Glucose dehydrogenase mutants and uses thereof

Abstract

The application belongs to the technical field of functional enzyme modification, and particularly relates to a glucose dehydrogenase mutant and application thereof. The glucose dehydrogenase mutant provided by the application is derived from Bacillus oleivorans, and can be used to construct a coenzyme cycle regeneration catalytic system with ketoreductase, and is used for catalyzing preparation of (R)-4-chloro-3-hydroxy-butyric acid ethyl ester.

Description

Technical Field

[0001] This invention relates to the field of functional enzyme modification technology, specifically to a glucose dehydrogenase mutant derived from Bacillus oleivorans, its encoded nucleic acid molecule, recombinant vector, recombinant cell, coenzyme recycling catalytic system and its preparation method, and its use in the catalytic preparation of (R)-4-chloro-3-hydroxy-butyrate ethyl ester. Background Technology

[0002] L-L-carnitine is a very important chiral active substance. In the human body, it is mainly responsible for transporting long-chain fatty acids from the outer mitochondrial membrane to the inner mitochondrial membrane for β-oxidation of fats. Normal people can synthesize enough in their bodies to meet their needs, but infants and young children lack the ability to synthesize it themselves. In addition, people who exercise and work out have a great demand for products containing L-L-carnitine, which has created a market demand for L-L-carnitine.

[0003] The chemical preparation of L-carnitine from (R)-4-chloro-3-hydroxybutyrate ethyl ester is a highly effective method. (R)-4-chloro-3-hydroxybutyrate ethyl ester is a valuable and important chiral compound, typically prepared using ethyl 4-chloroacetoacetate as a substrate via chiral catalysts. However, when using biocatalysts such as ketone reductases and alcohol dehydrogenases, the coenzyme regeneration system carried by the chosen *E. coli* expression host is not sufficiently developed, resulting in insufficient coenzyme production for the reaction. Therefore, it is necessary to add reduced coenzymes NADH / NADPH as electron donors. However, NADH / NADPH is expensive, and adding it directly to the enzyme-catalyzed reaction system would significantly increase industrial production costs. Therefore, designing and constructing an enzyme-catalyzed reaction system that can effectively achieve coenzyme recycling and efficiently catalyze the conversion of ethyl 4-chloroacetoacetate to (R)-4-chloro-3-hydroxybutyrate ethyl ester would be of great significance. Summary of the Invention

[0004] The purpose of this invention is to provide a glucose dehydrogenase mutant derived from Bacillus oleivorans and to construct a coenzyme recycling catalytic system, aiming to match the glucose dehydrogenase mutant with ketone reductase to efficiently catalyze the production of (R)-4-chloro-3-hydroxy-butyrate ethyl ester without the need for additional NADH.

[0005] To achieve the above-mentioned objectives, this invention obtains glucose dehydrogenase from Bacillus oleivorans and further mutates it to obtain a glucose dehydrogenase mutant with significantly improved conversion rate and reaction rate.

[0006] Specifically, in a first aspect, the present invention provides a glucose dehydrogenase mutant, the amino acid sequence of which is shown in SEQ ID NO:1.

[0007] Secondly, the present invention provides a nucleic acid molecule encoding the glucose dehydrogenase mutant, the nucleotide sequence of which is shown in SEQ ID NO:2.

[0008] Thirdly, the present invention provides a recombinant vector comprising a nucleic acid molecule encoding the glucose dehydrogenase mutant.

[0009] Fourthly, the present invention provides a recombinant cell comprising the recombinant vector described herein.

[0010] Fifthly, the present invention provides a coenzyme cycle regeneration catalytic system comprising the recombinant cells described in the present invention, the recombinant cells comprising a recombinant co-expression vector, the recombinant co-expression vector comprising the nucleic acid molecule described in claim 2 and a nucleic acid molecule encoding ketone reductase as shown in SEQ ID NO:3.

[0011] Sixthly, the present invention provides a method for preparing a coenzyme recycling catalytic system, comprising:

[0012] The recombinant cells provided by the present invention are induced and cultured to obtain a culture;

[0013] And optionally, the step of isolating the glucose dehydrogenase mutant and ketone reductase of the present invention from the culture;

[0014] The amino acid sequence of the ketone reductase is shown in SEQ ID NO:4.

[0015] In a seventh aspect, the present invention provides the use of the glucose dehydrogenase mutant, the nucleic acid molecule, the recombinant vector, the recombinant cell, the coenzyme recycling catalytic system and / or the coenzyme recycling catalytic system prepared by the preparation method in the catalytic preparation of (R)-4-chloro-3-hydroxy-butyrate ethyl ester.

