Glucose dehydrogenase mutant and its application

Abstract

The present invention provides a glucose dehydrogenase mutant and its application. The glucose dehydrogenase mutant includes: (a) a protein having an amino acid sequence shown in SEQ ID NO: 1; or (b) a protein having an amino acid mutation at at least one of the following sites in the amino acid sequence in (a): E96, A47, V72, K137, V11, V88, V140, I183, A100, D202, D255 or Q170 and having glucose dehydrogenase activity; (c) a protein having more than 80% homology with the amino acid sequence defined in any one of (a) and (b) and having glucose dehydrogenase function. The glucose dehydrogenase mutant of the present application has good tolerance and high activity under extreme conditions, can be well used for industrial amplification, has low cost and high yield.

Description

Technical Field

[0001] The invention relates to the technical field of enzyme reactions, in particular to a glucose dehydrogenase mutant and an application thereof. Background Art

[0002] In the development of medicine, pesticide and fine chemical industries, organic synthesis faces more and more challenges. In drug molecules, often only one stereoisomer has therapeutic effect, while other stereoisomers have no therapeutic effect or even have side effects. In traditional organic synthesis, protection and deprotection steps are usually introduced to make up for the lack of chemical and regional selectivity of the reaction. Enzyme-catalyzed reactions usually have higher stereoselectivity and are usually carried out under neutral and room temperature conditions with less environmental pollution. Therefore, the application of enzyme-catalyzed technology in organic synthesis has very important practical application value.

[0003] Glucose dehydrogenase (GDH) is a member of the short-chain alcohol dehydrogenase family. In the presence of coenzymes, it can catalyze the conversion of D-glucose into D-glucono-δ-lactone, and the generated D-glucono-δ-lactone will be further spontaneously hydrolyzed into gluconic acid.

[0004] In the patent (CN 107058362 A), the disclosed esterase gene est816 and its recombinant esterase have efficient soluble expression in the Escherichia coli expression system and the Pichia pastoris expression system, and the recombinant esterase has a strong degradation effect on pyrethroid pesticides (including chlorfenapyr, cypermethrin, cypermethrin and deltamethrin), with a degradation rate of more than 90%, and has broad application prospects in the pyrethroid pesticide residues; in the patent (CN 106929493 A), a lactonase is disclosed, in which the amino acid at position 167 in SEQ ID NO: 5 is mutated from valine to histidine, thereby improving the degradation efficiency of α-zearalenol.

[0005] Wild-type biocatalysts, i.e. wild-type glucose dehydrogenase, often have poor reactivity, tolerance (tolerance to factors such as high temperature and high concentration of organic solvents) and selectivity for non-natural substrates. In actual use, the amount of enzyme used is large and unstable. This application aims to obtain a glucose dehydrogenase that can stably exert high enzyme activity to obtain greater application prospects. Summary of the invention

[0006] The main purpose of the present invention is to provide a glucose dehydrogenase mutant and its application, so as to solve the problem that the enzyme activity of wild-type glucose dehydrogenase in the prior art is poor.

[0007] To achieve the above object, according to the first aspect of the present invention, a glucose dehydrogenase mutant is provided, comprising: (a) a protein having an amino acid sequence as shown in SEQ ID NO: 1; or (b) a protein having an amino acid mutation at at least one of the following sites of the amino acid sequence in (a): E96, A47, V72, K137, V11, V88, V140, I183, A100, D202, D255 or Q170 and having glucose dehydrogenase activity; (c) a protein having more than 80% homology with the amino acid sequence defined in any one of (a) and (b) and having glucose dehydrogenase function.

[0008] 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 letters before the numbers represent the original amino acids, and the letters after the numbers represent the mutated amino acids; preferably, in (c), the protein has more than 85%, preferably more than 90%, more preferably more than 95%, and further preferably more than 99% homology with the amino acid sequence defined in (a) or (b) and has the function of glucose dehydrogenase.

[0009] Furthermore, the mutation of the above-mentioned 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; E96Q+V140V; 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. ;

[0010] In order to achieve the above object, according to the second aspect of the present invention, a DNA molecule is provided, which encodes the above-mentioned glucose dehydrogenase mutant.

[0011] Furthermore, the above-mentioned DNA molecule is selected from: 1) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2; 2) a polynucleotide having more than 80%, more preferably more than 90%, and even more preferably more than 95% homology with the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2.

