect of the present invention.
  • the backbone of the recombinant expression vector is plasmid pET21a.
  • the fourth aspect of the present invention provides a transformant, which is the introduction of the isolated nucleic acid as described in the second aspect of the present invention or the recombinant expression vector as described in the third aspect of the present invention into a host.
  • the host is Escherichia coli; for example, Escherichia coli BL21.
  • the fifth aspect of the present invention provides a method for preparing the transaminase as described in the first aspect of the present invention, the method comprising culturing the transaminase as described in the fourth aspect of the present invention under conditions suitable for expressing the transaminase The transformant.
  • the method specifically includes: (1) inoculating the transformant containing the transaminase into an antibiotic-containing medium such as LB medium for shaking culture to obtain a seed solution; (2) inoculating the transaminase in (1) (3) add IPTG to the culture medium in (2) to induce overnight, and collect the thalli after centrifugation; (4) wash and resuspend (3) ), centrifuge after crushing to obtain the crude enzyme liquid containing the transaminase.
  • the antibiotic may preferably be 50 ⁇ g/mL kanamycin.
  • the shaking culture conditions described in (1) may preferably be 37° C. for 12 hours.
  • the inoculum amount of the seed solution in (2) may preferably be 2% by volume.
  • the shaking culture condition may preferably be 37° C. until the OD 600 is 0.75-0.85 such as 0.8.
  • the final concentration of IPTG in (3) may preferably be 0.05-5mM.
  • the overnight induction temperature may preferably be 15-30°C, for example, 18°C.
  • the conditions of the centrifugation may preferably be 4000-12000 rpm, such as 10000 rpm, and centrifugation for 5-30 min, such as 10 min.
  • the transformant expresses the transaminase
  • it can be extracted by conventional technical means in the art, for example, a crude enzyme solution can be prepared, and after the crude enzyme solution is prepared, conventional concentration and replacement can be carried out, or the crude enzyme solution can be further subjected to ion exchange chromatography One or more of purification steps such as affinity chromatography, hydrophobic chromatography and molecular sieve chromatography to purify the transaminase.
  • the following steps can be used: (1) inoculate the transformant containing the transaminase into an antibiotic-containing medium such as LB medium for shaking culture to obtain a seed solution; (2) inoculate the transaminase in (1) The seed liquid is transferred to an antibiotic-containing medium such as LB medium for shaking culture; (3) Add IPTG to the medium in (2) to induce overnight, and collect the bacteria after centrifugation; (4) Wash and resuspend (3) The thalline collected in the method is crushed and then centrifuged to obtain the crude enzyme solution containing the transaminase.
  • the crude enzyme solution is obtained by homogenizing the bacteria collected after centrifugation and the buffer in a ratio (for example, 1:7).
  • the sixth aspect of the present invention provides an enzyme preparation comprising the transaminase as described in the first aspect of the present invention.
  • the enzyme preparation may generally include a transaminase cofactor such as pyridoxal phosphate.
  • the enzyme preparation can usually be an enzyme preparation obtained from a transaminase-containing transformant host cell obtained in culture or its culture fluid, or a product obtained by processing it; wherein the product refers to An extract obtained from a transformant host cell, an isolated product obtained by isolating or purifying transaminase in the extract, or an immobilized product obtained by immobilizing a transformed cell and its extract or an isolated product of the extract.
  • the seventh aspect of the present invention provides an enzyme combination comprising at least two of the transaminases described in the first aspect of the present invention.
  • the eighth aspect of the present invention provides a method for preparing (R)-3-amino-1-morpholine-4-(2,4,5-trifluorophenyl)-1-butanone , which comprises in the presence of an amino donor, using the transaminase described in the first aspect of the present invention, the enzyme preparation described in the sixth aspect of the present invention or the enzyme combination described in the seventh aspect of the present invention to catalyze the substrate in a reaction solvent 1-morpholine-4-(2,4,5-trifluorophenyl)-1,3-butanedione (referred to as morpholine diketone) to obtain (R)-3-amino-1-morpholine-4- (2,4,5-Trifluorophenyl)-1-butanone step.
  • a reaction solvent 1-morpholine-4-(2,4,5-trifluorophenyl)-1,3-butanedione referred to as morpholine diketone
  • the reaction solvent is isopropanol and water.
  • the amino donor is isopropylamine hydrochloride.
  • the molar ratio of the amino donor to the substrate is 1:1 ⁇ 10:1.
  • the concentration of the substrate is 5-100 g/L such as 10 g/L.
  • the mass ratio of the transaminase to the substrate is 1:1-6:1, such as 3:1; wherein the transaminase exists in the form of bacteria or protein.
  • the prepared reaction system also includes a transaminase cofactor such as pyridoxal phosphate, and its concentration is preferably 0.5-5 mM such as 1 mM.
  • a transaminase cofactor such as pyridoxal phosphate
  • the reaction temperature is 30-60°C, such as 45°C.
  • the rotational speed during the reaction is 100-300 rpm, such as 200 rpm.
  • the transaminase is in the form of crude enzyme solution of transaminase.
  • the preparation of described crude enzyme liquid can comprise the following steps:
  • phosphate buffer 1:7 M/V (g/mL), and centrifuge to obtain it;
  • the phosphate buffer is, for example, 50 mM phosphate buffer, pH 6.0.
  • the ninth aspect of the present invention provides a method for preparing sitagliptin or sitagliptin phosphate, which includes preparing (R)-3 according to the preparation method described in the eighth aspect of the present invention -Amino-1-morpholine-4-(2,4,5-trifluorophenyl)-1-butanone step.
