Glucose dehydrogenase mutant and use thereof

Abstract

Provided in the present invention are a glucose dehydrogenase mutant and use thereof. The glucose dehydrogenase mutant comprises: (a) a protein having an amino acid sequence shown in SEQ ID NO: 1; or (b) a protein that has been subjected to amino acid mutation at at least one of the following sites of the amino acid sequence in (a): E96, A47, V72, K137, V11, V88, V140, I183, A100, D202, D255, or Q170, and has a glucose dehydrogenase activity function; and (c) a protein having at least 80% homology with the amino acid sequence defined in any one of (a) and (b) and having a glucose dehydrogenase function. The glucose dehydrogenase mutant of the present application has better tolerance and higher activity under extreme conditions, is suitable for industrial scale-up, and has low cost and high yield.

Description

Glucose dehydrogenase mutants and their applications

[0001] This application is based on and claims priority to Chinese application CN application number 202411430303.7 filed on October 14, 2024, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0002] This invention relates to the field of enzyme reaction technology, and more specifically, to a glucose dehydrogenase mutant and its applications. Background Technology

[0003] In the development of the pharmaceutical, pesticide, and fine chemical industries, organic synthesis faces increasing challenges. In drug molecules, often only one stereoisomer has therapeutic effects, while the other stereoisomers have no therapeutic effects or even side effects. In traditional organic synthesis, protecting and deprotecting steps are usually introduced to compensate for the lack of chemical and regioselectivity of the reaction. However, bio-enzyme catalysis reactions usually have high stereoselectivity, are usually carried out under neutral and room temperature conditions, and have less environmental pollution. Therefore, the application of bio-enzyme catalysis technology in organic synthesis has very significant practical value.

[0004] Glucose dehydrogenase (GDH) is a member of the short-chain alcohol dehydrogenase family. In the presence of a coenzyme, it can catalyze the conversion of D-glucose into D-gluconic acid-δ-lactone, and the generated D-gluconic acid-δ-lactone will be further spontaneously hydrolyzed into gluconic acid.

[0005] In patent (CN 107058362 A), the disclosed esterase gene est816 and its recombinant esterase exhibit highly efficient soluble expression in Escherichia coli and Pichia pastoris expression systems. Furthermore, the recombinant esterase demonstrates strong degradation of pyrethroid pesticides (including lambda-cyhalothrin, cypermethrin, fenvalerate, and deltamethrin), with a degradation rate exceeding 90%, showing broad application prospects in addressing pyrethroid pesticide residues. In patent (CN 106929493 A), a lactonease is disclosed, in which the amino acid at position 167 of SEQ ID NO: 5 is mutated from valine to histidine, improving the degradation efficiency of α-zearalenone.

[0006] Wild-type biocatalysts, namely wild-type glucose dehydrogenases, often exhibit poor reactivity, tolerance (to high temperatures and high concentrations of organic solvents), and selectivity for non-natural substrates. In practical applications, they require large quantities and are unstable. This application aims to obtain a glucose dehydrogenase that can stably exert high enzyme activity, thus achieving greater application potential. Summary of the Invention

[0007] The main objective of this invention is to provide a glucose dehydrogenase mutant and its application, in order to solve the problem of poor enzyme activity of wild-type glucose dehydrogenase in the prior art.

[0008] To achieve the above objectives, according to a first aspect of the present invention, a glucose dehydrogenase mutant is provided, comprising: (a) a protein having the amino acid sequence shown in SEQ ID NO: 1; or (b) a protein having glucose dehydrogenase activity by amino acid mutation at at least one of the following sites in the amino acid sequence of (a): E96, A47, V72, K137, V11, V88, V140, I183, A100, D202, D255 or Q170; and (c) a protein having more than 80% homology with the amino acid sequence defined in either (a) or (b).

[0009] Further, the amino acid mutations in (b) above are each independently selected from the following: E96Q; A47S or A47M or A47T or A47V or A47I or A47R; V72I or V72M or V72Q; K137I or K137C or K137R or K137V; V11A or V11C or V11Q or V11T; V88I or V88L; V140C or V140I or V140R; I 183C or I183V; A100P or A100R; D202T; D255C; Q170Y; wherein the letter before the number represents the original amino acid and the letter after the number represents the mutant amino acid; preferably, in (c), the protein has more than 85%, more preferably more than 90%, more preferably more than 95%, and even more preferably more than 99% homology with the amino acid sequence defined in (a) or (b) and has glucose dehydrogenase function.