[0012] In order to achieve the above object, according to the third aspect of the present invention, a recombinant plasmid is provided, wherein the recombinant plasmid is connected to the above DNA molecule.

[0013] In order to achieve the above object, according to a fourth aspect of the present invention, a host cell is provided, into which the above recombinant plasmid is transformed.

[0014] In order to achieve the above object, according to a fifth aspect of the present invention, a method for energy cycling using the above-mentioned glucose dehydrogenase mutant is provided, the method comprising: + Under the action of the above-mentioned glucose dehydrogenase mutant, NADH is formed.

[0015] In order to achieve the above object, according to the sixth aspect of the present invention, a method for preparing non-natural amino acids is provided, the preparation method comprising using the above-mentioned glucose dehydrogenase mutant in amino acid dehydrogenase, glucose and NAD + Under the action of, reacting the α-keto acid substrate shown in formula I to obtain a non-natural amino acid;

[0016] Formula I;

[0017] R is selected from substituted or unsubstituted aryl, C2-C8 substituted or unsubstituted heterocyclic group, C3-C8 substituted or unsubstituted cycloalkyl group, C1-C 10 Alkyl, C2-C 10 Olefin or C2-C 10 The substituents of the substituted aryl, substituted heterocyclic group and substituted cycloalkyl group are each independently selected from halogen, O atom or C1-C5 alkyl group.

[0018] Furthermore, the above R is selected from substituted aryl, C3-C6 unsubstituted cycloalkyl or C2-C8 olefin; the substituent of the substituted aryl is selected from halogen.

[0019] Furthermore, the α-keto acid substrate is selected from , , , , , , , or .

[0020] By applying the technical solution of the present invention, the glucose dehydrogenase mutant of the present 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 DESCRIPTION

[0021] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0022] As mentioned in the background technology, wild-type glucose dehydrogenase has low synthesis efficiency for non-natural amino acids and poor tolerance, especially when the reaction substrate can only be dissolved under high temperature or organic solvent conditions, and efficient industrial production cannot be carried out. Therefore, in this application, the inventor attempts to transform glucose dehydrogenase by enzyme evolution, thereby improving the enzyme activity of glucose dehydrogenase and its tolerance under extreme conditions, so that it can improve production efficiency in industrial production, and thus proposes a series of protection schemes of this application.

[0023] In a first typical embodiment of the present application, a glucose dehydrogenase mutant is provided, comprising: (a) a protein having an amino acid sequence as shown in SEQ ID NO: 1; or (b) a protein having a glucose dehydrogenase activity function after an 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; (c) a protein having more than 80% homology with the amino acid sequence defined in any one of (a) and (b) and having a glucose dehydrogenase function.

[0024] The amino acid sequence shown in the above SEQ ID NO: 1 is derived from the glucose dehydrogenase of Lysinibacillus sphaericus. The model structure of the enzyme was analyzed by computer simulation of homology modeling of the amino acid sequence, and the active site for the non-natural amino acid synthesis reaction was predicted. It was found that 12 amino acid residues included: E96, A47, V72, K137, V11, V88, V140, I183, A100, D202, D255 and Q170. These amino acid sites may affect the catalytic activity and stability of the protein. By mutating the above amino acid sites, a protein having glucose dehydrogenase or even glucose dehydrogenase function enhancement can be obtained. For the above-obtained protein, changes can be made at non-critical mutation sites and active sites, and a protein having more than 80% homology with the above amino acid sequence and having glucose dehydrogenase function can be obtained.

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

[0026] MYSDLEGKVIVITGASTGLGKAMALRFGEEKAKVIVNFRSDENEANAVVEGVKKAGGDAIAVKGDVTVEEDVINLVQTAVNKFGTLDVMINNAGIENPVASHEMPLSDWNRVINTNLTGAFLGSREAIKYF VENDIKGSVINMSSVHEMIPWPLFVHYAASKGGIKLMTQTLALEYAPKGIRINNIGPGAINTPINAEKFADPAKRADVESMVPMGYIGKPEEIAAVAAWLASSQASYVTGITLFADGGMTLYPDFQAGRG.

