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CN-121991913-A - Reductive amino enzyme mutant and application thereof

CN121991913ACN 121991913 ACN121991913 ACN 121991913ACN-121991913-A

Abstract

The invention discloses a reductive amination enzyme mutant and application thereof. The present invention provides a reductive amino acid enzyme mutant comprising amino acid residue differences in one or more of positions 49, 114, 120, 130, 207 and 237 as compared to the amino acid sequence shown in SEQ ID NO. 2. The mutant provided by the invention synthesizes chiral amine compounds, has mild reaction conditions, does not generate harmful byproducts, is environment-friendly, and can reach higher conversion rate and selectivity on the preparation scale.

Inventors

  • HAN SHURAN
  • LIU MEIQI
  • ZHOU DAN
  • LI JIE
  • SUN FENGLAI

Assignees

  • 上海合全药物研发有限公司

Dates

Publication Date
20260508
Application Date
20241104

Claims (11)

  1. 1. A mutant of a reducing amino acid enzyme, characterized in that the mutant of a reducing amino acid enzyme comprises one or more amino acid residue differences at positions 49, 114, 120, 130, 207 and 237 compared to the amino acid sequence shown in SEQ ID NO. 2.
  2. 2. The reductive amination enzyme mutant according to claim 1, comprising one or more of the amino acid residue differences selected from the group consisting of H49K, H114D, T120S, L C, A207L, A compared to the amino acid sequence shown in SEQ ID NO. 2; Preferably, the reductive amino acid enzyme mutant comprises the following combination of amino acid residue differences A207L compared to the amino acid sequence shown in SEQ ID NO. 2; Further preferably, the reductive amino acid enzyme mutant further comprises one or more of the following amino acid residue differences, preferably in combination with H114D/T120S/A237I, H49K, H114D, T120S, L C and A237I, compared to the amino acid sequence as shown in SEQ ID NO. 2; More preferably, the reductive amino acid enzyme mutant further comprises amino acid residue differences L130C and/or H49K, as compared to the amino acid sequence shown in SEQ ID NO. 2; Even more preferably, the amino acid sequence of the reductive amination mutant is shown as SEQ ID NO. 3, 4, 5 or 6.
  3. 3. An isolated nucleic acid encoding the reductive amino enzyme mutant of claim 1 or 2.
  4. 4. A recombinant expression vector comprising the isolated nucleic acid of claim 3, preferably wherein the backbone of the recombinant expression vector is pET-30a.
  5. 5. A transformant comprising the isolated nucleic acid according to claim 3 or the recombinant expression vector according to claim 4, preferably a host cell selected from the group consisting of E.coli cells, insect cells, yeast cells and mammalian cells, preferably E.coli, such as BL21 (DE 3), used in the construction of the transformant.
  6. 6. A method for producing a reductive amination mutant comprising culturing the transformant according to claim 5, to obtain the reductive amination mutant.
  7. 7. An enzyme preparation comprising a reductive amino enzyme mutant according to claim 1 or 2.
  8. 8. An enzyme composition comprising a reductive amination enzyme or a reductive amination enzyme mutant according to claim 1 or 2, and a coenzyme and a glucose dehydrogenase; Wherein the coenzyme is preferably nicotinamide adenine dinucleotide phosphate or nicotinamide adenine dinucleotide; The reducing amino enzyme is preferably derived from Aspergillus kawachii (Aspergillus udagawae); for example, the amino acid sequence of the reducing amino enzyme is selected from the group consisting of: (1) As shown in SEQ ID NO. 2; (2) Has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No. 2; Preferably, the amino acid sequence of the glucose dehydrogenase is preferably SEQ ID NO 7 or 8; More preferably, the mass ratio of the reduced amino enzyme or reduced amino enzyme mutant, coenzyme and glucose dehydrogenase is (10-30): (0.2-1.2): (1-6): e.g. 10:0.4:2, 30:1.2:6, 12:1.2:6, 10:0.2:1 or 15:0.3:3.
