Monooxygenase mutants and their use

Abstract

The present invention provides a monooxygenase mutant and its use. The monooxygenase mutant includes: (a) a protein having the amino acid sequence shown in SEQ ID NO: 1, or (b) a protein in which an amino acid mutation occurs at at least one site such as W60, Y65, D71, Y77, D163, V166, S178, T179, T199, G200, S201, R222 in the amino acid sequence of (a) and which has a monooxygenase function, and (c) a protein having a homology of 80% or more with the amino acid sequence limited to any one of (a) and (b) and which has a monooxygenase function. It can solve the problem of the prior art that the monooxygenase activity is low and is suitable for the field of enzyme catalysis.

Description

Technical Field

[0001] The present invention relates to the field of enzyme catalysts, and specifically to a monooxygenase mutant and its use.

Background Art

[0002] Chiral sulfoxides are a type of chiral organic sulfur compound and are widely used as chiral auxiliaries or ligands in asymmetric catalysis and as bioactive molecules in the pharmaceutical industry. Many chiral sulfoxides contain one or more chiral centers, and the pharmacological activities, metabolic processes, metabolic rates, and toxicities of different chiral drugs are significantly different. In many cases, one enantiomer is effective, while the other enantiomer has low or no effect. For example, in the case of omeprazole and lansoprazole, which are chiral sulfoxide gastrointestinal drugs, the enantiomeric purity has a great influence on the effectiveness of these drugs. Esomeprazole, the levorotatory form of omeprazole, is more effective than the dextrorotatory form, and dextrorotatory lansoprazole is more effective than the levorotatory form. Therefore, whether to construct a compound containing a chiral center with high stereoselectivity has important significance in pharmaceutical research and development.

[0003] Chiral sulfoxides can be synthesized using chemical and biological methods. Chemical methods include methods such as chiral auxiliary induction, chiral resolution, and asymmetric catalysis. However, the synthesis process has several obvious drawbacks, such as the use of harmful oxidants and organometallic reagents. Furthermore, it is insufficient to efficiently achieve high optical purity sulfoxides, and the removal of isomers downstream often causes problems. In contrast, the use of biological methods to achieve the synthesis of chiral sulfoxides has the "green" advantages of relatively mild reaction conditions and no generation of toxic waste, as well as better enantioselectivity and diastereoselectivity, and is becoming increasingly popular among people. However, wild-type monooxygenases generally have problems such as a narrow substrate range, low activity, low stability, insufficient selectivity, and the generation of sulfones as by-products. Monooxygenases can be modified by directed evolution to improve various properties of the enzyme and enable its use in industrial production.

Summary of the Invention

Problems to be Solved by the Invention

[0004] The main object of the present invention is to provide a monooxygenase mutant and its use to solve the problems of the prior art, such as low activity of monooxygenase, insufficient chirality of the product, and a large amount of impurity sulfone.

Means for Solving the Problems

[0005] To achieve the above object, according to one aspect of the present invention, (a) a protein having the amino acid sequence shown in SEQ ID NO: 1, or (b) A protein in which at least one site of W60, Y65, D71, Y77, D163, V166, S178, T179, T199, G200, S201, R222, T223, H337, P338, K342, R343, S344, R365, I393, F395, D396, A397, L448, D505, S506, Y508 or R509 in the amino acid sequence of (a) has an amino acid mutation and has a monooxygenase function, (c) Provided is a monooxygenase mutant comprising a protein having a homology of 80% or more with the amino acid sequence limited to any one of (a) and (b) and having a monooxygenase function.

[0006] Furthermore, the amino acid mutations in (b) are each independently D163V, D163A, or D163E; V166S, V166Y, V166M, V166C, V166D, V166H, V166I, V166N, V166K, V166M, V166L, V166A, V166T, V166P, V166G, or V166F; H337Y, H337M, H337K, H337P, H337F, H337A, H337L, H337E, H337D, or H337R; P338A, P338M, P338L, or P338Y; K342A, K342E, K342L, or K342Y; R343A, R343Y, R343H, or R343V; S344F, S344Y, S344L, S344M, S344K, S344A, S344T, S344N, S344R, S344D, or S344E; R365A, R365G, R365D, R365T, or R365Y; I393A, I393K, I393P, I393W, I393R, I393C, I393M, I393T, I393V, I393Y, I393G, or I393L; F395S, F395Q, or F395V; D396A, D396R, D396S, D396F, D396H, D396Q, or D396K; selected from A397V, A397L, A397I, A397M, A397R, A397H, or A397W, wherein the letter before the number represents the original amino acid and the letter after the number represents the mutant amino acid, preferably, the said (c) has a homology of 85% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 99% or more with the amino acid sequence limited to (a) or (b), and is a protein having a monooxygenase function.

