Amidase mutant and its application

Abstract

The present invention provides an amidase mutant and its application, including: (a) a protein having an amino acid sequence shown in SEQ ID NO: 1; or (b) a protein having an amidase function after amino acid mutation at least one of the following sites: L81, E62, M311, N180, P121, R58, E32, T230, N82, N190, D192, D217, H219, K77, M78, P79, F80, F61, D185, D59, L182, T183, P184, I227, C228, G229, A231 or V232 of the amino acid sequence shown in (a); or (c) a protein having an amidase function with a homology of more than 80% with the amino acid sequence defined in any one of (a) and (b). The amidase mutant can solve the problem of low stereoselectivity and catalytic activity of amidase in the prior art, and is applicable to the field of enzyme catalysis.

Description

Technical Field

[0001] The present invention relates to the field of enzyme catalysis, and in particular to an amidase mutant and application thereof. Background Art

[0002] Chiral carboxylic acids and amide derivatives are key intermediates for synthesizing a large number of chiral bioactive molecules and are widely used in fine chemicals, medicine, agricultural chemicals and functional materials. The chemical synthesis of chiral carboxylic acids and amide derivatives has limitations such as low total product yield, poor selectivity and high synthesis process cost, which is not suitable for industrial-scale production.

[0003] Amidase (EC 3.5.1.X) is an important hydrolase that catalyzes the hydrolysis of amide compounds to produce carboxylic acids and ammonia. The substrate spectrum of amidase is wide, covering aliphatic, aromatic, heterocyclic compounds, etc.; amidase generally has good stereoselectivity, among which S-type amidase is the most common, but R-type amidase has also been continuously discovered in recent years. These characteristics of amidase give it an incomparable advantage in the kinetic resolution of racemic amides to prepare chiral carboxylic acids and chiral amide derivatives, and it has attracted more and more attention from researchers. A series of new biosynthetic processes for synthesizing chiral carboxylic acids and / or amide derivatives using amidase as a catalyst have been developed, including important pharmaceutical intermediates such as S-2,2-dimethylcyclopropanecarboxamide, 1-cyanocyclohexylacetic acid, R-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid, and 2-chloronicotinic acid.

[0004] Wild-type amidase (obtained in nature) has certain limitations, and its deficiencies in catalytic activity, stereoselectivity and stability limit its application in industrial production. However, with the continuous development of protein engineering and a deeper understanding of the structure and function of amidase, the application of enzyme evolution technology makes it possible to change the catalytic properties of enzymes. Patent CN107937376B discloses an amidase from Pantoea sp. for the synthesis of 2-chloronicotinic acid. Its activity is increased by 2-3 times through enzyme evolution, and its tolerance to the substrate-chloronicotinamide exceeds 200mM. In addition, Tang, XL, et al. directed evolution of amidase from Pantoea sp. has improved the catalytic activity of amidase. The activity of single mutant G175A and double mutant G175A / A305T has increased by 3.2 times and 3.7 times, respectively, and the Kcat / Km value has increased to 3.1 times and 10.0 times that of the wild type (Appl Environ Microbio 2019 85(5):e02471-18). Therefore, it is very necessary to evolve the wild-type amidase, change its catalytic performance, and promote enzyme catalysis to be more and more adapted to the needs of corresponding industrial production. Summary of the invention

[0005] The main purpose of the present invention is to provide an amidase mutant and its application, so as to solve the problem of low stereoselectivity and low catalytic activity of wild-type amidase in the prior art.

[0006] In order to achieve the above-mentioned object, according to the first aspect of the present invention, there is provided an amidase mutant, comprising: (a) a protein having the amino acid sequence shown in SEQ ID NO: 1; or (b) a protein having an amino acid mutation at least one of the following sites of the amino acid sequence shown in (a): L81, E62, M311, N180, P121, R58, E32, T230, N82, N190, D192, D217, H219, K77, M78, P79, F80, F61, D185, D59, L182, T183, P184, I227, C228, G229, A231 or V232, and having amidase function; or (c) a protein having more than 80% homology with the amino acid sequence defined in any one of (a) and (b) and having amidase function.

[0007] Further, the amino acid mutations in (b) above are each independently selected from the following: L81A or L81C or L81V; P79Q; F80G; E62Q or E62K or E62M or E62T or E62A or E62R or E62C or E62V or E62P or E62H or E62N or E62S; L76V; K77T; D185H; R58M or R 58K or R58L or R58H or R58A; I227L or I227V or I227A or I227W or I227E or I227S or I227M or I227D or I227C or I227Y; T230C or T230V or T230A or T230L; N180V or N180D or N180K; P121H or P121K or P121N or P121A or P121R or P121E or P121Y or P121S; M311C or M311R or M311H or M311S; V232I; R58L or R58M or R58K or R58Q or R58P or R58A or R58V or R58F; E32D or E32H or E32M or E32Q or E32A or E32C or E32V or E32F or E32I or E32P or E32T; wherein the letters before the numbers represent the original amino acids, and the letters after the numbers represent the mutated amino acids; preferably, in (c), the protein has more than 85%, preferably more than 90%, more preferably more than 95%, and further preferably more than 99% homology with the amino acid sequence defined in (a) or (b) and has amidase function.

