CN-122012475-A - Amide hydrolase mutant, biological material, screening method, catalyst and application

Abstract

The invention discloses an amidohydrolase mutant, biological material, screening method, catalyst and application, wherein the amidohydrolase mutant has 317 th or 317 th and 63 th amino acid residue mutation or 317 th, 63 th and 65 th amino acid residue mutation compared with wild amidohydrolase. Compared with wild type amide hydrolase, the catalytic efficiency of the mutant can reach 16 times at most, the yield of catalyzing isobutyl glutarimide to generate R-isobutyl glutarimide reaches 99%, the stereoselectivity ee value is more than 99%, the reaction condition is mild, the reaction can be carried out only at 20-45 ℃, and the mutant has higher industrial application value.

Inventors

• REN XINKUN
• WANG JIAN
• QU ZHAN
• CAO JIANAN
• WANG ZHE
• MENG SHUANGHE
• WU JIAXIN

Assignees

• 南京大学

Dates

Publication Date

20260512

Application Date

20251231

Claims (10)

1. 1. An amidase mutant, characterized by having a mutation of amino acid residue 317, or a mutation of amino acid residues 317 and 63, or a mutation of amino acid residues 317, 63 and 65 based on the amino acid sequence of the wild type amidase shown in SEQ ID NO. 1.
2. 2. The amidohydrolase mutant of claim 1, wherein, The 317 th amino acid residue mutated protein is obtained by mutating a cysteine residue into any one of a glycine residue, an alanine residue, a valine residue, a leucine residue, an isoleucine residue, a methionine residue, a proline residue, a tryptophan residue, a serine residue, a tyrosine residue, a phenylalanine residue, an asparagine residue, a glutamine residue, a threonine residue, an aspartic acid residue, a glutamic acid residue, a lysine residue, an arginine residue, and a histidine residue; The 317 th and 63 th amino acid residues are mutated by mutating 317 th cysteine residue to tyrosine residue, and 63 th methionine residue is mutated to any one amino acid residue of glycine residue, alanine residue, valine residue, leucine residue, isoleucine residue, proline residue, tryptophan residue, serine residue, tyrosine residue, cysteine residue, phenylalanine residue, asparagine residue, glutamine residue, threonine residue, aspartic acid residue, glutamic acid residue, lysine residue, arginine residue and histidine residue; The 317, 63 and 65 amino acid residues are obtained by mutating 317 th cysteine residue to tyrosine residue, mutating 63 th methionine residue to tyrosine residue, and mutating 65 th phenylalanine residue to any one of glycine residue, alanine residue, valine residue, leucine residue, isoleucine residue, methionine residue, proline residue, tryptophan residue, serine residue, tyrosine residue, cysteine residue, asparagine residue, glutamine residue, threonine residue, aspartic acid residue, glutamic acid residue, lysine residue, arginine residue and histidine residue.
3. 3. The amidohydrolase mutant of claim 2, wherein, The 317 th amino acid residue mutated protein is obtained by mutating a cysteine residue into a tyrosine residue; The 317 th and 63 th amino acid residues are mutated proteins, which are obtained by mutating 317 th cysteine residue into tyrosine residue and mutating 63 rd methionine residue into tyrosine residue; The 317 th, 63 rd and 65 th amino acid residues are mutated proteins, which are obtained by mutating 317 th cysteine residue to tyrosine residue and 63 rd methionine residue to tyrosine residue and 65 th phenylalanine residue to serine residue, and the amino acid sequence is shown as SEQ ID NO. 3.
4. 4. A nucleic acid molecule, characterized in that it comprises a corresponding base mutation based on the wild-type amidase nucleic acid molecule as shown in sequence SEQ ID No. 2, encoding an amino acid sequence of the amidase mutant according to any one of claims 1-3.
5. 5. A recombinant vector comprising the nucleic acid molecule of claim 4.
6. 6. A recombinant microorganism comprising the nucleic acid molecule of claim 4 or the recombinant vector of claim 5.
7. 7. A method for screening an amidohydrolase mutant according to any one of claims 1 to 3, comprising: (1) Taking the three-dimensional structure of the wild type amidohydrolase as a template, performing visual analysis, and screening key sites influencing enzyme activity and stereoselectivity; (2) Respectively carrying out saturation mutation on the key sites obtained by screening, and screening to obtain preferred mutation sites; (3) Taking the preferable mutation site obtained in the step 2 as a fixed mutation site, carrying out saturation mutation on the rest key sites obtained in the step 1, and screening to obtain the preferable mutation site; (4) And (3) taking the preferable mutation sites obtained in the steps (2) and (3) as fixed mutation sites, carrying out saturation mutation on the rest key sites obtained in the step (1), and screening to obtain the preferable mutation sites, wherein the preferable mutation sites are used as amide hydrolase mutants.
8. 8. A catalyst, which is characterized in that, the catalyst comprising the amidohydrolase mutant of any one of claims 1-3.
9. 9. Use of an amidohydrolase mutant according to any of claims 1 to 3 or a catalyst according to claim 8 for the synthesis of (R) - (-) -3- (carbamoylmethyl) -5-methylhexanoic acid.
10. 10. The use according to claim 9, wherein the reaction temperature of the use is 20-45 ℃ and the reaction pH is 7-9.