  9. 9. Use of a reductive amidase, a reductive amidase mutant according to claim 1 or 2, an isolated nucleic acid according to claim 3, a recombinant expression vector according to claim 4, a transformant according to claim 5, an enzyme preparation according to claim 7 or an enzyme composition according to claim 8 for reducing a compound I according to the following structure I to a compound II according to the following structure II; ; Wherein R 1 is selected from hydrogen and hydroxy, R 2 is selected from methyl, ethyl, propyl, tert-butyl, phenyl, benzyl and thiophene; preferably, the application satisfies one or more of the following conditions: (1) The reducing amino enzyme is derived from Aspergillus awamori (Aspergillus udagawae), for example, the amino acid sequence of the reducing amino enzyme is selected from the group consisting of: (a) As shown in SEQ ID NO. 2; (b) Has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No. 2; (2) The compound I is any one of the following compounds: 、 、 、 、 、 、 、 、 And ; (3) The compound II is any one of the following compounds: 、 、 、 、 、 、 、 、 And 。
  10. 10. A process for preparing a compound of formula II, comprising the steps of contacting and reacting an amino donor with a compound of formula I using a reductive amino enzyme, a reductive amino enzyme mutant according to claim 1 or 2, an enzyme preparation according to claim 7 or an enzyme composition according to claim 8 to obtain a compound of formula II; ; Wherein R 1 is selected from hydrogen and hydroxy, R 2 is selected from methyl, ethyl, propyl, tert-butyl, phenyl, benzyl and thiophene; Preferably, the amino donor is an ammonium salt solution, such as an ammonium chloride buffer, and the molar ratio of the ammonium chloride to the compound of formula I is preferably (10-15): 1, more preferably 12:1; And/or the mass ratio of the reduced aminopeptidase or reduced aminopeptidase mutant to the compound of formula I is preferably (0.01-10): 1, more preferably (0.5-2): 1, e.g. 0.5:1, 0.2:1, 1:1 or 0.16:1; And/or, the compound I and the compound II are as claimed in claim 9; And/or, the reducing amino enzyme is from aspergillus kawachii (Aspergillus udagawae), preferably the amino acid sequence of the reducing amino enzyme is selected from the following group: (1) As shown in SEQ ID NO. 2; (2) Has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 2.
  11. 11. The method according to claim 10, wherein the method comprises the steps of contacting and reacting an amino donor with a compound represented by formula I in a solvent in the presence of a coenzyme, glucose dehydrogenase and D-glucose using the reductive amidase or the reductive amidase mutant to produce a compound represented by formula II; preferably, the preparation method is selected from one or more of the following conditions: (1) The pH of the reaction is 7-11, preferably 8.5; (2) The coenzyme is nicotinamide adenine dinucleotide phosphate or nicotinamide adenine dinucleotide, preferably, the coenzyme is nicotinamide adenine dinucleotide phosphate; (3) The solvent is an organic solvent such as dimethyl sulfoxide; (4) The mass volume ratio of the compound shown in the formula I to the solvent is 0.1-1 mg/mu L, preferably 0.2 mg/mu L; (5) The mass ratio of the coenzyme to the compound of formula I is (0.01-0.5): 1, preferably (0.05-0.1): 1, for example 0.02:1 or 0.1:1; (6) The mass ratio of the glucose dehydrogenase to the compound of formula I is (0.01-0.5): 1, preferably (0.05-0.1): 1, for example 0.1:1; (7) The mass ratio of D-glucose to the compound of formula I is (0.01-10): 1, preferably (1-6): 1, for example 3:1; (8) The reaction temperature is 20-35 ℃, preferably 30 ℃; (9) The amino acid sequence of the glucose dehydrogenase is shown as SEQ ID NO. 7 or 8, and the mass ratio of the reducing amino enzyme or the reducing amino enzyme mutant, the coenzyme and the glucose dehydrogenase is preferably (10-30): (0.2-1.2): (1-6): e.g. 10:0.4:2, 30:1.2:6, 12:1.2:6, 10:0.2:1 or 15:0.3:3; (10) The preparation method further comprises the steps of adding methanol to terminate the reaction after the reaction liquid is subjected to shaking table reaction overnight, adjusting the pH of the reaction liquid to 8.0-10.0 (preferably 9.0), centrifuging, and passing through a membrane to obtain the reaction liquid, and detecting by a high-efficiency gas chromatograph.

