Amidase mutants and uses thereof

Abstract

The present invention provides amidase mutants and uses thereof, including (a) a protein having the amino acid sequence set forth in SEQ ID NO: 1, or (b) a protein having an amino acid mutation at at least one of 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 in the amino acid sequence set forth in (a) and having amidase function, or (c) a protein having 80% or more homology to the amino acid sequence defined in either (a) or (b) and having amidase function. It can solve the problems of the prior art amidases, such as low stereoselectivity and catalytic activity, and is suitable for the field of enzyme catalysis.

Description

[Technical Field]

[0001] The present invention relates to the field of enzyme catalysis, and in particular to amidase mutants and their uses.

[0002] This application is based on and claims priority from a Chinese application bearing application number 202211539071.X and filed on December 2, 2022, the disclosure of which is incorporated herein in its entirety. [Background technology]

[0003] Chiral carboxylic acid and amide derivatives are important intermediates for the synthesis of large quantities of chiral bioactive molecules and are widely used in fields such as fine chemicals, pharmaceuticals, agrochemicals, and functional materials. However, the chemical synthesis of chiral carboxylic acid and amide derivatives has limitations such as low overall product yields, low selectivity, and high synthesis costs, making them unsuitable for industrial-scale production.

[0004] Amidases (EC 3.5.1.X) are important hydrolases that catalyze the hydrolysis of amide compounds to produce carboxylic acids and ammonia. Their substrate spectrum is broad, covering aliphatic, aromatic, and heterocyclic compounds. Amidases generally possess excellent stereoselectivity, and although the majority of amidases are S-type, R-type amidases have also been discovered in recent years. These characteristics of amidases give them unparalleled advantages in the kinetic resolution of racemic amides to produce chiral carboxylic acid and chiral amide derivatives, attracting increasing attention from researchers. Using amidases as catalysts, a series of novel biosynthetic processes have been developed to synthesize chiral carboxylic acid and / or amide derivatives, 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.

[0005] Wild-type amidases (obtained in nature) have certain limitations, including poor catalytic activity, stereoselectivity, and stability, limiting their application in industrial production. However, with the continued development of protein engineering and a deeper understanding of amidase structure and function, it is now possible to modify the catalytic properties of enzymes through the application of enzyme evolution techniques. Patent CN107937376B discloses an amidase derived from Pantoea for synthesizing 2-chloronicotinic acid, the activity of which was improved 2-3-fold through enzyme evolution, and the tolerance to the substrate chloronicotinamide exceeded 200 mM. Tang, XL, et al. also used directed evolution to improve the catalytic activity of amidase from Pantoea sp. The activities of the single mutant G175A and the double mutant G175A / A305T were improved by 3.2-fold and 3.7-fold, respectively, with Kcat / Km values ​​3.1-fold and 10.0-fold higher than those of the wild-type (Appl Environ Microbio 2019 85(5):e02471-18). Therefore, there is a great need to evolve wild-type amidases to alter their catalytic performance, enhance enzyme catalysis, and make them more adaptable to the corresponding needs of industrial production. Summary of the Invention [Problem to be solved by the invention]

[0006] The main object of the present invention is to provide amidase mutants and their uses to solve the problems of prior art wild-type amidases, namely low stereoselectivity and low catalytic activity. [Means for solving the problem]

[0007] To achieve the above object, according to a first aspect of the present invention, there is provided an amidase mutant comprising: (a) a protein having the amino acid sequence set forth in SEQ ID NO: 1; (b) a protein having an amino acid mutation at at least one site selected from L81, E62, M311, N180, P121, R58, E32, T230, N82, N190, D192, D217, H219, K77, M78, P79, F80, F61, D185, D59, L182, T183, P184, 1227, C228, G229, A231, and V232 in the amino acid sequence set forth in (a), and having amidase function; or (c) a protein having 80% or more homology to the amino acid sequence defined in either (a) or (b), and having amidase function.