[0008] Further, the mutation of the amidase mutant includes any of the following amino acid mutations: L81A; L81C; L81V; P79Q; F80G; E62Q; E62K; E62M; E62T; L76V; K77T; D185H; R58M; I227L; I227V; T230C; L81A+E62T; L81A+E62K; L81A+E62M; L81A+I227V; L81A+T230C; L81A+A231R; L81A+L76V; L81A+I227V; L81A+N180V; L81A+N180D; L81A+N180K; L81A+P121H; L81A+ P121K; L81A+P121N; L81A+P121A; L81A+M311C; L81A+M311R; L81A+M311H; L 81A+M311S; L81A+E62T+P121A; L81A+E62T+P121H; L81A+E62T+P121N; L81A +E62T+P121R; L81A+E62T+I227A; L81A+E62T+M311C; L81A+E62T+M311R; L8 1A+E62T+M311H; L81A+E62T+M311S; L81A+E62T+T230C; L81A+E62T+T230V; L81A+E62T+V232I; L81A+E62T+R58L; L81A+E62T+R58M; L81A+E62T+R58K; L81A+E62T+R58Q; L81A+E62T+E32D; L81A+E62T+E32H; L81A+E62T+E32M; L8 1A+E62T+M311C+E32Q; L81A+E62T+M311C+E32M; L81A+E62T+M311C+E32D; L 81A+E62T+M311C+E32A; L81A+E62T+M311C+E32C; L81A+E62T+M311C+E32V; L81A+E62T+M311C+P121E; L81A+E62T+M311C+P121A; L81A+E62T+M311C+P1 21H; L81A+E62T+M311C+P121R; L81A+E62T+M311C+P121N; L81A+E62R+M311 C; L81A+E62A+M311C; L81A+E62C+M311C; L81A+E62V+M311C; L81A+E62P+M3 11C; L81A+E62M+M311C; L81A+E62T+M311C+R58K; L81A+E62T+M311C+R58L;L81A+E62T+M311C+R58H;L81A+E62T+M311C+R58A;L81A+E62T+M311C+I227A;L81A+E62T+M311C+I227L;L81A+E62T+M311C+I227W;L81A+E62T+M311C+I227E;L81A+E62T+M311C+I227S;L81A+E62T+M311C+T230V;L81A+E62T+M311C+T230C;L81A+E62T+M311C+T230A;L81A+E62T+M311C+T230L;L81A+E62A+M311C+T230C;L81A+E62H+M311C+T230C;L81A+E62N+M311C+T230C;L81A+E62R+M311C+T230C;L81A+E62S+M311C+T230C;L81A+E62Q+M311C+T230C;L81A+E62T+M311C+T230C+R58K;L81A+E62T+M311C+T230C+R58P;L81A+E62T+M311C+T230C+R58A;L81A+E62T+M311C+T230C+R58V;L81A+E62T+M311C+T230C+R58F;L81A+E62T+M311C+T230C+R58M;L81A+E62T+M311C+T230C+I227V;L81A+E62T+M311C+T230C+I227M;L81A+E62T+M311C+T230C+I227A;L81A+E62T+M311C+T230C+I227D;L81A+E62T+M311C+T230C+I227C;L81A+E62T+M311C+T230C+I227Y;L81A+E62T+M311C+T230C+P121A;L81A+E62T+M311C+T230C+P121R;L81A+E62T+M311C+T230C+P121E;L81A+E62T+M311C+T230C+P121H;L81A+E62T+M311C+T230C+P121N;L81A+E62T+M311C+T230C+P121Y;L81A+E62A+M311C+T230C+E32F;L81A+E62A+M311C+T230C+E32I;L81A+E62A+M311C+T230C+E32P;L81A+E62A+M311C+T230C+E32T;L81A+E62A+M311C+T230C+I227V; L81A+E62A+M311C+T230C+I227L; L81A+E62A+M311C+T230C+I227A; L81A+E62A+M311 C+T230C+T230V; L81A+E62A+M311C+T230C+P121A; L81A+E62A+M311C+T230C+P121R; L81A+E62A+M311C+T230C+P121S. ;

[0009] In order to achieve the above object, according to the second aspect of the present invention, a DNA molecule encoding the above amidase mutant is provided.

[0010] In order to achieve the above object, according to the third aspect of the present invention, a recombinant plasmid connected with the above DNA molecule is provided.

[0011] In order to achieve the above object, according to the fourth aspect of the present invention, a host cell transformed with the above recombinant plasmid is provided.

[0012] In order to achieve the above object, according to the fifth aspect of the present invention, a method for preparing chiral carboxylic acids and amide derivatives is provided, the preparation method comprising using the above amidase mutant to catalyze the hydrolysis and resolution reaction of the amide substrate shown in formula I to obtain chiral carboxylic acids and amide derivatives, R1 is selected from aryl, heteroaryl, alkyl, alkylene, cycloalkyl, hydroxyl, amino or H, and when selected from hydroxyl, amino or H, it is preferably hydroxyl; R2 is selected from aryl, heteroaryl, alkyl, alkylene, cycloalkyl, hydroxyl, amino or H, and when selected from hydroxyl, amino or H, it is preferably hydroxyl; R3 is selected from aryl, heteroaryl, alkyl, alkylene, cycloalkyl, hydroxyl, amino or H, and when selected from hydroxyl, amino or H, it is preferably hydroxyl; or, R1 can form a ring or heterocycle with R2 or R3, and the number of C atoms in the ring or heterocycle is selected from 3 to 6; aryl, heteroaryl, alkyl, alkylene, cycloalkyl or ring are each independently unsubstituted or substituted, and when substituted, the substituents are each independently selected from methyl, methylene, methoxy, amino or halogen, preferably halogen, more preferably F; heteroaryl or heterocycle are each independently unsubstituted or substituted, and the heteroatoms therein are each independently selected from N, O or S, and preferably N. Further, the above-mentioned amide substrate is selected from:

