CN110283800A - Glucose oxidation enzyme mutant, double enzyme coexpression vectors and its application - Google Patents

Abstract

The invention discloses a glutamic acid oxidase mutant with significantly improved enzyme activity obtained by performing site-directed mutation on L-glutamic acid oxidase, and co-expressing the mutant enzyme and catalase to construct a recombinant engineering strain , using L-glutamate as a substrate to prepare α-ketoglutarate under normal temperature and pressure conditions. The process adopted in the present invention is simple, has the characteristics of high conversion rate, easy separation and purification, mild production conditions, little environmental pollution, etc., and has good industrial application prospect.

Description

Glutamate oxidase mutant, dual-enzyme co-expression vector and application thereof

technical field

The invention relates to a glutamic acid oxidase mutant, a double-enzyme co-expression carrier and applications thereof, belonging to the technical field of bioengineering.

Background technique

α-Ketoglutarate (α-KG) is an important organic acid, which plays an important role in the metabolism of organism cells, and is also an important precursor for the synthesis of various sugars, amino acids and proteins. It has broad application prospects in industries such as feed, chemical and cosmetics. In recent years, α-KG has been widely used in sports nutrition drinks and health products. After α-KG is digested and absorbed by the human body, excessive ammonia in the body can be combined with α-KG to reduce the problems caused by ammonia poisoning, and at the same time participate in the nitrogen metabolism and other metabolic processes. In addition, α-KG can also be used as a physical enhancer. After being combined with ornithine, it can increase the level of growth hormone and insulin, and can inhibit the decomposition of muscle fibers and repair muscle damage on the premise of reducing protein consumption. Has high health value.

At present, the production methods of α-KG include chemical synthesis, microbial fermentation and biocatalysis. Traditional α-KG is produced by chemical synthesis, but the strong acid and alkali, cyanide and other harmful reagents used in the chemical synthesis process not only easily cause environmental pollution, but also limit its application in food, cosmetics and pharmaceutical industries. The microbial fermentation method has a long fermentation period, and there are many by-products such as pyruvic acid and fumaric acid in the fermentation product, which will increase the difficulty and cost of subsequent α-KG extraction, and industrial large-scale production has not yet been realized. Enzymatic production of α-KG has the advantages of short reaction cycle and high conversion rate. Niu Panqing et al. obtained a Streptomyces mutant strain with high glutamate oxidase production through mutagenesis. Under optimal conditions, the α-KG yield can reach 24 h. up to 38.1 g/L (Niu Panqing, et al. Acta Bioengineering, 2014, 8: 1318-22); using genetic engineering, the glutamic acid oxidase gene was heterologously expressed in Escherichia coli and catalyzed by enzymatic , the yield of α-KG was 104.7 g/L in 24 hours (Niu PQ, et al. JBiotechnol, 2014, 179:56-62). The use of purified enzymes for in vitro catalysis not only requires tedious protein purification steps, but also requires the addition of a large amount of expensive catalase to assist catalysis during the reaction process, which greatly increases the industrial cost. Compared with isolated enzymes for in vitro catalysis, whole-cell biocatalysts are easier to prepare, have more stable catalytic performance, and are not easily affected by external temperature, pH and other factors; no exogenous cofactors are required during the conversion process, and no toxic and harmful products are generated , environment-friendly and has industrial application prospect.

L-glutamate oxidase (LGOX) is a flavoproteinase with flavin adenine dinucleotide as the prosthetic group, which has high substrate stereoselectivity, high catalytic efficiency, and Under mild conditions, it can oxidize L-glutamic acid to generate hydrogen peroxide, ammonia and α-ketoglutarate without adding exogenous cofactors. The study found that the enzyme mainly exists in Streptomyces, but the enzyme-producing ability varies greatly. Discovering and identifying glutamate oxidase with high activity or substrate specificity, and obtaining an enzyme catalytic system that meets the requirements of physiological research and production applications will provide favorable conditions for the industrial production of α-KG.

Contents of the invention

The purpose of the present invention is to optimize the co-expression of L-glutamic acid oxidase and catalase derived from microorganisms by means of genetic engineering, construct a co-expression recombinant bacterial strain with excellent catalytic performance, and use the bacterial strain to establish a high-efficiency production The whole cell transformation method of α-KG is shown in Figure 1.