[0008] Furthermore, the amino acid mutations in (b) above are each independently selected from the group consisting of L81A, L81C, or L81V; P79Q; F80G; E62Q, E62K, E62M, E62T, E62A, E62R, E62C, E62V, E62P, E62H, E62N, or E62S; L76V; K77T; D185H; R58M, R58K, R58L, and R58S. 8H, or R58A;I227L, I227V, I227A, I227W, I227E, I227S, I227M, I227D, I227C, or I227Y;T230C, T230V, T230A, or T230L;N180V, N180D, or N180K;P121H, P121K, P121N, P121A, P121R, P1 21E, P121Y, or P121S; M311C, M311R, M311H, or M311S; V232I; R58L, R58M, R58K, R58Q, R58P, R58A, R58V, or R58F; E32D, E32H, E32M, E32Q, E32A, E32C, E32V, E32F, E32I, E32P, or E32T, where the letter before the number represents the original amino acid and the letter after the number represents the mutated amino acid, and preferably (c) is a protein having 85% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 99% or more homology to the amino acid sequence defined in (a) or (b) and having amidase function.

[0009] Furthermore, the mutations of the above amidase mutants include any of the following amino acid mutations:

[0010] 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. ;

[0011] To achieve the above object, according to a second aspect of the present invention, there is provided a DNA molecule encoding the above amidase mutant.

[0012] To achieve the above object, according to a third aspect of the present invention, there is provided a recombinant plasmid in which the above DNA molecule is ligated.

[0013] To achieve the above object, according to a fourth aspect of the present invention, there is provided a host cell transformed with the above recombinant plasmid.

[0014] To achieve the above object, according to a fifth aspect of the present invention, there is provided a method for producing chiral carboxylic acid and amide derivatives, comprising the step of catalyzing the hydrolytic resolution reaction of an amide substrate represented by formula I using the above amidase mutant to obtain chiral carboxylic acid and amide derivatives.

[0015] [ka] wherein R1 is selected from aryl, heteroaryl, alkyl, alkylene, cycloalkyl, hydroxy, amino, or H, and when selected from hydroxy, amino, or H, is preferably hydroxy; R2 is selected from aryl, heteroaryl, alkyl, alkylene, cycloalkyl, hydroxy, amino, or H, and when selected from hydroxy, amino, or H, is preferably hydroxy; R3 is selected from aryl, heteroaryl, alkyl, alkylene, cycloalkyl, hydroxy, amino, or H, and when selected from hydroxy, amino, or H, is preferably hydroxy; or R1 may form a ring or heterocycle together with R2 or R3, the number of carbon atoms in the ring or heterocycle being selected from 3 to 6, the aryl, heteroaryl, alkyl, alkylene, cycloalkyl, or ring being each independently unsubstituted or substituted, and if substituted, the substituents being each independently selected from methyl, methylene, methoxy, amino, or halogen, preferably halogen, more preferably F, the heteroaryl or heterocycle being each independently unsubstituted or substituted, and the heteroatoms being each independently selected from N, O, or S, preferably N.

[0016] Furthermore, the amide-based substrate is selected from the following:

[0017] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [Effects of the Invention]

[0018] According to the technical solution of the present invention, an amidase mutant (SEQ ID NO: 1) derived from Yersinia frederiksenii was used as a parent, and protein engineering modifications such as saturation mutation and combinatorial mutation were performed to obtain an amidase mutant with significantly improved catalytic activity and stereoselectivity in the synthesis of chiral carboxylic acid and amide derivatives, which is more suitable for industrial production.

[0019] The drawings in the specification that form a part of this application are intended to provide a further understanding of the invention, and the illustrative embodiments of the invention and their descriptions are intended to illustrate the invention and are not intended to unduly limit the invention. [Brief explanation of the drawings]

[0020] [Figure 1]FIG. 1 shows a schematic diagram of the original amidase protein structure that is the target of protein engineering modification according to the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0021] It should be noted that, unless there is a contradiction, the examples and features in the examples of the present application can be combined with each other. The present invention will be described in more detail below with reference to examples.

[0022] As described in the background art, chiral carboxylic acid and amide derivatives are important intermediates for synthesizing large amounts of chiral bioactive molecules, but chemical methods for synthesizing chiral carboxylic acid and amide derivatives have many limitations. On the other hand, biological methods for synthesizing chiral carboxylic acid and chiral amide derivatives using amidases have certain advantages over chemical methods. However, wild-type amidases still have certain limitations, and their applications in industrial production are limited due to deficiencies in catalytic activity, stereoselectivity, and stability.