[0007] Furthermore, the mutation of the monooxygenase mutant includes any one of the following amino acid mutations. D163V; D163A; D163E; A397V; A397L; A397I; V166S; V166Y; V166M; I393A; I393K; I393P; D396A; D396R; D163A + A397L; D163A + A397V; D163A + A397I; D163A + A397M; D163A + A397R; D163A + A397H; D163A + A397W; D163A + I393W; D163A + I393R; D163A + I393C; D163A + I393L; D163A + I393M; D163A + I393T; D163A + I393V; D163A + I393Y; D163A + I393G; D163A + V166C; D163A + V166D; D163A + V166H; D163A + V166I; D163A + V166N; D163A + V166K; D163A + V166M; D163A + V166L; D163A + H337Y; D163A + H337M; D163A + H337K; D163A + H337P; D163A + H337F; D163A + S344F; D163A + S344Y; D163A + S344L; D163A + S344M; D163A + S344K; D163A + S344A; D163A + S344T; D163A + A397L + I393R; D163A + A397L + I393C; D163A + A397L + I393L; D163A + A397L + I393M; D163A + A397L + I393T; D163A + A397L + I393V; D163A + A397L + V166I; D163A + A397L + V166C; D163A + A397L + V166H; D163A + A397L + V166A; D163A + A397L + V166Y; D163A + A397L + V166D; D163A + A397L + V166T; D163A + A397L + V166N; D163A+A397L+S344F; D163A+A397L+S344M; D163A+A397L+S344L; D163A+A397L+S344Y; D163A+A397L+S344K; D163A+A397L+S344N; D163A+A397L+S344R; D163A+A397L+I393L; D163A+A397L+I393L+V166C; D163A+A397L+I393L+V166H; D163A+A397L+I393L+V166D; D163A+A397L+I393L+V166I; D163A+A397L+I393L+V166N; D163A+A397L+I393L+V166P; D163A+A397L+I393L+V166G; D163A+A397L+I393L+V166F; D163A+A397L+I393L+V166L; D163A+A397L+I393L+H337Y; D163A+A397L+I393L+H337A; D163A+A397L+I393L+H337L; D163A+A397L+I393L+H337E; D163A+A397L+I393L+H337D; D163A+A397L+I393L+S344A; D163A+A397L+I393L+S344D; D163A+A397L+I393L+S344F; D163A+A397L+I393L+S344L; D163A+A397L+I393L+S344R; D163A+A397L+I393L+S344Y; D163A+A397L+I393L+V166I+H337Y; D163A+A397L+I393L+V166I+H337A; D163A+A397L+I393L+V166I+H337L; D163A+A397L+I393L+V166I+H337R; D163A+A397L+I393L+V166I+H337F; D163A+A397L+I393L+V166I+S344M; D163A+A397L+I393L+V166I+S344Y; D163A+A397L+I393L+V166I+S344L; D163A+A397L+I393L+V166I+S344F; D163A+A397L+I393L+V166I+S344A; D163A+A397L+I393L+V166I+S344E; D163A+A397L+I393L+V166I+S344T; D163A+A397L+I393L+V166I+S344Y+P338A; D163A+A397L+I393L+V166I+S344Y+P338M; D163A+A397L+I393L+V166I+S344Y+P338L; D163A+A397L+I393L+V166I+S344Y+P338Y; D163A+A397L+I393L+V166I+S344Y+K342A; D163A+A397L+I393L+V166I+S344Y+K342E; D163A+A397L+I393L+V166I+S344Y+K342L; D163A+A397L+I393L+V166I+S344Y+K342Y; D163A+A397L+I393L+V166I+S344Y+R343A; D163A+A397L+I393L+V166I+S344Y+R343A; D163A + A397L + I393L + V166I + S344Y + R343Y; D163A + A397L + I393L + V166I + S344Y + R343H; D163A + A397L + I393L + V166I + S344Y + R343V; D163A + A397L + I393L + V166I + S344Y + R365A; D163A + A397L + I393L + V166I + S344Y + R365G; D163A + A397L + I393L + V166I + S344Y + R365D; D163A + A397L + I393L + V166I + S344Y + R365T: D163A + A397L + I393L + V166I + S344Y + R365Y; D163A + A397L + I393L + V166I + S344Y + D396S; D163A + A397L + I393L + V166I + S344Y + D396F; D163A + A397L + I393L + V166I + S344Y + D396H; D163A + A397L + I393L + V166I + S344Y + F164A; D163A + A397L + I393L + V166I + S344Y + F164L; D163A + A397L + I393L + V166I + S344Y + F164M; D163A + A397L + I393L + V166I + S344Y + D165A; D163A + A397L + I393L + V166I + S344Y + D165M; D163A + A397L + I393L + V166I + S344Y + D165Y; D163A + A397L + I393L + V166I + S344Y + K162A; D163A + A397L + I393L + V166I + S344Y + K162S; D163A + A397L + I393L + V166I + S344Y + K162G; D163A+A397L+I393L+V166I+S344Y+G394A; D163A+A397L+I393L+V166I+S344Y+G394L; D163A+A397L+I393L+V166I+S344Y+G394T; D163A+A397L+I393L+V166I+S344Y+F395S; D163A+A397L+I393L+V166I+S344Y+F395Q; D163A+A397L+I393L+V166I+S344Y+F395V; D163A+A397L+I393L+V166I+S344Y+D396Q; D163A+A397L+I393L+V166I+S344Y+D396S; D163A+A397L+I393L+V166I+S344Y+D396F; D163A+A397L+I393L+V166I+S344Y+D396H; D163A+A397L+I393L+V166I+S344Y+D396K

[0008] To achieve the above object, according to a second aspect of the present invention, there is provided a DNA molecule encoding the above monooxygenase mutant.