  • the tenth aspect of the present invention provides a transaminase as described in the first aspect of the present invention, the enzyme preparation described in the sixth aspect of the present invention or the enzyme combination described in the seventh aspect of the present invention to prepare Use of (R)-3-amino-1-morpholine-4-(2,4,5-trifluorophenyl)-1-butanone, sitagliptin or sitagliptin phosphate.
  • the sitagliptin phosphate is sitagliptin phosphate monohydrate.
  • the specific structural formula of the 1-morpholine-4-(2,4,5-trifluorophenyl)-1,3-butanedione (also referred to as morpholinodione in the present invention) is as follows :
  • the reagents and raw materials used in the present invention are all commercially available.
  • the positive and progressive effects of the present invention are: when the transaminase of the present invention is used to catalyze ketoamide substrates to produce sitagliptin intermediates, the conversion rate is high, the stability is good, and the stereoselectivity is high, thereby reducing production costs and facilitating industrialization Production.
  • codons corresponding to the amino acids are also conventional in the art, and the corresponding relationship between specific amino acids and codons is shown in Table 2.
  • pET21a was purchased from Novagen; NdeI enzyme and HindIII enzyme were purchased from Thermo Fisher; Escherichia coli BL21 competent cells were purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.
  • Morpholindione reference substance retention time 1.408min
  • the raw material of morpholinodione substrate and the reference substance (R)-3-amino-1-morpholine-4-(2,4,5-trifluorophenyl)-1-butanone were synthesized by our company.
  • the method refers to WO2019011236A1; 3-amino-1-morpholine-4-(2,4,5-trifluorophenyl)-1-butanone racemate is synthesized by the laboratory itself, and aminated by morpholinodione and Prepared by catalytic hydrogenation.
  • Chiral HPLC method detects product ee value method as follows:
  • transaminase gene in Table 3 the whole gene was synthesized.
  • the gene synthesis company is Suzhou Jinweizhi Biotechnology Co., Ltd. (Floor C3, Bio-Nano Technology Park, No. 218, Xinghu Street, Suzhou Industrial Park).
  • the recombinant plasmid is transformed into host Escherichia coli BL21 competent cells to obtain engineering strains containing transaminase Enz.1-Enz.3 genes.
  • transaminases Enz.4-Enz.36 obtained by performing site-directed mutation according to the Enz.1 gene in Table 4, the restriction site NdeI, HindIII, and the vector pET21a were connected to obtain transaminases Enz.4-Enz. Recombinant plasmids for 36 genes. Each recombinant plasmid was transformed into host Escherichia coli BL21 competent cells respectively, and engineering strains Enz.4-Enz.36 containing transaminase genes in Table 4 were obtained.
  • 152CTG in the table indicates that the codon encoding the 152nd amino acid residue is replaced by TGC with CTG
  • 155CGT indicates that the codon encoding the 155th amino acid residue is replaced by CAA with CGT
  • 150ACC indicates the codon encoding the 150th amino acid residue
  • the codons of other amino acid residues in this table are replaced by ACC in a similar manner.
  • Enzyme number Amino acid sequence number (SEQ ID NO:) Nucleotide sequence number (SEQ ID NO:) Enz.1 1 2 Enz.2 3 4 Enz.3 5 6
  • Each transaminase of embodiment 2 is used for catalyzing morpholinodione
  • the thalline that embodiment 1 obtains and the phosphate buffer (pH7.0) of 50mM are homogeneous by 1:7 (M/V), get supernatant and obtain transaminase crude enzyme liquid.
  • the method for detecting the conversion rate refers to the above-mentioned conversion rate UPLC method part. After testing, it was found that the retention times of each substrate and product were consistent with their respective reference substances. The test results are shown in the following tables 6-1, 6-2, 6-3 and 6-4.
  • the method of detecting the ee value refer to the above-mentioned method for detecting the ee value by chiral HPLC. After testing, the ee value of the products catalyzed by each mutant enzyme can reach more than 99%.