[0013] By applying the technical scheme of the present invention, an amidase mutant derived from Yersinia frederiksenii (SEQID NO: 1) was used as the parent, and protein engineering modifications such as saturation mutation and combination mutation were carried out to obtain an amidase mutant with greatly improved catalytic activity and stereoselectivity in the synthesis of chiral carboxylic acids and amide derivatives, which is more suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0015] Figure 1 A schematic diagram of the original amidase protein structure subjected to protein engineering according to the present invention is shown. DETAILED DESCRIPTION

[0016] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0017] As mentioned in the background technology, chiral carboxylic acids and amide derivatives are key intermediates for synthesizing a large number of chiral bioactive molecules, but there are many limitations in the chemical synthesis of chiral carboxylic acids and amide derivatives, and the biological method of amidase synthesis of chiral carboxylic acids and chiral amide derivatives has certain advantages over the chemical method. However, wild-type amidase still has certain limitations, and its catalytic activity, stereoselectivity and stability and other deficiencies limit its application in industrial production.

[0018] Therefore, in the present application, the inventors attempted to modify the amidase by enzyme evolution, thereby improving the catalytic performance of the amidase, so that the production efficiency can be improved in industrial production, and thus proposed a series of protection schemes of the present application.

[0019] In a first typical embodiment of the present application, an amidase mutant is provided, comprising: (a) a protein having the amino acid sequence shown in SEQ ID NO: 1; or (b) a protein having an amino acid mutation at at least one of the following sites of the amino acid sequence in (a): L81, E62, M311, N180, P121, R58, E32, T230, N82, N190, D192, D217, H219, K77, M78, P79, F80, F61, D185, D59, L182, T183, P184, I227, C228, G229, A231 or V232, and having amidase function; or (c) a protein having more than 80% homology with the amino acid sequence defined in any one of (a) and (b) and having amidase function.

[0020] The amino acid sequence shown in SEQ ID NO: 1 is an amidase derived from Yersinia frederiksenii. Figure 1 As shown in the figure, 28 sites were selected near the active site and substrate channel for modification: N82, N190, D192, D217, H219, M311, P121, N180, K77, M78, P79, F80, L81, E32, F61, E62, D185, R58, D59, L182, T183, P184, I227, C228, G229, T230, A231, V232. These amino acid sites may affect the stereoselectivity of the protein. By mutating the above amino acid sites, proteins with amidase function or even enhanced amidase function can be obtained. For the above-obtained protein, changes can be made at non-critical mutation sites and active sites, and proteins with more than 80% homology to the above amino acid sequence and amidase function can be obtained.

[0021] The sequence of SEQ ID NO: 1 is as follows:

[0022] MKWLEESIMVKRGVGAGRKPVTHHLTEEMQKEFHYTIGPYSTPVLTIEPGDRVIVDTRD

[0023] AFEGAINSEQDIPSQLLKMPFLNPQNGPIMVNGAEKGDVIAVYIESMLPRGVNPHGICAMIPH

[0024] FGGLTGTDLTAMLNDPLPEKVRMIKLDSEKVYWSERHTLPYKPHIGTLSVSPEIDSINSLTPD

[0025] NHGGNMDVPDIGPGSITYLPVRAPGRLLFIGDAHACQGDGEICGTAVEFASITTIKVDLIKNW

[0026] QLSWPRMENAETIMSIGSARPLEDATRIAYRDLIYWLVADFGFDQWDAYMLLSQCGKVRLG

[0027] NMVDPKYTVGAMLNKELLAP.

[0028] In a preferred embodiment, the amino acid mutations in (b) are each independently selected from the following: L81A or L81C or L81V; P79Q; F80G; E62Q or E62K or E62M or E62T or E62A or E62R or E62C or E62V or E62P or E62H or E62N or E62S; L76V; K77T; D185H; R58 M or R58K or R58L or R58H or R58A; I227L or I227V or I227A or I227W or I227E or I227S or I227M or I227D or I227C or I227Y; T230C or T230V or T230A or T230L; N180V or N180D or N180K; P121H or P121 K or P121N or P121A or P121R or P121E or P121Y or P121S; M311C or M311R or M311H or M311S; V232I; R58L or R58M or R58K or R58Q or R58P or R58A or R58V or R58F; E32D or E32H or E32M or E32Q or E32A or E32C or E32V or E32F or E32I or E32P or E32T; wherein the letters before the numbers represent the original amino acids, and the letters after the numbers represent the mutated amino acids; preferably, in (c), the protein has more than 85%, preferably more than 90%, more preferably more than 95%, and further preferably more than 99% homology with the amino acid sequence defined in (a) or (b) and has amidase function.

[0029] As used herein, amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y) and valine (Val; V).

[0030] Substitution and replacement rules. Generally speaking, amino acids with similar properties will have similar effects after replacement. For example, conservative amino acid replacements may occur in the above homologous proteins. "Conservative amino acid replacements" include but are not limited to:

[0031] Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;

[0032] The hydrophobic amino acids with bulky side chains (Phe, Tyr, Trp) are replaced by other hydrophobic amino acids with bulky side chains;

[0033] Amino acids with positively charged side chains (Arg, His, Lys) are replaced by other amino acids with positively charged side chains;

[0034] Amino acids with polar, uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar, uncharged side chains.