[0010] Furthermore, the mutations in the aforementioned glucose dehydrogenase mutants include any one of the following amino acid mutations: E96Q; E96Q+A47S; E96Q+A47M; E96Q+A47T; E96Q+A47V; E96Q+A47R; E96Q+V72I; E96Q+V72M; E96Q+V72Q; E96Q+K137I; E96Q+K137C; E96Q+K137R; E96Q+K137V; E96Q+V11A; E96Q+V11C; E96Q+V11Q; E96Q+V11T; E96Q+V88I; E96Q+V88L; E96Q+V140C; E96Q+V140I; E 96Q+V140R; E96Q+I183C; E96Q+I183V; E96Q+A100P; E96Q+A100R; E96Q+D2 02T; E96Q+D255C; E96Q+A100R+A47S; E96Q+A100R+A47M; E96Q+A100R+A47T ;E96Q+A100R+A47V; E96Q+A100R+A47R; E96Q+A100R+V72Q; E96Q+A100R+V 72I; E96Q+A100R+V72M; E96Q+A100R+K137I; E96Q+A100R+K137C; E96Q+A10 0R+K137V; E96Q+A100R+V11A; E96Q+A100R+V11C; E96Q+A100R+V11T; E96Q +A100R+V88L; E96Q+A100R+V140C; E96Q+A100R+V140I; E96Q+A100R+I183C ;E96Q+A100R+I183V; E96Q+A100R+Q170Y; E96Q+A100R+D202T; E96Q+A100 R+D255C; E96Q+A100R+V72I+A47S; E96Q+A100R+V72I+A47M; E96Q+A100R+V 72I+A47V; E96Q+A100R+V72I+A47R; E96Q+A100R+V72I+A47I; E96Q+A100R +V72I+K137I; E96Q+A100R+V72I+K137V; E96Q+A100R+V72I+K137C; E96Q+A 100R+V72I+V11A; E96Q+A100R+V72I+V11C; E96Q+A100R+V72I+V88L; E96Q +A100R+V72I+V140C; E96Q+A100R+V72I+I183C; E96Q+A100R+V72I+I183V;E96Q+A100R+V72I+Q170Y; E96Q+A100R+V72I+D202T; E96Q+A100R+V72I+D255C. ;

[0011] To achieve the above objectives, according to a second aspect of the present invention, a DNA molecule is provided that encodes the aforementioned glucose dehydrogenase mutant.

[0012] Furthermore, the DNA molecule is selected from: 1) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2; 2) a polynucleotide having 80% or more, preferably 90% or more, and more preferably 95% or more homology with the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2.

[0013] To achieve the above objectives, according to a third aspect of the present invention, a recombinant plasmid is provided, wherein the recombinant plasmid is linked with the aforementioned DNA molecule.

[0014] To achieve the above objectives, according to a fourth aspect of the present invention, a host cell is provided, wherein the host cell is transformed with the above-described recombinant plasmid.

[0015] To achieve the above objectives, according to a fifth aspect of the present invention, a method for energy cycling using the aforementioned glucose dehydrogenase mutant is provided, the method comprising: glucose and NAD... + Under the action of the aforementioned glucose dehydrogenase mutant, NADH is formed.

[0016] To achieve the above objectives, according to a sixth aspect of the present invention, a method for preparing non-natural amino acids is provided, the method comprising using the above-mentioned glucose dehydrogenase mutant in the reaction of amino acid dehydrogenase, glucose, and NAD+. + Under the influence of [the substance], the α-keto acid substrate shown in Formula I is reacted to obtain non-natural amino acids;

[0017] R is selected from substituted aryl or unsubstituted aryl, C2-C8 substituted heterocyclic or unsubstituted heterocyclic, C3-C8 substituted cycloalkyl or unsubstituted cycloalkyl, C1-C 10 Alkyl, C2-C 10 olefinic group or C2-C 10 The naphthyl group; the substituted aryl, substituted heterocyclic, and substituted cycloalkyl groups are each independently selected from halogens, O atoms, or C1-C5 alkyl groups.

[0018] Furthermore, the R mentioned above is selected from substituted aryl groups, unsubstituted cycloalkyl groups of C3-C6, or olefinic groups of C2-C8; the substituents of substituted aryl groups are selected from halogens.