One object of the present invention is to provide a glutamic acid oxidase mutant with improved enzymatic activity, which is the amino acid sequence corresponding to the 280th amino acid of SEQ ID NO:1 mutated from serine S to threonine T; or the The glutamate oxidase mutant is an amino acid sequence that further includes a double-site mutation of histidine H to leucine L at the 533rd amino acid corresponding to SEQ ID NO:1. In one embodiment, the glutamate oxidase mutant comprises the amino acid sequence shown in SEQ ID NO: 5, and the coding nucleotide sequence of the glutamate oxidase mutant may be SEQ ID NO: 6 The nucleotide sequence shown; In another embodiment, described glutamate oxidase mutant comprises the aminoacid sequence shown in SEQ ID NO:7, the coding nucleotide of described glutamate oxidase mutant The sequence may be the nucleotide sequence shown in SEQ ID NO:8. The glutamate oxidase mutant coding nucleotide sequence of the present invention can be derived from the wild-type L-glutamic acid oxidase coding nucleotide sequence shown in SEQ ID NO: 2, such as the mutant's The coding nucleotide sequence can be obtained by mutating the nucleotide sequence shown in SEQ ID NO: 2 or by substituting nucleotides at specific positions.

In one embodiment, the wild-type L-glutamic acid oxidase is derived from Streptomyces mobaraensis .

In one embodiment, the amino acid sequence of the glutamate oxidase mutant may also include 1-10 substitutions, deletions or insertions of other amino acid residues, but still maintain L-glutamate oxidase activity.

The present invention also provides a recombinant strain expressing the above-mentioned glutamic acid oxidase mutant, which is obtained by introducing the coding nucleotide sequence of the mutant or a vector containing the coding nucleotide sequence into a host strain. The recombinant strain can be used to produce α-ketoglutarate.

The second object of the present invention is to provide a dual-enzyme co-expression vector for producing α-KG. The co-expression vector is to simultaneously introduce the coding genes of L-glutamate oxidase and catalase into one plasmid.

According to the present invention, the L-glutamic acid oxidase is derived from the genus Streptomyces, and the catalase is derived from the genus Enterobacter. According to the present invention, the type of vector used for dual-enzyme co-expression is not particularly limited, and can be various expression vectors commonly used in the art capable of expressing a gene of interest in a bacterial strain, such as an Escherichia coli-Corynebacterium glutamicum shuttle expression plasmid pXMJ19, or pDXW series vectors, or pBL1 series vectors, or Escherichia coli expression plasmid pET series vectors, or pBAD series vectors, or Bacillus subtilis expression plasmid pHT01, or pMA5 vectors, etc. In a preferred embodiment, the expression vector is pXMJ19 plasmid. In one embodiment, the L-glutamic acid oxidase contains the amino acid sequence shown in SEQ ID NO:1, and its coding nucleotide sequence is shown in SEQ ID NO:2. In another embodiment, the L-glutamate oxidase is the glutamate oxidase mutant as described in the first object, and the amino acid of the mutant is as shown in SEQ ID NO:5 or SEQ ID NO:7 Its encoding nucleotide sequence is shown in SEQ ID NO:6 or SEQ ID NO:8.

In one embodiment, the catalase in the dual-enzyme co-expression vector contains the amino acid sequence shown in SEQ ID NO:3, and its encoding nucleotide sequence is shown in SEQ ID NO:4.

The invention provides multiple single-plasmid double-enzyme co-expression systems, which simultaneously express the coding gene of L-glutamic acid oxidase and the coding gene of catalase in one plasmid. In one embodiment, the dual-enzyme co-expression vector uses a single promoter mode to express a promoter, the gene encoding L-glutamate oxidase and the gene encoding hydrogen peroxide in tandem, and the two The two coding genes can be connected in any order; in another embodiment, the dual-enzyme co-expression vector adopts a dual-promoter mode, adding promoters before the coding genes of L-glutamate oxidase and catalase respectively. son. The promoter can be a strong tac promoter or a strong trc promoter. In one embodiment, a single promoter format is preferred and the promoter is a strong tac promoter.

In an embodiment of the present invention, in the single promoter mode, the conservative RBS-related sequence can be added between the two coding genes for segmentation; or in the double-promoter mode, the RBS-related sequence can be increased between the promoter and the coding gene sequence.