[0035] A person skilled in the art may also perform conservative substitutions on amino acids according to amino acid substitution rules well known to those skilled in the art, such as the "blosum62 scoring matrix" in the prior art.

[0036] In the present application, the applicant continues to explore the above active sites and finds that when the active sites are mutated to different amino acids, the corresponding protein activities are also different, and specific mutations can enhance the activity of amidase. Through experimental exploration, it is found that the above specific mutations in the active sites can obtain proteins with enhanced activity. For the amino acid mutation sites of amidase proteins, flexible selection and combination can be performed in the above mutations.

[0037] "Sequence identity" between two polypeptide sequences indicates the percentage of identical amino acids between the sequences, and methods for evaluating the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, amino acid sequence identity is typically measured using sequence analysis software. For example, the BLAST program of the NCBI database can be used to determine identity. For determination of sequence identity, see, for example: Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987 and Sequence Analysis Primer, Gribskov, M. and Devereux, J., edu., M Stockton Press, New York, 1991.

[0038] In a preferred embodiment, the mutation of the amidase mutant comprises any of the following amino acid mutations:

[0039] L81A;L81C;L81V;P79Q;F80G;E62Q;E62K;E62M;E62T;L76V;K77T;D185H;R58M;I227L;I227V;T230C;L81A+E62T;L81A+E62K;L81A+E62M;L81A+I227V;L81A+T230C;L81A+A231R;L81A+L76V;L81A+I227V;L81A+N180V;L81A+N180D;L81A+N180K;L81A+P121H;L81A+P121K;L81A+P121N;L81A+P121A;L81A+M311C;L81A+M311R;L81A+M311H;L81A+M311S;L81A+E62T+P121A;L81A+E62T+P121H;L81A+E62T+P121N;L81A+E62T+P121R;L81A+E62T+I227A;L81A+E62T+M311C;L81A+E62T+M311R;L81A+E62T+M311H;L81A+E62T+M311S;L81A+E62T+T230C;L81A+E62T+T230V;L81A+E62T+V232I;L81A+E62T+R58L;L81A+E62T+R58M;L81A+E62T+R58K;L81A+E62T+R58Q;L81A+E62T+E32D;L81A+E62T+E32H;L81A+E62T+E32M;L81A+E62T+M311C+E32Q;L81A+E62T+M311C+E32M;L81A+E62T+M311C+E32D;L81A+E62T+M311C+E32A;L81A+E62T+M311C+E32C;L81A+E62T+M311C+E32V;L81A+E62T+M311C+P121E;L81A+E62T+M311C+P121A;L81A+E62T+M311C+P121H;L81A+E62T+M311C+P121R;L81A+E62T+M311C+P121N;L81A+E62R+M311C;L81A+E62A+M311C;L81A+E62C+M311C;L81A+E62V+M311C;L81A+E62P+M311C;L81A+E62M+M311C;L81A+E62T+M311C+R58K;L81A+E62T+M311C+R58L;L81A+E62T+M311C+R58H;L81A+E62T+M311C+R58A;L81A+E62T+M311C+I227A;L81A+E62T+M311C+I227L;L81A+E62T+M311C+I227W;L81A+E62T+M311C+I227E;L81A+E62T+M311C+I227S;L81A+E62T+M311C+T230V;L81A+E62T+M311C+T230C;L81A+E62T+M311C+T230A;L81A+E62T+M311C+T230L;L81A+E62A+M311C+T230C;L81A+E62H+M311C+T230C;L81A+E62N+M311C+T230C;L81A+E62R+M311C+T230C;L81A+E62S+M311C+T230C;L81A+E62Q+M311C+T230C;L81A+E62T+M311C+T230C+R58K;L81A+E62T+M311C+T230C+R58P;L81A+E62T+M311C+T230C+R58A;L81A+E62T+M311C+T230C+R58V;L81A+E62T+M311C+T230C+R58F;L81A+E62T+M311C+T230C+R58M;L81A+E62T+M311C+T230C+I227V;L81A+E62T+M311C+T230C+I227M;L81A+E62T+M311C+T230C+I227A;L81A+E62T+M311C+T230C+I227D;L81A+E62T+M311C+T230C+I227C;L81A+E62T+M311C+T230C+I227Y;L81A+E62T+M311C+T230C+P121A;L81A+E62T+M311C+T230C+P121R;L81A+E62T+M311C+T230C+P121E;L81A+E62T+M311C+T230C+P121H;L81A+E62T+M311C+T230C+P121N;L81A+E62T+M311C+T230C+P121Y;L81A+E62A+M311C+T230C+E32F;L81A+E62A+M311C+T230C+E32I;L81A+E62A+M311C+T230C+E32P;L81A+E62A+M311C+T230C+E32T;L81A+E62A+M311C+T230C+I227V;L81A+E62A+M311C+T230C+I227L; L81A+E62A+M311C+T230C+I227A; L81A+E62A+M311C+T230C+T230V; L81A+E62A+M311C+T230C+P121A; L81A+E62A+M311C+T230C+P121R; L81A+E62A+M311C+T230C+P121S. ;

[0040] The above amino acid mutations were all experimentally investigated in the examples of the present application, and all had amidase activity. Compared with the parent having the amino acid sequence shown in SEQ ID NO: 1, amidase mutants with higher stereoselectivity and catalytic activity can be obtained, which can be used for industrial scale-up production.