[0019] Furthermore, the above-mentioned α-keto acid substrates are selected from...

[0020] By applying the technical solution of this invention, the glucose dehydrogenase mutant of this application has good tolerance and high activity under extreme conditions (high temperature and organic solvent environment, such as ethanol, tetrahydrofuran, DMSO), can efficiently synthesize non-natural amino acids, can be well used for industrial scale-up, has low cost and high yield, and realizes true green chemistry. Detailed Implementation

[0021] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0022] As mentioned in the background section, wild-type glucose dehydrogenase has low efficiency in synthesizing non-natural amino acids and poor tolerance, especially when the reaction substrate can only be dissolved under conditions of high temperature or organic solvents, making efficient industrial production impossible. Therefore, in this application, the inventors attempted to modify glucose dehydrogenase through enzyme evolution to improve its enzyme activity and tolerance to extreme conditions, thereby increasing production efficiency in industrial production. This application presents a series of protective solutions.

[0023] In a first typical embodiment of this application, a glucose dehydrogenase mutant is provided, comprising: (a) a protein having the amino acid sequence shown in SEQ ID NO: 1; or (b) a protein having glucose dehydrogenase activity by an amino acid mutation at at least one of the following sites in the amino acid sequence of (a): E96, A47, V72, K137, V11, V88, V140, I183, A100, D202, D255, or Q170; and (c) a protein having more than 80% homology with the amino acid sequence defined in either (a) or (b).

[0024] The amino acid sequence shown in SEQ ID NO:1 is derived from glucose dehydrogenase from Lysinibacillus sphaericus. Computer simulation analysis of the enzyme's model structure using homology modeling of this amino acid sequence predicted the active site for non-natural amino acid synthesis reactions, identifying 12 amino acid residues including: E96, A47, V72, K137, V11, V88, V140, I183, A100, D202, D255, and Q170. These amino acid sites may affect the protein's catalytic activity and stability. Mutating these amino acid sites can yield proteins with glucose dehydrogenase function, or even enhanced glucose dehydrogenase function. For the obtained proteins, changes can be made at non-critical mutation sites and active sites to obtain proteins with over 80% homology to the above amino acid sequence and possessing glucose dehydrogenase function.

[0025] The sequence of SEQ ID NO:1 is as follows:

[0026] In a preferred embodiment, the amino acid mutations in (b) are each independently selected from the following: E96Q; A47S or A47M or A47T or A47V or A47I or A47R; V72I or V72M or V72Q; K137I or K137C or K137R or K137V; V11A or V11C or V11Q or V11T; V88I or V88L; V140C or V140I or V140R ; I183C or I183V; A100P or A100R; D202T; D255C; Q170Y; wherein, the letter before the number represents the original amino acid, and the letter after the number represents the mutant amino acid; preferably, in (c), the protein has more than 85%, more preferably more than 90%, more preferably more than 95%, and even more preferably more than 99% homology with the amino acid sequence defined in (a) or (b) and has glucose dehydrogenase function.

[0027] As used herein, the amino acid residue abbreviations are 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).

[0028] Substitution and replacement rules generally apply to amino acids with similar properties; the effects of substitution are similar. For example, conserved amino acid substitutions can occur in the aforementioned homologous proteins. "Conserved amino acid substitutions" include, but are not limited to:

[0029] Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;

[0030] Hydrophobic amino acids with large side chains (Phe, Tyr, Trp) are replaced by other hydrophobic amino acids with large side chains;

[0031] Amino acids with positively charged side chains (Arg, His, Lys) are replaced by other amino acids with positively charged side chains;

[0032] Amino acids with polar, uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar, uncharged side chains.

[0033] Those skilled in the art can also perform conservative substitutions of amino acids based on amino acid substitution rules well known to them, such as the "blosum62 score matrix" in the prior art.

[0034] In this application, the applicant further investigated the aforementioned active site and discovered that mutations to different amino acids at the active site resulted in varying protein activities, with specific mutations enhancing glucose dehydrogenase activity. Experimental investigations revealed that specific mutations at the active site could yield proteins with enhanced activity. For the amino acid mutation sites of glucose dehydrogenase proteins, flexible selection and combinations of the aforementioned mutations are possible.