Description

Amide hydrolase mutant, biological material, screening method, catalyst and application Technical Field The invention relates to an amidohydrolase, in particular to an amidohydrolase mutant, a biological material, a screening method, a catalyst and application. Background Pregabalin is a gamma-aminobutyric acid (GABA) analogue, has antiepileptic, analgesic and anxiolytic activities, can effectively relieve pain due to high clinical use safety, and is widely applied to the treatment of neuropathic pain, epilepsy and other diseases. In 2021, pregabalin was included in the fourth national drug set for centralized purchase due to its large usage. According to the analysis of Data Bridge MARKET RESEARCH, the global pregabalin market size of 2023 was about $8.2 billion, and it was expected that $10.9 billion would be reached in 2031 with a annual compound growth rate of 3.53%. As market demand continues to rise, efficient synthesis of pregabalin is becoming a research hotspot. Currently, the synthetic methods of pregabalin mainly comprise a chemical method and a biological enzyme method. The chemical process is relatively mature, for example, cyclic imine is generated by the reaction of 2-cyanoacetamide and isovaleraldehyde, R/S-mixed monoamide is obtained by alkaline hydrolysis, R- (+) -1-phenethylamine is used for chiral resolution in chloroform to obtain R-monoamide, and S-pregabalin is finally synthesized by Hofmann rearrangement reaction. However, this method has significant drawbacks including the use of large amounts of organic solvents, severe environmental pollution, the racemic product and the need for chiral resolution, resulting in further reduced yields. Therefore, how to directly and efficiently synthesize chiral intermediates becomes a technical problem to be solved. Compared with the chemical method, the biological enzyme method has the advantages of high chiral selectivity, environmental protection and the like, and is widely paid attention to. The biological enzyme method can accurately catalyze and synthesize the chiral intermediate (R) - (-) -3- (carbamoylmethyl) -5-methylhexanoic acid of pregabalin, thereby avoiding the problem of low efficiency caused by chiral resolution links in the traditional chemical method and meeting the development requirement of green low carbon. Therefore, the chiral drug intermediate synthesis technology based on biological enzyme is considered as the core development direction of efficient synthesis of pregabalin, and has great application value and prospect. However, the existing biological enzyme method has higher reaction temperature and limits the application to a certain extent. Disclosure of Invention The invention aims to provide an amidohydrolase mutant with mild reaction conditions, a second aim to provide biological materials related to the mutant and a screening method, and a third aim to provide application of the mutant in catalytic synthesis of (R) - (-) -3- (carbamoylmethyl) -5-methylhexanoic acid. The technical scheme is that the amidohydrolase mutant has 317 th amino acid residue mutation, 317 th and 63 th amino acid residue mutation, or 317 th, 63 rd and 65 th amino acid residue mutation protein based on the amino acid sequence of the wild amidohydrolase shown in SEQ ID NO. 1. Preferably, the amidohydrolase mutant comprises: A protein in which the 317 th amino acid residue is mutated, which is obtained by mutating a cysteine residue to any one of a glycine residue, an alanine residue, a valine residue, a leucine residue, an isoleucine residue, a methionine residue, a proline residue, a tryptophan residue, a serine residue, a tyrosine residue, a phenylalanine residue, an asparagine residue, a glutamine residue, a threonine residue, an aspartic acid residue, a glutamic acid residue, a lysine residue, an arginine residue, and a histidine residue; a protein in which amino acid residues 317 and 63 are mutated, which is obtained by mutating a cysteine residue 317 to a tyrosine residue and mutating a methionine residue 63 to any one of a glycine residue, an alanine residue, a valine residue, a leucine residue, an isoleucine residue, a proline residue, a tryptophan residue, a serine residue, a tyrosine residue, a cysteine residue, a phenylalanine residue, an asparagine residue, a glutamine residue, a threonine residue, an aspartic acid residue, a glutamic acid residue, a lysine residue, an arginine residue and a histidine residue; Proteins in which amino acid residues 317, 63 and 65 are mutated are obtained by mutating a cysteine residue 317 to a tyrosine residue, mutating a methionine residue 63 to a tyrosine residue, and mutating a phenylalanine residue 65 to any one of a glycine residue, an alanine residue, a valine residue, a leucine residue, an isoleucine residue, a methionine residue, a proline residue, a tryptophan residue, a serine residue, a tyrosine residue, a cysteine residue, an asparagine residue, a glutamine residue, a threonine residue, a