[0041] In a second typical embodiment of the present application, a DNA molecule is provided, which encodes the above-mentioned amidase mutant.

[0042] In a third typical embodiment of the present application, a recombinant plasmid is provided, wherein the recombinant plasmid is connected to the above-mentioned DNA molecule.

[0043] The DNA can encode the amidase mutant and can be connected to a recombinant plasmid to form a circular DNA. Both the DNA and the recombinant plasmid can be transcribed and translated under the action of RNA polymerase, ribosome, tRNA, etc. to obtain the amidase mutant.

[0044] In a fourth typical embodiment of the present application, a host cell is provided, wherein the above-mentioned recombinant plasmid is transformed. The host cell can be a prokaryotic cell or a eukaryotic cell. Specifically, the prokaryotic cell can be Escherichia coli, and the eukaryotic cell can be yeast.

[0045] By using the host cell, the recombinant plasmid can be replicated in the host cell, and the DNA molecules carried on the recombinant plasmid can also be transcribed and translated to obtain a large number of amidase mutants. By using the existing technology, the host cell can be crushed for protein purification, crushed for crude enzyme catalysis or other methods to obtain amidase mutants, and the subsequent synthesis reaction of chiral carboxylic acid and chiral amide derivatives can be catalyzed. The host cell is a non-plant-derived host cell.

[0046] In a fifth typical embodiment of the present application, a method for preparing a chiral carboxylic acid and an amide derivative is provided, the preparation method comprising using the above-mentioned amidase mutant to catalyze the hydrolysis and resolution reaction of the amide substrate shown in formula I to obtain a chiral carboxylic acid and an amide derivative, R1 is selected from aryl, heteroaryl, alkyl, alkylene, cycloalkyl, hydroxyl, amino or H, and when selected from hydroxyl, amino or H, it is preferably hydroxyl; R2 is selected from aryl, heteroaryl, alkyl, alkylene, cycloalkyl, hydroxyl, amino or H, and when selected from hydroxyl, amino or H, it is preferably hydroxyl; R3 is selected from aryl, heteroaryl, , alkyl, alkylene, cycloalkyl, hydroxyl, amino or H, when selected from hydroxyl, amino or H, preferably hydroxyl; or, R1 can form a ring or heterocycle with R2 or R3, and the number of C atoms in the ring or heterocycle is selected from 3 to 6; aryl, heteroaryl, alkyl, alkylene, cycloalkyl or ring are each independently unsubstituted or substituted, and when substituted, the substituents are each independently selected from methyl, methylene, methoxy, amino or halogen, preferably halogen, more preferably F; heteroaryl or heterocycle are each independently unsubstituted or substituted, and the heteroatoms therein are each independently selected from N, O or S, preferably N.

[0047]

[0048] The preparation method is used to catalyze the hydrolysis and resolution reaction of the amide substrate shown in formula I by the amidase mutant to obtain chiral carboxylic acid and amide derivatives. The amidase mutant can perform a hydrolysis and resolution reaction on the amide substrate. The catalytic activity and stereoselectivity of the amidase are greatly improved, the production efficiency can be improved, the demand for industrial production of chiral carboxylic acid and amide derivatives can be met, and the industrial production cost can be reduced.

[0049] In a preferred embodiment, the amide substrate is selected from:

[0050]

[0051] In this application, the inventors selected 28 sites near the active site and substrate channel for modification based on the three-dimensional structure of the protein that has been resolved (5ubu.1): N82, N190, D192, D217, H219, M311, P121, N180, K77, M78, P79, F80, L81, E32, F61, E62, D185, R58, D59, L182, T183, P184, I227, C228, G229, T230, A231, V232. The protein modification methods include saturation mutation and combined mutation.

[0052] Saturation mutagenesis is a method of modifying the coding gene of the target protein to obtain mutants in which the amino acids at the target site are replaced by 19 other amino acids in a short period of time. This method is not only a powerful tool for protein-directed modification, but also an important means of studying the structure-function relationship of proteins. Saturation mutagenesis often produces more ideal evolutionary bodies than single-point mutations. These problems that cannot be solved by the site-directed mutagenesis method are precisely the unique features of the saturation mutagenesis method. The construction of saturation mutagenesis adopts the method of whole plasmid PCR, and then the PCR product is digested with DPNI enzyme to remove the template and transformed into Escherichia coli BL21 (DE3). The screening of mutants often requires high-throughput screening methods. Therefore, we developed the following method to screen mutant tolerance.

[0053] The following high-throughput screening method was developed to screen the mutant library:

[0054] 1. Mutant culture: Add 300 μL LB medium to each well of a 96-well plate, inoculate the single clone on the agar plate into a deep-well 96-well plate, and culture overnight at 37°C and 200 rpm; use Qpix to transfer the overnight cultured bacterial solution to another 96-well plate, add 800 μL LB medium to each well, and culture at 37°C and 200 rpm for 5 hours. When the OD600 of the bacterial solution in the 96-well plate reaches 0.6-0.9, use Qpix to add IPTG solution to the 96-well plate again to make the final concentration of IPTG in the well plate 0.1 mM, and induce overnight at 25°C and 200 rpm for about 16 hours; centrifuge at 4000 rpm for 5 minutes, discard the supernatant, and use whole cells for reaction.