[0027] 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 letters before the numbers represent the original amino acids, and the letters after the numbers represent the mutated amino acids; preferably, in (c), the protein has more than 85%, preferably more than 90%, more preferably more than 95%, and further preferably more than 99% homology with the amino acid sequence defined in (a) or (b) and has the function of glucose dehydrogenase.

[0028] As used herein, amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y) and valine (Val; V).

[0029] Substitution and replacement rules. Generally speaking, amino acids with similar properties will have similar effects after replacement. For example, conservative amino acid replacements may occur in the above homologous proteins. "Conservative amino acid replacements" include but are not limited to:

[0030] hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;

[0031] Substitution of hydrophobic amino acids with bulky side chains (Phe, Tyr, Trp) by other hydrophobic amino acids with bulky side chains;

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

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

[0034] A person skilled in the art may also perform conservative substitutions on amino acids according to amino acid substitution rules well known to those skilled in the art, such as the "blosum62 scoring matrix" in the prior art.

[0035] In the present application, the applicant continues to explore the above-mentioned active site and finds that the active site is mutated into different amino acids, and the corresponding protein activity is also different, and specific mutations can enhance the activity of glucose dehydrogenase. Through experimental exploration, it is found that the above-mentioned specific mutations are performed on the active site, and proteins with enhanced activity can be obtained. For the amino acid mutation sites of glucose dehydrogenase protein, flexible selection and combination can be performed in the above-mentioned mutations.

[0036] In a preferred embodiment, the mutation of the glucose dehydrogenase mutant comprises any 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+V140V; 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. ;

[0037] The above amino acid mutations were all experimentally investigated in the examples of the present application, and all had glucose dehydrogenase activity. Compared with the parent having 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, which can be used for industrial scale-up production.

[0038] In a second typical embodiment of the present application, a DNA molecule is provided, which encodes the above-mentioned glucose dehydrogenase mutant.

[0039] In a preferred embodiment, the DNA molecule is selected from: 1) a polynucleotide consisting of the nucleotide sequence as shown in SEQ ID NO: 2; 2) a polynucleotide having more than 80%, more preferably more than 90%, and further preferably more than 95% homology with the polynucleotide consisting of the nucleotide sequence as shown in SEQ ID NO: 2.

[0040] The sequence of SEQ ID NO:2 is as follows:

[0041] .

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

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

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

[0045] By using the above host cells, the recombinant plasmid can be replicated in the host cells, and the DNA molecules carried on the recombinant plasmid can also be transcribed and translated to obtain a large number of glucose dehydrogenase mutants. By using existing technologies, the host cells can be crushed for protein purification, crushed for crude enzyme catalysis or other methods to obtain glucose dehydrogenase mutants, and then the substrate can be catalyzed to obtain non-natural amino acids.

[0046] In a fifth typical embodiment of the present 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 above-mentioned glucose dehydrogenase mutant, NADH is formed. The glucose dehydrogenase mutant of the present application has good activity and tolerance in any process involving energy cycle (NAD→NADH).

[0047] In a sixth typical embodiment of the present application, a method for preparing a non-natural amino acid is provided, the preparation method comprising using the above-mentioned glucose dehydrogenase mutant in amino acid dehydrogenase, glucose and NAD + Under the action of, reacting the α-keto acid substrate shown in formula I to obtain a non-natural amino acid;

[0048] Formula I;

[0049] R is selected from substituted or unsubstituted aryl, C2-C8 substituted or unsubstituted heterocyclic group, C3-C8 substituted or unsubstituted cycloalkyl group, C1-C 10 Alkyl, C2-C 10 Olefin or C2-C 10 The substituents of the substituted aryl, substituted heterocyclic group and substituted cycloalkyl group are each independently selected from halogen, O atom and C1-C5 alkyl group.

[0050] Using the preparation method, using the glucose dehydrogenase mutant in glucose and NAD + On the basis of its existence, it provides NADH for the synthesis of non-natural amino acids from substrates, catalyzing the synthesis of non-natural amino acids. Since the activity and tolerance of the glucose dehydrogenase mutant are improved, it can catalyze under industrial production conditions such as extreme environments, which can improve production efficiency and reduce industrial production costs.

[0051] In a preferred embodiment, R is selected from substituted aryl, C3-C6 unsubstituted cycloalkyl or C2-C8 olefin; and the substituent of the substituted aryl is selected from halogen.