  • Enzyme number Amino acid sequence number (SEQ ID NO:) Nucleotide sequence number (SEQ ID NO:) Conversion rate(%) Enz.1 1 2 34.71 Enz.2 3 4 3.82 Enz.3 5 6 1.10

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Abstract

提供一种转氨酶及其用于制备西他列汀或西他列汀中间体的用途。所述转氨酶的氨基酸序列与如SEQ ID NO:1所示的氨基酸序列相比包含一个、两个或三个残基位置处的氨基酸残基差异。还提供了一种编码所述转氨酶的核酸、包括所述核酸的重组表达载体和包含所述核酸或重组表达载体的转化体以及它们的应用。还提供了一种包含所述转氨酶的酶制剂或所述转氨酶的组合。还提供了一种制备（R）-3-氨基-1-吗啉-4-（2,4,5-三氟苯基）-1-丁酮、西他列汀或西他列汀磷酸盐的方法，所述方法使用了所述转氨酶，具有转化率高、稳定性好、立体选择性高，进而降低了生产成本，有利于工业化生产的进步效果。

Description

转氨酶及其用于制备西他列汀或其中间体的用途
本申请要求申请日为2021/10/14的中国专利申请2021111978361的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明属于生物技术领域，具体涉及一种转氨酶、该转氨酶用于制备西他列汀或其中间体的用途，以及一种西他列汀或其中间体(R)-3-氨基-1-吗啉-4-(2,4,5-三氟苯基)-1-丁酮的制备方法。
背景技术
糖尿病是由于胰岛素分泌改变，导致胰岛素缺乏及作用减弱，或者胰岛素活性降低，或者在两者共同影响下，出现的代谢性疾病，以高血糖为特征，并同时伴有蛋白质、糖及脂肪的代谢紊乱。糖尿病及其并发症对人类健康的危害程度居心血管疾病、肿瘤之后的第三位，成为危害人类健康的重要疾病。在糖尿病的四种类型中，II型糖尿病占90％以上，多见于30岁以上的中老年人，病因主要是由于机体对胰岛素不敏感。
西他列汀磷酸盐(Sitagliptin phosphate)是2006年FDA批准上市的第一个二肽基酶-IV(DPP-4)抑制剂，用于治疗II型糖尿病。其单用或与二甲双胍、吡格列酮合用都有明显的降血糖作用，且服用安全，耐受性好，不良反应少。
WO2019011236A1中报道了1-吗啉-4-(2,4,5-三氟苯基)-1,3-丁二酮(本发明中又简称为吗啉双酮)可以经转氨酶(transaminase)催化制得(R)-3-氨基-1-吗啉-4-(2,4,5-三氟苯基)-1-丁酮，(R)-3-氨基-1-吗啉-4-(2,4,5-三氟苯基)-1-丁酮可以进一步得到Boc-(R)-3-氨基-4-(2,4,5-三氟苯基)丁酸(简称西他列汀-Boc丁酸)，西他列汀-Boc丁酸再经反应制得西他列汀磷酸盐，但是反应中酶活还需要进一步提高。
Figure PCTCN2022122803-appb-000001
发明内容
本发明所要解决的技术问题是为了克服现有技术中将转氨酶催化吗啉双酮底物以生产西他列汀中间体或西他列汀时转化率不高等缺陷，提供了一种转氨酶及其用于制备西他列汀中间体或西他列汀的用途。将本发明的转氨酶用于催化吗啉双酮底物以生产西他列汀中间体或西他列汀时转化率高、稳定性好，进而降低了生产成本，有利于工业化生产。
为解决上述技术问题，本发明第一方面提供了一种转氨酶，所述转氨酶的氨基酸序列与如SEQ ID NO:1所示的氨基酸序列相比包含选自以下一个、两个或三个残基位置处的氨基酸残基差异：
第150位氨基酸残基为A、L、Q、V、W、T、E或C；
第152位氨基酸残基为A、H、I、K、L、M、N、Q、S、T、V或Y；
第155位氨基酸残基为A、K、N或R。
换言之，所述转氨酶与如SEQ ID NO:1所示的氨基酸序列相比，具有S150A/L/Q/V/W/T/E/C、C152A/H/I/K/L/M/N/Q/S/T/V/Y和/或Q155A/K/N/R的改变。本发明中，所述改变不一定需要在SEQ ID NO:1的基础上进行突变，只要转氨酶最终实现与如SEQ ID NO:1所示的氨基酸序列相比，具有S150A/L/Q/V/W/T/E/C、C152A/H/I/K/L/M/N/Q/S/T/V/Y和/或Q155A/K/N/Q/R的氨基酸差异，则同样落入本发明保护的范围。
在一些实施方案中，转氨酶的氨基酸序列可在上述的特定位置以外的位置另外具有1-2、1-3、1-4、1-5、1-6、1-7、1-8、1-9、1-10、1-11、1-12、1-13、1-14、1-15、1-16、1-17、1-18、1-19或1-20个氨基酸残基的差异；这些残基的差异包括以保守氨基酸残基取代。通常这些取代不影响所述转氨酶的酶活。
在一些实施方案中，所述转氨酶的氨基酸序列与如SEQ ID NO:1所示的氨基酸序列相比包含选自以下两个残基位置处的氨基酸残基差异：第150位氨基酸残基为A、L、Q、V或W；第152位氨基酸残基为L、Q、T、A或S；第155位氨基酸残基为A或R。
在另一些实施方案中，所述转氨酶的氨基酸序列与如SEQ ID NO:1所示的氨基酸序列相比包含选自以下三个残基位置处的氨基酸残基差异：第150位氨基酸残基为A、V、W、T、E或C；第152位氨基酸残基为H、I、K、L、M、N、Q、S、T、V或Y；第155位氨基酸残基为A、K、N或R。
在一些优选的实施方案中，所述转氨酶的氨基酸序列与如SEQ ID NO:1所示的氨基酸序列相比包含选自以下两个残基位置处的氨基酸残基差异：第150位氨基酸残基为A、 L、V或W；第152位氨基酸残基为L、T、A或S。