[0023] Therefore, in this application, the present inventors attempted to modify amidase by enzyme evolution, thereby improving the catalytic performance of amidase and its production efficiency in industrial production. Therefore, in this application, a series of protection schemes are proposed.

[0024] A first exemplary embodiment of the present application provides an amidase mutant comprising: (a) a protein having the amino acid sequence set forth in SEQ ID NO: 1; (b) a protein having an amino acid mutation at at least one of 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 in the amino acid sequence of (a) and having amidase function; or (c) a protein having 80% or more homology to the amino acid sequence defined in either (a) or (b) and having amidase function.

[0025] The amino acid sequence shown in SEQ ID NO:1 above is an amidase derived from Yersinia frederiksenii. By analyzing the protein structure (see Figure 1) based on this amino acid sequence, 28 sites near the active site and substrate channel were selected 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, and V232. These amino acid sites may affect the stereoselectivity of the protein. Mutation of these amino acid sites can result in proteins with amidase function or enhanced amidase function. The protein obtained above can be modified at non-essential mutation sites or active sites to obtain a protein that has 80% or more homology with the above amino acid sequence and has amidase function.

[0026] The sequence of SEQ ID NO:1 is as follows:

[0027] TIFF2025533078000017.tif37164In one preferred embodiment, the amino acid mutations in (b) are each independently selected from the group consisting of L81A, L81C, or L81V; P79Q; F80G; E62Q, E62K, E62M, E62T, E62A, E62R, E62C, E62V, E62P, E62H, E62N, or E62S; L76V; K 77T;D185H;R58M, R58K, R58L, R58H, or R58A;I227L, I227V, I227A, I227W, I227E, I227S, I227M, I227D, I227C, or I227Y;T230C, T230V, T230A, or T230L;N180V, N180D, or N180K;P121H, P121K, P1 21N, P121A, P121R, P121E, P121Y, or P121S;M311C, M311R, M311H, or M311S;V232I;R58L, R58M, R58K, R58Q, R58P, R58A, R58V, or R58F;E32D, E32H, E32M, E32Q, E32A, E32C, E32V, E32F, E32I, E32P, or E32T, wherein the letter before the number represents the original amino acid and the letter after the number represents the mutated amino acid, and preferably (c) is a protein having 85% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 99% or more homology to the amino acid sequence defined in (a) or (b) and having amidase function.

[0028] As used herein, the abbreviations for amino acid residues are as follows: alanine (Ala, A), asparagine (Asn, N), aspartic acid (Asp, D), arginine (Arg, R), cysteine ​​(Cys, C), glutamic acid (Glu, E), glutamine (Gin, 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 (Vai, V).

[0029] The rule for substitutions and replacements is that, in general, amino acids with similar properties will have similar effects when replaced. For example, conservative amino acid substitutions may occur in the above-mentioned homologous proteins. "Conservative amino acid substitutions" include, but are not limited to, the following:

[0030] Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are substituted with other hydrophobic amino acids.

[0031] Hydrophobic amino acids with bulky side chains (Phe, Tyr, Trp) are replaced with other hydrophobic amino acids with bulky side chains.

[0032] Amino acids with positively charged side chains (Arg, His, Lys) are replaced with other amino acids with positively charged side chains.

[0033] Amino acids with polar but uncharged side chains (Ser, Thr, Asn, Gln) are substituted with other amino acids with polar but uncharged side chains.

[0034] Those skilled in the art can also make conservative substitutions of 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.

[0035] In this application, the applicant continued to search for the above-mentioned active site and discovered that when the active site is mutated to a different amino acid, the activity of the corresponding protein also changes, and that specific mutations enhance amidase activity. Experimental searches have revealed that by making the above-mentioned specific mutations in the active site, a protein with enhanced activity can be obtained. The amino acid mutation sites of the amidase protein can be flexibly selected and combined from the above-mentioned mutations.