In a specific embodiment, the gene encoding L-glutamate oxidase and the gene encoding hydrogen peroxide are expressed in tandem using a single strong tac promoter, wherein the gene encoding L-glutamate oxidase and the gene encoding catalase are separated by conserved RBS-related sequences; in another specific embodiment, a strong tac promoter and a conserved RBS-associated sequence; in yet another specific embodiment, a strong tac promoter and a conserved RBS-associated sequence are added before the L-glutamate oxidase coding gene using a double promoter pattern, and a trc-strong promoter is added before the catalase gene Promoter and conserved RBS-associated sequences.

The promoter and RBS-related sequences used in the co-expression system construction of the present invention are:

The third object of the present invention is to provide a dual-enzyme co-expression recombinant strain for producing α-KG, which contains the dual-enzyme co-expression vector as described in the second object. In one embodiment, the recombinant strain is obtained by transforming or introducing the dual-enzyme co-expression vector into a host strain. The host bacterial strain of the present invention is the bacterial strain that can express described glutamic acid oxidase and catalase, and described host bacterial strain can be selected from Corynebacterium, Escherichia or Bacillus, for example is glutamic acid rod Corynebacterium glutamicum , Escherichia coli or Bacillus subtilis ; preferably Corynebacterium glutamicum , more preferably Corynebacterium glutamicum ATCC 13032.

The fourth object of the present invention is to provide a whole-cell catalytic sludge, which includes the double-enzyme co-expression recombinant strain described in the third object; specifically, the recombinant bacterial strain is prepared by high-density liquid submerged fermentation .

In one embodiment, the whole-cell catalytic sludge is prepared by the following method:

(1) Inoculate the co-expression recombinant strain described in the third objective on the seed medium for culture;

(2) Then inoculate the seed solution in the fermentation medium for fermentation, optionally add isopropyl-β-D-thiogalactopyranoside (IPTG) to induce the expression of the target protein, and collect the bacterial cells to obtain the whole Cell Catalytic Sludge.

In a specific embodiment, the preparation method of the whole cell catalytic sludge is as follows:

The co-expressed recombinant strain was inoculated in 100 mL of seed culture medium (yeast powder 2.5 g/L, peptone 5 g/L, NaCl 5 g/L, brain heart infusion 18.5 g/L, sorbitol 91 g/L ) in a 500 mL Erlenmeyer flask, shake culture at 30°C for 15-18 h; transfer to 5 L fermentation medium (glucose 100 g/L, corn steep liquor 15 g/L) according to 5% inoculum , ammonium sulfate 20 g/L, magnesium sulfate 1 g/L, potassium dihydrogen phosphate 0.5 g/L, dipotassium hydrogen phosphate 0.1 g/L, sodium citrate 2 g/L, calcium carbonate 2 g/L; pH 7.0) In the fermenter, set the culture temperature at 30-32°C, control the pH value of the culture to 7.0, and control the speed at 300 r/min in the early stage of culture. When the dissolved oxygen drops below 20%, set the speed to be coupled with the dissolved oxygen. When the cell concentration OD ₆₀₀ grows to about 25~30, add isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 0.4 mM to induce the expression of the target protein, and set the induction temperature to 25~30. At 30°C, the pH value of the culture was controlled to be 7.0, and the induction time was about 24 hours. The final concentration OD ₆₀₀ of bacterial growth could reach about 150.0. After the whole induction process is completed, the above-mentioned fermentation culture is centrifuged, and the bacterial cells are collected to obtain the whole-cell catalytic sludge.

The fifth object of the present invention is to provide the application of the above-mentioned glutamate oxidase mutant, double-enzyme co-expression vector, recombinant strain or whole cell catalytic sludge in the preparation of α-ketoglutarate.

The sixth object of the present invention is also to provide a whole cell catalytic method for producing α-ketoglutarate, comprising culturing the recombinant strain co-expressing the double enzymes described in the third object.

In one embodiment, sodium glutamate monohydrate is added to the aqueous fermentation system, and the above-mentioned recombinant strain or the whole-cell catalytic sludge containing the recombinant strain is added to resuspend to carry out the catalytic reaction. Optionally, isopropyl-β-D-thiogalactopyranoside (IPTG) is added to the catalytic reaction to induce expression of the protein of interest. As a preferred embodiment, the present invention adopts a whole-cell catalytic method to prepare α-ketoglutarate without adding exogenous enzyme catalyst.