[0055] 2. 96-well plate high-throughput screening system: Add 200ul of phosphate buffer to each well of the 96-well plate, shake and resuspend, then add 2-20mg of substrate, shake at 30℃ constant temperature, 700rpm, and control different reaction times according to different screening purposes. After the reaction, add 1ml of acetonitrile to terminate the reaction, centrifuge and take the supernatant, and send it to UPLC for analysis.

[0056] Through the above-mentioned initial screening of mutants, mutants with improved traits are obtained. Then, the optimal mutant will be induced in a 2L shake flask (the best conditions for inducing expression: 25°C, 0.2mM IPTG overnight induction), and after centrifugation to obtain bacterial sludge, the crude enzyme solution is obtained by ultrasonic cell disruption, and finally a g-scale reaction is performed to confirm the optimal mutant traits. Single-point saturation mutations at the above 28 sites often do not have obvious improvement effects, so it is generally necessary to perform multiple rounds of iterative saturation mutations to obtain mutants with greatly improved traits.

[0057] On the basis of the mutants with improved activity obtained by saturation mutation, the beneficial amino acid sites obtained by screening can be combined to obtain mutants with better traits. The construction method of double-point mutations in combined mutations is the same as that of single-point mutations, and is constructed by whole-plasmid PCR. Multi-point mutations that mutate 2 or more sites at the same time are amplified by overlapping extension PCR to obtain mutant genes containing multiple point mutations. After restriction endonuclease digestion at both ends, they are connected to the expression vector, transformed into Escherichia coli cells, and coated in LB culture dishes containing 100 μg / mL ampicillin. Cultured at 37°C overnight to obtain combined mutants. After the above combined mutants are correctly identified by sequencing, they are directly subjected to 2L shake flask induction culture and reaction verification.

[0058] After multiple rounds of evolution, a series of amidase mutants were obtained, and it was found that the catalytic efficiency of these amidase mutants for a variety of amide compounds was greatly improved. Chiral carboxylic acids and amide derivatives with extremely high chiral purity were obtained, and these mutants were proven to be fully usable in the industrial production of chiral carboxylic acids and amide derivatives.

[0059] The present application is further described in detail below in conjunction with specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed in the present application.

[0060] Example 1

[0061] The saturation mutation of the above 28 sites was performed on the basis of the parent SEQ ID NO: 1, and the catalytic activity and ee value of the mutant were tested according to the following reaction conditions:

[0062] 1 mL reaction system included 100 mg substrate 1 or substrate 2, 10 mg enzyme powder (obtained by freeze-drying 100 mg fermentation wet bacterial sludge) and 100 mM pH 7.5 phosphate buffer. The reaction time was tracked at 30°C. After 2 hours of reaction, samples were taken and sent to HPLC to measure the yield. After 16 hours of reaction, samples were taken and sent to HPLC to measure the ee value.

[0063] The test results are shown in the table below.

[0064] Table 1:

[0065]

[0066]

[0067] Note: In the above table, - indicates that the yield of the wild-type parent is 10%, + indicates that the yield is greater than or equal to 20% and less than 30%, ++ indicates that the yield is greater than or equal to 30% and less than 40%, and +++ indicates that the yield is greater than or equal to 40% and less than 45%. In addition, the ee value in the above table is the ee value of R-carboxylic acid when the yield reaches ~50%. * indicates that the ee value is less than 80%, ** indicates that the ee value is greater than or equal to 80% and less than 90%, and *** indicates that the ee value is greater than or equal to 90% and less than 95%.

[0068] Example 2

[0069] The next round of saturation mutation and combination mutation was carried out on the basis of Example 1, and the activity of the combination mutation was screened according to the following reaction conditions. The results are shown in the table below.

[0070] 1 mL reaction system included 100 mg substrate 1 or substrate 2, 8 mg enzyme powder (obtained by freeze-drying 80 mg fermentation wet bacterial sludge) and 100 mM pH 7.5 phosphate buffer. The reaction time was tracked at 30°C. After 2 h of reaction, samples were taken and sent to HPLC to measure the yield. After 16 h, samples were taken and sent to HPLC to measure the ee value.

[0071] Table 2:

[0072]

[0073] Note: In the above table, + represents a yield greater than or equal to 20% and less than 30%, ++ represents a yield greater than or equal to 30% and less than 40%, and +++ represents a yield greater than or equal to 40% and less than 45%. In addition, the ee value in the above table is the ee value of R-carboxylic acid when the yield reaches 50%. * represents an ee value less than 80%, ** represents an ee value greater than or equal to 80% and less than 90%, *** represents an ee value greater than or equal to 90% and less than 95%, and **** represents an ee value greater than or equal to 95% and less than 98%.

[0074] Example 3

[0075] Saturation mutation and combined mutation were performed on the basis of Example 2, and the catalytic activity of the mutants was tested according to the following reaction conditions:

[0076] 1 mL reaction system included 100 mg substrate 1 or substrate 2, 5 mg enzyme powder (obtained by freeze-drying 50 mg fermentation wet bacterial sludge) and 100 mM pH 7.5 phosphate buffer. The reaction time was tracked at 30°C. After 2 h of reaction, samples were taken and sent to HPLC to measure the yield. After 16 h, samples were taken and sent to HPLC to measure the ee value.