[0036] "Sequence identity" between two polypeptide sequences refers to the percentage of identical amino acids between the sequences, and methods for assessing 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 usually measured using sequence analysis software. For example, identity can be determined using the BLAST program in the NCBI database. For determining 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.

[0037] In one preferred embodiment, the mutation of the amidase mutant comprises any of the following amino acid mutations:

[0038] 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. ;

[0039] All of the above amino acid mutations have been explored through testing in the examples of this application, and as a result, amidase mutants can be obtained that have amidase activity, have higher stereoselectivity and catalytic activity than the parent enzyme having the amino acid sequence shown in SEQ ID NO: 1, and can be used for industrial scale-up production.

[0040] In a second exemplary embodiment of the present application, a DNA molecule encoding the above-described amidase mutant is provided.

[0041] In a third exemplary embodiment of the present application, a recombinant plasmid is provided in which the above-described DNA molecules are ligated.

[0042] The DNA can encode the amidase mutant and can be ligated into a recombinant plasmid to form a circular DNA. The DNA and the recombinant plasmid can be transcribed and translated by the action of RNA polymerase, ribosomes, tRNA, etc. to obtain the amidase mutant.

[0043] In a fourth exemplary embodiment of the present application, a host cell is provided that has been transformed with the recombinant plasmid. The host cell may be a prokaryotic cell or a eukaryotic cell. Specifically, the prokaryotic cell may be Escherichia coli, and the eukaryotic cell may be yeast.

[0044] Using the above-mentioned host cells, the recombinant plasmid can be replicated within the host cells, and the DNA molecule carried on the recombinant plasmid can be transcribed and translated to obtain a large number of amidase mutants. Using conventional techniques, the host cells can be disrupted to purify the protein, or disrupted and then catalyzed with crude enzymes, or other methods can be used to obtain the amidase mutants, which can then catalyze the synthesis of chiral carboxylic acid and chiral amide derivatives. This host cell is not derived from a plant.

[0045] A fifth exemplary embodiment of the present application provides a method for producing chiral carboxylic acid and amide derivatives, comprising the step of catalyzing the hydrolytic resolution reaction of an amide substrate represented by formula I using the above-described amidase mutant to obtain chiral carboxylic acid and amide derivatives.

[0046] [ka] wherein R1 is selected from aryl, heteroaryl, alkyl, alkylene, cycloalkyl, hydroxy, amino, or H, and when selected from hydroxy, amino, or H, is preferably hydroxy; R2 is selected from aryl, heteroaryl, alkyl, alkylene, cycloalkyl, hydroxy, amino, or H, and when selected from hydroxy, amino, or H, is preferably hydroxy; R3 is selected from aryl, heteroaryl, alkyl, alkylene, cycloalkyl, hydroxy, amino, or H, and when selected from hydroxy, amino, or H, is preferably hydroxy; or R1 may form a ring or heterocycle together with R2 or R3, the number of carbon atoms in the ring or heterocycle being selected from 3 to 6, the aryl, heteroaryl, alkyl, alkylene, cycloalkyl, or ring being each independently unsubstituted or substituted, and if substituted, the substituents being each independently selected from methyl, methylene, methoxy, amino, or halogen, preferably halogen, more preferably F, the heteroaryl or heterocycle being each independently unsubstituted or substituted, and the heteroatoms being each independently selected from N, O, or S, preferably N. The above-mentioned preparation method utilizes the above-mentioned amidase mutant to catalyze the hydrolytic resolution of the amide substrate represented by Formula I to obtain chiral carboxylic acid and amide derivatives. The above-mentioned amidase mutant can catalyze the hydrolytic resolution of the above-mentioned amide substrate. The catalytic activity and stereoselectivity of the amidase are significantly improved, improving production efficiency, satisfying the needs for industrial production of chiral carboxylic acid and amide derivatives, and reducing industrial production costs.

[0047] In one preferred embodiment, the amide-based substrate is selected from:

[0048] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] Based on the solved three-dimensional structure of the protein (5ubu.1), we selected and modified 28 sites near the active site and substrate channel: N82, N190, D192, D217, H219, M311, P121, N180, K77, M78, P79, F80, L81, E32, F61, E62, D185, R58, D59, L182, T183, P184, 1227, C228, G229, T230, A231, and V232. Protein modifications included saturation and combinatorial mutations.