[0009] To achieve the above object, according to a third aspect of the present invention, there is provided a recombinant plasmid to which the above DNA molecule is ligated.

[0010] 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.

[0011] Furthermore, the host cell includes a prokaryotic cell, and preferably, the prokaryotic cell includes Escherichia coli.

[0012] In order to achieve the above object, according to a fifth aspect of the present invention, there is provided a method for producing a chiral sulfoxide compound, comprising the step of subjecting the above-mentioned monooxygenase mutant to an oxygen addition reaction with a thioether-based substrate represented by formula I and / or formula II to obtain a chiral sulfoxide compound.

Chemical formula

Chemical formula

[0013] Furthermore, the thioether-based substrate is selected from the following.

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

[0014] According to the technical solution of the present invention, a monooxygenase mutant (SEQ ID NO: 1) derived from Rhodococcus jostii RHA1 is used as the parent strain, and protein engineering modifications such as single-site specific mutations, saturation mutations, and combinatorial mutations are performed to produce a high-purity optically pure chiral sulfoxide compound, obtain a monooxygenase mutant with less impurity sulfone and high enzyme activity.

Mode for Carrying Out the Invention

[0015] Unless there is a contradiction, the examples of the present application and the features in the examples of the present application can be combined with each other. Hereinafter, the present invention will be described in more detail by way of examples.

[0016] As described in the background art, although the chemical synthesis of chiral sulfoxides has various drawbacks, synthesizing chiral sulfoxides using biological methods is not only more "green" but also the reaction is milder, making it a preferred method for synthesizing this substance. However, wild-type monooxygenases for biosynthesis mainly have the problem of low activity. Furthermore, there are also problems such as a narrow substrate range, low stability, insufficient selectivity, and the generation of by-product sulfones.

[0017] Therefore, in the present application, the inventors attempted to improve various properties of the enzyme by modifying the monooxygenase by directed evolution so that it can be used for a technical solution for the industrial production of chiral sulfoxides, and as a result, a series of protection solutions of the present application were proposed.

[0018] In the first representative embodiment of the present application, a monooxygenase mutant is provided, which includes: (a) a protein having the amino acid sequence shown in SEQ ID NO: 1, or (b) a protein in which at least one site of W60, Y65, D71, Y77, D163, V166, S178, T179, T199, G200, S201, R222, T223, H337, P338, K342, R343, S344, R365, I393, F395, D396, A397, L448, D505, S506, Y508, or R509 in the amino acid sequence of (a) has an amino acid mutation and has a monooxygenase function, and (c) a protein having a homology of 80% or more with the amino acid sequence limited to any one of (a) and (b) and having a monooxygenase function.

[0019] The amino acid sequence represented by SEQ ID NO: 1 is an amino acid mutant derived from Rhodococcus jostii RHA1. Through computer simulation of homology modeling of this amino acid sequence, as a result of analyzing the molecular docking results between the model structure and the thioether substrate, 28 amino acid residues including W60, Y65, D71, Y77, D163, V166, S178, T179, T199, G200, S201, R222, T223, H337, P338, K342, R343, S344, R365, I393, F395, D396, A397, L448, D505, S506, Y508, R509 were found. These amino acid sites are near the active center and may affect the catalytic properties of the protein. By mutating the above amino acid sites, a protein having a monooxygenase function, and thus a protein with enhanced monooxygenase function, can be obtained. Changes can be made to the non-essential mutation sites and active sites of the protein obtained above, and a protein having a homology of 80% or more with the above amino acid sequence and having a monooxygenase function can be obtained.

[0020] The sequence of SEQ ID NO: 1 is as follows: MTTSMKAANPMNFPSTSDTGIVDVLGVGAGFSGLYLSHRLTTAGWTFAGFEAGPSVGGTWFWNTYPGARCDVESIYYSYSFDEALQQEWTWSQRFAPQAEILSYINHVADRFDLRKHFTFNTRVVGATWNAAERLWEVQLDNGETRRGRYLISGAGGLSTPKDFDVPGLGNFTGLQVSTSRWNISLDDLAGKRVAVIGTGSSGVQAIPLIAEVAEHVTVFQRTPNYVMPARNAELPLERVDSIKDDYPAIREECRHSPGGIPDRPVTDKAFDVSAEERQRRYEAAYERSGFNGVGGEFADLLTDVEANRTASEFIHDKIREIVEDPATAELLVPRYHPLGAKRSVFGTDYYETYNRPNVSLVSLRDEPIETMTANAIVTSKGTYEADAVVLAIGFDAFTGPLYGLGLTGASGRKLQETWQDGIRTYLGMMTTDFPNFFMVAGPQSPALASNVVMTIEQAVDWIADLIEHARDSGATLVEATPEGQNDWVDITEETVAQTLYATTDSWYRGSNVEGKPNTFMGYVGGVGKYRRMCTEIAKRGYPGVRIDGETESPHLGPIHREIS

