A highly active ketopantolactone reductase mutant and its encoding gene and application

Abstract

The present invention discloses a highly active ketopantoic acid lactone reductase mutant, its encoding gene and application. The ketopantoic acid lactone reductase mutant disclosed in the present invention has the following mutations at the 126th amino acid residue and / or the 215th amino acid residue relative to the wild-type ketopantoic acid lactone reductase CviCPR shown in SEQ ID NO: 1: the leucine L at the 126th position is mutated to one of proline P, phenylalanine F, tyrosine Y and tryptophan W; the alanine A at the 215th position is mutated to one of aspartic acid D and glutamic acid E. The ketopantoic acid lactone reductase mutant provided by the present invention can catalyze the preparation of D-pantoic acid lactone from ketopantoic acid lactone with high activity and high stereoselectivity, and provides a potential high-efficiency biocatalyst for the industrial production of D-pantoic acid lactone prepared by the bioenzymatic asymmetric reduction route.

Description

Technical Field

[0001] The invention belongs to the technical field of biocatalysis, and relates to a ketopantolactone reductase mutant derived from Candida viswanathii, a nucleic acid molecule encoding the ketopantolactone reductase mutant, a recombinant vector, a recombinant cell, a preparation method of the ketopantolactone reductase mutant, and an application of the ketopantolactone reductase mutant in the preparation of D-pantolactone. Background Art

[0002] The structural formula of D-pantolactone ((R)-pantolactone) is shown in formula (I), which is a key intermediate for the synthesis of D-calcium pantothenate. At present, in industrial production, D-pantolactone is mainly synthesized by the isobutyraldehyde-formaldehyde-sodium cyanide method to first synthesize DL-pantolactone, and then prepared by a microbial enzymatic separation route catalyzed by D-pantolactone hydrolase. This route is mature, but there are problems such as complicated steps, large amount of extraction solvent, and high acid and alkali dosage. Therefore, it is of great significance to develop a simple, environmentally friendly and efficient bioenzymatic asymmetric reduction route.

[0003]

[0004] Ketopantoic acid lactone reductase is a key enzyme in the redox enzyme asymmetric synthesis of D-pantoic acid lactone. Its catalytic activity and enantioselectivity have an important influence on the efficiency of the enzyme-catalyzed reaction and the optical purity of the product. At present, the number of publicly reported ketopantoic acid lactone reductases and their gene resources is relatively small, which limits the study of their enzymatic properties, structure-activity relationship and their application in the efficient asymmetric synthesis of D-pantoic acid lactone. Kataoka et al. reported two conjugated polyketide reductases CPR-C1 and CPR-C2 from Candida parapsilosis IFO 0708 in 2003. Their heterologous expression level in Escherichia coli was low, and the recombinant cell specific activity was only 0.251U / mg, resulting in low efficiency of their enzyme-catalyzed reaction. Since 2018, only three ketopantolactone reductases have been reported for the asymmetric synthesis of D-pantolactone, including CorCPR, CduCPR, and CalCPR. Among them, CduCPR has the highest catalytic efficiency, but its spatiotemporal yield is only 307.5 g L -1 d -1 CN 114085820A and CN 113913399A respectively disclose novel ketopantolactone reductases CviCPR and CmaCPR, which can produce D-pantolactone by stereoselective asymmetric reduction of ketopantolactone with a spatial and temporal yield of 520-590 g L -1 d -1 .

[0005] In summary, in the existing oxidoreductase-based asymmetric synthesis of D-pantoic acid lactone technology route involving ketopantoic acid lactone reductase, there is still a problem of low spatiotemporal yield of the asymmetric reduction reaction of the key enzyme ketopantoic acid lactone reductase, which limits its application on an industrial scale. Therefore, the development of highly active ketopantoic acid lactone reductase and the green and efficient preparation of D-pantoic acid lactone have important industrial application value. Summary of the invention

[0006] The purpose of the present invention is to provide a ketopantolactone reductase mutant with improved activity, a coding gene thereof and an application thereof, so as to solve the problem of low product time-space yield in the existing technology of synthesizing D-pantolactone by catalysis of ketopantolactone reductase.

[0007] Specifically, the present invention uses the ketopantolactone reductase CviCPR (see CN 114085820A) derived from Candida viswanathii, which was constructed and preserved in the laboratory in the early stage, as the original sequence, whose amino acid sequence is shown in SEQ ID NO: 1 and nucleotide sequence is shown in SEQ ID NO: 2, and performs site-directed mutagenesis on it, thereby obtaining a ketopantolactone reductase mutant with significantly improved catalytic activity.

[0008] In a first aspect, the present invention provides a ketopantoic acid lactone reductase mutant with improved activity, which has the following mutations at amino acid residue 126 and / or amino acid residue 215 relative to the wild-type ketopantoic acid lactone reductase CviCPR shown in SEQ ID NO: 1:

[0009] The leucine L at position 126 mutates to one of proline P, phenylalanine F, tyrosine Y and tryptophan W; the alanine A at position 215 mutates to one of aspartic acid D and glutamic acid E.

[0010] In some embodiments, the ketopantoate lactone reductase mutant is a ketopantoate lactone reductase single-point mutant obtained by mutating the leucine L at position 126 of the wild-type ketopantoate lactone reductase CviCPR shown in SEQ ID NO:1 to proline P, denoted as CviCPR / L126P, and its amino acid sequence is shown in SEQ ID NO:15.

[0011] In some embodiments, the ketopantoate lactone reductase mutant is a ketopantoate lactone reductase single-point mutant obtained by mutating the leucine L at position 126 of the wild-type ketopantoate lactone reductase CviCPR shown in SEQ ID NO: 1 to phenylalanine F, denoted as CviCPR / L126F, and its amino acid sequence is shown in SEQ ID NO: 17;

[0012] In some embodiments, the ketopantoate lactone reductase mutant is a ketopantoate lactone reductase single-point mutant obtained by mutating the leucine L at position 126 of the wild-type ketopantoate lactone reductase CviCPR shown in SEQ ID NO: 1 to tyrosine Y, denoted as CviCPR / L126Y, and its amino acid sequence is shown in SEQ ID NO: 19;

[0013] In some embodiments, the ketopantoate lactone reductase mutant is a ketopantoate lactone reductase single-point mutant obtained by mutating the leucine L at position 126 of the wild-type ketopantoate lactone reductase CviCPR shown in SEQ ID NO: 1 to tryptophan W, denoted as CviCPR / L126W, and its amino acid sequence is shown in SEQ ID NO: 21;

[0014] In some embodiments, the ketopantoate lactone reductase mutant is a ketopantoate lactone reductase single-point mutant obtained by mutating alanine A at position 215 of the wild-type ketopantoate lactone reductase CviCPR shown in SEQ ID NO: 1 to aspartic acid D, denoted as CviCPR / A215D, and its amino acid sequence is shown in SEQ ID NO: 23;

[0015] In some embodiments, the ketopantoate lactone reductase mutant is a ketopantoate lactone reductase single point mutant obtained by mutating alanine A at position 215 of the wild-type ketopantoate lactone reductase CviCPR shown in SEQ ID NO: 1 to glutamic acid E, denoted as CviCPR / A215E, and its amino acid sequence is shown in SEQ ID NO: 25;

[0016] In some embodiments, the ketopantoate lactone reductase mutant is a ketopantoate lactone reductase double-point mutant obtained by mutating the leucine L at position 126 of the wild-type ketopantoate lactone reductase CviCPR shown in SEQ ID NO:1 to proline P, and mutating the alanine A at position 215 to aspartic acid D, denoted as CviCPR / L126P / A215D, and its amino acid sequence is shown in SEQ ID NO:27;

[0017] In some embodiments, the ketopantoate lactone reductase mutant is a ketopantoate lactone reductase double-point mutant obtained by mutating the leucine L at position 126 of the wild-type ketopantoate lactone reductase CviCPR shown in SEQ ID NO:1 to phenylalanine F, and mutating the alanine A at position 215 to aspartic acid D, denoted as CviCPR / L126F / A215D, and its amino acid sequence is shown in SEQ ID NO:29;

[0018] In some embodiments, the ketopantoate lactone reductase mutant is a ketopantoate lactone reductase double-point mutant obtained by mutating the leucine L at position 126 of the wild-type ketopantoate lactone reductase CviCPR shown in SEQ ID NO:1 to tyrosine Y, and mutating the alanine A at position 215 to aspartic acid D, denoted as CviCPR / L126Y / A215D, and its amino acid sequence is shown in SEQ ID NO:31;

[0019] In some embodiments, the ketopantoate lactone reductase mutant is a ketopantoate lactone reductase double-point mutant obtained by mutating the leucine L at position 126 of the wild-type ketopantoate lactone reductase CviCPR shown in SEQ ID NO:1 to tryptophan W, and mutating the alanine A at position 215 to aspartic acid D, denoted as CviCPR / L126W / A215D, and its amino acid sequence is shown in SEQ ID NO:33;

[0020] In some embodiments, the ketopantoate lactone reductase mutant is a ketopantoate lactone reductase double-point mutant obtained by mutating the leucine L at position 126 of the wild-type ketopantoate lactone reductase CviCPR shown in SEQ ID NO:1 to proline P, and mutating the alanine A at position 215 to glutamate E, denoted as CviCPR / L126P / A215E, and its amino acid sequence is shown in SEQ ID NO:35;

[0021] In some embodiments, the ketopantoate lactone reductase mutant is a ketopantoate lactone reductase double-point mutant obtained by mutating the leucine L at position 126 of the wild-type ketopantoate lactone reductase CviCPR shown in SEQ ID NO:1 to phenylalanine F, and mutating the alanine A at position 215 to glutamate E, denoted as CviCPR / L126F / A215E, and its amino acid sequence is shown in SEQ ID NO:37;

[0022] In some embodiments, the ketopantoate lactone reductase mutant is a ketopantoate lactone reductase double-point mutant obtained by mutating the leucine L at position 126 of the wild-type ketopantoate lactone reductase CviCPR shown in SEQ ID NO:1 to tyrosine Y, and mutating the alanine A at position 215 to glutamate E, denoted as CviCPR / L126Y / A215E, and its amino acid sequence is shown in SEQ ID NO:39;

[0023] In some embodiments, the ketopantoate lactone reductase mutant is a ketopantoate lactone reductase double-point mutant obtained by mutating the leucine L at position 126 of the wild-type ketopantoate lactone reductase CviCPR shown in SEQ ID NO:1 to tryptophan W, and mutating the alanine A at position 215 to glutamate E, and is recorded as CviCPR / L126W / A215E, and its amino acid sequence is shown in SEQ ID NO:41.

[0024] The ketopantoate lactone reductase mutant provided by the present invention can be artificially synthesized, or its encoding gene can be synthesized first and then expressed biologically, for example, by expressing it from a prokaryotic host (such as Escherichia coli) or a eukaryotic host (such as yeast, higher plants) using recombinant technology.

[0025] In some embodiments, the above-mentioned ketopantoic acid lactone reductase mutant is obtained by site-directed mutagenesis technology to obtain its encoding gene, and then the recombinant vector containing the encoding gene is introduced into Escherichia coli (such as E. coli BL21 (DE3)) to obtain a recombinant genetically engineered bacterium, and then the recombinant genetically engineered bacterium is induced to express to obtain the ketopantoic acid lactone reductase mutant.