[0077] Table 3:

[0078]

[0079] Note: In the above table, + represents a yield greater than or equal to 20% and less than 30%, ++ represents a yield greater than or equal to 30% and less than 40%, and +++ represents a yield greater than or equal to 40% and less than 45%. ++++ represents a yield greater than or equal to 45% and less than 50%. In addition, the ee value in the above table is the ee value of R-carboxylic acid when the yield reaches ~50%. * represents an ee value less than 80%, ** represents an ee value greater than or equal to 80% and less than 90%, *** represents an ee value greater than or equal to 90% and less than 95%, and **** represents an ee value greater than or equal to 95% and less than 98%.

[0080] Example 4

[0081] Saturation mutation and combined mutation were performed on the basis of Example 3, and the catalytic activity of the mutants was tested according to the following reaction conditions:

[0082] 1 mL reaction system included 100 mg substrate 1 or substrate 2, 2 mg enzyme powder (obtained by freeze-drying 20 mg fermentation wet bacterial sludge) and 100 mM pH 7.5 phosphate buffer. The reaction time was tracked at 30°C. After 1 h of reaction, samples were taken and sent to HPLC to measure the yield. After 8 h, samples were taken and sent to HPLC to measure the ee value.

[0083] Table 4:

[0084]

[0085] Note: In the above table, + represents a yield greater than or equal to 20% and less than 30%, ++ represents a yield greater than or equal to 30% and less than 40%, and +++ represents a yield greater than or equal to 40% and less than 45%. ++++ represents a yield greater than or equal to 45% and less than 50%. In addition, the ee value in the above table is the ee value of R-carboxylic acid when the yield reaches ~50%. * represents an ee value less than 80%, ** represents an ee value greater than or equal to 80% and less than 90%, *** represents an ee value greater than or equal to 90% and less than 95%, and **** represents an ee value greater than or equal to 95% and less than 98%.

[0086] Example 5

[0087] Based on Example 4, multiple rounds of saturation mutation and combination mutation were performed, and the catalytic activity of the mutants was tested according to the following reaction conditions:

[0088] 1 mL reaction system included 100 mg substrate 1 or substrate 2, 1 mg enzyme powder (obtained by freeze-drying 10 mg fermentation wet bacterial sludge) and 100 mM pH 7.5 phosphate buffer. The reaction time was tracked at 30°C. After 1 h of reaction, samples were taken and sent to HPLC to measure the yield. After 8 h, samples were taken and sent to HPLC to measure the ee value.

[0089] Table 5:

[0090]

[0091] Note: In the above table, + represents a yield greater than or equal to 20% and less than 30%, ++ represents a yield greater than or equal to 30% and less than 40%, and +++ represents a yield greater than or equal to 40% and less than 45%. ++++ represents a yield greater than or equal to 45% and less than 50%. In addition, the ee value in the above table is the ee value of R-carboxylic acid when the yield reaches ~50%. * represents an ee value less than 80%, ** represents an ee value greater than or equal to 80% and less than 90%, *** represents an ee value greater than or equal to 90% and less than 95%, **** represents an ee value greater than or equal to 95% and less than 98%, and ***** represents an ee value greater than or equal to 99%.

[0092] Example 6

[0093] Add 100 mL of 100 mmol / L phosphate buffer and 10 g of (Substrate 1), stir evenly, then add 1mL of enzyme solution of the amidase mutant L81A+E62A+M311C+T230C based on SEQ ID NO: 1 (prepared from 1g of wet bacterial mud), adjust pH to 7.5-8.0. Heat to 30℃ and stir, track the reaction time, and terminate the reaction system when the yield reaches about 50%. Then perform post-treatment respectively, and finally obtain the target product R-carboxylic acid and S-amide derivatives

[0094] HPLC detection showed that the purity of the R-carboxylic acid and S-amide derivatives were both greater than 99%, the ee values ​​were both greater than 99%, and the yields were 42% and 40%, respectively.

[0095] Example 7

[0096] Add 100 mL of 100 mmol / L phosphate buffer and 10 g of (Substrate 2), stir evenly, then add 1mL of enzyme solution of the amidase mutant L81A+E62A+M311C+T230C based on SEQ ID NO: 1 (prepared from 1g of wet bacterial mud), adjust pH to 7.5-8.0. Heat to 30℃ and stir, track the reaction time, and terminate the reaction system when the yield reaches about 50%. Then perform post-treatment respectively, and finally obtain the target product R-carboxylic acid and S-amide derivatives

[0097] HPLC detection showed that the purity of the R-carboxylic acid and S-amide derivatives were both greater than 99%, the ee values ​​were both greater than 99%, and the yields were 41% and 43%, respectively.

[0098] Example 8

[0099] Add 100 mL of 100 mmol / L phosphate buffer and 10 g of (Substrate 14), stir evenly, then add 1mL of enzyme solution of the amidase mutant L81A+E62A+M311C+T230C based on SEQ ID NO: 1 (prepared from 1g of wet bacterial mud), adjust pH to 7.5-8.0. Heat to 30℃ and stir, track the reaction time, and terminate the reaction system when the yield reaches 95%. Then perform post-treatment to finally obtain the target product carboxylic acid

[0100] HPLC detection showed that the purity of the product carboxylic acid was >99%, the ee value was >99%, and the yield was >84%.