[0016] Eighthly, the present invention provides a method for preparing ethyl (R)-4-chloro-3-hydroxy-butyrate, comprising:

[0017] Using the recombinant cells, the coenzyme recycling catalytic system, and / or the coenzyme recycling catalytic system prepared by the method of the present invention as catalysts, ethyl 4-chloroacetoacetate is catalyzed to obtain ethyl (R)-4-chloro-3-hydroxy-butyrate.

[0018] This invention provides for the first time a glucose dehydrogenase mutant derived from Bacillus oleivorans, which significantly improves conversion rate and reaction rate compared to the original glucose dehydrogenase. Furthermore, based on this glucose dehydrogenase mutant, this invention also constructs a coenzyme recycling catalytic system. This system combines the glucose dehydrogenase mutant with ketone reductase, resulting in a more matched rate between the two enzyme catalytic reactions, thereby further improving the catalytic activity and substrate conversion rate of the combined enzyme, which is beneficial for the efficient production of (R)-4-chloro-3-hydroxy-butyrate ethyl ester. Attached Figure Description

[0019] Figure 1 Schematic diagram of the reaction catalyzed by glucose dehydrogenase and ketone reductase to synthesize ethyl (R)-4-chloro-3-hydroxy-butyrate;

[0020] Figure 2 Gas chromatogram of ethyl 4-chloroacetoacetate standard;

[0021] Figure 3 Gas chromatogram of (R)-4-chloro-3-hydroxybutyrate ethyl ester standard;

[0022] Figure 4 This is a gas chromatogram of the reaction process in Example 5 of the present invention. Detailed Implementation

[0023] The glucose dehydrogenase mutant provided in this invention is obtained by modifying glucose dehydrogenase. Specifically, this invention, through gene mining, discovered a glucose dehydrogenase derived from the Bacillus oleivorans strain, which has the amino acid sequence shown in SEQ ID NO:4 and the coding nucleic acid sequence shown in SEQ ID NO:5. Furthermore, the inventors found that this glucose dehydrogenase has the potential for further modification and improvement. Therefore, this invention employs a semi-rational design approach to modify the protein. First, error-prone PCR is used to mutate the protein, resulting in a library of 96 mutant proteins. After high-throughput screening, the activity hotspots for glucose dehydrogenase modification were identified as G727C (D243H) and C1231G (P411A). Subsequently, site-directed saturation mutations were designed and performed at amino acid positions 243 and 411, respectively, yielding two promising positive mutants (D243E and P411R). Single-site and combined-site mutations were then co-transformed with ketone reductase and screened for enzyme catalysis. The optimal glucose dehydrogenase mutant, D243E, was obtained, showing significant improvements in overall catalytic performance, including conversion rate and reaction rate. Its amino acid sequence is shown in SEQ ID NO:1.

[0024] The amino acid sequence of a glucose dehydrogenase mutant derived from Bacillus oleivorans (SEQ ID NO:1):

[0025] MLHIPEKLLVREFIWCEVNNRFYKHLYQYADDRAEGPINVLLKAQSEQTNMILYLMNLFSISKPAFDEEEVRYMDEPHSLITEVIEREKELTLIYESYPYFLANFPNLSPLIHRLRYLQHE KLNELNKLKSQFQKFNHLETNERIDRDYWLEEGYELEKIASGFTFPTSIAFDDEGELFVGESGYSYGPAYAKARILNIRKDGQIQEIASGFEGPLTGIAWYKGYFYVITGGFDGKVYRVSK E GQKKVLISGLRSGADHFTSEIVFGPDNKMYFAVGTVTNSGVVGVDNEYYGWLGQRPTFHDIPARDLKLVGQNFVSDNPLTKINPNDKVSTGAFHPFGTASRRGEVVKGQLLANGVLYRANPDGSNLEIVADGFRNVFGLGFSPEGKLFATNNGFDFRGSRPIEGDWDP LYEIRPGWYGWPDFASGLPVTLPYFKPPGHPQPQFLLEQHPPLAAQPLIRFKPHAATQKFDFSKNERFGRRGEMFLAQIGSAPPITTGEQKPSGYRVVRAMPYTGQVRDFLVNLKPGKGGKGPERPVAVRFSPDGNFLYIVDFGLLGATATTAIPYADTGAIWRVKRK

[0026] Accordingly, the nucleotide sequence of the nucleic acid molecule encoding the glucose dehydrogenase mutant is shown in SEQ ID NO:2.