在一些优选的实施方案中，所述转氨酶的氨基酸序列与如SEQ ID NO:1所示的氨基酸序列相比包含选自以下两个残基位置处的氨基酸残基差异：第150位氨基酸残基为Q或V；第155位氨基酸残基为A或R。
在一些优选的实施方案中，所述转氨酶的氨基酸序列与如SEQ ID NO:1所示的氨基酸序列相比包含选自以下两个残基位置处的氨基酸残基差异：第152位氨基酸残基为L或Q；第155位氨基酸残基为R。
在一些具体实施方案中，所述转氨酶的氨基酸序列与如SEQ ID NO:1所示的氨基酸序列相比包含选自以下组的氨基酸残基差异，如下表格所示：
组 氨基酸残基位置及差异
1 C152L和Q155R
2 C152Q和Q155R
3 S150T、C152L和Q155R
4 S150W和C152T
5 S150A、C152L和Q155R
6 S150E、C152K和Q155N
7 S150W、C152H和Q155R
8 S150A和C152L
9 S150A、C152L和Q155K
10 S150A、C152T和Q155R
11 S150V和Q155A
12 S150C、C152S和Q155K
13 S150Q和Q155R
14 S150T、C152V和Q155R
15 S150V、C152S和Q155A
16 S150L和C152A
17 S150W、C152M和Q155R
18 S150C、C152L和Q155R
19 S150A、C152I和Q155R
20 S150W、C152I和Q155R
21 S150W、C152Y和Q155R
22 S150V、C152L和Q155R
23 S150V、C152S和Q155K
24 S150W、C152T和Q155K
25 S150C、C152T和Q155K
26 S150W、C152S和Q155K
27 S150V和C152S
28 S150V、C152T和Q155K
29 S150C、C152I和Q155R
30 S150C、C152Q和Q155R
31 S150V、C152I和Q155R
32 S150W、C152Q和Q155R
33 S150C、C152N和Q155R
。
在一些具体实施方案中，编码所述转氨酶的核苷酸序列与如SEQ ID NO:2所示的核苷酸序列相比包含选自以下组的核苷酸差异，如下表格所示：
组 核苷酸残基差异
1 152CTG-155CGT
2 152CAA-155CGT
3 150ACC-152CTG-155CGT
4 150TGG-152ACC
5 150GCG-152CTG-155CGT
6 150GAA-152AAG-155AAC
7 150TGG-152CAT-155CGC
8 150GCG-152CTG
9 150GCG-152CTG-155AAG
10 150GCG-152ACC-155CGT
11 150GTT-155GCT
12 150TGT-152AGC-155AAG
13 150CAA-155CGT
14 150ACC-152GTG-155CGC
15 150GTT-152AGT-155GCT
16 150CTG-152GCA
17 150TGG-152ATG-155CGC
18 150TGC-152CTG-155CGC
19 150GCA-152ATT-155CGC
20 150TGG-152ATT-155CGC
21 150TGG-152TAC-155CGC
22 150GTT-152CTG-155CGC
23 150GTT-152AGC-155AAA
24 150TGG-152ACC-155AAA
25 150TGT-152ACT-155AAA
26 150TGG-152AGT-155AAG
27 150GTT-152TCC-155CAG
28 150GTC-152ACA-155AAG
29 150TGT-152ATT-155CGA
30 150TGC-152CAG-155CGC
31 150GTA-152ATC-155CGG
32 150TGG-152CAA-155AGA
33 150TGC-152AAT-155CGT
。
上表中，152CTG表示编码第152位氨基酸残基的密码子由TGC替换为CTG，155CGT表示编码第155位氨基酸残基的密码子由CAA替换为CGT，150ACC表示编码第150位氨基酸残基的密码子由AGT替换为ACC，在本表中其他的氨基酸残基密码子的替换表示方式与之类似。
为解决上述技术问题，本发明第二方面提供了一种分离的核酸，其编码如本发明第一方面所述的转氨酶。
为解决上述技术问题，本发明第三方面提供了一种重组表达载体，其包括如本发明第二方面所述的分离的核酸。
较佳地，所述重组表达载体的骨架为质粒pET21a。
为解决上述技术问题，本发明第四方面提供了一种转化体，其为在宿主中导入如本发明第二方面所述的分离的核酸或者如本发明第三方面所述的重组表达载体。
较佳地，所述宿主为大肠杆菌(Escherichia coli)；例如大肠杆菌BL21。
为解决上述技术问题，本发明第五方面提供了一种制备如本发明第一方面所述的转氨酶的方法，所述方法包括在适于表达所述转氨酶的条件下培养如本发明第四方面所述的转化体。
在一些实施方案中，所述方法具体包括：(1)将含所述转氨酶的转化体接种至含抗生素的培养基例如LB培养基中振荡培养，得种子液；(2)将(1)中的种子液转接至含抗生素的培养基例如LB培养基中振荡培养；(3)向(2)中的培养基中加入IPTG诱导过夜，离心后收集菌体；(4)洗涤并重悬(3)中收集的菌体，破碎后离心，即得含所述转氨酶的粗酶液。其中，所述抗生素可以优选为50μg/mL卡那霉素。其中，(1)中所述振荡培养的条件可以优选为37℃、12h。其中，(2)中所述种子液的接种量可以优选为2％体积。其中，所述振荡培养的条件可以优选为37℃、直至OD 600为0.75-0.85例如0.8。其中，(3)中所述IPTG的终浓度可以优选为0.05-5mM。其中，所述诱导过夜的温度可以优选为15-30℃例如18℃。其中，所述离心的条件可以优选为4000-12000rpm例如10000rpm、离心5-30min例如10min。