[0026] In a second aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned ketopantoate lactone reductase mutant;

[0027] Preferably, the nucleic acid molecule is a gene as shown in 1)-14) below:

[0028] 1) The DNA molecule shown in SEQ ID NO: 16, encoding CviCPR / L126P;

[0029] 2) the DNA molecule shown in SEQ ID NO: 18, encoding CviCPR / L126F;

[0030] 3) the DNA molecule shown in SEQ ID NO: 20, encoding CviCPR / L126Y;

[0031] 4) the DNA molecule shown in SEQ ID NO:22, encoding CviCPR / L126W;

[0032] 5) the DNA molecule shown in SEQ ID NO:24, encoding CviCPR / A215D;

[0033] 6) the DNA molecule shown in SEQ ID NO:26, encoding CviCPR / A215E;

[0034] 7) The DNA molecule shown in SEQ ID NO:28, encoding CviCPR / L126P / A215D;

[0035] 8) The DNA molecule shown in SEQ ID NO:30, encoding CviCPR / L126F / A215D;

[0036] 9) The DNA molecule shown in SEQ ID NO:32, encoding CviCPR / L126Y / A215D;

[0037] 10) The DNA molecule shown in SEQ ID NO:34, encoding CviCPR / L126W / A215D;

[0038] 11) The DNA molecule shown in SEQ ID NO:36, encoding CviCPR / L126P / A215E;

[0039] 12) The DNA molecule shown in SEQ ID NO:38, encoding CviCPR / L126F / A215E;

[0040] 13) The DNA molecule shown in SEQ ID NO:40, encoding CviCPR / L126Y / A215E;

[0041] 14) The DNA molecule shown in SEQ ID NO:42, which encodes CviCPR / L126W / A215E.

[0042] Particularly preferably, the present invention provides a ketopantoate lactone reductase combination mutant CviCPR / L126W / A215E with significantly improved enzyme activity, as described above, whose amino acid sequence is shown in SEQ ID NO:41, and whose nucleotide sequence is shown in SEQ ID NO:42.

[0043] The nucleic acid molecule provided by the present invention can usually be obtained by PCR amplification or artificial synthesis.

[0044] In a third aspect, the present invention provides a recombinant vector comprising any of the above-mentioned nucleic acid molecules;

[0045] The recombinant vector includes a cloning vector and an expression vector. The cloning vector is used to replicate related sequences, and the expression vector is used to express related genes.

[0046] In some preferred embodiments, the recombinant vector further comprises a nucleic acid molecule encoding glucose dehydrogenase; the advantage is that the obtained recombinant vector can simultaneously express the ketopantoic acid lactone reductase mutant and the glucose dehydrogenase, and in the process of the ketopantoic acid lactone reductase mutant catalyzing the asymmetric reduction of ketopantoic acid to prepare D-pantoic acid lactone, the glucose dehydrogenase catalyzes the reduction of NADP + Reduced to NADPH, NADPH provides reducing power for the asymmetric reduction reaction catalyzed by ketopantoic acid lactone reductase, thereby avoiding the exogenous addition of expensive cofactors and effectively reducing production costs; more preferably, the glucose dehydrogenase is derived from Bacillus megaterium, and its amino acid sequence is shown in SEQ ID NO:43, and its nucleotide sequence is shown in SEQ ID NO:44.

[0047] In some embodiments, in the above-mentioned recombinant vector, the nucleic acid molecule encoding any of the above-mentioned ketopantoic acid lactone reductase mutants and the nucleic acid molecule encoding glucose dehydrogenase are respectively inserted between the NcoI and NotI restriction sites of the first group of multiple cloning sites of the pACYCDuet-1 vector and between the BglII and PacI restriction sites of the second group of multiple cloning sites, and the other sequences remain unchanged, to obtain a recombinant plasmid that co-expresses the ketopantoic acid lactone reductase mutant and the glucose dehydrogenase BmGDH. The recombinant plasmid can be a recombinant plasmid encoding a single point mutant of ketopantoic acid lactone reductase: pACYCDuet-1-CviCPR / L126P-BmGDH, pACYCDuet-1-CviCPR / L126F-BmGDH, pACYCDuet-1-CviCPR / L126Y-BmGDH, pACYCDuet-1-CviCPR / L126W-BmGDH, pACYCDuet-1-CviCPR / A215D- BmGDH, pACYCDuet-1-CviCPR / A215E-BmGDH; or recombinant plasmids encoding double-point mutants of ketopantolactone reductase: pACYCDuet-1-CviCPR / L126P / A215D-BmGDH, pACYCDuet-1-CviCPR / L126F / A215D-BmGDH, pACYCDuet-1-CviCPR / L126Y / A215D-BmGDH, pACYCDuet-1-CviCPR / L126Y / A215D-BmGDH, pACYCDuet-1-CviCPR / L126P / A215D-BmGDH uet-1-CviCPR / L126W / A215D-BmGDH, pACYCDuet-1-CviCPR / L126P / A215E-BmGDH, pACYCDuet-1-CviCPR / L1 26F / A215E-BmGDH, pACYCDuet-1-CviCPR / L126Y / A215E-BmGDH, pACYCDuet-1-CviCPR / L126W / A215E-BmGDH.

[0048] In a fourth aspect, the present invention provides a recombinant cell comprising the above-mentioned recombinant vector.

[0049] In some preferred embodiments, the recombinant cell induces the production of any of the above-mentioned ketopantoate lactone reductase mutants; more preferably, the recombinant cell also induces the production of glucose dehydrogenase; further preferably, the glucose dehydrogenase is derived from Bacillus megaterium, and its amino acid sequence is shown in SEQ ID NO:43, and its nucleotide sequence is shown in SEQ ID NO:44.

[0050] In some preferred embodiments, the method for constructing the recombinant cell is as follows:

[0051] The recombinant vector is transformed into a host cell, and after induction, a recombinant cell expressing the ketopantoate lactone reductase mutant is obtained.

[0052] Further, the recombinant vector is any of the above-mentioned recombinant vectors, and the host cell is a prokaryotic cell or a eukaryotic cell, such as Escherichia coli, yeast, etc., preferably Escherichia coli BL21 (DE3). Any of the above-mentioned recombinant vectors is transformed into E. coli BL21 (DE3), and after induction, a recombinant genetically engineered bacterium (recombinant cell) that simultaneously expresses the above-mentioned ketopantolactone reductase mutant and glucose dehydrogenase from Bacillus megaterium is obtained.

[0053] In some embodiments, the recombinant genetically engineered bacteria are E. coli BL21 (DE3) / pACYCDuet-1-CviCPR / L126P-BmGDH, E. coli BL21 (DE3) / pACYCDuet-1-CviCPR / L126F-BmGDH, E. coli BL21 (DE3) / pACYCDuet-1-CviCPR / L126Y-BmGDH, E. coli BL21 (DE3) / pACYCDuet-1-CviCPR / L126W-BmGDH, E. coli BL21 (DE3) / pACYCDuet-1-CviCPR / A215D-BmGDH, E. coli BL21 (DE3) / pACYCDuet-1-CviCPR / A215E-BmGDH; E. coli BL21(DE3) / pACYCDuet-1-CviCPR / L126P / A215D-BmGDH, E.coli BL21(DE3) / pACYCDuet-1-CviCPR / L126F / A215D-BmGDH, E.coli BL21(DE3) / pACYCDuet-1-CviCPR / L126Y / A215D-BmGDH, E.coli BL21(DE3) / pACYCDuet-1-CviCPR / L126W / A215D-BmGDH, E.coli BL21(DE3) / pACYCDuet-1-CviCPR / L126P / A215E-BmGDH, E.coli BL21(DE3) / pACYCDuet-1-CviCPR / L126F / A215E-BmGDH, E.coli BL21(DE3) / pACYCDuet-1-CviCPR / L126Y / A215E-BmGDH, E.coli BL21(DE3) / pACYCDuet-1-CviCPR / L126W / A215E-BmGDH.

[0054] In a fifth aspect, the present invention provides a method for preparing ketopantolactone reductase, comprising:

[0055] Inducing and culturing any of the above-mentioned recombinant cells to obtain a culture;

[0056] Any of the above-mentioned ketopantoate lactone reductase mutants is isolated from the culture.

[0057] In some embodiments, the above method further comprises a step of isolating glucose dehydrogenase from the culture. Among them, the method of inducing culture of recombinant cells, the method of isolating ketopantoic acid lactone reductase mutants from the culture, and the method of isolating glucose dehydrogenase from the culture are all conventional methods in the art.

[0058] In a sixth aspect, the present invention provides the use of any of the above-mentioned ketopantoic acid lactone reductase mutants, any of the above-mentioned nucleic acid molecules, any of the above-mentioned recombinant vectors and / or any of the above-mentioned recombinant cells in the preparation of D-pantoic acid lactone, specifically for catalyzing the asymmetric synthesis of D-pantoic acid lactone.

[0059] In a seventh aspect, the present invention provides a method for preparing D-pantoic acid lactone, comprising: using any of the above-mentioned ketopantoic acid lactone reductase mutants, any of the above-mentioned recombinant cells and / or ketopantoic acid lactone reductase prepared by any of the above-mentioned methods as a catalyst, catalyzing ketopantoic acid lactone to obtain D-pantoic acid lactone. The reaction principle involved in the preparation method of D-pantoic acid lactone is as follows: Figure 1 As shown, ketopantolactone is the substrate and glucose is the cosubstrate.

[0060] In some preferred embodiments, the preparation method of D-pantoic acid lactone is as follows:

[0061] A recombinant cell expressing any of the above-mentioned ketopantolactone reductase mutants and glucose dehydrogenase from Bacillus megaterium is contacted with a substrate ketopantolactone and a cosubstrate glucose to perform an asymmetric reduction reaction, and then the generated D-pantolactone is collected from the reaction product.

[0062] In some embodiments, in the above-mentioned method for preparing D-pantolactone, the pH of the catalytic reaction is 6-8, such as pH 6, pH 6.5, pH 7, pH 7.5 and pH 8, or a value or range between any two of these values; and / or

[0063] The temperature of the catalytic reaction is 30°C-50°C, such as 30°C, 35°C, 40°C, 45°C and 50°C, or a value or range between any two of these values.

[0064] In some embodiments, in any of the above methods for preparing D-pantolactone, the catalytic reaction is carried out in a phosphate buffer; and / or

[0065] In the catalytic reaction, the cumulative final concentration of the ketopantolactone is 1 mM-400 mM; and / or

[0066] In the catalytic reaction, the mass ratio of the catalyst to the ketopantoic acid lactone is (0.01-0.1):1, for example, 0.01:1, 0.02:1, 0.03:1, 0.04:1, 0.05:1, 0.06:1, 0.07:1, 0.08:1, 0.09:1 or 0.1:1, or a value or range between any two of these values.

[0067] In some embodiments, the method for preparing D-pantolactone described in any of the above comprises catalyzing the preparation of D-pantolactone from ketopantolactone using any of the above recombinant cells (recombinant genetically engineered bacteria);

[0068] Specifically, the bacterial cells obtained by centrifugation after fermentation and culture of the recombinant genetically engineered bacteria, the immobilized bacterial cells, the enzyme extracted after ultrasonic disruption of the bacterial cells, the immobilized enzyme or the pure enzyme can be used as a catalyst, ketopantolactone as a substrate, glucose as a co-substrate, a reaction solution with a pH of 6-8 (for example, potassium phosphate buffer (100 mM, pH 6.5)) as a reaction medium, and the reaction is carried out at 30-50° C. and 150-300 rpm to obtain a reaction solution containing D-pantolactone;

[0069] Preferably, the concentration of ketopantolactone is 1-400 mM, the concentration of glucose is 1-600 mM, and the amount of catalyst used based on the mass of substrate ketopantolactone is 0.01-0.1 g cell / g ketopantolactone;

[0070] During the reaction, the pH was monitored online to adjust the addition of Na2CO3 and control the pH at about 6.5. The pH of the reaction solution is related to many factors such as the catalytic performance of the enzyme and the physical and chemical properties of the substrate. After investigation, it was found that the reaction effect was better when the pH was 6-8, and the preferred pH was 6.5.