[0027] The nucleotide sequence encoding a glucose dehydrogenase mutant derived from Bacillus oleivorans (SEQ ID NO:2):

[0028] atgttgcacatacctgaaaaattactggtgagggagtttatttggtgtgaagtaaataatcgattttacaagcatttatatcaatatgcggatgatcgtgctgaaggtccaatcaatgtcttgttgaaagctcaatcggagcaaaccaacatgattctttatttaatgaatttgttttcgatatcaaagcctgctttcgatgaggaagaagtaagatatatggatgagccccattcgttgattaccgaagtaatagaaagggaaaaagagctgacattaatctatgaatcttatccgtattttttagctaatttccccaacctttctccgcttattcaccgcttacgttatcttcaacacgaa aagttgaatgagttaaataagttaaaatctcaatttcaaaagtttaatcatttggagactaatgaaagaatcgatagggattattggctggaagaggggtatgagttagaaaaaatagcttcaggctttacgtttccaacaagtatagcctttgatgatgagggtgaactatttgttggagagtcgggatattcctatgggcccgcctatgcaaaagccaggattctaaacatcaggaaagatgggcagatccaagaaattgcttcaggttttgaaggaccactgaccggaattgcatggtataaaggatacttttatgtgattacaggaggttttgatggaaaggtatatcgagtaagcaaa gaa

[0029] The amino acid sequence of glucose dehydrogenase derived from Bacillus oleivorans (SEQ ID NO:5):

[0030] MLHIPEKLLVREFIWCEVNNRFYKHLYQYADDRAEGPINVLLKAQSEQTNMILYLMNLFSISKPAFDEEEVRYMDEPHSLITEVIEREKELTLIYESYPYFLANFPNLSPLIHRLRYLQHE KLNELNKLKSQFQKFNHLETNERIDRDYWLEEGYELEKIASGFTFPTSIAFDDEGELFVGESGYSYGPAYAKARILNIRKDGQIQEIASGFEGPLTGIAWYKGYFYVITGGFDGKVYRVSK D GQKKVLISGLRSGADHFTSEIVFGPDNKMYFAVGTVTNSGVVGVDNEYYGWLGQRPTFHDIPARDLKLVGQNFVSDNPLTKINPNDKVSTGAFHPFGTASRRGEVVKGQLLANGVLYRANPDGSNLEIVADGFRNVFGLGFSPEGKLFATNNGFDFRGSRPIEGDWD P LYEIRPGWYGWPDFASGLPVTLPYFKPPGHPQPQFLLEQHPPLAAQPLIRFKPHAATQKFDFSKNERFGRRGEMFLAQIGSAPPITTGEQKPSGYRVVRAMPYTGQVRDFLVNLKPGKGGKGPERPVAVRFSPDGNFLYIVDFGLLGATATTAIPYADTGAIWRVKRK

[0031] The nucleotide sequence encoding glucose dehydrogenase derived from Bacillus oleivorans (SEQ ID NO:6):

[0032] atgttgcacatacctgaaaaattactggtgagggagtttatttggtgtgaagtaaataatcgattttacaagcatttatatcaatatgcggatgatcgtgctgaaggtccaatcaatgtcttgttgaaagctcaatcggagcaaaccaacatgattctttatttaatgaatttgttttcgatatcaaagcctgctttcgatgaggaagaagtaagatatatggatgagccccattcgttgattaccgaagtaatagaaagggaaaaagagctgacattaatctatgaatcttatccgtattttttagctaatttccccaacctttctccgcttattcaccgcttacgttatcttcaacacgaa aagttgaatgagttaaataagttaaaatctcaatttcaaaagtttaatcatttggagactaatgaaagaatcgatagggattattggctggaagaggggtatgagttagaaaaaatagcttcaggctttacgtttccaacaagtatagcctttgatgatgagggtgaactatttgttggagagtcgggatattcctatgggcccgcctatgcaaaagccaggattctaaacatcaggaaagatgggcagatccaagaaattgcttcaggttttgaaggaccactgaccggaattgcatggtataaaggatacttttatgtgattacaggaggttttgatggaaaggtatatcgagtaagcaaa gat

[0033] The glucose dehydrogenase mutant provided in this embodiment of the invention can be artificially synthesized, or its encoding gene can be synthesized first and then expressed biologically, for example, by using recombinant technology to express it from a prokaryotic host (e.g., Escherichia coli) or a eukaryotic host (e.g., yeast, higher plants).

[0034] In some embodiments, the glucose dehydrogenase mutant is obtained by introducing a recombinant vector containing its encoding gene into an expression host (e.g., E. coli BL21(DE3)) to obtain a recombinant genetically engineered bacterium, and then inducing expression of the recombinant genetically engineered bacterium to obtain the glucose dehydrogenase mutant.