[0021] In a preferred embodiment, the amino acid mutations in (b) are, independently of each other, D163V, D163A, or D163E; V166S, V166Y, V166M, V166C, V166D, V166H, V166I, V166N, V166K, V166M, V166L, V166A, V166T, V166P, V166G, or V166F; H337Y, H337M, H337K, H337P, H337F, H337A, H337L, H337E, H337D, or H337R; P338A, P338M, P338L, or P338Y; K342A, K342E, K342L, or K342Y; R343A, R343Y, R343H, or R343V; S344F, S344Y, S344L, S344M, S344K, S344A, S344T, S344N, S344R, S344D, or S344E; R365A, R365G, R365D, R365T, or R365Y; I393A, I393K, I393P, I393W, I393R, I393C, I393M, I393T, I393V, I393Y, I393G, or I393L; F395S, F395Q, or F395V; D396A, D396R, D396S, D396F, D396H, D396Q, or D396K; A397V, A397L, A397I, A397M, A397R, A397H, or A397W, where the letter before the number represents the original amino acid and the letter after the number represents the mutated amino acid. Preferably, (c) has a homology of 85% or more, preferably 90% or more, more preferably 95%, 96%, 97% or 98% or more, still more preferably 99%, 99.9% or more with the amino acid sequence limited in (a) or (b) and is a protein having a monooxygenase function.

[0022] In the present application, as a result of the applicant's continued exploration of the above active site, it was found that when the active site mutates to different amino acids, the activity of the corresponding protein also differs, and that specific mutations can improve the activity of the monooxygenase. Through experimental exploration, it was found that a protein with enhanced activity can be obtained by introducing the above specific mutation into the active site. The amino acid mutation sites of the monooxygenase protein can be flexibly selected from among the above mutations and can be combined.

[0023] In a preferred embodiment, the mutation of the monooxygenase mutant comprises any one of the following amino acid mutations. D163V; D163A; D163E; A397V; A397L; A397I; V166S; V166Y; V166M; I393A; I393K; I393P; D396A; D396R; D163A + A397L; D163A + A397V; D163A + A397I; D163A + A397M; D163A + A397R; D163A + A397H; D163A + A397W; D163A + I393W; D163A + I393R; D163A + I393C; D163A + I393L; D163A + I393M; D163A + I393T; D163A + I393V; D163A + I393Y; D163A + I393G; D163A + V166C; D163A + V166D; D163A + V166H; D163A + V166I; D163A + V166N; D163A + V166K; D163A + V166M; D163A + V166L; D163A + H337Y; D163A + H337M; D163A + H337K; D163A + H337P; D163A + H337F; D163A + S344F; D163A + S344Y; D163A + S344L; D163A + S344M; D163A + S344K; D163A + S344A; D163A + S344T; D163A + A397L + I393R; D163A + A397L + I393C; D163A + A397L + I393L; D163A + A397L + I393M; D163A + A397L + I393T; D163A + A397L + I393V; D163A + A397L + V166I; D163A + A397L + V166C; D163A + A397L + V166H; D163A + A397L + V166A; D163A + A397L + V166Y; D163A + A397L + V166D; D163A + A397L + V166T; D163A + A397L + V166N; D163A + A397L + S344F; D163A + A397L + S344M; D163A + A397L + S344L; D163A + A397L + S344Y; D163A + A397L + S344K; D163A + A397L + S344N; D163A + A397L + S344R; D163A + A397L + I393L; D163A + A397L + I393L + V166C; D163A + A397L + I393L + V166H; D163A + A397L + I393L + V166D;D163A+A397L+I393L+V166I; D163A+A397L+I393L+V166N; D163A+A397L+I393L+V166P; D163A+A397L+I393L+V166G; D163A+A397L+I393L+V166F; D163A+A397L+I393L+V166L; D163A+A397L+I393L+H337Y; D163A+A397L+I393L+H337A; D163A+A397L+I393L+H337L; D163A+A397L+I393L+H337E; D163A+A397L+I393L+H337D; D163A+A397L+I393L+S344A; D163A+A397L+I393L+S344D; D163A+A397L+I393L+S344F; D163A+A397L+I393L+S344L; D163A+A397L+I393L+S344R; D163A+A397L+I393L+S344Y; D163A+A397L+I393L+V166I+H337Y; D163A+A397L+I393L+V166I+H337A; D163A+A397L+I393L+V166I+H337L; D163A+A397L+I393L+V166I+H337R; D163A+A397L+I393L+V166I+H337F; D163A+A397L+I393L+V166I+S344M; D163A+A397L+I393L+V166I+S344Y; D163A+A397L+I393L+V166I+S344L; D163A+A397L+I393L+V166I+S344F; D163A+A397L+I393L+V166I+S344A; D163A+A397L+I393L+V166I+S344E; D163A+A397L+I393L+V166I+S344T; D163A+A397L+I393L+V166I+S344Y+P338A; D163A+A397L+I393L+V166I+S344Y+P338M; D163A+A397L+I393L+V166I+S344Y+P338L; D163A+A397L+I393L+V166I+S344Y+P338Y; D163A+A397L+I393L+V166I+S344Y+K342A; D163A+A397L+I393L+V166I+S344Y+K342E;D163A+A397L+I393L+V166I+S344Y+K342L; D163A+A397L+I393L+V166I+S344Y+K342Y; D163A+A397L+I393L+V166I+S344Y+R343A; D163A+A397L+I393L+V166I+S344Y+R343A; D163A+A397L+I393L+V166I+S344Y+R343Y; D163A+A397L+I393L+V166I+S344Y+R343H; D163A+A397L+I393L+V166I+S344Y+R343V; D163A+A397L+I393L+V166I+S344Y+R365A; D163A+A397L+I393L+V166I+S344Y+R365G; D163A+A397L+I393L+V166I+S344Y+R365D; D163A+A397L+I393L+V166I+S344Y+R365T; D163A+A397L+I393L+V166I+S344Y+R365Y; D163A+A397L+I393L+V166I+S344Y+D396S; D163A+A397L+I393L+V166I+S344Y+D396F; D163A+A397L+I393L+V166I+S344Y+D396H; D163A+A397L+I393L+V166I+S344Y+F164A; D163A+A397L+I393L+V166I+S344Y+F164L; D163A+A397L+I393L+V166I+S344Y+F164M; D163A+A397L+I393L+V166I+S344Y+D165A; D163A+A397L+I393L+V166I+S344Y+D165M; D163A+A397L+I393L+V166I+S344Y+D165Y; D163A+A397L+I393L+V166I+S344Y+K162A; D163A+A397L+I393L+V166I+S344Y+K162S; D163A+A397L+I393L+V166I+S344Y+K162G; D163A+A397L+I393L+V166I+S344Y+G394A; D163A+A397L+I393L+V166I+S344Y+G394L; D163A+A397L+I393L+V166I+S344Y+G394T;D163A+A397L+I393L+V166I+S344Y+F395S; D163A+A397L+I393L+V166I+S344Y+F395Q; D163A+A397L+I393L+V166I+S344Y+F395V; D163A+A397L+I393L+V166I+S344Y+D396Q; D163A+A397L+I393L+V166I+S344Y+D396S; D163A+A397L+I393L+V166I+S344Y+D396F; D163A+A397L+I393L+V166I+S344Y+D396H; D163A+A397L+I393L+V166I+S344Y+D396K。;