[0071] It should be understood that the ketopantoic acid lactone reductase mutant of the present invention can be used in the form of engineered bacteria whole cells, can be used in the form of unpurified crude enzymes, can be used in the form of partially purified or completely purified enzymes. The ketopantoic acid lactone reductase mutant of the present invention can also be made into a catalyst in the form of an immobilized enzyme or an immobilized cell using immobilization techniques known in the art.

[0072] Preferably, the ketopantoate lactone reductase mutant is CviCPR / L126W / A215E or a recombinant genetically engineered bacterium E. coli BL21 (DE3) / pACYCDuet-1-CviCPR / L126W / A215E-BmGDH expressing the ketopantoate lactone reductase mutant.

[0073] The culture medium used when the recombinant genetically engineered bacteria express the mutant can be a culture medium in the art that can grow the recombinant genetically engineered bacteria and produce the ketopantoic acid lactone reductase mutant of the present invention, preferably LB culture medium: yeast powder 5g / L, peptone 10g / L, NaCl 10g / L, pH 7.0.

[0074] There are no special requirements for the culture method and culture conditions, as long as the genetically engineered bacteria can grow normally and express ketopantoic acid lactone reductase. Specifically, the following steps are included:

[0075] (1) Plate culture: streak the recombinant genetically engineered bacteria of the present invention onto a solid LB plate containing a screening antibiotic and culture at 37° C. overnight;

[0076] (2) Seed culture: Pick a single colony from the plate obtained in step (1) in a clean bench, inoculate it into LB liquid culture medium containing screening antibiotics, and culture it at 37°C overnight;

[0077] (3) Induction culture: In a clean bench, the seed culture solution obtained in step (2) was inoculated into LB liquid medium containing screening antibiotics and cultured at 37°C until OD 600 When the value reaches 0.6-0.8, add the inducer IPTG with a final concentration of 0.2 mM and induce the culture at 16°C for 12 h;

[0078] (4) Collecting bacteria: The bacterial solution obtained by the induction culture in step (3) is centrifuged at 8000 rpm for 10 min to separate wet bacteria. The wet bacteria are washed twice with physiological saline to obtain the catalyst used in the catalytic reaction.

[0079] Since the substrate ketopantoic acid lactone is extremely easy to hydrolyze, in order to reduce the spontaneous hydrolysis of the substrate during the catalytic process, the addition of the substrate and the co-substrate is preferably carried out in a flow-addition mode, thereby continuously increasing the cumulative concentration of ketopantoic acid lactone in the reaction system, while avoiding the spontaneous hydrolysis caused by the accumulation of high-concentration ketopantoic acid lactone and its inhibitory effect on the catalyst activity.

[0080] The present invention finds that positions 126 and 215 of the ketopantolactone reductase CviCPR from Candida viswanathii play a key role in improving the activity. In particular, the ketopantolactone reductase mutant CviCPR / L126W / A215E provided by the present invention can catalyze the preparation of D-pantolactone from ketopantolactone with high activity, has the characteristics of being green, efficient, economical and environmentally friendly, and provides a potential biocatalyst for the asymmetric synthesis of D-pantolactone by redox enzyme method.

[0081] The beneficial effects of the present invention compared with the prior art are as follows:

[0082] (1) The ketopantolactone reductase mutant CviCPR / L126W / A215E provided by the present invention can catalyze high concentration ketopantolactone (400 mM) to generate D-pantolactone, with a substrate conversion rate of 99%, a product optical purity (ee) of 99%, and a spatiotemporal yield of D-pantolactone of 706 g L -1 d -1 .

[0083] (2) The present invention improves the catalytic activity of ketopantolactone reductase through enzyme engineering, which has important industrial application value for the green and efficient preparation of D-pantolactone. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 The schematic diagram of the asymmetric synthesis of D-pantolactone from ketopantolactone of the present invention is shown in FIG.

[0085] Figure 2 This is the gas chromatogram of D-pantolactone standard.

[0086] Figure 3 This is the gas chromatogram of L-pantolactone standard.

[0087] Figure 4 This is the gas chromatogram of the ketopantolactone standard.

[0088] Figure 5 This is the reaction progress curve of the recombinant genetically engineered bacteria E. coli BL21 (DE3) / pACYCDuet-1-CviCPR-BmGDH expressing the wild-type ketopantolactone reductase CviCPR in Example 4 of the present invention catalyzing the production of D-pantolactone from ketopantolactone.

[0089] Figure 6 This is the reaction progress curve of the recombinant genetically engineered bacteria E. coli BL21 (DE3) / pACYCDuet-1-CviCPR / L126W / A215E-BmGDH expressing the ketopantolactone reductase mutant CviCPR / L126W / A215E in Example 4 of the present invention catalyzing the production of D-pantolactone from ketopantolactone.

[0090] Figure 7 This is a gas chromatogram of the reaction liquid in which the recombinant genetically engineered bacteria E. coli BL21(DE3) / pACYCDuet-1-CviCPR / L126W / A215E-BmGDH catalyzes ketopantolactone to produce D-pantolactone. DETAILED DESCRIPTION

[0091] The present invention will be further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional operations in the art, or according to the experimental methods recommended by the kit and instrument manufacturers. The reagents and biological materials used in the examples can be obtained from commercial sources unless otherwise specified.

[0092] In the following example, plasmid pACYCDuet-1-CviCPR-BmGDH was constructed according to Example 2 of the Chinese invention patent application with publication number CN 114085820 A and invention name “Ketopantolactone reductase from Candida viswanathii”. The plasmid co-expressed ketopantolactone reductase CviCPR from Candida viswanathii and glucose dehydrogenase BmGDH from Bacillus megaterium, wherein the amino acid sequence of ketopantolactone reductase CviCPR is shown in SEQ ID NO: 1, and the nucleotide sequence is shown in SEQ ID NO: 2, and the amino acid sequence of glucose dehydrogenase BmGDH is shown in SEQ ID NO: 43, and the nucleotide sequence is shown in SEQ ID NO: 44. The ketopantolactone reductase CviCPR derived from Candida viswanathii is the wild-type ketopantolactone reductase in the following examples.

[0093] Ketopantolactone is a product of Sigma-Aldrich Company with catalog number 305847.

[0094] Example 1 Construction of a recombinant plasmid of a ketopantolactone reductase mutant

[0095] 1. Using the recombinant plasmid pACYCDuet-1-CviCPR-BmGDH containing the target gene fragment as a template, a single point mutation was introduced into the gene sequence of the wild-type ketopantolactone reductase by designing primers. The primer sequences used are shown in Table 1.

[0096] Table 1 Primers for ketopantolactone reductase mutants

[0097]

[0098]

[0099] By whole plasmid amplification, recombinant plasmids encoding single point mutants of ketopantolactone reductase were obtained: pACYCDuet-1-CviCPR / L126P-BmGDH, pACYCDuet-1-CviCPR / L126F-BmGDH, pACYCDuet-1-CviCPR / L126Y-BmGDH, pACYCDuet-1-CviCPR / L126W-BmGDH, pACYCDuet-1-CviCPR / A215D-BmGDH and pACYCDuet-1-CviCPR / A215E-BmGDH.

[0100] The ketopantoate lactone reductase expressed by pACYCDuet-1-CviCPR / L126P-BmGDH is a single-point mutant of ketopantoate lactone reductase obtained by mutating the leucine L at position 126 of the wild-type ketopantoate lactone reductase to proline P, denoted as CviCPR / L126P, whose amino acid sequence is shown in SEQ ID NO:15, and whose nucleotide sequence is shown in SEQ ID NO:16.

[0101] The ketopantoate lactone reductase expressed by pACYCDuet-1-CviCPR / L126F-BmGDH is a single-point mutant of ketopantoate lactone reductase obtained by mutating the leucine L at position 126 of the wild-type ketopantoate lactone reductase to phenylalanine F, denoted as CviCPR / L126F, whose amino acid sequence is shown in SEQ ID NO:17, and whose nucleotide sequence is shown in SEQ ID NO:18.

[0102] The ketopantoic acid lactone reductase expressed by pACYCDuet-1-CviCPR / L126Y-BmGDH is a single-point mutant of ketopantoic acid lactone reductase obtained by mutating the leucine L at position 126 of the wild-type ketopantoic acid lactone reductase to tyrosine Y, denoted as CviCPR / L126Y, whose amino acid sequence is shown in SEQ ID NO:19, and whose nucleotide sequence is shown in SEQ ID NO:20.

[0103] The ketopantoate lactone reductase expressed by pACYCDuet-1-CviCPR / L126W-BmGDH is a single-point mutant of ketopantoate lactone reductase obtained by mutating the leucine L at position 126 of the wild-type ketopantoate lactone reductase to tryptophan W, denoted as CviCPR / L126W, whose amino acid sequence is shown in SEQ ID NO:21, and whose nucleotide sequence is shown in SEQ ID NO:22.

[0104] The ketopantoate lactone reductase expressed by pACYCDuet-1-CviCPR / A215D-BmGDH is a single-point mutant of ketopantoate lactone reductase obtained by mutating the 215th alanine A of the wild-type ketopantoate lactone reductase to aspartic acid D, denoted as CviCPR / A215D, and its amino acid sequence is shown in SEQ ID NO:23, and its nucleotide sequence is shown in SEQ ID NO:24.

[0105] The ketopantoate lactone reductase expressed by pACYCDuet-1-CviCPR / A215E-BmGDH is a single-point mutant of ketopantoate lactone reductase obtained by mutating alanine A at position 215 of the wild-type ketopantoate lactone reductase to glutamate E, denoted as CviCPR / A215E, whose amino acid sequence is shown in SEQ ID NO:25, and whose nucleotide sequence is shown in SEQ ID NO:26.

[0106] 2. The above-mentioned recombinant plasmids encoding the single point mutants at amino acid position 126, pACYCDuet-1-CviCPR / L126P-BmGDH, pACYCDuet-1-CviCPR / L126F-BmGDH, pACYCDuet-1-CviCPR / L126Y-BmGDH, and pACYCDuet-1-CviCPR / L126W-BmGDH, were used as templates and the single point mutant primers at position 215 were used for polymerase chain reaction (PCR) (whole plasmid amplification) to obtain recombinant plasmids encoding double point mutants of ketopantoic acid lactone reductase: pACYCDuet-1-CviCPR / L126P / A215D-BmGDH, pACYCDuet-1-CviCPR / L126F-BmGDH, pACYCDuet-1-CviCPR / L126Y-BmGDH, and pACYCDuet-1-CviCPR / L126W-BmGDH. CDuet-1-CviCPR / L126F / A215D-BmGDH, pACYCDuet-1-CviCPR / L126Y / A215D-BmGDH, pACYCDuet-1-CviCPR / L126W / A215D-BmGDH, pACYCDuet-1-CviCPR / L 126P / A215E-BmGDH, pACYCDuet-1-CviCPR / L126F / A215E-BmGDH, pACYCDuet-1-CviCPR / L126Y / A215E-BmGDH, pACYCDuet-1-CviCPR / L126W / A215E-BmGDH.

[0107] The ketopantoate lactone reductase expressed by pACYCDuet-1-CviCPR / L126P / A215D-BmGDH is a double-point mutant of ketopantoate lactone reductase obtained by mutating the leucine L at position 126 of the wild-type ketopantoate lactone reductase to proline P, and mutating the alanine A at position 215 to aspartic acid D, denoted as CviCPR / L126P / A215D, whose amino acid sequence is shown in SEQ ID NO:27, and whose nucleotide sequence is shown in SEQ ID NO:28.