[0035] The nucleic acid molecules of the glucose dehydrogenase mutant provided in this embodiment of the invention can usually be obtained by PCR amplification or artificial synthesis.

[0036] This invention also provides a recombinant vector comprising a nucleic acid molecule encoding the glucose dehydrogenase mutant. Specifically, the recombinant vector comprises a cloning vector and an expression vector, wherein the cloning vector is used to replicate the relevant sequence, and the expression vector is used to express the relevant gene.

[0037] In some embodiments, the recombinant vector further comprises a nucleic acid molecule, as shown in SEQ ID NO:3, encoding ketone reductase (derived from Lachnellulahyalina), for co-expression of glucose dehydrogenase mutant and ketone reductase.

[0038] The nucleotide sequence of the nucleic acid molecule encoding the ketoreductase (SEQ ID NO:3): atgaaggtctttctgagcggaggaagtggcttcatcgccgcccacgtcctcgacatcctactcgagcatggccatactgtcatcacctcggttcgttcccaagagaaagccaacaagatcacagaggcgcaccccaacacgcctgcctcccagctcgagttccggattgtcaaagacatagcacaggagggggcctttacgaagccatcaagattgacggcctggaagcggtgattcacacagcctcgcctttccatttcaacgtcacagatgtcaagaaagacttgcttgaccctgccataatcggcacaacaggtatcctgaaagccatcaagaagaatgctcccagcgtgaagagagtcgtcatcacgagttcctttgccaacatcgtcaatccaagtaagggaaactcctggaccgagcacacgtacagcgaggaggactggaaccccatcacggaagaagaggcggtgctgaaccccagcaatggatacagagccagcaagacgttcgccgagaaagctgcgtgggagtttgccgagaaggagaaaccaaactttacgttgagcactatgtgccctcctttagttataggtccaattgtccactacctcaacagcctcgatagcctcaacacctctaaccagcgaaccgccaacctcatgaccggcaagaacaagtctgaaatccccgacaccggtacctacatctggatcgatgtgcgagatctcgccctcgcccacgtcaaagccatcgagctcccagaagccgcgaacaagcgattcttcatcaccgctggttacttctccaaccaggagatcgctgagattatccgcaagaacttccccgcgctcgaaaaggaattgccggcgaaggacgtcaagggtggagattacccgaaagagggattatataaggagtga

[0039] Accordingly, the amino acid sequence of the ketone reductase encoded by the nucleic acid molecule encoding ketone reductase is shown in SEQ ID NO:4.

[0040] Amino acid sequence of ketoreductase from Lachnellula hyalina (SEQ ID NO:4):MKVFLSGGSGFIAAHVLDILLEHGHTVITSVRSQEKANKITEAHPNTPASQLEFRIVKDIAQEGAFDEAIKIDGLEAVIHTASPFHFNVTDVKKDLLDPAIIGTTGILKAIKKNAPSVKRVVITSSFANIVNPSKGNSWTEHTYSEEDWN PITEEEAVLNPSNGYRASKTFAEKAAWEFAEKEKPNFTLSTMCPPLVIGPIVHYLNSLDSLNTSNQRTANLMTGKNKSEIPDTGTYIWIDVRDLALAHVKAIELPEAANKRFFITAGYFSNQEIAEIIRKNFPALEKELPAKDVKGGDYPKEGLYKE

[0041] The glucose dehydrogenase mutant and the ketone reductase described in this invention can be cloned into expression vectors, and then transformed into expression hosts for expression. In some embodiments, the expression vector is pET-28a(+).

[0042] In some embodiments, the recombinant vector is pACYCDuet-1-TE-GE or pACYCDuet-1-TE-GEM, which are obtained by replacing the sequences between the Ned1 and Xho 1 & EcoRI and HindIII restriction sites of pACYCDuet-1 with nucleic acid molecules encoding the above-mentioned ketoreductase and glucose dehydrogenase mutants, respectively, while keeping the remaining sequences unchanged.

[0043] In some embodiments, the recombinant vector is a recombinant co-expression vector that clones the encoding nucleic acid molecules of the glucose dehydrogenase mutant and the ketone reductase.

[0044] Accordingly, embodiments of the present invention also provide a recombinant cell comprising the recombinant vector described above.

[0045] In some implementations, the recombinant cells are obtained by converting the recombinant co-expression vector into an expression host.

[0046] In some embodiments, the recombinant cells are prokaryotic or eukaryotic cells, such as Escherichia coli or yeast, with E. coli expression host E. coli BL21(DE3) being preferred.