[0049] Saturation mutagenesis is a method for rapidly obtaining mutants in which each amino acid at the target site is replaced with one of the other 19 amino acids by modifying the gene encoding the target protein. This method is not only a powerful tool for directed modification of target proteins, but also an important tool for studying the relationship between protein structure and function. Saturation mutagenesis often yields more ideal evolved mutants than single point mutations. These problems, which cannot be solved by site-directed mutagenesis, are precisely what saturation mutagenesis excels at. Saturation mutagenesis is constructed using whole-plasmid PCR, and then the PCR product is digested with DPNI enzyme to remove the template and transformed into E. coli BL21(DE3). Because screening for mutants often requires high-throughput screening methods, we developed the following method for screening for mutant resistance.

[0050] The following high-throughput screening method for screening the mutation library was developed.

[0051] 1. Culturing mutants: Add 300 μL of LB medium to each well of a 96-well plate, inoculate a single clone from the agar plate into a deep-well 96-well plate, and grow overnight at 37°C and 200 rpm. Using a Qpix, transfer the overnight culture to another 96-well plate with 800 μL of LB medium added to each well. Grow at 37°C and 200 rpm for 5 hours. When the OD600 of the 96-well plate reaches 0.6-0.9, add IPTG solution to the 96-well plate using a Qpix to a final IPTG concentration of 0.1 mM. Induce overnight at 200 rpm at 25°C for approximately 16 hours. Centrifuge at 4000 rpm for 5 minutes. Discard the supernatant and use the whole cells for the reaction.

[0052] 2. 96-well plate high-throughput screening system: Add 200 μl of phosphate buffer to each well of a 96-well plate and shake to resuspend. Then add 2–20 mg of substrate and react at 700 rpm in a constant-temperature shaker at 30°C. The reaction time can be varied depending on the screening objective. After the reaction is complete, add 1 ml of acetonitrile to the system to terminate the reaction. After centrifugation, collect the supernatant and subject it to UPLC for analysis.

[0053] Preliminary screening of the above mutants yielded mutants with improved properties. The optimal mutants were then induced and cultured in 2L shake flasks (optimal conditions for inducible expression: 25°C, overnight induction with 0.2 mM IPTG). The resulting slime was centrifuged and then sonicated to obtain a crude enzyme solution. Finally, a G-scale reaction was performed to confirm the properties of the optimal mutations. Because single-point saturation mutagenesis at the 28 sites described above often yields little improvement, multiple rounds of iterative saturation mutagenesis are usually required to obtain mutants with significantly improved properties.

[0054] After obtaining mutants with improved activity through saturation mutagenesis, beneficial amino acid sites identified through screening can be combined to obtain mutants with even better properties. Similar to single-point mutations, double-point mutations in combinatorial mutagenesis are constructed using whole-plasmid PCR. Multi-point mutations, which simultaneously mutate two or more sites, are amplified by overlap extension PCR to obtain mutant genes containing multiple point mutations. After digesting both ends with restriction enzymes, the resulting genes are ligated into expression vectors and transformed into E. coli. The cells are plated on LB plates containing 100 μg / mL ampicillin and grown overnight at 37°C to obtain combinatorial mutants. After accurate identification of the above combinatorial mutants by sequencing, they are directly induced and cultured in 2L shake flasks to confirm their activity.

[0055] Through multiple rounds of evolution, a series of amidase mutants were obtained, and it was found that these amidase mutants exhibited significantly improved catalytic efficiency toward various amide compounds. Chiral carboxylic acid and amide derivatives with extremely high chiral purity were obtained, demonstrating that these mutants are suitable for industrial production of chiral carboxylic acid and amide derivatives.

[0056] The present application will now be described in more detail with reference to specific examples, which should not be construed as limiting the scope of protection claimed by the present application.

[0057] Example 1 Based on the parent SEQ ID NO:1, saturation mutagenesis was carried out at the above 28 sites, and the catalytic activity and ee value of the mutants were detected under the following reaction conditions.