In a specific embodiment, the preferred final concentration of monosodium glutamate monohydrate (MSG) in the fermentation system is 270 g/L. In one embodiment, the concentration of the whole cell catalytic sludge in the fermentation system is 10-20 g/L, such as 12 g/L, 15 g/L, 18 g/L. In one embodiment, the control speed is 400 r/min, the dissolved oxygen is set at 25-40%, the reaction temperature is 35°C, and the catalytic reaction time is 24-48 h.

The term "corresponding to" in the present invention has the meaning commonly understood by those of ordinary skill in the art. Specifically, "corresponding to" means that after two sequences are aligned for homology or sequence identity, one sequence corresponds to the specified position in the other sequence. Therefore, for example, in terms of "mutation from serine S to threonine T corresponding to the 280th amino acid of SEQ ID NO: 1", if a 6×His tag is added to one end of the amino acid sequence shown in SEQ ID NO: 1, Then the 280th position corresponding to the amino acid sequence shown in SEQ ID NO: 1 in the obtained mutant may be the 286th position in the amino acid sequence of the mutant. Those of ordinary skill in the art can use any method known in the art for determining sequence homology or identity to determine or compare sequence homology or identity, including but not limited to Computational Molecular Biology (Computational Molecular Biology), Lesk, Edited by A.M., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects (Biocomputing: Informatics and Genome Projects), edited by Smith, D.W., Academic Press, New York, 1993; Computer Analysis of Sequence Data of Sequence Data), Part I, Griffin, A.M. and Griffin, H.G. eds., Humana Press, New Jersey, 1994 et al.

Beneficial effects of the present invention

The present invention successfully constructs a recombinant engineering strain that efficiently co-expresses glutamate oxidase and catalase, and provides a biological preparation method of α-ketoglutarate without adding catalase from an external source, The method has high substrate conversion rate and less by-products, simple and convenient preparation method, mild production conditions, little environmental pollution, and good technical application prospect. The L-glutamic acid oxidase used in the present invention has excellent catalytic performance, and can efficiently catalyze the substrate L-glutamic acid to generate α-ketoglutarate. The catalase used in the present invention can convert the The hydrogen peroxide by-product is rapidly degraded, thereby promoting the production of α-ketoglutarate. In the present invention, Corynebacterium glutamicum, a recognized food safety grade microorganism, can be selected as the production strain. As a traditional main industrial fermentation microorganism, this strain has been used for the production of monosodium glutamate for decades. Therefore, this strain is used to produce α-ketopentyl Diacids can effectively avoid food safety hazards.

Description of drawings

Figure 1 is a schematic diagram of α-ketoglutarate production based on whole cell catalysis.

Fig. 2 is the SDS-PAGE electrophoresis diagram of the whole cell soluble protein of the co-expressed recombinant strain.

Figure 3 is the activity analysis of glutamate oxidase mutants.

Fig. 4 is an analysis and detection diagram of α-ketoglutarate content in the whole cell catalytic solution.

Detailed ways

The following implementation further illustrates the content of the present invention, but should not be construed as limiting the present invention. In the following examples, both Escherichia coli DH5α and Corynebacterium glutamicum are commercially available, Escherichia coli DH5α is used for cloning of all genes in the present invention, and Corynebacterium glutamicum is used for gene protein expression and whole cell transformation in the present invention Production of alpha-ketoglutarate. Escherichia coli DH5α competent cells and Corynebacterium glutamicum competent cells were prepared according to conventional methods. For the test methods not indicated in the following examples, the specific conditions were carried out according to the conventional conditions, such as the conditions described in "Molecular Cloning: Laboratory Manual", or according to the conditions suggested by the manufacturers of the corresponding biological reagents.

Example 1 Construction of glutamate oxidase and catalase co-expression vector

It should be understood by those skilled in the art that Corynebacterium glutamicum and Streptomyces moyuanensis all have different degrees of codon preference when expressing proteins. Codon optimization of the glutamic acid oxidase gene from Streptomyces Maoyuan can make the target protein more efficiently expressed in the expression system of Corynebacterium glutamicum, and the codon optimization method is well known to those skilled in the art , which will not be repeated here.