[0108] The ketopantoate lactone reductase expressed by pACYCDuet-1-CviCPR / L126F / A215D-BmGDH is a double-point mutant of ketopantoate lactone reductase obtained by mutating the leucine L at position 126 of the wild-type ketopantoate lactone reductase to phenylalanine F and the alanine A at position 215 to aspartic acid D, denoted as CviCPR / L126F / A215D, whose amino acid sequence is shown in SEQ ID NO:29, and whose nucleotide sequence is shown in SEQ ID NO:30.

[0109] The ketopantoic acid lactone reductase expressed by pACYCDuet-1-CviCPR / L126Y / A215D-BmGDH is a double-point mutant of ketopantoic acid lactone reductase obtained by mutating the leucine L at position 126 of the wild-type ketopantoic acid lactone reductase to tyrosine Y and the alanine A at position 215 to aspartic acid D, denoted as CviCPR / L126Y / A215D, whose amino acid sequence is shown in SEQ ID NO:31, and whose nucleotide sequence is shown in SEQ ID NO:32.

[0110] The ketopantoic acid lactone reductase expressed by pACYCDuet-1-CviCPR / L126W / A215D-BmGDH is a double-point mutant of ketopantoic acid lactone reductase obtained by mutating the leucine L at position 126 of the wild-type ketopantoic acid lactone reductase to tryptophan W and the alanine A at position 215 to aspartic acid D, denoted as CviCPR / L126W / A215D, whose amino acid sequence is shown in SEQ ID NO:33, and whose nucleotide sequence is shown in SEQ ID NO:34.

[0111] The ketopantoate lactone reductase expressed by pACYCDuet-1-CviCPR / L126P / A215E-BmGDH is a double-point mutant of ketopantoate lactone reductase obtained by mutating the leucine L at position 126 of the wild-type ketopantoate lactone reductase to proline P, and mutating the alanine A at position 215 to glutamate E, denoted as CviCPR / L126P / A215E, whose amino acid sequence is shown in SEQ ID NO:35, and whose nucleotide sequence is shown in SEQ ID NO:36.

[0112] The ketopantoate lactone reductase expressed by pACYCDuet-1-CviCPR / L126F / A215E-BmGDH is a double-point mutant of ketopantoate lactone reductase obtained by mutating the leucine L at position 126 of the wild-type ketopantoate lactone reductase to phenylalanine F and the alanine A at position 215 to glutamate E, denoted as CviCPR / L126F / A215E, whose amino acid sequence is shown in SEQ ID NO:37, and whose nucleotide sequence is shown in SEQ ID NO:38.

[0113] The ketopantoate lactone reductase expressed by pACYCDuet-1-CviCPR / L126Y / A215E-BmGDH is a double-point mutant of ketopantoate lactone reductase obtained by mutating the leucine L at position 126 of the wild-type ketopantoate lactone reductase to tyrosine Y and the alanine A at position 215 to glutamate E, denoted as CviCPR / L126Y / A215E, whose amino acid sequence is shown in SEQ ID NO:39, and whose nucleotide sequence is shown in SEQ ID NO:40.

[0114] The ketopantoate lactone reductase expressed by pACYCDuet-1-CviCPR / L126W / A215E-BmGDH is a double-point mutant of ketopantoate lactone reductase obtained by mutating the leucine L at position 126 of the wild-type ketopantoate lactone reductase to tryptophan W and the alanine A at position 215 to glutamate E, denoted as CviCPR / L126W / A215E, whose amino acid sequence is shown in SEQ ID NO:41, and whose nucleotide sequence is shown in SEQ ID NO:42.

[0115] Example 2 Construction of a recombinant genetically engineered bacterium expressing a ketopantolactone reductase mutant

[0116] The recombinant plasmids of the single-point mutant and double-point mutant of ketopantoic acid lactone reductase obtained in Example 1 were transformed into E. coli BL21 (DE3) competent cells, and after recovery, they were spread on LB plates containing 25 μg / mL chloramphenicol for overnight culture.

[0117] 5-10 clones were picked from the LB plate cultured overnight, inoculated into LB liquid culture medium respectively, and cultured at 37°C for 8 h. The cultured bacterial solution was sequenced and verified to obtain recombinant genetically engineered bacteria with correct sequences of ketopantoic acid lactone reductase mutants: E. coli BL21 (DE3) / pACYCDuet-1-CviCPR / L126P-BmGDH, E. coli BL21 (DE3) / pACYCDuet-1-CviCPR / L126F-BmGDH, E. coli BL21 (DE3) / pACYCDuet-1-CviCPR / L126Y-BmGDH, E. coli BL21 (DE3) / pACYCDuet-1-CviCPR / L126W-BmGDH, E. coli BL21 (DE3) / pACYCDuet-1-CviCPR / A215D-BmGDH, E. coli BL21(DE3) / pACYCDuet-1-CviCPR / A215E-BmGDH; E.coli BL21(DE3) / pACYCDuet-1-CviCPR / L126P / A215D-BmGDH, E.coli BL21(DE3) / pACYCDuet-1-CviCPR / L126F / A215D-BmGDH, E.coli BL21(DE3) / pACYCDuet-1-CviCPR / L126Y / A215D-BmGDH, E.coli BL21(DE3) / pACYCDuet-1-CviCPR / L126W / A215D-BmGDH, E.coli BL21(DE3) / pACYCDuet-1-CviCPR / L126P / A215E-BmGDH, E.coli BL21(DE3) / pACYCDuet-1-CviCPR / L126F / A215E-BmGDH, E.coli BL21(DE3) / pACYCDuet-1-CviCPR / L126Y / A215E-BmGDH, E. coli BL21(DE3) / pACYCDuet-1-CviCPR / L126W / A215E-BmGDH.

[0118] Using pACYCDuet-1-CviCPR-BmGDH, according to the above method, a wild-type ketopantolactone reductase recombinant genetic engineering bacterium E. coli BL21 (DE3) / pACYCDuet-1-CviCPR-BmGDH was obtained.

[0119] Example 3 Activity and selectivity screening of ketopantolactone reductase mutants

[0120] The recombinant genetically engineered bacteria obtained in Example 2 were fermented in LB medium and IPTG was used to induce gene expression to obtain a bacterial solution expressing various ketopantoic acid lactone reductases and glucose dehydrogenase BmGDH from Bacillus megaterium. Wet bacterial cells were obtained by centrifugation to be used as a catalyst.

[0121] The obtained wet cells were added to 10 mL potassium phosphate buffer (100 mM, pH 6.5) and 10% (w / v) substrate ketopantoic acid lactone, respectively, to make the wet cell concentration reach 1 g / L, and reacted at 30°C and 200 rpm for 9 hours. Ethyl acetate was added to extract the reaction solution, and samples were taken to analyze the conversion rate of ketopantoic acid lactone and the optical purity of D-pantoic acid lactone. The specific gas chromatography (SHIMADZU GC2030) method was used, and the detection conditions were as follows: the chromatographic column was BGB-174 (30m×250μm×0.25μm), FID detector (250°C), nitrogen was used as carrier gas (30mL / min), hydrogen was 40mL / min, the injection volume was 1μL, the split ratio was 30:1, the injection port was 250°C, and the column temperature was maintained at 175°C for 8 minutes. The chromatogram of the standard product is as follows: Figure 2 to Figure 4 As shown in Table 2, under the analytical conditions, the retention times of D-pantoic acid lactone, L-pantoic acid lactone and ketopantoic acid lactone are 5.983 min, 6.116 min and 6.397 min, respectively. The functional screening results are shown in Table 2.

[0122] Table 2 Activity and selectivity screening results of ketopantolactone reductase

[0123]

[0124]

[0125] As can be seen from Table 2, the activity of the single-point mutants CviCPR / L126W and CviCPR / A215E relative to the wild-type CviCPR (i.e., the conversion rate of the substrate ketopantoic acid lactone to D-pantoic acid lactone) is improved, and a high selectivity is maintained. The activity of the double-point mutant CviCPR / L126W / A215E obtained by combining the two is further significantly improved, and a high selectivity is maintained, indicating that the mutant CviCPR / L126W / A215E can efficiently and stereospecifically asymmetrically reduce the substrate ketopantoic acid lactone to generate D-pantoic acid lactone, and its catalytic reaction conversion rate is significantly better than that of the wild-type CviCPR.

[0126] Example 4 Ketopantolactone reductase mutant CviCPR / L126W / A215E catalyzes the synthesis of D-pantolactone from ketopantolactone

[0127] The recombinant genetically engineered bacteria E. coli BL21 (DE3) / pACYCDuet-1-CviCPR-BmGDH and E. coli BL21 (DE3) / pACYCDuet-1-CviCPR / L126W / A215E-BmGDH obtained in Example 2 were fermented and cultured in LB medium and IPTG was used to induce gene expression to obtain a recombinant genetically engineered bacterial liquid expressing ketopantoic acid lactone reductase and glucose dehydrogenase BmGDH from Bacillus megaterium. The wet cells were obtained by centrifugation as a catalyst.

[0128] The obtained wet cells of E. coli BL21 (DE3) / pACYCDuet-1-CviCPR-BmGDH and E. coli BL21 (DE3) / pACYCDuet-1-CviCPR / L126W / A215E-BmGDH were added into potassium phosphate buffer (100 mM, pH 6.5) to make the wet cell concentration reach 5.1 g / L, and the reaction was carried out by continuous addition of potassium phosphate buffer (100 mM, pH 6.5) containing 1 M substrate ketopantolactone and 1.1 M glucose. The reaction temperature was 30 ° C and the rotation speed was 200 rpm. The addition of Na2CO3 was adjusted by online monitoring of pH feedback to control the pH at about 6.5. During the reaction, samples were taken at intervals, and the reaction solution was extracted with ethyl acetate. After dilution, the concentration of the substrate ketopantolactone and the optical purity of the product D-pantolactone were analyzed by gas phase analysis, and the reaction progress curve was obtained. The reaction progress curve of the recombinant genetically engineered bacteria E. coli BL21 (DE3) / pACYCDuet-1-CviCPR-BmGDH catalyzing the generation of D-pantolactone from ketopantolactone is shown in the figure. Figure 5 As shown, the reaction process curve of the recombinant genetic engineering bacteria E. coli BL21 (DE3) / pACYCDuet-1-CviCPR / L126W / A215E-BmGDH catalyzing ketopantoic acid lactone to produce D-pantoic acid lactone is as shown in Figure 6 shown.

[0129] The gas chromatography detection conditions of the substrate ketopantolactone and the product D-pantolactone and its isomer L-pantolactone are the same as those in Example 3.

[0130] The gas chromatogram of the reaction solution of the recombinant genetically engineered bacteria E. coli BL21 (DE3) / pACYCDuet-1-CviCPR / L126W / A215E-BmGDH catalyzing ketopantoic acid lactone to produce D-pantoic acid lactone is shown in Figure 7 shown.

[0131] The reaction catalyzed by the recombinant genetically engineered bacteria E. coli BL21(DE3) / pACYCDuet-1-CviCPR / L126W / A215E-BmGDH was completed in 1.75 h, and the cumulative concentration of the substrate ketopantoic acid lactone reached 400 mM. Figure 4 and Figure 7 It can be seen that no chromatographic peak was detected at the retention time of 6.397 min for the substrate ketopantoic acid lactone, which indicates that the substrate ketopantoic acid lactone was basically completely converted into D-pantoic acid lactone, with a substrate conversion rate of ≥99%, and an optical purity (ee) of D-pantoic acid lactone of 99%. After calculation, the spatiotemporal yield of D-pantoic acid lactone was 706 g L -1 d -1 .