所述转化体表达转氨酶后，可采用本领域常规技术手段进行提取，例如可制备粗酶液，粗酶液制备后可进行常规的浓缩、置换，也可将粗酶液进一步经离子交换层析、亲和层析、疏水层析和分子筛层析等纯化步骤中的一种或多种以提纯所述转氨酶。在一些实施方案中，可采用以下步骤：(1)将含所述转氨酶的转化体接种至含抗生素的培养基例 如LB培养基中振荡培养，得种子液；(2)将(1)中的种子液转接至含抗生素的培养基例如LB培养基中振荡培养；(3)向(2)中的培养基中加入IPTG诱导过夜，离心后收集菌体；(4)洗涤并重悬(3)中收集的菌体，破碎后离心，即得含所述转氨酶的粗酶液。在某一较佳实施例中，采用将离心后收集菌体和缓冲液按比例(例如1:7)均质获得粗酶液。
为解决上述技术问题，本发明第六方面提供了一种酶制剂，其包含如本发明第一方面所述的转氨酶。
较佳地，所述的酶制剂通常可包括转氨酶的辅助因子例如吡哆醛磷酸。本发明中，所述的酶制剂通常可以是从培养物中获得的含转氨酶的转化体宿主细胞或其培养液得到的酶制剂，或者用其加工后得到的制品；其中，所述制品是指由转化体宿主细胞得到的提取物，通过对提取物中的转氨酶进行分离或纯化得到的分离产品，或通过固定化转化体细胞及其提取物或提取物的分离产品而得到的固定化制品。
为解决上述技术问题，本发明第七方面提供了一种酶组合，其包含如本发明第一方面所述的转氨酶中的至少两种。
为解决上述技术问题，本发明第八方面提供了一种制备(R)-3-氨基-1-吗啉-4-(2,4,5-三氟苯基)-1-丁酮的方法，其包括在氨基供体存在时，在反应溶剂中用本发明第一方面所述的转氨酶、本发明第六方面所述的酶制剂或本发明第七方面所述的酶组合来催化底物1-吗啉-4-(2,4,5-三氟苯基)-1,3-丁二酮(简称吗啉双酮)得(R)-3-氨基-1-吗啉-4-(2,4,5-三氟苯基)-1-丁酮的步骤。
较佳地，所述反应溶剂为异丙醇和水。
较佳地，所述氨基供体为异丙胺盐酸盐。
较佳地，所述氨基供体与所述底物的摩尔比为1:1～10:1。
较佳地，所述底物的浓度为5～100g/L例如10g/L。
较佳地，所述转氨酶与所述底物的质量比为1:1～6:1，例如3:1；其中所述转氨酶以菌体或蛋白形式存在。
较佳地，所述制备的反应体系中还包括转氨酶的辅助因子例如吡哆醛磷酸，其浓度优选为0.5～5mM例如1mM。
较佳地，所述反应的温度为30-60℃，例如45℃。
较佳地，所述反应时的转速为100-300rpm，例如200rpm。
在某一较佳实施例中，所述转氨酶为转氨酶的粗酶液形式。所述粗酶液的制备可以包括以下步骤：
将含有转氨酶基因的工程菌在液体培养基例如LB液体培养基中37℃培养至OD600达到0.6～0.8时，加入终浓度为0.5mM的IPTG，20～30℃例如18℃诱导培养16～24h后，将培养液8000～14000rpm例如10000rpm、离心5～30min例如10min收集菌体；
将表达转氨酶的菌体与磷酸缓冲液按1:7M/V(g/mL)均质、离心即得；所述磷酸缓冲液例如为50mM的磷酸缓冲液，pH6.0。
为解决上述技术问题，本发明第九方面提供了一种制备西他列汀或西他列汀磷酸盐的方法，其包括根据如本发明第八方面所述的制备方法制备(R)-3-氨基-1-吗啉-4-(2,4,5-三氟苯基)-1-丁酮的步骤。
为解决上述技术问题，本发明第十方面提供了一种如本发明第一方面所述的转氨酶、本发明第六方面所述的酶制剂或本发明第七方面所述的酶组合来在制备(R)-3-氨基-1-吗啉-4-(2,4,5-三氟苯基)-1-丁酮、西他列汀或西他列汀磷酸盐中的用途。
本发明一些具体实施方案中，所述西他列汀磷酸盐为西他列汀磷酸盐一水合物。
本发明中，所述的1-吗啉-4-(2,4,5-三氟苯基)-1,3-丁二酮(本发明中又简称为吗啉双酮)，具体结构式如下：
Figure PCTCN2022122803-appb-000002
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：将本发明转氨酶用于催化酮酰胺底物以生产西他列汀中间体时转化率高、稳定性好、立体选择性高，进而降低了生产成本，有利于工业化生产。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
本发明中的实验方法如无特别说明均为常规方法，基因克隆操作具体可参加J.萨姆布鲁克等编的《分子克隆实验指南》。
本发明中的氨基酸简写符号如无特殊说明均为本领域常规，具体简写符号对应的氨基酸如表1所示。
表1
Figure PCTCN2022122803-appb-000003
所述氨基酸对应的密码子也为本领域常规，具体氨基酸与密码子的对应关系如表2所示。
表2
Figure PCTCN2022122803-appb-000004
Figure PCTCN2022122803-appb-000005
pET21a购买自Novagen公司；NdeI酶、HindIII酶购买自Thermo Fisher公司，大肠杆菌BL21感受态细胞购买自北京鼎国昌盛生物技术有限责任公司。
转化率UPLC方法：色谱柱：ZORBAX Eclipse plus C18(RRHD，1.8μm，50×2.1mm)；流动相A：0.1％TFA水溶液；流动相B：0.1％TFA甲醇溶液；梯度洗脱：60％A+40％B(0.01min)，30％A+70％B(1.5min)，100％B(1.6min)，100％B(2.0min)，60％A+40％B(2.1min)，60％A+40％B(3.5min)；柱温：35℃；流速：0.5mL/min；进样量：1μL。