[0024] All of the above amino acid mutations were experimentally studied in the examples of this application. All of them have monooxygenase activity. Compared with the parental strain having the amino acid sequence shown in SEQ ID NO: 1, a monooxygenase mutant capable of obtaining a high-purity optically pure chiral sulfoxide, having high enzyme activity, and generating less impurity sulfone can be obtained.

[0025] In the second representative embodiment of this application, a DNA molecule encoding the above monooxygenase mutant is provided.

[0026] In the third representative embodiment of this application, a recombinant plasmid to which the above DNA molecule is ligated is provided.

[0027] The above DNA can encode the above monooxygenase mutant and can be ligated to a recombinant plasmid to form circular DNA. Both the above DNA and the recombinant plasmid can obtain the above monooxygenase mutant through transcription and translation by the action of RNA polymerase, ribosome, tRNA, etc.

[0028] In the fourth representative embodiment of this application, a prokaryotic host cell transformed with the above recombinant plasmid is provided.

[0029] Using the above-mentioned prokaryotic host cell, a recombinant plasmid can be replicated in the prokaryotic host cell, and a DNA molecule carried on the recombinant plasmid can be transcribed and translated to obtain a large amount of monooxygenase mutants. Using conventional techniques, the prokaryotic host cell can be disrupted to purify the protein, and the monooxygenase mutants can be obtained by methods such as catalyzing with a crude enzyme after disruption, and then the sulfoxide compound can be catalyzed. This host cell is a non-plant-derived prokaryotic host cell.

[0030] In the fifth representative embodiment of the present application, a method for producing a chiral sulfoxide compound is provided. The production method includes the step of subjecting the above-mentioned monooxygenase mutant to an oxygen addition reaction with a thioether substrate represented by Formula I and / or Formula II to obtain a chiral sulfoxide compound, where R1 is selected from alkyl, cycloalkyl, aryl or heteroaryl, the number of carbon atoms of the alkyl is selected from 1 to 8, the number of carbon atoms of the cycloalkyl, aryl or heteroaryl is selected from 5 to 10, R2 is selected from alkyl, cycloalkyl, aryl or heteroaryl, the number of carbon atoms of the alkyl is selected from 1 to 8, the number of carbon atoms of the cycloalkyl, aryl or heteroaryl is selected from 5 to 10, or R1 and R2 together with a sulfur atom form a heterocyclyl, carbocyclic group or heteroaryl, the number of carbon atoms of the heterocyclyl, carbocyclic group or heteroaryl is selected from 5 to 10, the heteroatoms in the heterocyclyl or heteroaryl are each independently at least one selected from nitrogen, oxygen, and sulfur, and the aryl in the aryl, heteroaryl in the heteroaryl, carbocyclic group in the carbocyclic group or heterocyclyl in the heterocyclyl is each independently unsubstituted or substituted, and the substituent is at least one group selected from halogen, alkoxy or alkyl.