[0132] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

[0133] sequence

[0134] SEQ ID NO:1

[0135] MPSQTHPVKLTREFKTKSGADVSIAGTGTKWKKDSKDDDINQELVDQILLALKSGFRHIDTAEVYNTQAEVGEAVKQLGIPREDLWITTKYNPGWTYKSSSASPNASIDKALQQLGTDYIDLYLIHQPFFTEESTHGFSLEDTWKILIDYKK QGKIREIGVSNFAEEHIERLKKILDPEFYPVVNQIESHPFLQDQSKGITAYSQANGILVEAFSPLTPVSRVDKNALTGYLEELSKKYNKTGGQILLRWTLQRGVLPITTSAKEERIKEALDVFDFELTKEEFDKITEIGQANPHRVFFHEEFKDL

[0136] SEQ ID NO:2

[0137] ATGCCTTCACAAACTCACCCTGTCAAATTAACCAGAGAATTTAAGACCAAGTCGGGCGCAGACGTCTCAATTGCTACCGGAACAGGCACCAAGTGGAAAAAAGACTCCAAGGACGACGACATCAACCAGGAATTGGTCGACCAGATCTTGCTTGCGCTCAAGTCTGGGTTCAGACACATTGACACCGCTGAAGTGTACAACACGCAGGCTGAAGTCGGCGAGGCCGTCAAGCAACTGGGCATCCCAAGAGAAGACCTCTGGATCACCACCAAGTACAACCCGGGCTGGAGAACCATCAAGTCGAGCAGCGCCAGTCCAAATGCGTCTATTGACAAGGCATTGCAGCAGTTGGGTACCGATTACATTGACTTGTACTTGATCCACCAGCCTTTCTTCACGGAGGAAAGCACCCATGGGTTCTCGTTGGAAGACACCTGGAAGATCTTGATTGACTACAAGAAGCAGGGCAAGATCAGAGAAATTGGCGTTTCTAACTTCGCTGAGGAACACATCGAGAGATTGAAGAAGATCCTGGACCCAGAGTTCTACCCCGTGGTCAACCAGATCGAGAGCCATCCATTCCTTCAAGACCAGTCCAAGGGCATCACCGCATACTCGCAAGCCAACGGCATCTTAGTCGAAGCATTCTCGCCATTGACGCCTGTTTCAAGAGTCGACAAGAACGCATTGACTGGTTACTTGGAGGAATTGTCCAAGAAGTACAACAAGACCGGTGGCCAGATCTTGCTCAGATGGACGTTGCAGAGAGGTGTGTTGCCAATTACTACCTCGGCCAAAGAAGAGAGAATCAAAGAGGCATTGGATGTTTTTGACTTTGAGTTGACCAAAGAGGAGTTTGACAAGATCACCGAAATCGGTCAGGCCAACCCACACCGTGTCTTCTTCCATGAGGAGTTCAAGGATTTGTAA

[0138] SEQ ID NO:15

[0139] MPSQTHPVKLTREFKTKSGADVSIATGTGTKWKKDSKDDDINQELVDQILLALKSGFRHIDTAEVYNTQAEVGEAVKQLGIPREDLWITTKYNPGWRITKSSSASPNASIDKALQQLGTDYIDLYPIHQPFFTEESTHGFSLEDTWKILIDYKKQGKIREIGVSNFAEEHIERLKILDPEFYPVVNQIESHPFLQDQSKGITAYSQANGILVEAFSPLTPVSRVDKNALTGYLEELSKKYNKTGGQILLRWTLQRGVLPITTSAKEERIKEALDVFDFELTKEEFDKITEIGQANPHRVFFHEEFKDL

[0140] SEQ ID NO:16

[0141] ATGCCTTCACAAACTCACCCTGTCAAATTAACCAGAGAATTTAAGACCAAGTCGGGCGCAGACGTCTCAATTGCTACCGGAACAGGCACCAAGTGGAAAAAAGACTCCAAGGACGACGACATCAACCAGGAATTGGTCGACCAGATCTTGCTTGCGCTCAAGTCTGGGTTCAGACACATTGACACCGCTGAAGTGTACAACACGCAGGCTGAAGTCGGCGAGGCCGTCAAGCAACTGGGCATCCCAAGAGAAGACCTCTGGATCACCACCAAGTACAACCCGGGCTGGAGAACCATCAAGTCGAGCAGCGCCAGTCCAAATGCGTCTATTGACAAGGCATTGCAGCAGTTGGGTACCGATTACATTGACTTGTACCCGATCCACCAGCCTTTCTTCACGGAGGAAAGCACCCATGGGTTCTCGTTGGAAGACACCTGGAAGATCTTGATTGACTACAAGAAGCAGGGCAAGATCAGAGAAATTGGCGTTTCTAACTTCGCTGAGGAACACATCGAGAGATTGAAGAAGATCCTGGACCCAGAGTTCTACCCCGTGGTCAACCAGATCGAGAGCCATCCATTCCTTCAAGACCAGTCCAAGGGCATCACCGCATACTCGCAAGCCAACGGCATCTTAGTCGAAGCATTCTCGCCATTGACGCCTGTTTCAAGAGTCGACAAGAACGCATTGACTGGTTACTTGGAGGAATTGTCCAAGAAGTACAACAAGACCGGTGGCCAGATCTTGCTCAGATGGACGTTGCAGAGAGGTGTGTTGCCAATTACTACCTCGGCCAAAGAAGAGAGAATCAAAGAGGCATTGGATGTTTTTGACTTTGAGTTGACCAAAGAGGAGTTTGACAAGATCACCGAAATCGGTCAGGCCAACCCACACCGTGTCTTCTTCCATGAGGAGTTCAAGGATTTGTAA

[0142] SEQ ID NO:17

[0143] MPSQTHPVKLTREFKTKSGADVSIATGTGTKWKKDSKDDDINQELVDQILLALKSGFRHIDTAEVYNTQAEVGEAVKQLGIPREDLWITTKYNPGWRTIKSSSASPNASIDKALQQLGTDYIDLYFIHQPFFTEESTHGFSLEDTWKILIDYKKQGKIREIGVSNFAEEHIERLKKILDPEFYPVVNQIESHPFLQDQSKGITAYSQANGILVEAFSPLTPVSRVDKNALTGYLEELSKKYNKTGGQILLRWTLQRGVLPITTSAKEERIKEALDVFDFELTKEEFDKITEIGQANPHRVFFHEEFKDL

[0144] SEQ ID NO:18

[0145] ATGCCTTCACAAACTCACCCTGTCAAATTAACCAGAGAATTTAAGACCAAGTCGGGCGCAGACGTCTCAATTGCTACCGGAACAGGCACCAAGTGGAAAAAAGACTCCAAGGACGACGACATCAACCAGGAATTGGTCGACCAGATCTTGCTTGCGCTCAAGTCTGGGTTCAGACACATTGACACCGCTGAAGTGTACAACACGCAGGCTGAAGTCGGCGAGGCCGTCAAGCAACTGGGCATCCCAAGAGAAGACCTCTGGATCACCACCAAGTACAACCCGGGCTGGAGAACCATCAAGTCGAGCAGCGCCAGTCCAAATGCGTCTATTGACAAGGCATTGCAGCAGTTGGGTACCGATTACATTGACTTGTACTTTATCCACCAGCCTTTCTTCACGGAGGAAAGCACCCATGGGTTCTCGTTGGAAGACACCTGGAAGATCTTGATTGACTACAAGAAGCAGGGCAAGATCAGAGAAATTGGCGTTTCTAACTTCGCTGAGGAACACATCGAGAGATTGAAGAAGATCCTGGACCCAGAGTTCTACCCCGTGGTCAACCAGATCGAGAGCCATCCATTCCTTCAAGACCAGTCCAAGGGCATCACCGCATACTCGCAAGCCAACGGCATCTTAGTCGAAGCATTCTCGCCATTGACGCCTGTTTCAAGAGTCGACAAGAACGCATTGACTGGTTACTTGGAGGAATTGTCCAAGAAGTACAACAAGACCGGTGGCCAGATCTTGCTCAGATGGACGTTGCAGAGAGGTGTGTTGCCAATTACTACCTCGGCCAAAGAAGAGAGAATCAAAGAGGCATTGGATGTTTTTGACTTTGAGTTGACCAAAGAGGAGTTTGACAAGATCACCGAAATCGGTCAGGCCAACCCACACCGTGTCTTCTTCCATGAGGAGTTCAAGGATTTGTAA

[0146] SEQ ID NO:19

[0147] MPSQTHPVKLTREFKTKSGADVSIATGTGTKWKKDSKDDDINQELVDQILLALKSGFRHIDTAEVYNTQAEVGEAVKQLGIPREDLWITTKYNPGWRTIKSSSASPNASIDKALQQLGTDYIDLYYIHQPFFTEESTHGFSLEDTWKILIDYKKQGKIREIGVSNFAEEHIERLKKILDPEFYPVVNQIESHPFLQDQSKGITAYSQANGILVEAFSPLTPVSRVDKNALTGYLEELSKKYNKTGGQILLRWTLQRGVLPITTSAKEERIKEALDVFDFELTKEEFDKITEIGQANPHRVFFHEEFKDL

[0148] SEQ ID NO:20

[0149] ATGCCTTCACAAACTCACCCTGTCAAATTAACCAGAGAATTTAAGACCAAGTCGGGCGCAGACGTCTCAATTGCTACCGGAACAGGCACCAAGTGGAAAAAAGACTCCAAGGACGACGACATCAACCAGGAATTGGTCGACCAGATCTTGCTTGCGCTCAAGTCTGGGTTCAGACACATTGACACCGCTGAAGTGTACAACACGCAGGCTGAAGTCGGCGAGGCCGTCAAGCAACTGGGCATCCCAAGAGAAGACCTCTGGATCACCACCAAGTACAACCCGGGCTGGAGAACCATCAAGTCGAGCAGCGCCAGTCCAAATGCGTCTATTGACAAGGCATTGCAGCAGTTGGGTACCGATTACATTGACTTGTACTATATCCACCAGCCTTTCTTCACGGAGGAAAGCACCCATGGGTTCTCGTTGGAAGACACCTGGAAGATCTTGATTGACTACAAGAAGCAGGGCAAGATCAGAGAAATTGGCGTTTCTAACTTCGCTGAGGAACACATCGAGAGATTGAAGAAGATCCTGGACCCAGAGTTCTACCCCGTGGTCAACCAGATCGAGAGCCATCCATTCCTTCAAGACCAGTCCAAGGGCATCACCGCATACTCGCAAGCCAACGGCATCTTAGTCGAAGCATTCTCGCCATTGACGCCTGTTTCAAGAGTCGACAAGAACGCATTGACTGGTTACTTGGAGGAATTGTCCAAGAAGTACAACAAGACCGGTGGCCAGATCTTGCTCAGATGGACGTTGCAGAGAGGTGTGTTGCCAATTACTACCTCGGCCAAAGAAGAGAGAATCAAAGAGGCATTGGATGTTTTTGACTTTGAGTTGACCAAAGAGGAGTTTGACAAGATCACCGAAATCGGTCAGGCCAACCCACACCGTGTCTTCTTCCATGAGGAGTTCAAGGATTTGTAA

[0150] SEQ ID NO:21

[0151] MPSQTHPVKLTREFKTKSGADVSIATGTGTKWKKDSKDDDINQELVDQILLALKSGFRHIDTAEVYNTQAEVGEAVKQLGIPREDLWITTKYNPGWRTIKSSSASPNASIDKALQQLGTDYIDLYWIHQPFFTEESTHGFSLEDTWKILIDYKKQGKIREIGVSNFAEEHIERLKKILDPEFYPVVNQIESHPFLQDQSKGITAYSQANGILVEAFSPLTPVSRVDKNALTGYLEELSKKYNKTGGQILLRWTLQRGVLPITTSAKEERIKEALDVFDFELTKEEFDKITEIGQANPHRVFFHEEFKDL