[0047] More specifically, the method for constructing the recombinant cells includes the following steps:

[0048] (i) Obtained by screening for glucose dehydrogenase-positive mutant genes;

[0049] (ii) Construction of recombinant co-expression vectors;

[0050] (iii) The recombinant co-expression vector is transformed into the host strain;

[0051] (iv) Positive clones were obtained by screening on plate resistance medium.

[0052] Accordingly, embodiments of the present invention also provide a biocatalyst comprising the glucose dehydrogenase mutant and the ketone reductase described in the present invention.

[0053] This invention also provides a coenzyme recycling catalytic system comprising the recombinant cells described herein, wherein the recombinant cells comprise the recombinant co-expression vector.

[0054] The coenzyme recycling catalytic system provided in this invention can catalyze the production of (R)-4-chloro-3-hydroxybutyrate ethyl 4-acetoacetate without the need for additional NADH. Furthermore, by combining a modified glucose dehydrogenase mutant with ketone reductase, compared to the original glucose dehydrogenase and ketone reductase combination system, the combined enzyme recycling catalytic system exhibits a more balanced reaction rate between the two enzyme steps, resulting in further improvements in the combined enzyme catalytic activity and substrate conversion rate.

[0055] The coenzyme recycling catalytic system provided by this invention can be obtained by purifying the glucose dehydrogenase mutant and ketone reductase described in this invention separately and then mixing them in a certain proportion. In some embodiments, the coenzyme recycling catalytic system is a whole-cell coenzyme recycling system, which has a lower cost advantage and better enzyme stability retention than free enzymes. Furthermore, since the catalytic reaction system involves the optimization of enzyme addition ratio, immobilization and other conditions, it is preferable to use a single expression strain (recombinant cell) containing the recombinant co-expression expression vector, so that the catalytic reaction rates of glucose dehydrogenase mutant and ketone reductase are more matched, resulting in better mass transfer effect than the dual-strain coupling reaction.

[0056] Accordingly, embodiments of the present invention provide a method for preparing a coenzyme cycling regeneration catalytic system, comprising: inducing culture of the recombinant cells described in the present invention to obtain a culture; and optionally isolating the glucose dehydrogenase mutant and ketone reductase described in the present invention from the culture;

[0057] The amino acid sequence of the ketone reductase is shown in SEQ ID NO:4.

[0058] Specifically, the recombinant cell comprises a recombinant co-expression vector, which comprises the nucleic acid molecule of claim 2 and a nucleic acid molecule encoding ketone reductase as shown in SEQ ID NO:3.

[0059] The embodiments of the present invention do not have special requirements for the culture method and conditions during the induction culture, as long as the recombinant cells grow normally. In some embodiments, the culture medium used is a protein-expressing culture medium in the art, preferably TB medium.

[0060] In some embodiments, the induction culture is performed at 18°C ​​using IPTG at a final concentration of 0.5 mM to induce expression.

[0061] The method for isolating glucose dehydrogenase mutants and ketone reductase from cultures can be a conventional method in the art.

[0062] The coenzyme recycling catalytic system provided in this embodiment of the invention can either induce and culture the recombinant cells, freeze-dry the recombinant cells according to conventional methods in the art to obtain lyophilized bacterial powder, and then catalyze the lyophilized bacterial powder with a substrate, or induce and culture the recombinant cells, and then isolate the enzyme expressed by the culture for use in catalytic reactions.

[0063] This invention also provides the use of the glucose dehydrogenase mutant, the nucleic acid molecule, the recombinant vector, the recombinant cell, the coenzyme recycling catalytic system, and / or the coenzyme recycling catalytic system prepared by the method in the catalytic preparation of (R)-4-chloro-3-hydroxy-butyrate ethyl ester.

[0064] Accordingly, embodiments of the present invention also provide a method for preparing (R)-4-chloro-3-hydroxy-butyrate ethyl ester, comprising:

[0065] Using the recombinant cells, the coenzyme NADH recycling catalytic system, and / or the coenzyme recycling catalytic system prepared by the method described in this invention as catalysts, ethyl 4-chloroacetoacetate is catalyzed to obtain (R)-4-chloro-3-hydroxy-butyrate ethyl ester.

[0066] The preparation method provided in this invention can efficiently catalyze the conversion of ethyl 4-chloroacetoacetate to ethyl (R)-4-chloro-3-hydroxybutyrate, with a conversion rate of 84.7% in 24 hours, which is 1.619 times the conversion rate (52.3%) of the coenzyme cycle regeneration system constructed from the original glucose dehydrogenase (wild type).