[0058] A 1-mL reaction system contained 100 mg of substrate 1 or substrate 2, 10 mg of enzyme powder (lyophilized from 100 mg of fermented wet slime), and 100 mM pH 7.5 phosphate buffer. The reaction time was monitored at 30°C. After 2 hours, a sample was taken and subjected to HPLC to measure the yield. After 16 hours, a sample was taken and subjected to HPLC to measure the ee value.

[0059] The detection results are shown in the table below.

[0060] [Table 1] Note: In the table above, - indicates a 10% yield relative to the wild-type parent, + indicates a yield of 20% or greater but less than 30%, ++ indicates a yield of 30% or greater but less than 40%, and +++ indicates a yield of 40% or greater but less than 45%. The ee values ​​in the table above represent the ee values ​​of the R-carboxylic acid when the yield reaches approximately 50%. * indicates an ee value of less than 80%, ** indicates an ee value of 80% or greater but less than 90%, and *** indicates an ee value of 90% or greater but less than 95%.

[0061] Example 2 Based on Example 1, the next round of saturation mutagenesis and combinatorial mutagenesis was carried out, and activity screening of the combinatorial mutations was carried out under the following reaction conditions, and the results are shown in the table below.

[0062] Each 1 mL reaction mixture contained 100 mg of substrate 1 or 2, 8 mg of enzyme powder (lyophilized from 80 mg of fermented wet slime), and 100 mM pH 7.5 phosphate buffer. The reaction time was monitored at 30°C. After 2 hours, a sample was taken and subjected to HPLC to measure the yield. After 16 hours, a sample was taken and subjected to HPLC to measure the ee value.

[0063] [Table 2] Note: In the table above, + indicates a yield of 20% or more but less than 30%, ++ indicates a yield of 30% or more but less than 40%, and +++ indicates a yield of 40% or more but less than 45%. The ee values ​​in the table above represent the ee values ​​of the R-carboxylic acid when the yield reaches approximately 50%. * indicates an ee value of less than 80%, ** indicates an ee value of 80% or more but less than 90%, *** indicates an ee value of 90% or more but less than 95%, and **** indicates an ee value of 95% or more but less than 98%.

[0064] Example 3 Based on Example 2, saturation mutagenesis and combinatorial mutagenesis were carried out, and the catalytic activity of the mutants was detected under the following reaction conditions.

[0065] Each 1 mL reaction mixture contained 100 mg of substrate 1 or 2, 5 mg of enzyme powder (lyophilized from 50 mg of fermented wet slime), and 100 mM pH 7.5 phosphate buffer. The reaction time was monitored at 30°C. After 2 hours, a sample was taken and subjected to HPLC to measure the yield. After 16 hours, a sample was taken and subjected to HPLC to measure the ee value.

[0066] [Table 3] Note: In the table above, + indicates a yield of 20% or more but less than 30%, ++ indicates a yield of 30% or more but less than 40%, +++ indicates a yield of 40% or more but less than 45%, and ++++ indicates a yield of 45% or more but less than 50%. The ee values ​​in the table above represent the ee values ​​of the R-carboxylic acid when the yield reaches approximately 50%. * indicates an ee value of less than 80%, ** indicates an ee value of 80% or more but less than 90%, *** indicates an ee value of 90% or more but less than 95%, and **** indicates an ee value of 95% or more but less than 98%.

[0067] Example 4 Based on Example 3, saturation mutagenesis and combinatorial mutagenesis were carried out, and the catalytic activity of the mutants was detected under the following reaction conditions.

[0068] A 1-mL reaction system contained 100 mg of substrate 1 or substrate 2, 2 mg of enzyme powder (lyophilized from 20 mg of fermented wet slime), and 100 mM pH 7.5 phosphate buffer. The reaction time was monitored at 30°C. After 1 hour, a sample was taken and subjected to HPLC to measure the yield. After 8 hours, a sample was taken and subjected to HPLC to measure the ee value.