吗啉双酮对照品保留时间：1.408min；
(R)-3-氨基-1-吗啉-4-(2,4,5-三氟苯基)-1-丁酮对照品保留时间：0.696min；
其中，吗啉双酮底物原料和对照品(R)-3-氨基-1-吗啉-4-(2,4,5-三氟苯基)-1-丁酮由本公司自行合成，合成方法参考WO2019011236A1；3-氨基-1-吗啉-4-(2,4,5-三氟苯基)-1-丁酮消旋体由实验室自行合成，由吗啉双酮进行氨基化和催化加氢制得。对照品(R)-3-氨基-1-吗啉-4-(2,4,5-三氟苯基)-1-丁酮构型的确定：(R)-3-氨基-1-吗啉-4-(2,4,5-三氟苯基)-1-丁酮可进一步反应制得(3R)-N-叔丁氧羰基-3-氨基-4-(2,4,5-三氟苯基)-丁酸(参考WO2019011236A1)，以(3R)-N-叔丁氧羰基-3-氨基-4-(2,4,5-三氟苯基)-丁酸标准品(采购于安徽海康药业有限责任公司)做对照，可以确定为R构型。
构型确定的HPLC方法：
色谱柱：Daicel Chiralpak AD-H(4.6mm*250mm,5μm)；流动相：正己烷：异丙醇＝90:10；检测波长：210nm；流速：1.0ml/min；进样体积：10μl；柱温：25℃；运行时间：40min。
手性HPLC方法检测产物ee值方法如下：
色谱柱：Daicel ChiralpakAD-H柱4.6mm×250mm，5μm；流动相：正己烷:异丙醇:二乙胺＝40:60:0.1；检测器：UV 268nm；柱温：25℃；流速：0.8mL/min；进样量：10μL。
3-氨基-1-吗啉-4-(2,4,5-三氟苯基)-1-丁酮消旋体对照品保留时间：10.290min和28.087min；
(R)-3-氨基-1-吗啉-4-(2,4,5-三氟苯基)-1-丁酮对照品保留时间：28.093min。
实施例1转氨酶的制备
根据表3中转氨酶基因，全基因进行合成基因SEQ ID NO:2、4、6，酶切位点NdeI、 HindIII，连接载体pET21a，获得含有转氨酶基因的重组质粒。基因合成公司为苏州金唯智生物科技有限公司(苏州工业园区星湖街218号生物纳米科技园C3楼)。重组质粒转化至宿主大肠杆菌BL21感受态细胞，得到含有转氨酶Enz.1～Enz.3基因的工程菌株。
同样地，将表4中根据Enz.1基因进行定点突变得到的转氨酶Enz.4～Enz.36的基因，酶切位点NdeI、HindIII，连接载体pET21a，获得分别含有转氨酶Enz.4～Enz.36的基因的各重组质粒。各重组质粒分别转化至宿主大肠杆菌BL21感受态细胞，得到表4含有转氨酶基因的工程菌株Enz.4～Enz.36。表格中的152CTG表示编码第152位氨基酸残基的密码子由TGC替换为CTG，155CGT表示编码第155位氨基酸残基的密码子由CAA替换为CGT，150ACC表示编码第150位氨基酸残基的密码子由AGT替换为ACC，在本表中其他的氨基酸残基密码子的替换表示方式与之类似。
将上述含有转氨酶基因的工程菌在经平皿划线活化后，挑单菌落接种至含50μg/ml卡那霉素的5ml LB液体培养基中，37℃震荡培养12h。按2％(v/v)接种量转接至150ml同样含50μg/ml卡那霉素的新鲜LB液体培养基中，37℃震荡至OD600达到0.8左右时，加入IPTG至其终浓度为0.5mM，18℃诱导培养16h。培养结束后，将培养液10,000rpm离心10min，弃上清液，收集菌体，置于-80℃超低温冰箱中保存，待用。
表3
酶编号 氨基酸序列编号(SEQ ID NO:) 核苷酸序列编号(SEQ ID NO:)
Enz.1 1 2
Enz.2 3 4
Enz.3 5 6
表4
Figure PCTCN2022122803-appb-000006
Figure PCTCN2022122803-appb-000007
Figure PCTCN2022122803-appb-000008
Enz.1的氨基酸序列(SEQ ID NO:1)
Figure PCTCN2022122803-appb-000009
Enz.1的核苷酸序列(SEQ ID NO:2)
Figure PCTCN2022122803-appb-000010
Enz.2的氨基酸序列(SEQ ID NO:3)
Figure PCTCN2022122803-appb-000011
Enz.2的核苷酸序列(SEQ ID NO:4)
Figure PCTCN2022122803-appb-000012
Figure PCTCN2022122803-appb-000013
Enz.3的氨基酸序列(SEQ ID NO:5)
Figure PCTCN2022122803-appb-000014
Enz.3的核苷酸序列(SEQ ID NO:6)
Figure PCTCN2022122803-appb-000015
实施例2各转氨酶用于催化吗啉双酮
将实施例1得到的菌体和50mM的磷酸缓冲液(pH7.0)按1:7(M/V)均质，取上 清液获得转氨酶粗酶液。
5mL反应体系中(表5)，加入2.5mL异丙醇，50mg吗啉双酮，0.1mL 12.5mg/mL的PLP(吡哆醛磷酸)水溶液，0.2mL 4M异丙胺盐酸盐，0.5mL 1M三乙醇胺，1.1mL粗酶液，45℃、200rpm摇床反应，反应18小时取样进行灭酶处理后离心，取上清液用甲醇稀释20倍后检测转化率和ee值。
其中，检测转化率的方法见上述的转化率UPLC方法部分。经过检测发现，各底物和产物的保留时间与各自的对照品一致。检测结果如下表6-1、6-2、6-3、6-4所示。检测ee值的方法见上述手性HPLC检测ee值的方法，经检测，各突变体酶催化所得产物ee值都能达到99％以上。