Chemical formula

Chemical formula

[0031] According to the above manufacturing method, by using the above monooxygenase mutant, in the reaction of the thioether-based substrate of formula I and / or formula II, an oxygen addition reaction is carried out on the thioether-based substrate represented by formula I and / or formula II to obtain a chiral sulfoxide compound. The above monooxygenase mutant can perform chiral catalysis on the above thioether-based substrate to synthesize a desired chiral sulfoxide compound. It greatly improves the activity and selectivity of monooxygenase, avoids the generation of sulfoxide impurities in the catalytic process, enhances the production efficiency, reduces the cost of industrial production, and is more suitable for industrial production.

[0032] In one preferred embodiment, the thioether-based substrate is selected from the following.

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0033] In the present application, the inventors used homology modeling to perform molecular docking on different thioether substrates according to the model structure, analyzed the docking results, and selected 28 residues near the active center that may affect the catalytic properties of the protein. These residues include W60, Y65, D71, Y77, D163, V166, S178, T179, T199, G200, S201, R222, T223, H337, P338, K342, R343, S344, R365, I393, F395, D396, A397, L448, D505, S506, Y508, R509. Saturated mutagenesis and site-directed mutagenesis were performed on these residues.

[0034] Among these, saturated mutagenesis is a method for modifying the gene encoding the target protein to obtain mutants in a short time in which the amino acids at the target site are each replaced by 19 other amino acids. This method is not only a powerful tool for the directed modification of proteins but also an important means for studying the relationship between the structure and function of proteins. Saturated mutagenesis often yields more ideal evolved forms than single-point mutagenesis. These problems that cannot be solved by the site-directed mutagenesis method are exactly what the saturated mutagenesis method excels at. The mutants obtained by saturated mutagenesis were identified by sequencing, and their activities against various substrates and resistance at high temperatures were tested.

[0035] Site-directed mutagenesis: Refers to introducing desirable changes (usually changes characterizing the advantageous direction), including addition of bases, deletion, point mutations, etc., into a target DNA fragment (which may be a genome or a plasmid) by methods such as polymerase chain reaction (PCR). Site-directed mutagenesis can rapidly and efficiently improve the traits and characteristics of the target protein expressed by DNA and is a very useful means in genetic research work. The method of introducing site-directed mutagenesis using whole plasmid PCR is simple and effective and is currently a widely used method. Its principle is as follows. After annealing a pair of primers (forward and reverse) containing the mutation site together with the template plasmid, polymerase is used for "circular extension". So-called circular extension means that the polymerase extends the primer according to the template, stops back at the 5' end of the primer after one cycle, and then repeats the cycles of heating, annealing, and extension. This reaction, unlike rolling circle amplification, does not form multiple tandem copies. The extension products of the forward primer and the reverse primer anneal to form a paired nicked open plasmid. Since the extension product is digested by DpnI enzyme and the original template plasmid is derived from conventional Escherichia coli and modified by Dam methylation, it is sensitive to DpnI and is cleaved. However, the plasmid with the mutant sequence synthesized in vitro is not methylated and is not cleaved, so it can be successfully transformed in subsequent transformation and clones of the mutant plasmid can be obtained.

[0036] Develop the following high-throughput screening methods for screening mutant libraries.

[0037] 1. Culturing of mutants: Add 300 μL of LB medium to each well of a 96-well plate, inoculate a single clone on an agar plate into a deep-well 96-well plate, and culture overnight at 37 °C and 200 rpm. Using Qpix, transfer the overnight-cultured bacterial solution to another 96-well plate with 800 μL of LB medium added to each well, incubate at 37 °C and 200 rpm for 5 h, and when the OD 600 of the bacterial solution in the 96 wells reaches 0.6 - 0.9, use Qpix again to add IPTG solution to the 96-well plate to make the final concentration of IPTG in the well plate 0.1 mM, induce at 25 °C and 200 rpm for about 16 h, centrifuge at 4000 rpm for 5 min, discard the supernatant, and perform the reaction using the whole cells.

[0038] 2. High-throughput screening system for 96-well plates: Add the reaction system to the 96-well plate obtained in Step 1. Supplement 0.6 mg of NADP+ and 0.5 wt of alcohol dehydrogenase with PB (0.2 M) at pH 8.0 to a total volume of 300 μL and mix uniformly. Dissolve 3 mg of the substrate in 20 μL of isopropyl alcohol to prepare a substrate solution. Add the prepared substrate to the 96-well plate reaction system, mix uniformly, react at 20 °C and 200 rpm for 16 h using a thermostatic shaker, add acetonitrile to the 96-well plate reaction system that has reacted overnight to stop the reaction, centrifuge, use a multi-channel pipette to aspirate the supernatant into a shallow-well plate dedicated for HPLC detection, seal with tin foil, and perform high-throughput HPLC analysis to select mutants with improved characteristics.

[0039] Determine the sequences of the mutants selected from the mutant library, select appropriate mutants based on the results, and perform amplification reaction verification. After multiple rounds of evolution, the inventors obtained a series of monooxygenase mutants in which both the activity and selectivity of these mutants for various thioether-based substrates were significantly improved and the impurity sulfone was significantly reduced. These mutants can be used in industrial production and extremely greatly improve the catalytic efficiency.

[0040] Hereinafter, the present application will be described in more detail with reference to specific examples, but these examples should not be understood as limiting the scope of protection required by the present application.