[0035] In a preferred embodiment, the mutation of the glucose dehydrogenase mutant includes any one of the following amino acid mutations: E96Q; E96Q+A47S; E96Q+A47M; E96Q+A47T; E96Q+A47V; E96Q+A47R; E96Q+V72I; E96Q+V72M; E96Q+V72Q; E96Q+K137I; E96Q+K137C; E96Q+K137R; E96Q+K137V; E96Q+V11A; E96Q+V11C; E96Q+V11Q; E96Q+V11T; E96Q+V88I; E96Q+V88L; E96Q+V140C; E96Q+V140I; E 96Q+V140R; E96Q+I183C; E96Q+I183V; E96Q+A100P; E96Q+A100R; E96Q+D20 2T; E96Q+D255C; E96Q+A100R+A47S; E96Q+A100R+A47M; E96Q+A100R+A47T; E 96Q+A100R+A47V; E96Q+A100R+A47R; E96Q+A100R+V72Q; E96Q+A100R+V72I ;E96Q+A100R+V72M; E96Q+A100R+K137I; E96Q+A100R+K137C; E96Q+A100R+K 137V; E96Q+A100R+V11A; E96Q+A100R+V11C; E96Q+A100R+V11T; E96Q+A100 R+V88L; E96Q+A100R+V140C; E96Q+A100R+V140I; E96Q+A100R+I183C; E96Q+ A100R+I183V; E96Q+A100R+A100P; E96Q+A100R+A100R; E96Q+A100R+Q170Y ;E96Q+A100R+D202T; E96Q+A100R+D255C; E96Q+A100R+V72I+A47S; E96Q+A1 00R+V72I+A47M; E96Q+A100R+V72I+A47V; E96Q+A100R+V72I+A47R; E96Q+A 100R+V72I+A47I; E96Q+A100R+V72I+K137I; E96Q+A100R+V72I+K137V; E96Q +A100R+V72I+K137C; E96Q+A100R+V72I+V11A; E96Q+A100R+V72I+V11C; E9 6Q+A100R+V72I+V88L; E96Q+A100R+V72I+V140C; E96Q+A100R+V72I+I183C;E96Q+A100R+V72I+I183V; E96Q+A100R+V72I+A100P; E96Q+A100R+V72I+A100R; E96Q+A100R+V72I+Q170Y; E96Q+A100R+V72I+D202T; E96Q+A100R+V72I+D255C. ;

[0036] All of the above-mentioned amino acid mutations were experimentally investigated in the embodiments of this application, and all of them have glucose dehydrogenase activity. Compared with the parent with the amino acid sequence shown in SEQ ID NO: 1, glucose dehydrogenase mutants with high tolerance to extreme environments and high enzyme activity can be obtained and can be used for industrial scale-up production.

[0037] In a second typical embodiment of this application, a DNA molecule is provided that encodes the above-described glucose dehydrogenase mutant.

[0038] In a preferred embodiment, the DNA molecule is selected from: 1) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2; 2) a polynucleotide having 80% or more, preferably 90% or more, and more preferably 95% or more homology with the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2.

[0039] The sequence of SEQ ID NO:2 is as follows:

[0040] In a third typical embodiment of this application, a recombinant plasmid is provided, wherein the recombinant plasmid is linked to the aforementioned DNA molecule.

[0041] The aforementioned DNA can encode the glucose dehydrogenase mutant and can be ligated to a recombinant plasmid to form a circular DNA. Both the aforementioned DNA and the recombinant plasmid can be transcribed and translated under the action of RNA polymerase, ribosomes, tRNA, etc., to obtain the aforementioned glucose dehydrogenase mutant.

[0042] In a fourth typical embodiment of this application, a host cell is provided, wherein the recombinant plasmid described above is transformed within the host cell. This host cell is not of plant origin and can be a prokaryotic cell or a eukaryotic cell. Specifically, the prokaryotic cell can be *Escherichia coli*, and the eukaryotic cell can be yeast.

[0043] Using the aforementioned host cells, recombinant plasmids can be replicated within the host cells, and the DNA molecules carried on the recombinant plasmids can be transcribed and translated to obtain a large number of glucose dehydrogenase mutants. Using existing technologies, glucose dehydrogenase mutants can be obtained by cleaving and purifying host cells, followed by crude enzyme catalysis or other methods, and then used for subsequent catalysis of substrates to obtain non-natural amino acids.