表5 5mL反应体系
Figure PCTCN2022122803-appb-000016
表6-1
酶编号 氨基酸序列编号(SEQ ID NO:) 核苷酸序列编号(SEQ ID NO:) 转化率(％)
Enz.1 1 2 34.71
Enz.2 3 4 3.82
Enz.3 5 6 1.10
表6-2第一批酶突变体的转化率
酶编号 氨基酸差异(相对于SEQ ID NO:1) 转化率(％)
Enz.1 / 33.38
Enz.4 C152L-Q155R 48.77
Enz.5 C152Q-Q155R 42.30
Enz.6 S150T-C152L-Q155R 41.27
Enz.7 S150W-C152T 40.84
Enz.8 S150A-C152L-Q155R 49.44
Enz.9 S150E-C152K-Q155N 40.56
Enz.10 S150W-C152H-Q155R 44.33
Enz.11 S150A-C152L 39.35
Enz.12 S150A-C152L-Q155K 41.41
Enz.13 S150A-C152T-Q155R 38.33
Enz.14 S150V-Q155A 38.09
Enz.15 S150C-C152S-Q155K 44.58
Enz.16 S150Q-Q155R 41.96
Enz.17 S150T-C152V-Q155R 31.63
Enz.18 S150V-C152S-Q155A 39.63
Enz.19 S150L-C152A 39.51
表6-3第二批酶突变体的转化率
酶编号 氨基酸差异(相对于SEQ ID NO:1) 转化率(％)
Enz.1 / 33.35
Enz.20 S150W-C152M-Q155R 45.09
Enz.21 S150C-C152L-Q155R 45.85
表6-4第三批酶突变体的转化率
酶编号 氨基酸差异(相对于SEQ ID NO:1) 转化率(％)
Enz.1 / 35.5
Enz.22 S150A-C152I-Q155R 41.71
Enz.23 S150W-C152I-Q155R 47.86
Enz.24 S150W-C152Y-Q155R 36.21
Enz.25 S150V-C152L-Q155R 57.5
Enz.26 S150V-C152S-Q155K 54.74
Enz.27 S150W-C152T-Q155K 58.06
Enz.28 S150C-C152T-Q155K 44.38
Enz.29 S150W-C152S-Q155K 53.76
Enz.30 S150V-C152S-Q155Q 40.56
Enz.31 S150V-C152T-Q155K 51.94
Enz.32 S150C-C152I-Q155R 37.4
Enz.33 S150C-C152Q-Q155R 50.3
Enz.34 S150V-C152I-Q155R 39.63
Enz.35 S150W-C152Q-Q155R 39.48
Enz.36 S150C-C152N-Q155R 40.04
由上表6-2～6-4可知，以上酶突变体的转化率大多都比Enz.1的转化率提高了10％以上。

Claims (13)

  1. 一种转氨酶，其特征在于，所述转氨酶的氨基酸序列与如SEQ ID NO:1所示的氨基酸序列相比包含选自以下一个、两个或三个残基位置处的氨基酸残基差异：
    第150位氨基酸残基为A、L、Q、V、W、T、E或C；
    第152位氨基酸残基为A、H、I、K、L、M、N、Q、S、T、V或Y；
    第155位氨基酸残基为A、K、N或R。
  2. 如权利要求1所述的转氨酶，其特征在于，所述转氨酶的氨基酸序列与如SEQ ID NO:1所示的氨基酸序列相比包含选自以下两个残基位置处的氨基酸残基差异：第150位氨基酸残基为A、L、Q、V或W；第152位氨基酸残基为A、L、Q、T或S；第155位氨基酸残基为A或R；
    或者，所述转氨酶的氨基酸序列与如SEQ ID NO:1所示的氨基酸序列相比包含选自以下三个残基位置处的氨基酸残基差异：第150位氨基酸残基为A、V、W、T、E或C；第152位氨基酸残基为H、I、K、L、M、N、Q、S、T、V或Y；第155位氨基酸残基为A、K、N或R。
  3. 如权利要求1或2所述的转氨酶，其特征在于，所述转氨酶的氨基酸序列与如SEQ ID NO:1所示的氨基酸序列相比包含选自以下两个残基位置处的氨基酸残基差异：第150位氨基酸残基为A、L、V或W；第152位氨基酸残基为A、L、T或S；
    或者，所述转氨酶的氨基酸序列与如SEQ ID NO:1所示的氨基酸序列相比包含选自以下两个的残基位置处的氨基酸残基差异：第150位氨基酸残基为Q或V；第155位氨基酸残基为A或R；
    或者，所述转氨酶的氨基酸序列与如SEQ ID NO:1所示的氨基酸序列相比包含选自以下两个的残基位置处的氨基酸残基差异：第152位氨基酸残基为L或Q；第155位氨基酸残基为R。
  4. 如权利要求1或2所述的转氨酶，其特征在于，所述转氨酶的氨基酸序列与如SEQ ID NO:1所示的氨基酸序列相比包含选自以下组的氨基酸残基差异，如下表格所示：
    组 氨基酸残基位置及差异 1 C152L和Q155R 2 C152Q和Q155R 3 S150T、C152L和Q155R