[0069] [Table 4] Note: In the table above, + indicates a yield of 20% or more but less than 30%, ++ indicates a yield of 30% or more but less than 40%, +++ indicates a yield of 40% or more but less than 45%, and ++++ indicates a yield of 45% or more but less than 50%. The ee values ​​in the table above represent the ee values ​​of the R-carboxylic acid when the yield reaches approximately 50%. * indicates an ee value of less than 80%, ** indicates an ee value of 80% or more but less than 90%, *** indicates an ee value of 90% or more but less than 95%, and **** indicates an ee value of 95% or more but less than 98%.

[0070] Example 5 Based on Example 4, saturation mutagenesis and combinatorial mutagenesis were carried out multiple times, and the catalytic activity of the mutants was detected under the following reaction conditions.

[0071] A 1-mL reaction system contained 100 mg of substrate 1 or substrate 2, 1 mg of enzyme powder (lyophilized 10 mg of fermented wet slime), and 100 mM pH 7.5 phosphate buffer. The reaction time was monitored at 30°C. After 1 hour, a sample was taken and subjected to HPLC to measure the yield. After 8 hours, a sample was taken and subjected to HPLC to measure the ee value.

[0072] [Table 5] Note: In the table above, + indicates a yield of 20% or more but less than 30%, ++ indicates a yield of 30% or more but less than 40%, +++ indicates a yield of 40% or more but less than 45%, and ++++ indicates a yield of 45% or more but less than 50%. The ee values ​​in the table above represent the ee values ​​of the R-carboxylic acid when the yield reaches approximately 50%. * indicates an ee value of less than 80%, ** indicates an ee value of 80% or more but less than 90%, *** indicates an ee value of 90% or more but less than 95%, **** indicates an ee value of 95% or more but less than 98%, and ***** indicates an ee value of 99% or more.

[0073] Example 6 100 mL of 100 mmol / L phosphate buffer solution in a 250 mL four-neck flask. [ka] 10 g of the enzyme solution was added at room temperature and stirred uniformly. Next, 1 mL of the enzyme solution (prepared with 1 g of wet slime) of the amidase mutant L81A+E62A+M311C+T230C, which had been mutated based on SEQ ID NO: 1, was added, and the pH was adjusted to 7.5-8.0. The temperature was raised to 30°C and the mixture was stirred. The reaction time was monitored, and the reaction system was stopped when the yield reached about 50%. After that, post-treatment was performed, and finally, the desired product, R-carboxylic acid, was obtained. [ka] [ka] obtained.

[0074] HPLC detection showed that the purity of the R-carboxylic acid and S-amide derivatives was over 99%, the ee values ​​were over 99%, and the yields were 42% and 40%, respectively.

[0075] Example 7 100 mL of 100 mmol / L phosphate buffer solution in a 250 mL four-neck flask. [ka] 10 g of the enzyme solution was added at room temperature and stirred uniformly. Next, 1 mL of the enzyme solution (prepared with 1 g of wet slime) of the amidase mutant L81A+E62A+M311C+T230C, which had been mutated based on SEQ ID NO: 1, was added, and the pH was adjusted to 7.5-8.0. The temperature was raised to 30°C and the mixture was stirred. The reaction time was monitored, and the reaction system was stopped when the yield reached about 50%. After that, post-treatment was performed, and finally, the desired product, R-carboxylic acid, was obtained. [ka] [ka] obtained.

[0076] As a result of HPLC detection, the purity of the R-carboxylic acid and S-amide derivatives both exceeded 99%, the ee values ​​both exceeded 99%, and the yields were 41% and 43%, respectively.

[0077] Example 8 100 mL of 100 mmol / L phosphate buffer solution in a 250 mL four-neck flask. [ka] 10 g of the enzyme solution was added at room temperature and stirred uniformly. Next, 1 mL of the enzyme solution (prepared with 1 g of wet slide) of the amidase mutant L81A+E62A+M311C+T230C, which had been mutated based on SEQ ID NO: 1, was added and the pH was adjusted to 7.5-8.0. The temperature was raised to 30°C and the mixture was stirred. The reaction time was monitored, and the reaction system was stopped when the yield reached 95%. After that, workup was performed, and finally, the desired product, carboxylic acid, was obtained. [ka] obtained.

[0078] As a result of HPLC detection, the product carboxylic acid had a purity of >99%, an ee value of >99%, and a yield of >84%.