In the present invention, the glutamic acid oxidase gene from Streptomyces mobaraensis ( S. mobaraensis DSM40903) is optimized according to the codon usage frequency of Corynebacterium glutamicum. The nucleotide sequence is shown in SEQ ID No: 2. Jinweizhi Biotechnology Co., Ltd. carried out gene synthesis, and used Lgox-5F and Lgox-3R as a primer pair to obtain the glutamic acid oxidase gene sequence with a conservative RBS-related sequence added to the front of the start codon through PCR amplification. Using rbs-katE-5F and rbs-katE-3R as a primer pair, using Escherichia coli ( E. coli MG1655) genome as a template, obtain hydrogen peroxide with a conserved RBS-related sequence added to the front of the start codon by PCR amplification Enzyme gene sequence. Using the above two gene sequences as templates and using Lgox-5F and rbs-katE-3R as primers, the fusion fragments of glutamate oxidase and catalase (rbs- lgox-rbs-katE). Using pXMJ19-5F and pXMJ19-3R as a primer pair and plasmid pXMJ19 as a template, the pXMJ19 plasmid backbone containing the AarI restriction site was obtained by PCR. Using the fragment assembly method based on GoldenGate cloning, the fusion fragment rbs-lgox-rbs-katE and the pXMJ19 plasmid backbone were connected into a single promoter tandem expression plasmid. The recombinant plasmid obtained above was named pXMJ19-tac-lgox-katE, and sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing confirmation.

The pXMJ19-tac-lgox-katE plasmid was selected as a template, and the pXMJ19-tac-lgox-katE plasmid backbone containing the AarI restriction site was obtained by PCR using the pXMJ19-lk-5F and pXMJ19-lk-3R primer pairs. The tac-fusionF and tac-fusionR primers are annealed and fused to form a double-stranded DNA fragment containing cohesive ends, that is, the tac promoter sequence; trc-fusionF and trc-fusionR primers are annealed and fused to form a double-stranded DNA fragment containing cohesive ends Strand DNA fragment, namely trc promoter sequence. After the plasmid backbone was treated with AarI endonuclease, it was ligated with the tac promoter sequence and trc promoter sequence obtained above to obtain two dual-promoter expression plasmids pXMJ19-tac-lgox-tac-katE and pXMJ19-tac -lgox-trc-katE, send the obtained plasmid to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing confirmation.

The primer sequences used in the above-mentioned implementation cases are:

In this example, the PCR amplification reaction system is: 10 μL 5 × HF Phusion buffer, 2.5 μL 2.5mM dNTP, 2.5 μL 10 μM Primer 1, 2.5 μL 10 μM Primer 2, 0.5 μL Template, 0.5 μL DMSO, 0.5 μL Phusion DNA Polymerase, supplemented with ddH ₂ O to 50 μL. The PCR reaction conditions were as follows: pre-denaturation at 98°C for 1 min, denaturation at 98°C for 10 s, annealing at 60°C for 20 s, extension at 72°C for 1 min, 35 cycles; extension at 72°C for 8 min, and storage at 16°C.

In this example, the Golden Gate assembly system is: 1.5 μL 10 × T4 Ligase buffer, 1 μL T4 Ligase, 1 μL AarI endonuclease, 0.5 μL 100 × BSA, 0.2 μL Oligo, 200 ng gene fragment or plasmid backbone, Supplement ddH ₂ O to 15 μL. The Golden Gate assembly procedure is: 37°C, 3 min; 22°C, 4 min; cycle 30 times; 22°C, 20 min; 50°C, 2 min; 80°C, 2 min; 16°C, 5 min min. After the reaction, the reaction solution was directly transformed into Escherichia coli DH5α competent, and after plate resistance screening, the transformants were picked for verification.

In this example, the digestion system was: 5 μL 10 × FastDigest buffer, 2 μL AarI endonuclease; 0.5 μL 100 × BSA, 200 ng DNA fragments, supplemented with ddH ₂ O to 50 μL. The digestion reaction conditions were 37°C and the digestion time was 2 h.

The expression vector pXMJ19 selected in this example is an inducible expression vector. The plasmid itself contains a strong promoter tac-related sequence (including the operator sequence lacO). initial gene expression.