[0052] In a preferred embodiment, the substrate is selected from (Substrate 1), (Substrate 2), (Substrate 3), (Substrate 4), (Substrate 5), (Substrate 6), (Substrate 7), (Substrate 8) or (Substrate 9).

[0053] The present application is further described in detail below in conjunction with specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed in the present application.

[0054] Each glucose dehydrogenase mutant obtained in the present application is obtained by saturation mutation. Saturation mutation is a method of obtaining mutants in which the amino acids at the target site are replaced by other 19 amino acids in a short period of time by modifying the coding gene of the target protein. This method is not only a powerful tool for directed modification of proteins, but also an important means for studying the structure-function relationship of proteins. Saturation mutation can often obtain more ideal evolutionary bodies than single-point mutation. These problems that cannot be solved by the site-directed mutagenesis method are precisely the unique features of the saturation mutation method. The construction of saturation mutation adopts the method of whole plasmid PCR, and then the PCR product is digested with DpnI enzyme to remove the template and transformed into Escherichia coli BL21 (DE3).

[0055] The specific process of culturing the glucose dehydrogenase mutant enzyme solution in the following embodiments of the present application is as follows:

[0056] Take 4 ml of BL21(DE3) mutant strains containing different mutation sites and inoculate them into a 2 L Erlenmeyer flask containing 400 mL LB medium. After shaking culture at 37°C and 200 rpm for 2-3 h, the OD 600 When the pH value was 0.6-0.8, add IPTG with a final concentration of 0.06 mM, induce at 25℃ for 18 h, and collect the cells by centrifugation at 4℃. Resuspend the cells with 10 mL 0.1 M Tris-HCl (pH 8.0) per gram of bacterial mud, break them by ultrasonication, centrifuge, collect the supernatant, and obtain the crude enzyme solution for catalytic reaction.

[0057] The reaction system for enzyme activity detection in the following examples of the present application does not add the specific substrate that the wild-type glucose dehydrogenase acts on in the process of preparing non-natural amino acids. Since glucose dehydrogenase functions as a coenzyme in the process of preparing non-natural amino acids, it mainly synthesizes NADH to meet the needs of the main enzyme for synthesizing 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 examples mainly detect the activity and tolerance of glucose dehydrogenase in the synthesis cycle of NAD→NADH, and then characterize its activity and tolerance in the process of synthesizing non-natural amino acids.

[0058] Example 1

[0059] The enzyme solution was treated at 60°C and 40% ethanol for 1 h, and then added to 100 mM glucose and 10 mM NAD + The 0.3 mL reaction system was tested at 340 nm for 10 min to observe the change in NADH OD340 to characterize the enzyme activity. The results are shown in Table 1.

[0060] Table 1

[0061]

[0062] The activity is reduced and increased by times compared with the parent, --- reduced by 10-50 times, -- reduced by 5-10 times, - reduced by 1-5 times, + increased by 1-2 times, ++ increased by 2-10 times, +++ increased by 10-50 times, ++++ increased by more than 50 times.

[0063] Based on Example 1, mutations were continued to be performed to improve activity and tolerance.

[0064] Example 2

[0065] The enzyme solution was treated at 65°C and 50% ethanol for 1 h, and then added to 100 mM glucose and 10 mM NAD + The 0.3 mL reaction system was detected at 340 nm for 10 min, and the change of NADH OD340 was observed to characterize the enzyme activity. The results are shown in Table 2.

[0066] Table 2

[0067]

[0068] The activity is reduced and increased by times compared with the parent, --- reduced by 10-50 times, -- reduced by 5-10 times, - reduced by 1-5 times, + increased by 1-2 times, ++ increased by 2-10 times, +++ increased by 10-50 times, ++++ increased by more than 50 times.

[0069] Mutations were continued based on Example 2 to further improve activity and tolerance.

[0070] Example 3

[0071] The enzyme solution was treated at 65°C and 50% ethanol for 3 h, and then added to 100 mM glucose and 10 mM NAD + The 0.3 mL reaction system was detected at 340 nm for 10 min, and the change of NADH OD340 was observed to characterize the enzyme activity. The results are shown in Table 3.