[0067] It should be understood that the glucose dehydrogenase mutant and ketone reductase provided in the embodiments of the present invention can be used in whole-cell engineered bacteria, or in the form of unpurified crude enzyme, or in the form of partially purified or fully purified enzyme; the glucose dehydrogenase mutant and ketone reductase of the present invention can also be made into immobilized enzymes or catalysts in immobilized cell form using immobilization techniques known in the art.

[0068] In some embodiments, the concentration of ethyl 4-chloroacetoacetate is 0.2-0.6 M, preferably 0.45 M.

[0069] In some embodiments, the recombinant cells are prepared into a lyophilized powder and used as a catalyst. The method for preparing the lyophilized powder can be a conventional method in the art, such as collecting the induced recombinant cells (bacterial cells) by centrifugation, washing, and then freezing overnight at -20°C. The cells are then freeze-dried using a vacuum freeze dryer at -20°C and a vacuum of 15 Pa to obtain the lyophilized bacterial cell powder. In some specific embodiments, the mass ratio of the lyophilized bacterial cell powder to the ethyl 4-chloroacetoacetate is (0.1-0.2):1, preferably 0.15:1.

[0070] As a catalytic reaction known in the art, it is understood that glucose and NAD+ may be added, for example, during the catalytic preparation of (R)-4-chloro-3-hydroxy-butyrate ethyl ester. In some embodiments, the concentration of glucose is equal to the concentration of ethyl 4-chloroacetoacetate; the concentration of NAD+ is 0.01-0.05 mM, preferably 0.03 mM.

[0071] In some embodiments, the reaction temperature of the catalytic reaction is 25-40°C, preferably 35°C; the initial pH is 6.0-7.5, preferably 7.0, which can be adjusted using Tris or hydrochloric acid (e.g., 500 mM Tris and 1 M hydrochloric acid); and the reaction time is 24-36 h, preferably 24 h.

[0072] In order to enable those skilled in the art to clearly understand the above-described implementation details and operations of the present invention, and to demonstrate the significant improvement in the performance of the glucose dehydrogenase mutant and its uses in the embodiments of the present invention, the above technical solutions are illustrated below through examples.

[0073] Unless otherwise specified, the experimental methods used in the following examples are generally performed according to conventional experimental methods in the field of molecular biology, including but not limited to the methods described in *Molecular Cloning: A Laboratory Manual* by M.R. Green and *Molecular Biology* by Robert F. Weaver, or the experimental methods recommended by the reagent kit and instrument manufacturers. Unless otherwise specified, all reagents and biological materials used in the examples are commercially available.

[0074] Bacillus oleivorans has been published in the literature Sasikala, et al. "Bacillus oleivorans sp nov. a diesel oil-degrading and solvent-tolerant bacterium." International Journal of Systematic and Evolutionary Microbiology 65. Pt.4 (2015): 1310-1315. and is available to the public from Wanhua Chemical Group Co., Ltd.

[0075] Example 1: Gene mining of glucose dehydrogenase

[0076] (11) Extract genomic DNA from Bacillus oleivorans;

[0077] (12) Using the genomic DNA obtained in step (11) as a template, PCR was performed using primer 1 (ggatccgaattcatgttgcacat, SEQ ID NO:7) and primer 2 (aagcttctatttccgtttaactcgcc, SEQ ID NO:8) to obtain a PCR amplification fragment containing the wild-type glucose dehydrogenase gene. The nucleotide sequence of this glucose dehydrogenase gene is shown in SEQ ID NO:6, and the amino acid sequence of the glucose dehydrogenase it encodes is shown in SEQ ID NO:5.

[0078] Example 2: Obtaining mutants of random glucose dehydrogenase using error-prone PCR technology

[0079] Using the PCR amplification fragment from Example 1 as a template, and primers 1 and 2 as primers, error-prone PCR was performed according to the following reaction system to obtain 96 mutants of the glucose dehydrogenase gene.

[0080] Error-prone PCR reaction system: 5 μl of 10× amplification buffer, 4 μl of a mixture of four dNTPs (2.5 mM), 1.5 μl of each primer, 1.5 μl of template DNA, 1 μL of Taq DNA polymerase, and Mn... 2+ Add 5mM and double-distilled water to a final volume of 50μl.

[0081] PCR reaction procedure: (1) Pre-denaturation: 94℃, 3min; (2) Denaturation: 94℃, 30s; Annealing: 58℃, 30s; Extension: 72℃, 1.5min, 30 cycles; (3) Post-extension: 72℃, 10min; (4) Incubate at 4℃.

[0082] Example 3: High-throughput screening yielded glucose dehydrogenase mutants, which were then subjected to site-directed saturation mutagenesis.