[0152] SEQ ID NO:22

[0153] ATGCCTTCACAAACTCACCCTGTCAAATTAACCAGAGAATTTAAGACCAAGTCGGGCGCAGACGTCTCAATTGCTACCGGAACAGGCACCAAGTGGAAAAAAGACTCCAAGGACGACGACATCAACCAGGAATTGGTCGACCAGATCTTGCTTGCGCTCAAGTCTGGGTTCAGACACATTGACACCGCTGAAGTGTACAACACGCAGGCTGAAGTCGGCGAGGCCGTCAAGCAACTGGGCATCCCAAGAGAAGACCTCTGGATCACCACCAAGTACAACCCGGGCTGGAGAACCATCAAGTCGAGCAGCGCCAGTCCAAATGCGTCTATTGACAAGGCATTGCAGCAGTTGGGTACCGATTACATTGACTTGTACTGGATCCACCAGCCTTTCTTCACGGAGGAAAGCACCCATGGGTTCTCGTTGGAAGACACCTGGAAGATCTTGATTGACTACAAGAAGCAGGGCAAGATCAGAGAAATTGGCGTTTCTAACTTCGCTGAGGAACACATCGAGAGATTGAAGAAGATCCTGGACCCAGAGTTCTACCCCGTGGTCAACCAGATCGAGAGCCATCCATTCCTTCAAGACCAGTCCAAGGGCATCACCGCATACTCGCAAGCCAACGGCATCTTAGTCGAAGCATTCTCGCCATTGACGCCTGTTTCAAGAGTCGACAAGAACGCATTGACTGGTTACTTGGAGGAATTGTCCAAGAAGTACAACAAGACCGGTGGCCAGATCTTGCTCAGATGGACGTTGCAGAGAGGTGTGTTGCCAATTACTACCTCGGCCAAAGAAGAGAGAATCAAAGAGGCATTGGATGTTTTTGACTTTGAGTTGACCAAAGAGGAGTTTGACAAGATCACCGAAATCGGTCAGGCCAACCCACACCGTGTCTTCTTCCATGAGGAGTTCAAGGATTTGTAA

[0154] SEQ ID NO:23

[0155] MPSQTHPVKLTREFKTKSGADVSIATGTGTKWKKDSKDDDINQELVDQILLALKSGFRHIDTAEVYNTQAEVGEAVKQLGIPREDLWITTKYNPGWRTIKSSSASPNASIDKALQQLGTDYIDLYLIHQPFFTEESTHGFSLEDTWKILIDYKKQGKIREIGVSNFAEEHIERLKKILDPEFYPVVNQIESHPFLQDQSKGITAYSQANGILVEDFSPLTPVSRVDKNALTGYLEELSKKYNKTGGQILLRWTLQRGVLPITTSAKEERIKEALDVFDFELTKEEFDKITEIGQANPHRVFFHEEFKDL

[0156] SEQ ID NO:24

[0157] ATGCCTTCACAAACTCACCCTGTCAAATTAACCAGAGAATTTAAGACCAAGTCGGGCGCAGACGTCTCAATTGCTACCGGAACAGGCACCAAGTGGAAAAAAGACTCCAAGGACGACGACATCAACCAGGAATTGGTCGACCAGATCTTGCTTGCGCTCAAGTCTGGGTTCAGACACATTGACACCGCTGAAGTGTACAACACGCAGGCTGAAGTCGGCGAGGCCGTCAAGCAACTGGGCATCCCAAGAGAAGACCTCTGGATCACCACCAAGTACAACCCGGGCTGGAGAACCATCAAGTCGAGCAGCGCCAGTCCAAATGCGTCTATTGACAAGGCATTGCAGCAGTTGGGTACCGATTACATTGACTTGTACTTGATCCACCAGCCTTTCTTCACGGAGGAAAGCACCCATGGGTTCTCGTTGGAAGACACCTGGAAGATCTTGATTGACTACAAGAAGCAGGGCAAGATCAGAGAAATTGGCGTTTCTAACTTCGCTGAGGAACACATCGAGAGATTGAAGAAGATCCTGGACCCAGAGTTCTACCCCGTGGTCAACCAGATCGAGAGCCATCCATTCCTTCAAGACCAGTCCAAGGGCATCACCGCATACTCGCAAGCCAACGGCATCTTAGTCGAAGATTTCTCGCCATTGACGCCTGTTTCAAGAGTCGACAAGAACGCATTGACTGGTTACTTGGAGGAATTGTCCAAGAAGTACAACAAGACCGGTGGCCAGATCTTGCTCAGATGGACGTTGCAGAGAGGTGTGTTGCCAATTACTACCTCGGCCAAAGAAGAGAGAATCAAAGAGGCATTGGATGTTTTTGACTTTGAGTTGACCAAAGAGGAGTTTGACAAGATCACCGAAATCGGTCAGGCCAACCCACACCGTGTCTTCTTCCATGAGGAGTTCAAGGATTTGTAA

[0158] SEQ ID NO:25

[0159] MPSQTHPVKLTREFKTKSGADVSIATGTGTKWKKDSKDDDINQELVDQILLALKSGFRHIDTAEVYNTQAEVGEAVKQLGIPREDLWITTKYNPGWRTIKSSSASPNASIDKALQQLGTDYIDLYLIHQPFFTEESTHGFSLEDTWKILIDYKKQGKIREIGVSNFAEEHIERLKKILDPEFYPVVNQIESHPFLQDQSKGITAYSQANGILVEEFSPLTPVSRVDKNALTGYLEELSKKYNKTGGQILLRWTLQRGVLPITTSAKEERIKEALDVFDFELTKEEFDKITEIGQANPHRVFFHEEFKDL

[0160] SEQ ID NO:26

[0161] ATGCCTTCACAAACTCACCCTGTCAAATTAACCAGAGAATTTAAGACCAAGTCGGGCGCAGACGTCTCAATTGCTACCGGAACAGGCACCAAGTGGAAAAAAGACTCCAAGGACGACGACATCAACCAGGAATTGGTCGACCAGATCTTGCTTGCGCTCAAGTCTGGGTTCAGACACATTGACACCGCTGAAGTGTACAACACGCAGGCTGAAGTCGGCGAGGCCGTCAAGCAACTGGGCATCCCAAGAGAAGACCTCTGGATCACCACCAAGTACAACCCGGGCTGGAGAACCATCAAGTCGAGCAGCGCCAGTCCAAATGCGTCTATTGACAAGGCATTGCAGCAGTTGGGTACCGATTACATTGACTTGTACTTGATCCACCAGCCTTTCTTCACGGAGGAAAGCACCCATGGGTTCTCGTTGGAAGACACCTGGAAGATCTTGATTGACTACAAGAAGCAGGGCAAGATCAGAGAAATTGGCGTTTCTAACTTCGCTGAGGAACACATCGAGAGATTGAAGAAGATCCTGGACCCAGAGTTCTACCCCGTGGTCAACCAGATCGAGAGCCATCCATTCCTTCAAGACCAGTCCAAGGGCATCACCGCATACTCGCAAGCCAACGGCATCTTAGTCGAAGAATTCTCGCCATTGACGCCTGTTTCAAGAGTCGACAAGAACGCATTGACTGGTTACTTGGAGGAATTGTCCAAGAAGTACAACAAGACCGGTGGCCAGATCTTGCTCAGATGGACGTTGCAGAGAGGTGTGTTGCCAATTACTACCTCGGCCAAAGAAGAGAGAATCAAAGAGGCATTGGATGTTTTTGACTTTGAGTTGACCAAAGAGGAGTTTGACAAGATCACCGAAATCGGTCAGGCCAACCCACACCGTGTCTTCTTCCATGAGGAGTTCAAGGATTTGTAA

[0162] SEQ ID NO:27

[0163] MPSQTHPVKLTREFKTKSGADVSIATGTGTKWKKDSKDDDINQELVDQILLALKSGFRHIDTAEVYNTQAEVGEAVKQLGIPREDLWITTKYNPGWRTIKSSSASPNASIDKALQQLGTDYIDLYPIHQPFFTEESTHGFSLEDTWKILIDYKKQGKIREIGVSNFAEEHIERLKKILDPEFYPVVNQIESHPFLQDQSKGITAYSQANGILVEDFSPLTPVSRVDKNALTGYLEELSKKYNKTGGQILLRWTLQRGVLPITTSAKEERIKEALDVFDFELTKEEFDKITEIGQANPHRVFFHEEFKDL

[0164] SEQ ID NO:28

[0165] ATGCCTTCACAAACTCACCCTGTCAAATTAACCAGAGAATTTAAGACCAAGTCGGGCGCAGACGTCTCAATTGCTACCGGAACAGGCACCAAGTGGAAAAAAGACTCCAAGGACGACGACATCAACCAGGAATTGGTCGACCAGATCTTGCTTGCGCTCAAGTCTGGGTTCAGACACATTGACACCGCTGAAGTGTACAACACGCAGGCTGAAGTCGGCGAGGCCGTCAAGCAACTGGGCATCCCAAGAGAAGACCTCTGGATCACCACCAAGTACAACCCGGGCTGGAGAACCATCAAGTCGAGCAGCGCCAGTCCAAATGCGTCTATTGACAAGGCATTGCAGCAGTTGGGTACCGATTACATTGACTTGTACCCGATCCACCAGCCTTTCTTCACGGAGGAAAGCACCCATGGGTTCTCGTTGGAAGACACCTGGAAGATCTTGATTGACTACAAGAAGCAGGGCAAGATCAGAGAAATTGGCGTTTCTAACTTCGCTGAGGAACACATCGAGAGATTGAAGAAGATCCTGGACCCAGAGTTCTACCCCGTGGTCAACCAGATCGAGAGCCATCCATTCCTTCAAGACCAGTCCAAGGGCATCACCGCATACTCGCAAGCCAACGGCATCTTAGTCGAAGATTTCTCGCCATTGACGCCTGTTTCAAGAGTCGACAAGAACGCATTGACTGGTTACTTGGAGGAATTGTCCAAGAAGTACAACAAGACCGGTGGCCAGATCTTGCTCAGATGGACGTTGCAGAGAGGTGTGTTGCCAATTACTACCTCGGCCAAAGAAGAGAGAATCAAAGAGGCATTGGATGTTTTTGACTTTGAGTTGACCAAAGAGGAGTTTGACAAGATCACCGAAATCGGTCAGGCCAACCCACACCGTGTCTTCTTCCATGAGGAGTTCAAGGATTTGTAA

[0166] SEQ ID NO:29

[0167] MPSQTHPVKLTREFKTKSGADVSIATGTGTKWKKDSKDDDINQELVDQILLALKSGFRHIDTAEVYNTQAEVGEAVKQLGIPREDLWITTKYNPGWRTIKSSSASPNASIDKALQQLGTDYIDLYFIHQPFFTEESTHGFSLEDTWKILIDYKKQGKIREIGVSNFAEEHIERLKKILDPEFYPVVNQIESHPFLQDQSKGITAYSQANGILVEDFSPLTPVSRVDKNALTGYLEELSKKYNKTGGQILLRWTLQRGVLPITTSAKEERIKEALDVFDFELTKEEFDKITEIGQANPHRVFFHEEFKDL