    4 S150W和C152T 5 S150A、C152L和Q155R 6 S150E、C152K和Q155N 7 S150W、C152H和Q155R 8 S150A和C152L 9 S150A、C152L和Q155K 10 S150A、C152T和Q155R 11 S150V和Q155A 12 S150C、C152S和Q155K 13 S150Q和Q155R 14 S150T、C152V和Q155R 15 S150V、C152S和Q155A 16 S150L和C152A 17 S150W、C152M和Q155R 18 S150C、C152L和Q155R 19 S150A、C152I和Q155R 20 S150W、C152I和Q155R 21 S150W、C152Y和Q155R 22 S150V、C152L和Q155R 23 S150V、C152S和Q155K 24 S150W、C152T和Q155K 25 S150C、C152T和Q155K 26 S150W、C152S和Q155K 27 S150V和C152S 28 S150V、C152T和Q155K 29 S150C、C152I和Q155R
    30 S150C、C152Q和Q155R 31 S150V、C152I和Q155R 32 S150W、C152Q和Q155R 33 S150C、C152N和Q155R
    ；
    较佳地，编码所述转氨酶的核苷酸序列与如SEQ ID NO:2所示的核苷酸序列相比包含选自以下组的核苷酸差异，如下表格所示：
    组 核苷酸残基差异 1 152CTG-155CGT 2 152CAA-155CGT 3 150ACC-152CTG-155CGT 4 150TGG-152ACC 5 150GCG-152CTG-155CGT 6 150GAA-152AAG-155AAC 7 150TGG-152CAT-155CGC 8 150GCG-152CTG 9 150GCG-152CTG-155AAG 10 150GCG-152ACC-155CGT 11 150GTT-155GCT 12 150TGT-152AGC-155AAG 13 150CAA-155CGT 14 150ACC-152GTG-155CGC 15 150GTT-152AGT-155GCT 16 150CTG-152GCA 17 150TGG-152ATG-155CGC 18 150TGC-152CTG-155CGC
    19 150GCA-152ATT-155CGC 20 150TGG-152ATT-155CGC 21 150TGG-152TAC-155CGC 22 150GTT-152CTG-155CGC 23 150GTT-152AGC-155AAA 24 150TGG-152ACC-155AAA 25 150TGT-152ACT-155AAA 26 150TGG-152AGT-155AAG 27 150GTT-152TCC-155CAG 28 150GTC-152ACA-155AAG 29 150TGT-152ATT-155CGA 30 150TGC-152CAG-155CGC 31 150GTA-152ATC-155CGG 32 150TGG-152CAA-155AGA 33 150TGC-152AAT-155CGT
    。
  5. 一种分离的核酸，其编码如权利要求1-4任一项所述的转氨酶。
  6. 一种重组表达载体，其包括如权利要求5所述的分离的核酸；
    较佳地，所述重组表达载体的骨架为质粒pET21a。
  7. 一种转化体，其为在宿主中导入如权利要求5所述的分离的核酸或者如权利要求6所述的重组表达载体；
    较佳地，所述宿主为大肠杆菌；例如大肠杆菌BL21。
  8. 一种制备如权利要求1-4任一项所述的转氨酶的方法，其特征在于，所述方法包括在适于表达所述转氨酶的条件下培养如权利要求7所述的转化体。
  9. 一种酶制剂，其包含如权利要求1-4任一项所述的转氨酶；
    较佳地，所述酶制剂包括转氨酶的辅助因子例如吡哆醛磷酸。
  10. 一种酶组合，其包含如权利要求1-4任一项所述的转氨酶中的至少两种。
  11. 一种制备(R)-3-氨基-1-吗啉-4-(2,4,5-三氟苯基)-1-丁酮的方法，其包括在氨基供体 存在时，在反应溶剂中用如权利要求1-4任一项所述的转氨酶、权利要求9所述的酶制剂或权利要求10所述的酶组合来催化底物1-吗啉-4-(2,4,5-三氟苯基)-1,3-丁二酮得(R)-3-氨基-1-吗啉-4-(2,4,5-三氟苯基)-1-丁酮的步骤；
    较佳地：所述反应溶剂为异丙醇和水；
    和/或，所述氨基供体为异丙胺盐酸盐；
    和/或，所述氨基供体与所述底物的摩尔比为1:1～10:1；
    和/或，所述底物的浓度为5～100g/L例如10g/L；
    和/或，所述转氨酶与所述底物的质量比为1:1～6:1，例如3:1；其中所述转氨酶以菌体或蛋白形式存在；
    和/或，所述制备的反应体系中还包括转氨酶的辅助因子例如吡哆醛磷酸，其浓度优选为0.5～5mM例如1mM；
    和/或，所述反应的温度为30-60℃，例如45℃；
    和/或，所述反应的转速为100-300rpm，例如200rpm。
  12. 一种制备西他列汀或西他列汀磷酸盐的方法，其包括根据如权利要求11所述的方法制备(R)-3-氨基-1-吗啉-4-(2,4,5-三氟苯基)-1-丁酮的步骤。
  13. 一种如权利要求1-4任一项所述的转氨酶、权利要求9所述的酶制剂或权利要求10所述的酶组合在制备(R)-3-氨基-1-吗啉-4-(2,4,5-三氟苯基)-1-丁酮、西他列汀或西他列汀磷酸盐中的用途。
PCT/CN2022/122803 2021-10-14 2022-09-29 转氨酶及其用于制备西他列汀或其中间体的用途 WO2023061237A1 (zh)

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