[0083] The glucose dehydrogenase gene PCR amplification fragment obtained in Example 2 was digested with EcoRI and HindIII into the pET-28a(+) vector to obtain recombinant expression plasmids. After sequencing was confirmed to be correct, the recombinant expression plasmids were transformed into the expression host E. coli BL21(DE3) by heat shock. The selected positive clones were cultured in 5 ml TB medium for 8 h, and then IPTG was added to a final concentration of 0.5 mM and induced at 18°C ​​for 12 h. 500 μL of bacterial culture was centrifuged and resuspended in 1 mL of pH 7.0 PBS. 100 μL of the solution was transferred to a 96-well plate, and 100 μL of glucose dehydrogenase reaction premix (0.5 mM NAD+; 20 mM glucose; pH 7.0 PBS solution) was added. The plate was incubated at 37°C and 120 rpm for 2 h, and the absorbance was measured at 340 nm.

[0084] Plasmids were extracted from the strains (recombinant bacteria 35 and 83) corresponding to the reaction solutions with high absorbance values ​​and sequenced to obtain the mutation sites encoding glucose dehydrogenase: amino acid D at position 243 was mutated to H, and amino acid P at position 411 was mutated to A. For these two amino acid sites, site-directed saturation mutagenesis was designed using NNK on each codon. The screening method was as described in the first paragraph of this embodiment, resulting in two positive mutants with good catalytic effects: amino acid D at position 243 was mutated to E, and amino acid P at position 411 was mutated to R.

[0085] Site-directed saturation mutagenesis full plasmid PCR reaction system: 10 μl DNA polymerase mixture, primer 3 at position 243 (aaggtatatcgagtaagcaaannkggaca, SEQ ID NO:9), primer 4 (cgctgattaatacctttttctgtccnnkttt, SEQ ID NO:10); primer 5 at position 411 (agggggattgggacnnktta, SEQ ID NO:11), primer 6 (ctcgtataannkgtcccaatcccc, SEQ ID NO:12), 1 μl each of primers and template, and double-distilled water to a final volume of 20 μl.

[0086] PCR reaction procedure: (1) Pre-denaturation: 96℃, 3min (2) Denaturation: 96℃, 30s; Annealing: 58℃, 30s; Extension: 72℃, 2.0min, 30 cycles; (3) Extension: 72℃, 5min; (4) Keep warm at 4℃.

[0087] Example 4: Screening for the optimal combination enzyme – ketone reductase & glucose dehydrogenase mutant combination

[0088] Using Ned1 & Xho I and EcoRI & HindIII double restriction sites, ketone reductase (its amino acid sequence is shown in SEQ ID NO:4, and its encoding nucleotide sequence is shown in SEQ ID NO:3), two glucose dehydrogenase mutants, and gene fragments with combined mutation sites were ligated into the pACYCDuet-1 co-expression plasmid vector to obtain three recombinant co-expression plasmids. The recombinant co-expression plasmid with accurate sequencing was transformed into the expression host E. coli BL21(DE3) using a heat shock transformation method. Single colonies selected by chloramphenicol were picked and transferred to 100 ml of TB medium and cultured in shake flasks at 37°C for 8 h. IPTG was added to a final concentration of 0.5 mM and the culture was induced at 18°C ​​for 12 h. After centrifugation, 2 ml of bacterial culture was centrifuged and the cells were washed with PBS. The washed cells were resuspended in 1 ml of PBS and 1 ml of premixed catalytic reaction solution (0.06 mM NAD+; 0.9 M ethyl 4-chloroacetoacetate; 0.9 M glucose; pH 7.0 PBS solution) was added. After reacting for 24 h, the conversion rate was determined by gas chromatography. The optimal mutant for glucose dehydrogenase was determined to be D243E, whose amino acid sequence is shown in SEQ ID NO:1 and whose encoding nucleotide sequence is shown in SEQ ID NO:2. Accordingly, the glucose dehydrogenase mutant D243E and the aforementioned ketone reductase constitute the optimal combination enzyme.

[0089] Example 5: Determination and comparison of product generation between the optimal and wild-type combinatorial enzymes

[0090] The wild-type combinatorial enzyme is a combination of the original glucose dehydrogenase obtained in Example 1 and the above-mentioned ketone reductase.