[0101] Example 9

[0102] Using the enzyme solution of the amidase mutant L81A+E62A+M311C+T230C mutated on the basis of SEQ ID NO: 1, referring to the catalytic synthesis steps of Examples 6 to 8, the substrates 3-13 were catalyzed. The results are shown in the following table:

[0103] Table 6:

[0104]

[0105] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: 1) The amidase mutant with improved activity and stereoselectivity is used to perform amide splitting reaction, and a series of chiral carboxylic acids and amide derivatives can be obtained with high activity and high selectivity. 2) The amidase mutant is used for biocatalysis, and the reaction conditions are mild, and no heavy metal catalysts and toxic reagents are required, thereby realizing green chemistry. 3) The amidase has high biocatalytic activity, high reaction substrate concentration, high product yield, greatly reducing the three wastes, and saving production costs.

[0106] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An amidase mutant, characterized in that The amidase mutant is a mutant having an amino acid mutation at the following positions of the amino acid sequence shown in SEQ ID NO: 1: L81A; L81A+E62T; L81A+E62K; L81A+E62M; L81A+I227V; L81A+T230C; L81A+A231R; L81A+L76V; L81A+I227V; L81A+N180V; L81A+N180D; L81A+N180K; L81A+P121H; L81A+P121K; L81A+P121N; L81A+P121A; L81A+M311C; L81A+M311R; L81A+M311H; L81A+M311S; L81A+E62T+P121A; L81A+E62T+P121H; L81A+E62T+P121N; L81A+E62T+P121R; L81A+E62T+I227A; L81A+E62T+M311C; L81A+E62T+M311R; L81A+E62T+M311H; L81A+E62T+M311S; L81A+E62T+T230C; L81A+E62T+T230V; L81A+E62T+V232I; L81A+E62T+R58L; L81A+E62T+R58M; L81A+E62T+R58K; L81A+E62T+R58Q; L81A+E62T+E32D; L81A+E62T+E32H; L81A+E62T+E32M; L81A+E62T+M311C+E32Q; L81A+E62T+M311C+E32M; L81A+E62T+M311C+E32D; L81A+E62T+M311C+E32A; L81A+E62T+M311C+E32C; L81A+E62T+M311C+E32V; L81A+E62T+M311C+P121E; L81A+E62T+M311C+P121A; L81A+E62T+M311C+P12 1H; L81A+E62T+M311C+P121R; L81A+E62T+M311C+P121N; L81A+E62R+M311C; L81A+E62A+M311C; L81A+E62C+M311C; L81A+E62V+M311C; L81A+E62P+M311C; L81A+E62M+M311C; L81A+E62T+M311C+R58K; L81A+E62T+M311C+R58L; L81A+E62T+M311C+R58H; L81A+E62T+M311C+R58A; L81A+E62T+M311C+I227A; L81A+E62T+M311C+I227L; L81A+E62T+M311C+I227W; L81A+E62T+M311C+I227E; L81A+E62T+M311C+I227S; L81A+E62T+M311C+T230V; L81A+E62T+M311C+T230C; L81A+E62T+M311C+T230A; L81A+E62T+M311C+T230L; L81A+E62A+M311C+T230C; L81A+E62H+M311C+T230C; L81A+E62N+M311C+T230C; L81A+E62R+M311C+T230C; L81A+E62S+M311C+T230C; L81A+E62Q+M311C+T230C; L81A+E62T+M311C+T230C+R58K; L81A+E62T+M311C+T230C+R58P; L81A+E62T+M311C+T230C+R58A; L81A+E62T+M3 11C+T230C+R58V; L81A+E62T+M311C+T230C+R58F; L81A+E62T+M311C+T230C+R58M; L81A+E62T+M311C+T230C+I2 27V; L81A+E62T+M311C+T230C+I227M; L81A+E62T+M311C+T230C+I227A; L81A+E62T+M311C+T230C+I227D; L81A+ E62T+M311C+T230C+I227C; L81A+E62T+M311C+T230C+I227Y; L81A+E62T+M311C+T230C+P121A; L81A+E62T+M311C +T230C+P121R; L81A+E62T+M311C+T230C+P121E; L81A+E62T+M311C+T230C+P121H; L81A+E62T+M311C+T230C+P1 21N; L81A+E62T+M311C+T230C+P121Y; L81A+E62A+M311C+T230C+E32F; L81A+E62A+M311C+T230C+E32I; L81A+E6 2A+M311C+T230C+E32P; L81A+E62A+M311C+T230C+E32T; L81A+E62A+M311C+T230C+I227V; L81A+E62A+M311C+T2 30C+I227L; L81A+E62A+M311C+T230C+I227A; L81A+E62A+M311C+T230C+T230V; L81A+E62A+M311C+T230C+P121A; L81A+E62A+M311C+T230C+P121R; L81A+E62A+M311C+T230C+P121S; The letters before the numbers represent the original amino acids, and the letters after the numbers represent the mutated amino acids.

2. A DNA molecule, characterized in that The DNA molecule encodes the amidase mutant according to claim 1.

3. A recombinant plasmid, characterized in that: The recombinant plasmid is connected to the DNA molecule according to claim 2.

4. A host cell, characterized in that The host cell is transformed with the recombinant plasmid according to claim 3.

5. A method for preparing chiral carboxylic acid and chiral amide derivatives, characterized in that: The preparation method comprises using the amidase mutant according to claim 1 to catalyze the hydrolysis and resolution reaction of the amide substrate shown below to obtain a chiral carboxylic acid and a chiral amide derivative. The amide substrate is selected from:

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