[0044] In a fifth typical embodiment of this application, a method for energy cycling using the above-mentioned glucose dehydrogenase mutant is provided, the method comprising: glucose and NAD. + Under the action of the aforementioned glucose dehydrogenase mutant, NADH is formed. The glucose dehydrogenase mutant of this application exhibits good activity and tolerance in any process involving the energy cycle (NAD→NADH).

[0045] In a sixth typical embodiment of this application, a method for preparing non-natural amino acids is provided. The method includes using the aforementioned glucose dehydrogenase mutant in the reaction of amino acid dehydrogenase, glucose, and NAD+. + Under the influence of [the substance], the α-keto acid substrate shown in Formula I is reacted to obtain non-natural amino acids;

[0046] R is selected from substituted aryl or unsubstituted aryl, C2-C8 substituted heterocyclic or unsubstituted heterocyclic, C3-C8 substituted cycloalkyl or unsubstituted cycloalkyl, C1-C 10 Alkyl, C2-C 10 olefinic group or C2-C 10 The naphthyl group; the substituted aryl group, substituted heterocyclic group, and substituted cycloalkyl group are each independently selected from halogens, O atoms, and C1-C5 alkyl groups.

[0047] Using the above preparation method, the glucose dehydrogenase mutant was used in the reaction of glucose and NAD+. + Based on its existing structure, it provides NADH for the synthesis of non-natural amino acids from substrates, catalyzing the synthesis of non-natural amino acids. Due to the enhanced activity and tolerance of the glucose dehydrogenase mutant, it can catalyze under extreme industrial production conditions, thereby improving production efficiency and reducing industrial production costs.

[0048] In a preferred embodiment, R is selected from substituted aryl, unsubstituted cycloalkyl (C3-C6), or olefin (C2-C8); the substituent of the substituted aryl is selected from halogens.

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

[0050] The present application will be further described in detail below with reference to specific embodiments, which should not be construed as limiting the scope of protection claimed in the present application.

[0051] The glucose dehydrogenase mutants obtained in this application were derived through saturation mutagenesis. Saturation mutagenesis is a method that modifies the coding gene of the target protein to obtain mutants in a short period of time in which the target amino acid is replaced by one of 19 other amino acids. This method is not only a powerful tool for targeted protein modification but also an important means of studying protein structure-function relationships. Saturation mutagenesis often yields more ideal evolutionary forms than single-point mutagenesis. And for problems that site-directed mutagenesis cannot solve, this is precisely where saturation mutagenesis excels. The saturation mutants were constructed using whole-plasmid PCR, followed by digestion of the PCR product with DpnI enzyme to remove the template, and transformation into *E. coli* BL21(DE3).

[0052] The specific process of culturing the glucose dehydrogenase mutant enzyme solution in the following embodiments of this application is as follows:

[0053] Take 4 ml of BL21(DE3) mutant strains containing different mutation sites, inoculate them into a 2L Erlenmeyer flask containing 400 mL of LB medium, and incubate at 37℃ with shaking at 200 rpm for 2-3 hours. OD 600 When the pH is 0.6-0.8, add IPTG to a final concentration of 0.06 mM, induce at 25℃ for 18 h, and collect the bacterial cells by centrifugation at 4℃ after induction. Resuspend the bacterial cells in 10 mL of 0.1 M Tris-HCl (pH 8.0) per gram of bacterial sludge, disrupt by sonication, centrifuge, collect the supernatant, and obtain the crude enzyme solution for the catalytic reaction.

[0054] The reaction system used for enzyme activity detection in the following embodiments of this application did not contain the specific substrates for the role of wild-type glucose dehydrogenase in the preparation of non-natural amino acids. Since glucose dehydrogenase functions as a coenzyme in the preparation of non-natural amino acids, it mainly synthesizes NADH to meet the needs of the main enzyme in the synthesis of non-natural amino acids. After the main enzyme consumes NADH to produce NAD, glucose dehydrogenase catalyzes NAD to form NADH, forming a cycle. Therefore, the following embodiments mainly characterize the activity and tolerance of glucose dehydrogenase in the NAD→NADH synthesis cycle by detecting its activity and tolerance in the synthesis of non-natural amino acids.

[0055] Example 1

[0056] The enzyme solution was treated at 60°C and 40% ethanol for 1 hour, respectively, and then added to solutions containing 100 mM glucose and 10 mM NAD+. +The 0.3 mL reaction system was tested at 340 nm for 10 min, and the change in OD340 of NADH was observed to characterize enzyme activity. The results are shown in Table 1.