[0168] SEQ ID NO:30

[0169] ATGCCTTCACAAACTCACCCTGTCAAATTAACCAGAGAATTTAAGACCAAGTCGGGCGCAGACGTCTCAATTGCTACCGGAACAGGCACCAAGTGGAAAAAAGACTCCAAGGACGACGACATCAACCAGGAATTGGTCGACCAGATCTTGCTTGCGCTCAAGTCTGGGTTCAGACACATTGACACCGCTGAAGTGTACAACACGCAGGCTGAAGTCGGCGAGGCCGTCAAGCAACTGGGCATCCCAAGAGAAGACCTCTGGATCACCACCAAGTACAACCCGGGCTGGAGAACCATCAAGTCGAGCAGCGCCAGTCCAAATGCGTCTATTGACAAGGCATTGCAGCAGTTGGGTACCGATTACATTGACTTGTACTTTATCCACCAGCCTTTCTTCACGGAGGAAAGCACCCATGGGTTCTCGTTGGAAGACACCTGGAAGATCTTGATTGACTACAAGAAGCAGGGCAAGATCAGAGAAATTGGCGTTTCTAACTTCGCTGAGGAACACATCGAGAGATTGAAGAAGATCCTGGACCCAGAGTTCTACCCCGTGGTCAACCAGATCGAGAGCCATCCATTCCTTCAAGACCAGTCCAAGGGCATCACCGCATACTCGCAAGCCAACGGCATCTTAGTCGAAGATTTCTCGCCATTGACGCCTGTTTCAAGAGTCGACAAGAACGCATTGACTGGTTACTTGGAGGAATTGTCCAAGAAGTACAACAAGACCGGTGGCCAGATCTTGCTCAGATGGACGTTGCAGAGAGGTGTGTTGCCAATTACTACCTCGGCCAAAGAAGAGAGAATCAAAGAGGCATTGGATGTTTTTGACTTTGAGTTGACCAAAGAGGAGTTTGACAAGATCACCGAAATCGGTCAGGCCAACCCACACCGTGTCTTCTTCCATGAGGAGTTCAAGGATTTGTAA

[0170] SEQ ID NO:31

[0171] MPSQTHPVKLTREFKTKSGADVSIATGTGTKWKKDSKDDDINQELVDQILLALKSGFRHIDTAEVYNTQAEVGEAVKQLGIPREDLWITTKYNPGWRTIKSSSASPNASIDKALQQLGTDYIDLYYIHQPFFTEESTHGFSLEDTWKILIDYKKQGKIREIGVSNFAEEHIERLKKILDPEFYPVVNQIESHPFLQDQSKGITAYSQANGILVEDFSPLTPVSRVDKNALTGYLEELSKKYNKTGGQILLRWTLQRGVLPITTSAKEERIKEALDVFDFELTKEEFDKITEIGQANPHRVFFHEEFKDL

[0172] SEQ ID NO:32

[0173] ATGCCTTCACAAACTCACCCTGTCAAATTAACCAGAGAATTTAAGACCAAGTCGGGCGCAGACGTCTCAATTGCTACCGGAACAGGCACCAAGTGGAAAAAAGACTCCAAGGACGACGACATCAACCAGGAATTGGTCGACCAGATCTTGCTTGCGCTCAAGTCTGGGTTCAGACACATTGACACCGCTGAAGTGTACAACACGCAGGCTGAAGTCGGCGAGGCCGTCAAGCAACTGGGCATCCCAAGAGAAGACCTCTGGATCACCACCAAGTACAACCCGGGCTGGAGAACCATCAAGTCGAGCAGCGCCAGTCCAAATGCGTCTATTGACAAGGCATTGCAGCAGTTGGGTACCGATTACATTGACTTGTACTATATCCACCAGCCTTTCTTCACGGAGGAAAGCACCCATGGGTTCTCGTTGGAAGACACCTGGAAGATCTTGATTGACTACAAGAAGCAGGGCAAGATCAGAGAAATTGGCGTTTCTAACTTCGCTGAGGAACACATCGAGAGATTGAAGAAGATCCTGGACCCAGAGTTCTACCCCGTGGTCAACCAGATCGAGAGCCATCCATTCCTTCAAGACCAGTCCAAGGGCATCACCGCATACTCGCAAGCCAACGGCATCTTAGTCGAAGATTTCTCGCCATTGACGCCTGTTTCAAGAGTCGACAAGAACGCATTGACTGGTTACTTGGAGGAATTGTCCAAGAAGTACAACAAGACCGGTGGCCAGATCTTGCTCAGATGGACGTTGCAGAGAGGTGTGTTGCCAATTACTACCTCGGCCAAAGAAGAGAGAATCAAAGAGGCATTGGATGTTTTTGACTTTGAGTTGACCAAAGAGGAGTTTGACAAGATCACCGAAATCGGTCAGGCCAACCCACACCGTGTCTTCTTCCATGAGGAGTTCAAGGATTTGTAA

[0174] SEQ ID NO:33

[0175] MPSQTHPVKLTREFKTKSGADVSIATGTGTKWKKDSKDDDINQELVDQILLALKSGFRHIDTAEVYNTQAEVGEAVKQLGIPREDLWITTKYNPGWRTIKSSSASPNASIDKALQQLGTDYIDLYWIHQPFFTEESTHGFSLEDTWKILIDYKKQGKIREIGVSNFAEEHIERLKKILDPEFYPVVNQIESHPFLQDQSKGITAYSQANGILVEDFSPLTPVSRVDKNALTGYLEELSKKYNKTGGQILLRWTLQRGVLPITTSAKEERIKEALDVFDFELTKEEFDKITEIGQANPHRVFFHEEFKDL

[0176] SEQ ID NO:34

[0177] ATGCCTTCACAAACTCACCCTGTCAAATTAACCAGAGAATTTAAGACCAAGTCGGGCGCAGACGTCTCAATTGCTACCGGAACAGGCACCAAGTGGAAAAAAGACTCCAAGGACGACGACATCAACCAGGAATTGGTCGACCAGATCTTGCTTGCGCTCAAGTCTGGGTTCAGACACATTGACACCGCTGAAGTGTACAACACGCAGGCTGAAGTCGGCGAGGCCGTCAAGCAACTGGGCATCCCAAGAGAAGACCTCTGGATCACCACCAAGTACAACCCGGGCTGGAGAACCATCAAGTCGAGCAGCGCCAGTCCAAATGCGTCTATTGACAAGGCATTGCAGCAGTTGGGTACCGATTACATTGACTTGTACTGGATCCACCAGCCTTTCTTCACGGAGGAAAGCACCCATGGGTTCTCGTTGGAAGACACCTGGAAGATCTTGATTGACTACAAGAAGCAGGGCAAGATCAGAGAAATTGGCGTTTCTAACTTCGCTGAGGAACACATCGAGAGATTGAAGAAGATCCTGGACCCAGAGTTCTACCCCGTGGTCAACCAGATCGAGAGCCATCCATTCCTTCAAGACCAGTCCAAGGGCATCACCGCATACTCGCAAGCCAACGGCATCTTAGTCGAAGATTTCTCGCCATTGACGCCTGTTTCAAGAGTCGACAAGAACGCATTGACTGGTTACTTGGAGGAATTGTCCAAGAAGTACAACAAGACCGGTGGCCAGATCTTGCTCAGATGGACGTTGCAGAGAGGTGTGTTGCCAATTACTACCTCGGCCAAAGAAGAGAGAATCAAAGAGGCATTGGATGTTTTTGACTTTGAGTTGACCAAAGAGGAGTTTGACAAGATCACCGAAATCGGTCAGGCCAACCCACACCGTGTCTTCTTCCATGAGGAGTTCAAGGATTTGTAA

[0178] SEQ ID NO:35

[0179] MPSQTHPVKLTREFKTKSGADVSIATGTGTKWKKDSKDDDINQELVDQILLALKSGFRHIDTAEVYNTQAEVGEAVKQLGIPREDLWITTKYNPGWRTIKSSSASPNASIDKALQQLGTDYIDLYPIHQPFFTEESTHGFSLEDTWKILIDYKKQGKIREIGVSNFAEEHIERLKKILDPEFYPVVNQIESHPFLQDQSKGITAYSQANGILVEEFSPLTPVSRVDKNALTGYLEELSKKYNKTGGQILLRWTLQRGVLPITTSAKEERIKEALDVFDFELTKEEFDKITEIGQANPHRVFFHEEFKDL

[0180] SEQ ID NO:36

[0181] ATGCCTTCACAAACTCACCCTGTCAAATTAACCAGAGAATTTAAGACCAAGTCGGGCGCAGACGTCTCAATTGCTACCGGAACAGGCACCAAGTGGAAAAAAGACTCCAAGGACGACGACATCAACCAGGAATTGGTCGACCAGATCTTGCTTGCGCTCAAGTCTGGGTTCAGACACATTGACACCGCTGAAGTGTACAACACGCAGGCTGAAGTCGGCGAGGCCGTCAAGCAACTGGGCATCCCAAGAGAAGACCTCTGGATCACCACCAAGTACAACCCGGGCTGGAGAACCATCAAGTCGAGCAGCGCCAGTCCAAATGCGTCTATTGACAAGGCATTGCAGCAGTTGGGTACCGATTACATTGACTTGTACCCGATCCACCAGCCTTTCTTCACGGAGGAAAGCACCCATGGGTTCTCGTTGGAAGACACCTGGAAGATCTTGATTGACTACAAGAAGCAGGGCAAGATCAGAGAAATTGGCGTTTCTAACTTCGCTGAGGAACACATCGAGAGATTGAAGAAGATCCTGGACCCAGAGTTCTACCCCGTGGTCAACCAGATCGAGAGCCATCCATTCCTTCAAGACCAGTCCAAGGGCATCACCGCATACTCGCAAGCCAACGGCATCTTAGTCGAAGAATTCTCGCCATTGACGCCTGTTTCAAGAGTCGACAAGAACGCATTGACTGGTTACTTGGAGGAATTGTCCAAGAAGTACAACAAGACCGGTGGCCAGATCTTGCTCAGATGGACGTTGCAGAGAGGTGTGTTGCCAATTACTACCTCGGCCAAAGAAGAGAGAATCAAAGAGGCATTGGATGTTTTTGACTTTGAGTTGACCAAAGAGGAGTTTGACAAGATCACCGAAATCGGTCAGGCCAACCCACACCGTGTCTTCTTCCATGAGGAGTTCAAGGATTTGTAA

[0182] SEQ ID NO:37

[0183] MPSQTHPVKLTREFKTKSGADVSIATGTGTKWKKDSKDDDINQELVDQILLALKSGFRHIDTAEVYNTQAEVGEAVKQLGIPREDLWITTKYNPGWRTIKSSSASPNASIDKALQQLGTDYIDLYFIHQPFFTEESTHGFSLEDTWKILIDYKKQGKIREIGVSNFAEEHIERLKKILDPEFYPVVNQIESHPFLQDQSKGITAYSQANGILVEEFSPLTPVSRVDKNALTGYLEELSKKYNKTGGQILLRWTLQRGVLPITTSAKEERIKEALDVFDFELTKEEFDKITEIGQANPHRVFFHEEFKDL