[0079] Example 9 Using the enzyme solution of the amidase mutant L81A+E62A+M311C+T230C, which was mutated based on SEQ ID NO:1, catalytic reactions were carried out for substrates 3 to 13 in accordance with the catalyst synthesis steps in Examples 6 to 8, and the results are shown in the table below.

[0080] [Table 6] From the above description, it can be seen that the above-described embodiments of the present invention achieve the following technical effects: 1) When an amidase mutant with improved activity and stereoselectivity is used to perform an amide resolution reaction, a series of chiral carboxylic acid and amide derivatives can be obtained with high activity and selectivity. 2) When this amidase mutant is used as a biocatalyst, the reaction conditions are mild and no heavy metal catalyst or toxic reagents are required, thereby realizing green chemistry. 3) This amidase has high biocatalytic activity, high concentration of reaction substrates, and high product yields, which significantly reduces exhaust, wastewater, and solid waste, thereby saving production costs.

[0081] The above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will appreciate that the present invention can be modified and changed in various ways. Any modifications, equivalent replacements, improvements, etc. made without departing from the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. an amidase mutant, (a) a protein having the amino acid sequence set forth in SEQ ID NO: 1, or (b) a protein in which an amino acid mutation occurs at at least one site selected from 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, and V232 in the amino acid sequence shown in (a), and which has amidase function; or (c) An amidase mutant, characterized in that it has a homology of 80% or more with the amino acid sequence defined in either (a) or (b) and contains a protein having amidase function.

2. The amino acid mutations in (b) each independently comprise: 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 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 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; Selected from E32D, or E32H, or E32M, or E32Q, or E32A, or E32C, or E32V, or E32F, or E32I, or E32P, or E32T; where the letter before the number represents the original amino acid, and the letter after the number represents the mutated amino acid, Preferably, (c) is a protein having 85% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 99% or more homology with the amino acid sequence defined in (a) or (b) and having amidase function.

3. The amidase mutant according to claim 2, characterized in that 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; 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+T227V; 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.

4. A DNA molecule encoding the amidase mutant according to any one of claims 1 to 3.

5. A recombinant plasmid comprising the DNA molecule of claim 4 ligated thereto.

6. A host cell transformed with the recombinant plasmid of claim 5.

7. A method for producing chiral carboxylic acid and chiral amide derivatives, comprising the steps of: A method for producing chiral carboxylic acid and chiral amide derivatives, comprising the step of catalyzing the hydrolytic resolution reaction of an amide substrate represented by formula I using the amidase mutant described in any one of claims 1 to 3 to obtain chiral carboxylic acid and chiral amide derivatives. 【Chemical 1】 (R １ is selected from aryl, heteroaryl, alkyl, alkylene, cycloalkyl, hydroxy, amino, or H, and when selected from said hydroxy, said amino, or said H, is preferably said hydroxy; R ２ is selected from aryl, heteroaryl, alkyl, alkylene, cycloalkyl, hydroxy, amino, or H, and when selected from said hydroxy, said amino, or said H, is preferably said hydroxy; R ３ is selected from aryl, heteroaryl, alkyl, alkylene, cycloalkyl, hydroxy, amino, or H, and when selected from said hydroxy, said amino, or said H, is preferably said hydroxy; Or, the R １ is the R ２ Or the R ３ and a ring or a heterocycle may be formed with the ring or the heterocycle, and the number of C atoms in the ring or the heterocycle is selected from 3 to 6; wherein the aryl, heteroaryl, alkyl, alkylene, cycloalkyl, or ring is 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; The heteroaryl or heterocycle is independently unsubstituted or substituted, and the heteroatoms are independently selected from N, O, or S, preferably N.

8. The method of claim 7, wherein the amide-based substrate is selected from the following: 【Chemistry 2】 【Chemistry 3】 【Chemistry 4】 【Chemistry 5】 【Chemistry 6】 【Chemistry 7】 【Chemistry 8】 【Chemistry 9】 【Chemistry 10】 【Chemistry 11】 【Chemistry 12】 【Chemistry 13】 【Chemistry 14】 【Chemistry 15】

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