[0041] Example 1 Using the above method, site-directed mutagenesis was carried out based on the sequence number 1 of the parent strain. The detailed mutation sites are shown in Table 1, and the catalytic activity of the mutant was detected according to the following reaction conditions.

[0042] The reaction system contained 100 mg of substrate 1 or substrate 2, 120 μL of isopropyl alcohol, NADP + 20 mg, 5 mg of alcohol dehydrogenase dry powder, and 1 mL of monooxygenase crude enzyme solution (prepared from 100 mg of wet bacterial sludge), and the total volume was supplemented to 6 mL with 0.2 M PB 8.0. The reaction was carried out at 200 rpm for 16 h in a 20 °C constant temperature shaker. After the reaction was completed, three volumes of acetonitrile were added to the system to stop the reaction, followed by centrifugation, sampling, and HPLC analysis of the conversion rate and ee value of the product. The results are shown in the following table.

[0043] The above-mentioned wet bacterial sludge was obtained by centrifuging the Escherichia coli of the corresponding mutant after fermentation, and the crude enzyme solution was obtained by adding 100 mM phosphate buffer at pH 8.0 to the obtained wet bacterial sludge and disrupting the cell wall by ultrasonic waves.

[0044] The detection results are shown in Table 1 below.

Table 1

[0045] Example 2 Based on Example 1, saturation mutations and combinatorial mutations were carried out, and the activity of the combinatorial mutations was screened according to the same reaction conditions as in Example 1. The results are shown in Table 2. [Table 2] Remarks: In the above table, + represents a conversion rate < 20%, ++ represents a conversion rate of 20% or more and less than 30%, +++ represents a conversion rate of 30% or more and 50% or less, - represents an ee value of 90% or less, -- represents an ee value of more than 90% and 95% or less, --- represents an ee value of more than 95% and 98% or less, ---- represents an ee value of more than 98% and 99.5% or less, **** represents an impurity sulfone content of more than 5%, *** represents an impurity sulfone content of less than 5% and more than 2%, ** represents an impurity sulfone content of less than 2% and more than 0.5%, and * represents an impurity sulfone content of 0.5% or less.

[0046] Example 3 Based on Example 2, saturation mutations and combinatorial mutations were carried out, and the catalytic activity of the mutants was detected according to the following reaction conditions.

[0047] The reaction system contained 100 mg of Substrate 1 or Substrate 2, 120 μL of isopropyl alcohol, 20 mg of NADP + 20 mg, 5 mg of dry powder of alcohol dehydrogenase, and 0.5 mL of crude monooxygenase enzyme solution (prepared from 50 mg of wet bacterial sludge), and the total volume was supplemented to 6 mL with 0.2 M PB 8.0. The reaction was carried out at 200 rpm for 16 h in a 20 °C constant temperature shaker. After the reaction was completed, three volumes of acetonitrile were added to the system to stop the reaction, and centrifugation was carried out for sampling, which was then subjected to HPLC to analyze the conversion rate and ee value of the product. The results are shown in Table 3. [Table 3] Note: In the above table, + represents a conversion rate < 20%, ++ represents a conversion rate of 20% or more and less than 30%, +++ represents a conversion rate of 30% or more and 50% or less, - represents an ee value of 90% or less, -- represents an ee value of more than 90% and 95% or less, --- represents an ee value of more than 95% and 98% or less, ---- represents an ee value of more than 98% and 99.5% or less, **** represents an impurity sulfone content of more than 5%, *** represents an impurity sulfone content of less than 5% and more than 2%, ** represents an impurity sulfone content of less than 2% and more than 0.5%, and * represents an impurity sulfone content of 0.5% or less.

[0048] Example 4 Based on Example 3, saturation mutation and combinatorial mutation were carried out, and activity screening was performed on the combinatorial mutation according to the same reaction conditions as in Example 3. The results are shown in Table 4.

Table 4

[0049] Example 5 Based on Example 4, saturation mutation and combinatorial mutation were carried out, and the catalytic activity of the mutant was detected according to the following reaction conditions.

[0050] Into the reaction system, 100 mg of Substrate 1 or Substrate 2, 120 μL of isopropanol, NADP +It contains 20 mg, 5 mg of alcohol dehydrogenase dry powder, and 0.25 mL of monooxygenase crude enzyme solution (produced from 25 mg of wet bacterial sludge), and the total volume is supplemented to 6 mL with 0.2 M PB 8.0. The reaction was carried out at 200 rpm for 16 h in a 20 °C constant temperature shaker. After the reaction was completed, three volumes of acetonitrile were added to the system to stop the reaction, followed by centrifugation, sampling, and HPLC analysis of the conversion rate and ee value of the product. The results are shown in Table 5 and Table 6.

Table 5

Table 6

[0051] Example 6 56 mL of 200 mmol / L phosphate buffer, 1.2 mL of isopropyl alcohol, NADP + 200 mg and 50 mg of alcohol dehydrogenase dry powder were added to a 250 mL four-necked flask at room temperature and adjusted to pH 8.0.

Chemical formula

Chemical Structure

[0052] As a result of HPLC detection, the purity was >99%, the ee value was >99%, and the yield was 82%.

[0053] Example 7 Add 56 mL of 200 mmol / L phosphate buffer, 1.2 mL of isopropanol, 200 mg of NADP + and 50 mg of dry powder of alcohol dehydrogenase to a 250 mL four-necked flask at room temperature, and adjust the pH to 8.0.