[0057] Table 1

[0058] The ratio of decrease to increase in activity compared to the parent material: --- decrease by 10-50 times, -- decrease by 5-10 times, - decrease by 1-5 times, + increase by 1-2 times, ++ increase by 2-10 times, +++ increase by 10-50 times, ++++ increase by more than 50 times.

[0059] Based on Example 1, further mutations were performed to improve activity and tolerance.

[0060] Example 2

[0061] The enzyme solution was treated at 65°C and 50% ethanol for 1 hour, respectively, and then added to solutions containing 100 mM glucose and 10 mM NAD+. + The 0.3 mL reaction system was tested at 340 nm for 10 min, and the change in OD340 of NADH was observed to characterize enzyme activity. The results are shown in Table 2.

[0062] Table 2

[0063] The ratio of decrease to increase in activity compared to the parent material: --- decrease by 10-50 times, -- decrease by 5-10 times, - decrease by 1-5 times, + increase by 1-2 times, ++ increase by 2-10 times, +++ increase by 10-50 times, ++++ increase by more than 50 times.

[0064] Based on Example 2, further mutations were performed to further improve activity and tolerance.

[0065] Example 3

[0066] The enzyme solution was treated at 65°C and 50% ethanol for 3 hours, respectively, and then added to solutions containing 100 mM glucose and 10 mM NAD+, respectively. + The 0.3 mL reaction system was tested at 340 nm for 10 min, and the change in OD340 of NADH was observed to characterize enzyme activity. The results are shown in Table 3.

[0067] Table 3

[0068] The ratio of decrease to increase in activity compared to the parent material: --- decrease by 10-50 times, -- decrease by 5-10 times, - decrease by 1-5 times, + increase by 1-2 times, ++ increase by 2-10 times, +++ increase by 10-50 times, ++++ increase by more than 50 times.

[0069] Example 4

[0070] The enzyme solutions were treated under other conditions, and then added to solutions containing 100 mM glucose and 10 mM NAD+, respectively. + The 0.3 mL reaction system was tested at 340 nm for 10 min, and the change in OD340 of NADH was observed to characterize enzyme activity. The results are shown in Table 4.

[0071] Table 4

[0072] The ratio of decrease to increase in activity compared to the parent material: --- decrease by 10-50 times, -- decrease by 5-10 times, - decrease by 1-5 times, + increase by 1-5 times, ++ increase by 5-10 times, +++ increase by 10-50 times, ++++ increase by more than 50 times.

[0073] A scale-up reaction was carried out with 1g of substrate.

[0074] Example 5

[0075] Based on the principle of evolution, the best enzyme, namely the enzyme mutant in Example 4, was obtained and scaled up. Add 1g to a 100mL reaction system, 5mg of the glucose dehydrogenase mutant from Example 4, 3eq of glucose, 5eq of ammonium chloride, 5mg of amino acid dehydrogenase, and NAD. + 100 mg of 0.1 M Tris-HCl (pH 9.0) was reacted at 37 °C. Samples were taken and analyzed over time. The specific conversion rates and ee values ​​are shown in Table 5 below.

[0076] Table 5:

[0077] As can be seen from the above description, the above embodiments of the present invention achieve the following technical effects: by evolving glucose dehydrogenase, mutants with improved activity and tolerance are obtained, which can perform efficient synthesis of non-natural amino acids under high temperature and high organic solvent conditions, with high product yield, greatly reducing waste, saving production costs, and realizing green chemistry.

[0078] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A glucose dehydrogenase mutant, characterized in that, include: (a) A protein having the amino acid sequence shown in SEQ ID NO: 1; or (b) At least one of the following sites in the amino acid sequence described in (a): E96, A47, V72, K137, V11, V88, V140, I183, A100, D202, D255 or Q170, a protein that has undergone an amino acid mutation and has glucose dehydrogenase activity. (c) A protein having more than 80% homology with the amino acid sequence defined in either (a) or (b) and having glucose dehydrogenase function.

2. The glucose dehydrogenase mutant of claim 1, wherein, The amino acid mutations in (b) are each independently selected from the following: E96Q; A47S or A47M or A47T or A47V or A47I or A47R; V72I, V72M, or V72Q; K137I or K137C or K137R or K137V; V11A or V11C or V11Q or V11T; V88I or V88L; V140C or V140I or V140R; I183C or I183V; A100P or A100R; D202T; D255C; Q170Y; In this context, the letter before the number represents the original amino acid, and the letter after the number represents the mutated amino acid. Preferably, in (c), the protein has 85% or more, 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 has glucose dehydrogenase function.