Description

Reductive amino enzyme mutant and application thereof Technical Field The invention relates to a reductive amination enzyme mutant and application thereof, belonging to the field of biotechnology and medical intermediate synthesis. Background Chiral amines are common building blocks in the pharmaceutical industry and can be used to obtain large amounts of biologically active compounds. They can be used in different applications, from the development of small molecule drugs to more complex therapeutic methods, for example for proteolytic chimeras or for biological binding of peptides or proteins. The pharmaceutical industry is continually looking for innovative ways to produce new bioactive compounds, which increases the need for new building blocks in drug design. The structural diversity of such compounds is advantageous for studying drug properties such as permeability, potency or secondary pharmacology. Current synthetic methods can widely cover available chemical space, but still have the opportunity to develop new building blocks. Biocatalysis is a powerful synthetic method that can be used to obtain new chemicals by exploiting mild reaction conditions and the catalytic nature of the conversion, and has high stereoselectivity driven by precise enzyme/substrate interactions. Importantly, biocatalytic synthesis can play a key role in drug discovery, as enzymes are tunable catalysts that can provide an emerging platform for excellent control of chemoselectivity, regioselectivity, and biocatalysis. Here, the present invention has found and evolved a reductive amino enzyme mutant and is useful for the synthesis of 4-substituted-cyclohexylamines. Disclosure of Invention In order to solve the technical problem that the prior art lacks a method for preparing chiral amine compounds by using a biological enzyme catalysis method, the invention provides a reductive amino enzyme mutant and application thereof. The invention adopts the reductive amination mutant to catalyze and synthesize the chiral amine compound for the first time, has mild reaction conditions, does not generate harmful byproducts, is environment-friendly, and can reach higher conversion rate and selectivity on the preparation scale. The technical problems are solved by the following technical scheme. In a first aspect the invention provides a reductive amino acid enzyme mutant comprising amino acid residue differences in one or more of positions 49, 114, 120, 130, 207 and 237 compared to the amino acid sequence as shown in SEQ ID NO. 2. In some embodiments of the invention, the reductive amino acid enzyme mutants comprise one or more amino acid residue differences selected from the group consisting of H49K, H114,114, 114D, T120,120, 120S, L130, 130C, A207,207, 207L, A237,237I, as compared to the amino acid sequence shown in SEQ ID NO. 2. In some embodiments of the invention, the reductive amino acid enzyme mutant comprises an amino acid residue difference of A207L, as compared to the amino acid sequence shown in SEQ ID NO. 2; Preferably, the reductive amino acid enzyme mutant further comprises one or more of the following amino acid residue differences, H49K, H114,114, 114D, T120, 120S, L130C and A237I, preferably further comprises the following amino acid residue difference combination, H114D/T120S/A237I, compared to the amino acid sequence as shown in SEQ ID NO. 2; more preferably, the reductive amino acid enzyme mutant further comprises an amino acid residue difference of L130C and/or H49K as compared to the amino acid sequence shown in SEQ ID NO. 2. In some embodiments of the invention, the amino acid sequence of the reductive amino acid enzyme mutant is as shown in SEQ ID NO. 3, 4, 5 or 6. In a second aspect the invention provides an isolated nucleic acid encoding a reductive amino enzyme mutant according to the first aspect of the invention. In a third aspect the invention provides a recombinant expression vector comprising an isolated nucleic acid according to the second aspect of the invention. In some embodiments of the invention, the backbone of the recombinant expression vector is pET-30a. In a fourth aspect the invention provides a transformant comprising an isolated nucleic acid according to the second aspect of the invention or a recombinant expression vector according to the third aspect of the invention. In some embodiments of the invention, the host cell used in the construction of the transformant is selected from the group consisting of E.coli cells, insect cells, yeast cells and mammalian cells, preferably E.coli, such as BL21 (DE 3). In a fifth aspect the present invention provides a method of preparing a reductive amination mutant comprising culturing a transformant according to the fourth aspect of the present invention to obtain the reductive amination mutant. In a sixth aspect the invention provides an enzyme preparation comprising a reductive amino enzyme mutant according to the first aspect of the invention. In a sevent