Chemical Structure

Chemical Structure

[0054] As a result of HPLC detection, the purity was >98%, the ee value was >99%, and the yield was 84%.

[0055] Example 8 56 mL of 200 mmol / L phosphate buffer, 1.2 mL of isopropyl alcohol, NADP + 200 mg and 50 mg of dry alcohol dehydrogenase powder were added to a 250 mL four-necked flask at room temperature and adjusted to pH 8.0.

Chemical formula

Chemical formula

[0056] As a result of HPLC detection, the purity was >99%, the ee value was >99%, and the yield was 87%.

[0057] Example 9 Using the enzyme solution of the monooxygenase mutant (D163A + A397L + I393L + V166I + S344Y) mutated based on SEQ ID NO: 1, referring to the catalytic synthesis steps of Examples 6 - 8, the reaction was catalyzed for substrates 4 - 11, and the results are shown in Table 7.

Table 7

[0058] 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, any modifications, equivalent substitutions, improvements, etc. made within the scope of the spirit and principle of the present invention shall all be included in the protection scope of the present invention.

Claims

1. (a) A protein having the amino acid sequence shown by SEQ ID NO: 1, or (b) A protein in which an amino acid mutation has occurred at at least one site of W60, Y65, D71, Y77, D163, V166, S178, T179, T199, G200, S201, R222, T223, H337, P338, K342, R343, S344, R365, I393, F395, D396, A397, L448, D505, S506, Y508 or R509 in the amino acid sequence of (a), and which has a monooxygenase function, or (c) A protein having a homology of 80% or more with the amino acid sequence described in any one of (a) and (b) and having a monooxygenase function, characterized in that it comprises a monooxygenase mutant.

2. The amino acid mutations in (b) are each independently D163V, D163A, or D163E; V166S, V166Y, V166M, V166C, V166D, V166H, V166I, V166N, V166K, V166M, V166L, V166A, V166T, V166P, V166G, or V166F; H337Y, H337M, H337K, H337P, H337F, H337A, H337L, H337E, H337D, or H337R; P338A, P338M, P338L, or P338Y; K342A, K342E, K342L, or K342Y; R343A, R343Y, R343H, or R343V; S344F, S344Y, S344L, S344M, S344K, S344A, S344T, S344N, S344R, S344D, or S344E; R365A, R365G, R365D, R365T, or R365Y; I393A, I393K, I393P, I393W, I393R, I393C, I393M, I393T, I393V, I393Y, I393G, or I393L; F395S, F395Q, or F395V; D396A, D396R, D396S, D396F, D396H, D396Q, or D396K; A397V, A397L, A397I, A397M, A397R, A397H, or A397W, provided that the letter before the number represents the original amino acid and the letter after the number represents the mutant amino acid. Preferably, the said (c) has a homology of 85% or more, preferably 90% or more, more preferably 95% or more, still more preferably 99% or more with the amino acid sequence limited to (a) or (b), and is a protein having a monooxygenase function. The monooxygenase mutant according to claim 1, characterized in that.

3. The mutation of the monooxygenase mutant includes any one of the following amino acid mutations. The monooxygenase mutant according to claim 2, characterized in that. 【Table 1】

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

5. A recombinant plasmid characterized in that the DNA molecule according to claim 4 is ligated.

6. A host cell characterized in that the recombinant plasmid according to claim 5 is transformed into a prokaryotic host cell.

7. A method for producing a chiral sulfoxide compound, comprising the step of reacting the monooxygenase mutant according to any one of claims 1 to 3 with a thioether-based substrate represented by formula I and / or formula II to obtain a chiral sulfoxide compound. 【Chemical 1】 【Chemical 2】 (R １ is selected from alkyl, cycloalkyl, aryl or heteroaryl, the number of carbon atoms of the alkyl is selected from 1 to 8, and the number of carbon atoms of the cycloalkyl, aryl or heteroaryl is selected from 5 to 10, R ２ is selected from alkyl, cycloalkyl, aryl or heteroaryl, the number of carbon atoms of the alkyl is selected from 1 to 8, and the number of carbon atoms of the cycloalkyl, aryl or heteroaryl is selected from 5 to 10, Or, said R １ and R ２ together with a sulfur atom form a heterocyclyl, a carbocyclic group or a heteroaryl, and the number of carbon atoms of said heterocyclyl, carbocyclic group or heteroaryl is selected from 5 to 10, The heteroatoms in the heterocyclyl or the heteroaryl are each independently at least one selected from nitrogen, oxygen, and sulfur. The aryl, heteroaryl, carbocyclic group, or heterocyclyl is each independently unsubstituted or substituted. In the case of substitution, the substituent is at least one group selected from halogen, alkoxy, or alkyl. )

8. The thioether-based substrate is selected from the following. The production method according to claim 7, characterized in that. [Chemical 3] 【Chemical Formula 4】 【Chemical Formula 5】 ​ [Chemical Formula 7] 【Chemical 8】 【Chemical Formula 9】 【Chemical Formula 10】 【Chemical Formula 11】 【Chemical 12】 【Chemical 13】

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