[0072] Table 3

[0073]

[0074] The activity is reduced and increased by times compared with the parent, --- reduced by 10-50 times, -- reduced by 5-10 times, - reduced by 1-5 times, + increased by 1-2 times, ++ increased by 2-10 times, +++ increased by 10-50 times, ++++ increased by more than 50 times.

[0075] Example 4

[0076] The enzyme solution was treated under other conditions and then added to 100 mM glucose and 10 mM NAD + The 0.3 mL reaction system was tested at 340 nm for 10 min to observe the change in NADH OD340 to characterize the enzyme activity. The results are shown in Table 4.

[0077] Table 4

[0078]

[0079] The activity is reduced and increased by times compared with the parent, --- reduced by 10-50 times, -- reduced by 5-10 times, - reduced by 1-5 times, + increased by 1-5 times, ++ increased by 5-10 times, +++ increased by 10-50 times, ++++ increased by more than 50 times.

[0080] Perform a scale-up reaction using 1 g of substrate.

[0081] Example 5

[0082] Based on the best enzyme obtained by evolution, i.e., the enzyme mutant in Example 4, an amplification reaction is performed, (Substrate 1), (Substrate 2), (Substrate 3), (Substrate 4), (Substrate 5), (Substrate 6), (Substrate 7), (Substrate 8) or (Substrate 9) 1 g, 100 mL reaction system, 5 mg of the glucose dehydrogenase mutant in Example 4, 3 eq of glucose, 5 eq of ammonium chloride, 5 mg of amino acid dehydrogenase, NAD + 100 mg, 0.1 M Tris-HCl pH 9.0, react at 37°C, follow the reaction time and take samples for detection. The specific conversion rate and ee value are shown in Table 5 below:

[0083] Table 5:

[0084]

[0085] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: by evolving glucose dehydrogenase, mutants with improved activity and tolerance are obtained, and efficient non-natural amino acids can be synthesized under high temperature and high organic solvent conditions, with high product yield, greatly reducing the three wastes, saving production costs, and realizing green chemistry.

[0086] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A glucose dehydrogenase mutant, characterized in that: The glucose dehydrogenase mutant is: A protein having glucose dehydrogenase activity after amino acid mutation in which the E96 position of the amino acid sequence shown in SEQ ID NO: 1 is mutated to Q and at least one of the following positions: A47S, A47M, A47T, A47V, A47I, A47R, V72I, V72M, V72Q, K137I, K137C, K137R, K137V, V11A, V11C, V11Q, V11T, V88I, V88L, V140C, V140I, V140R, I183C, I183V, A100P, A100R, D202T, D255C or Q170Y; Among them, the letters before the number represent the original amino acid, and the letters after the number represent the mutated amino acid; The mutation of the glucose dehydrogenase mutant is any one of the following amino acid mutations: 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。; 2. A DNA molecule, characterized in that The DNA molecule encodes the glucose dehydrogenase mutant according to claim 1.

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

4. A host cell of a non-animal or plant species, characterized in that The host cell is transformed with the recombinant plasmid according to claim 3.

5. A method for energy cycling using the glucose dehydrogenase mutant according to claim 1, characterized in that: The method comprises: Glucose and NAD + Under the action of the glucose dehydrogenase mutant according to claim 1, NADH is formed.

6. A method for preparing a non-natural amino acid, characterized in that: The preparation method comprises using the glucose dehydrogenase mutant according to claim 1 in amino acid dehydrogenase, glucose and NAD + Under the action of , reacting the α-keto acid substrate shown in formula I to obtain the non-natural amino acid; Formula I; R is selected from substituted or unsubstituted aryl, C2-C8 substituted or unsubstituted heterocyclic group, C3-C8 substituted or unsubstituted cycloalkyl group, C1-C 10 Alkyl, C2-C 10 Olefin or C2-C 10 The naphthyl group; The substituents of the substituted aryl group, the substituted heterocyclic group and the substituted cycloalkyl group are each independently selected from halogen, an O atom or a C1-C5 alkyl group.

7. The preparation method according to claim 6, characterized in that: The R is selected from a substituted aryl group, a C3-C6 unsubstituted cycloalkyl group or a C2-C8 olefin group; The substituent of the substituted aryl group is selected from halogen.

8. The preparation method according to claim 6, characterized in that: The α-keto acid substrate is selected from , , , , , , , or .

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