[0091] Following the method in Example 4, recombinant co-expression bacteria expressing the optimal combinatorial enzyme and the wild-type combinatorial enzyme were constructed, respectively. The induced recombinant co-expression bacteria were collected by centrifugation, washed, and then frozen overnight at -20°C. A vacuum freeze dryer was used to freeze-dry the bacteria at -20°C and a vacuum of 15 Pa to obtain lyophilized bacterial powder. The lyophilized bacterial powder was added to a reaction system (0.03 mM NAD+; 0.45 M ethyl 4-chloroacetoacetate; 0.45 M glucose; pH 7.0 PBS solution) at a mass ratio of 0.15:1, and catalytic reactions were carried out. After the reaction was complete, 400 μL of the supernatant was centrifuged and mixed with 800 μL of ethyl acetate. The mixture was then filtered through a membrane and used for gas chromatography analysis of the conversion rate. An Agilent DB-5 capillary column was used with nitrogen as the carrier gas and a hydrogen ion detector. The injection port temperature was 250 °C, the detector temperature was 250 °C, the column oven temperature was 90 °C, the injection volume was 1 μL, and the column flow rate was 1 mL / min.

[0092] like Figure 2 and Figure 3 As shown, the peak time for ethyl 4-chloroacetoacetate standard was 6.61 min, and the peak time for (R)-4-chloro-3-hydroxybutyrate standard was 7.12 min; Figure 4 As shown, the elution times of the reaction products in the gas phase spectrum of the reaction process in this embodiment are related to... Figure 3 The similarity indicates that the reaction product is (R)-4-chloro-3-hydroxybutyrate ethyl ester.

[0093] The results showed that the optimal combination enzyme constructed from the glucose dehydrogenase mutant with mutation at 243(DE) and co-expressed with ketone reductase had a better conversion rate: the conversion rate at 24h was 84.7%, which was 1.619 times that of the coenzyme recycling system constructed from the wild combination enzyme (52.3%).

[0094] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A glucose dehydrogenase mutant, characterized in that, The amino acid sequence of the glucose dehydrogenase mutant is shown in SEQ ID NO:

1.

2. A nucleic acid molecule encoding the glucose dehydrogenase mutant of claim 1, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:

2.

3. A recombinant vector, characterized in that, It includes the nucleic acid molecule as described in claim 2.

4. The recombinant vector according to claim 3, characterized in that, It further includes a nucleic acid molecule encoding a ketone reductase, as shown in SEQ ID NO:

3.

5. A recombinant cell, characterized in that, It includes the recombinant vector as described in claim 3.

6. A recombinant cell, characterized in that, It includes the recombinant vector as described in claim 4.

7. A coenzyme recycling catalytic system, characterized in that, The recombinant cell comprises the recombinant cell of claim 6, the recombinant cell comprising a recombinant co-expression vector comprising the nucleic acid molecule of claim 2 and a nucleic acid molecule encoding a ketone reductase as shown in SEQ ID NO:

3.

8. A method for preparing a coenzyme recycling catalytic system, characterized in that, include: The recombinant cells described in claim 5 were induced and cultured to obtain a culture. The step of isolating the glucose dehydrogenase mutant of claim 1 from the culture.

9. A method for preparing a coenzyme recycling catalytic system, characterized in that, include: The recombinant cells described in claim 6 were induced and cultured to obtain a culture. The steps of isolating the glucose dehydrogenase mutant and ketone reductase of claim 1 from the culture; The amino acid sequence of the ketone reductase is shown in SEQ ID NO:

4.

10. Use of the glucose dehydrogenase mutant of claim 1, the nucleic acid molecule of claim 2, the recombinant vector of claim 3 or 4, the recombinant cell of claim 5 or 6, the coenzyme recycling catalytic system of claim 7, and / or the coenzyme recycling catalytic system prepared by the preparation method of claim 8 or 9 in the catalytic preparation of (R)-4-chloro-3-hydroxy-butyrate ethyl ester.

11. A method for preparing ethyl (R)-4-chloro-3-hydroxy-butyrate, characterized in that, include: Using the recombinant cells of claim 6, the coenzyme recycling catalytic system of claim 7, and / or the coenzyme recycling catalytic system prepared by the preparation method of claim 9 as catalysts, ethyl 4-chloroacetoacetate is catalyzed to obtain ethyl (R)-4-chloro-3-hydroxy-butyrate.

12. The preparation method according to claim 11, characterized in that, The reaction temperature of the catalytic reaction is 25-40°C; the initial pH is 6.0-7.5; the concentration of ethyl 4-chloroacetoacetate is 0.2-0.6 M; the mass ratio of the lyophilized catalyst powder to ethyl 4-chloroacetoacetate is (0.1-0.2):1; and the reaction time is 24-36 h.

13. The preparation method according to claim 12, characterized in that, The reaction temperature of the catalytic reaction was 35°C; the initial pH was 7.0; the concentration of ethyl 4-chloroacetoacetate was 0.45 M; the mass ratio of the lyophilized catalyst powder to ethyl 4-chloroacetoacetate was 0.15:1; and the reaction time was 24 h.

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