[0184] SEQ ID NO:38

[0185] ATGCCTTCACAAACTCACCCTGTCAAATTAACCAGAGAATTTAAGACCAAGTCGGGCGCAGACGTCTCAATTGCTACCGGAACAGGCACCAAGTGGAAAAAAGACTCCAAGGACGACGACATCAACCAGGAATTGGTCGACCAGATCTTGCTTGCGCTCAAGTCTGGGTTCAGACACATTGACACCGCTGAAGTGTACAACACGCAGGCTGAAGTCGGCGAGGCCGTCAAGCAACTGGGCATCCCAAGAGAAGACCTCTGGATCACCACCAAGTACAACCCGGGCTGGAGAACCATCAAGTCGAGCAGCGCCAGTCCAAATGCGTCTATTGACAAGGCATTGCAGCAGTTGGGTACCGATTACATTGACTTGTACTTTATCCACCAGCCTTTCTTCACGGAGGAAAGCACCCATGGGTTCTCGTTGGAAGACACCTGGAAGATCTTGATTGACTACAAGAAGCAGGGCAAGATCAGAGAAATTGGCGTTTCTAACTTCGCTGAGGAACACATCGAGAGATTGAAGAAGATCCTGGACCCAGAGTTCTACCCCGTGGTCAACCAGATCGAGAGCCATCCATTCCTTCAAGACCAGTCCAAGGGCATCACCGCATACTCGCAAGCCAACGGCATCTTAGTCGAAGAATTCTCGCCATTGACGCCTGTTTCAAGAGTCGACAAGAACGCATTGACTGGTTACTTGGAGGAATTGTCCAAGAAGTACAACAAGACCGGTGGCCAGATCTTGCTCAGATGGACGTTGCAGAGAGGTGTGTTGCCAATTACTACCTCGGCCAAAGAAGAGAGAATCAAAGAGGCATTGGATGTTTTTGACTTTGAGTTGACCAAAGAGGAGTTTGACAAGATCACCGAAATCGGTCAGGCCAACCCACACCGTGTCTTCTTCCATGAGGAGTTCAAGGATTTGTAA

[0186] SEQ ID NO:39

[0187] MPSQTHPVKLTREFKTKSGADVSIATGTGTKWKKDSKDDDINQELVDQILLALKSGFRHIDTAEVYNTQAEVGEAVKQLGIPREDLWITTKYNPGWRTIKSSSASPNASIDKALQQLGTDYIDLYYIHQPFFTEESTHGFSLEDTWKILIDYKKQGKIREIGVSNFAEEHIERLKKILDPEFYPVVNQIESHPFLQDQSKGITAYSQANGILVEEFSPLTPVSRVDKNALTGYLEELSKKYNKTGGQILLRWTLQRGVLPITTSAKEERIKEALDVFDFELTKEEFDKITEIGQANPHRVFFHEEFKDL

[0188] SEQ ID NO:40

[0189] ATGCCTTCACAAACTCACCCTGTCAAATTAACCAGAGAATTTAAGACCAAGTCGGGCGCAGACGTCTCAATTGCTACCGGAACAGGCACCAAGTGGAAAAAAGACTCCAAGGACGACGACATCAACCAGGAATTGGTCGACCAGATCTTGCTTGCGCTCAAGTCTGGGTTCAGACACATTGACACCGCTGAAGTGTACAACACGCAGGCTGAAGTCGGCGAGGCCGTCAAGCAACTGGGCATCCCAAGAGAAGACCTCTGGATCACCACCAAGTACAACCCGGGCTGGAGAACCATCAAGTCGAGCAGCGCCAGTCCAAATGCGTCTATTGACAAGGCATTGCAGCAGTTGGGTACCGATTACATTGACTTGTACTATATCCACCAGCCTTTCTTCACGGAGGAAAGCACCCATGGGTTCTCGTTGGAAGACACCTGGAAGATCTTGATTGACTACAAGAAGCAGGGCAAGATCAGAGAAATTGGCGTTTCTAACTTCGCTGAGGAACACATCGAGAGATTGAAGAAGATCCTGGACCCAGAGTTCTACCCCGTGGTCAACCAGATCGAGAGCCATCCATTCCTTCAAGACCAGTCCAAGGGCATCACCGCATACTCGCAAGCCAACGGCATCTTAGTCGAAGAATTCTCGCCATTGACGCCTGTTTCAAGAGTCGACAAGAACGCATTGACTGGTTACTTGGAGGAATTGTCCAAGAAGTACAACAAGACCGGTGGCCAGATCTTGCTCAGATGGACGTTGCAGAGAGGTGTGTTGCCAATTACTACCTCGGCCAAAGAAGAGAGAATCAAAGAGGCATTGGATGTTTTTGACTTTGAGTTGACCAAAGAGGAGTTTGACAAGATCACCGAAATCGGTCAGGCCAACCCACACCGTGTCTTCTTCCATGAGGAGTTCAAGGATTTGTAA

[0190] SEQ ID NO:41

[0191] MPSQTHPVKLTREFKTKSGADVSIATGTGTKWKKDSKDDDINQELVDQILLALKSGFRHIDTAEVYNTQAEVGEAVKQLGIPREDLWITTKYNPGWRTIKSSSASPNASIDKALQQLGTDYIDLYWIHQPFFTEESTHGFSLEDTWKILIDYKKQGKIREIGVSNFAEEHIERLKKILDPEFYPVVNQIESHPFLQDQSKGITAYSQANGILVEEFSPLTPVSRVDKNALTGYLEELSKKYNKTGGQILLRWTLQRGVLPITTSAKEERIKEALDVFDFELTKEEFDKITEIGQANPHRVFFHEEFKDL

[0192] SEQ ID NO:42

[0193] ATGCCTTCACAAACTCACCCTGTCAAATTAACCAGAGAATTTAAGACCAAGTCGGGCGCAGACGTCTCAATTGCTACCGGAACAGGCACCAAGTGGAAAAAAGACTCCAAGGACGACGACATCAACCAGGAATTGGTCGACCAGATCTTGCTTGCGCTCAAGTCTGGGTTCAGACACATTGACACCGCTGAAGTGTACAACACGCAGGCTGAAGTCGGCGAGGCCGTCAAGCAACTGGGCATCCCAAGAGAAGACCTCTGGATCACCACCAAGTACAACCCGGGCTGGAGAACCATCAAGTCGAGCAGCGCCAGTCCAAATGCGTCTATTGACAAGGCATTGCAGCAGTTGGGTACCGATTACATTGACTTGTACTGGATCCACCAGCCTTTCTTCACGGAGGAAAGCACCCATGGGTTCTCGTTGGAAGACACCTGGAAGATCTTGATTGACTACAAGAAGCAGGGCAAGATCAGAGAAATTGGCGTTTCTAACTTCGCTGAGGAACACATCGAGAGATTGAAGAAGATCCTGGACCCAGAGTTCTACCCCGTGGTCAACCAGATCGAGAGCCATCCATTCCTTCAAGACCAGTCCAAGGGCATCACCGCATACTCGCAAGCCAACGGCATCTTAGTCGAAGAATTCTCGCCATTGACGCCTGTTTCAAGAGTCGACAAGAACGCATTGACTGGTTACTTGGAGGAATTGTCCAAGAAGTACAACAAGACCGGTGGCCAGATCTTGCTCAGATGGACGTTGCAGAGAGGTGTGTTGCCAATTACTACCTCGGCCAAAGAAGAGAGAATCAAAGAGGCATTGGATGTTTTTGACTTTGAGTTGACCAAAGAGGAGTTTGACAAGATCACCGAAATCGGTCAGGCCAACCCACACCGTGTCTTCTTCCATGAGGAGTTCAAGGATTTGTAA

[0194] SEQ ID NO:43

[0195] MYKDLEGKVVVITGSSTGLGKSMAIRFATEKAKVVVNYRSKEDEANSVLEEIKKVGGEAIAVKGDVTVESDVINLVQSAIKEFGKLDVMINNAGLENPVSSHEMSLSDWNKVIDTNLTGAFLGSREAIKYFVENDIKGTVINMSSVHEKIPWPLFVHYAASKGGMKLMTETLALEYAPKGIRVNNIGPGAINTPINAEKFADPEQRADVESMIPMGYIGEPEEIAAVAAWLASSEASYVTGITLFADGGMTQYPSFQAGRG

[0196] SEQ ID NO:44

[0197] ATGTATAAAGATTTAGAAGGAAAGTAGTGGTCATAACAGGTTCATCTACAGGTTTGGGAAAATCAATGGCGATTCGTTTTGCGACAGAAAAAGCTAAAGTAGTTGTGAATTATCGTTCTAAGGAAGACGAAGCTAACAGCGTTTTAGAAGAAATTAAAAAAGTTGGCGGAGAGGCTATTGCCGTTAAAGGTGACGTAACAGTTGAGTCTGATGTAATCAATTTAGTTCAATCTGCAATTAAAGAATTTGGAAAGTTAGACGTCATGATTAATAACGCAGGACTAGAAAATCCGGTTTCATCTCATGAAATGTCTTTAAGCGATTGGAATAAAGTAATTGATACGAACTTAACGGGAGCTTTCTTAGGTAGCCGTGAAGCGATTAAATATTTT GTTGAAAATGATATTAAGGGAACAGTTATTAACATGTCGAGTGTTCACGAGAAAATTCCTTGGCCATTATTTGTTCATTATGCAGCAAGTAAAGGCGGTATGAAGCTTATGACTGAAACACTGGCATTAGAATACGCTCCAAAAGGTATTCGTGTAAATAACATTGGACCGGGAGCGATTAATACACCGATTAACG CTGAGAAATTTGCTGATCCTGAGCAGCGTGCAGATGTAGAAAGCATGATTCCAATGGGATACATCGGAGAGCCGGAAGAAATTGCAGCAGTTGCTGCATGGCTAGCTTCTTCAGAGGCGAGTTATGTAACAGGAATTACGCTCTTTGCTGACGGCGGTATGACACACAGTACCCATCATTCCAAGCAGGACGCGGATAA

Claims

1. A ketopantoate lactone reductase mutant, whose amino acid sequence is shown in SEQ ID NO: 21, 23, 25, 29 or 41.

2. A nucleic acid molecule encoding the ketopantoate lactone reductase mutant according to claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that: The nucleic acid molecule is a DNA molecule shown in SEQ ID NO: 22, 24, 26, 30 or 42. A recombinant vector comprising the nucleic acid molecule according to claim 2 or 3.

5. The recombinant vector according to claim 4, characterized in that: The recombinant vector also contains a nucleic acid molecule encoding glucose dehydrogenase.

6. The recombinant vector according to claim 5, characterized in that: The glucose dehydrogenase is derived from Bacillus megaterium, and its amino acid sequence is shown in SEQ ID NO:43, and its nucleotide sequence is shown in SEQ ID NO:

44.

7. A recombinant cell comprising the recombinant vector according to any one of claims 4 to 6.

8. The recombinant cell according to claim 7, characterized in that: The recombinant cell induces production of the ketopantoate lactone reductase mutant according to claim 1.

9. The recombinant cell according to claim 8, characterized in that: The recombinant cells also induce the production of glucose dehydrogenase.

10. A method for preparing ketopantolactone reductase, comprising: Carrying out induction culture on the recombinant cell according to any one of claims 7 to 9 to obtain a culture; The ketopantoate lactone reductase mutant according to claim 1 is isolated from the culture.

11. The method according to claim 10, characterized in that: The method further comprises the step of isolating glucose dehydrogenase from the culture.

12. Use of the ketopantolactone reductase mutant according to claim 1, the nucleic acid molecule according to claim 2 or 3, the recombinant vector according to any one of claims 4 to 6 and / or the recombinant cell according to any one of claims 7 to 9 in the preparation of D-pantolactone.

13. A method for preparing D-pantolactone, comprising: Using the ketopantolactone reductase mutant according to claim 1, the recombinant cell according to any one of claims 7 to 9 and / or the ketopantolactone reductase prepared by the method according to claim 10 or 11 as a catalyst, ketopantolactone is catalyzed to obtain D-pantolactone.

14. The preparation method according to claim 13, characterized in that: The reaction pH of the catalytic reaction is 6-8; and / or The temperature of the catalytic reaction is 30°C-50°C.

15. The preparation method according to claim 13 or 14, characterized in that: The catalytic reaction is carried out in a phosphate buffer; and / or In the catalytic reaction, the cumulative final concentration of the ketopantolactone is 1 mM-400 mM; and / or In the catalytic reaction, the mass ratio of the catalyst to the ketopantoic acid lactone is (0.01-0.1):1.

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