3. The glucose dehydrogenase mutant of claim 2, wherein, The mutations in the glucose dehydrogenase mutant include any one of the following amino acid mutations: E96Q; E96Q+A47S; E96Q+A47M; E96Q+A47T; E96Q+A47V; E96Q+A47R; E96Q+V72I; E96Q+V72M; E96Q+V72Q; E96Q+K137I; E96Q+K137C; E96Q+K137R; E96Q+K137V; E96Q+V11A; E96Q+V11C; E96Q+V11Q; E96Q+V11T; E96Q+V88I; E96Q+V88L; E96Q+V140C; E96Q+V140I; E96Q+V140R; E96Q+I183C; E96Q+I183V; E96Q+A100P; E96Q+A100R; E96Q+D202T; E96Q+D255C; E96Q+A100R+A47S; E96Q+A100R+A47M; E96Q+A100R+A47T; E96Q+A100R+A47V; E96Q+A100R+A47R; E96Q+A100R+V72Q; E96Q+A100R+V72I; E96Q+A100R+V72M; E96Q+A100R+K137I; E96Q+A100R+K137C; E96Q+A100R+K137V; E96Q+A100R+V11A; E96Q+A100R+V11C; E96Q+A100R+V11T; E96Q+A100R+V88L; E96Q+A100R+V140C; E96Q+A100R+V140I; E96Q+A100R+I183C; E96Q+A100R+I183V; E96Q+A100R+Q170Y; E96Q+A100R+D202T; E96Q+A100R+D255C; E96Q+A100R+V72I+A47S; E96Q+A100R+V72I+A47M; E96Q+A100R+V72I+A47V; E96Q+A100R+V72I+A47R; E96Q+A100R+V72I+A47I; E96Q+A100R+V72I+K137I; E96Q+A100R+V72I+K137V; E96Q+A100R+V72I+K137C; E96Q+A100R+V72I+V11A; E96Q+A100R+V72I+V11C; E96Q+A100R+V72I+V88L; E96Q+A100R+V72I+V140C; E96Q+A100R+V72I+I183C; E96Q+A100R+V72I+I183V; E96Q+A100R+V72I+Q170Y; E96Q+A100R+V72I+D202T; E96Q+A100R+V72I+D255C.

4. A DNA molecule, characterized in that, The DNA molecule encodes the glucose dehydrogenase mutant according to any one of claims 1 to 3.

5. The DNA molecule of claim 4, wherein, The DNA molecules are selected from: 1) A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2; 2) A polynucleotide having 80% or more, preferably 90% or more, more preferably 95% or more homology with the polynucleotide sequence shown in SEQ ID NO:

2.

6. A recombinant plasmid, characterized in that, The recombinant plasmid is ligated with the DNA molecule as described in claim 4 or 5.

7. A host cell, characterized in that, The host cell is transformed with the recombinant plasmid as described in claim 6.

8. A method for energy cycle using the glucose dehydrogenase mutant according to any one of claims 1 to 3, characterized by, The method includes: glucose and NAD + NADH is formed under the action of the glucose dehydrogenase mutant of any one of claims 1 to 3.

9. A method for producing a non-natural amino acid, characterized by, The production method includes reacting an α-keto acid substrate represented by Formula I under the action of an amino acid dehydrogenase, glucose, and NAD + to obtain the unnatural amino acid. R is selected from substituted aryl or unsubstituted aryl, C2-C8substituted heterocyclyl or unsubstituted heterocyclyl, C3-C8substituted cycloalkyl or unsubstituted cycloalkyl, C1-C 10 alkyl, C2-C 10 alkenyl or C2-C 10 naphthyl; The substituted aryl group, the substituted heterocyclic group, and the substituted cycloalkyl group are each independently selected from halogens, O atoms, or C1-C5 alkyl groups.

10. The method of claim 9, wherein, The R is selected from substituted aryl, unsubstituted cycloalkyl (C3-C6), or olefin (C2-C8); The substituents of the substituted aryl group are selected from halogens.

11. The preparation method according to claim 9, characterized in that, The alpha-keto acid substrate is selected from

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