Example 2 Construction and Expression Analysis of Glutamate Oxidase and Catalase Co-expression Recombinant Strain

Extract the co-expression plasmids pXMJ19-tac-lgox-katE, pXMJ19-tac-lgox-tac-katE and pXMJ19-tac-lgox-trc-katE obtained in the above-mentioned Example 1, and transfer them to glutamine by electroporation acid Corynebacterium, the specific transformation method is: take 100 μL of Corynebacterium glutamicum competent cells, add about 200 ng of the above-mentioned co-expression plasmids respectively, mix well, transfer to a pre-cooled 2 mm electroporation cup, and store in ice Place it on the table for 20 minutes, adjust the voltage of the electrotransfer instrument to 2.1 kV for electric shock, immediately add 1 mL LBHIS liquid medium, place in a 46°C water bath for heat shock for 6 minutes, and revive culture at 32°C, 150rpm shaker After 2 h, spread LBHIS solid medium containing 15 μg/mL ampicillin, culture overnight at 32°C, and verify after a single colony grows.

The recombinant Corynebacterium glutamicum containing different co-expression plasmids obtained above was subjected to protein induction expression detection, and the expression of glutamate oxidase and catalase was analyzed. The specific method is as follows: single clones were picked and inoculated in shake flasks containing 100 mL LBHIS liquid medium, and cultured overnight at 32°C in a shaker at 200 r/min. Subsequently, according to the 2% inoculation amount, the overnight bacteria were retransferred into 100 mL LBHIS liquid medium, cultivated in a shaker at 32°C and 200 r/min until the bacterial cell concentration OD ₆₀₀ to about 1.0, and the final concentration of 0.4 mM IPTG induced the expression of the target protein, adjusted the culture temperature to 28°C, and continued to induce culture for 12 h. After the induction was completed, 1 mL of the bacterial liquid was taken for sonication, and then SDS-PAGE was used to detect the expression of the dual enzymes in the co-expression strains.

As shown in Figure 2, protein expression analysis found that the expression of catalase in the pXMJ19-tac-lgox-tac-katE co-expression strain was significantly higher than that of glutamate oxidase, indicating that tac was added before the gene encoding catalase The promoter is helpful for gene expression; in the pXMJ19-tac-lgox-trc-katE co-expression strain, the expression of catalase decreased significantly, suggesting that the trc promoter is not suitable for the dual-enzyme co-expression system; pXMJ19-tac Both glutamate oxidase and catalase have relatively high expression levels in -lgox-katE co-expression strains, and the protein concentrations of the two are basically similar.

Example 3 High-density fermentation culture to prepare whole-cell catalysis sludge

This embodiment is used to provide a method for high-density fermentation culture and sludge preparation of co-expressed recombinant strains. The method of fermentation culture may include the following steps: inoculate the co-expressed recombinant strain obtained in the above-mentioned Example 2 into the In the chloramphenicol-resistant seed medium, shake culture at 32°C and 200 r/min for 16 h to obtain the seed liquid of the co-expression recombinant strain; inoculate the seed liquid in the 5 In the fermenter of L fermentation medium, the culture temperature was set at 32°C, the stirring speed was 300 r/min, and the pH value of the culture was controlled to be 7.0. When the dissolved oxygen dropped below 20%, the speed was set to be coupled with the dissolved oxygen. Monitor the cell concentration OD ₆₀₀ , and when the cell grows to an OD ₆₀₀ of about 25, add a final concentration of 0.4 mM IPTG inducer to activate the expression of the target protein, set the culture temperature at about 28°C, and control the culture pH value to 7.0 to induce The time is about 24 hours, and the final cell concentration OD ₆₀₀ can reach about 150.0; centrifuge the above liquid fermentation culture at a speed of 8000×g for 10 min, pour off the supernatant of the fermentation broth, and collect the cells to obtain the recombinant corn Corynebacterium acidic acid sludge can be placed in a 4°C refrigerator for later use.

Those skilled in the art should understand that the antibiotics added in the seed medium and fermentation medium are used as selection markers for the fermentation of co-expressed recombinant strains. The concentration of the antibiotics is not particularly limited, and the working concentration is 15 μg/L . In this embodiment, the components of the seed medium are: yeast powder 2.5 g/L, peptone 5 g/L, NaCl 5 g/L, brain heart infusion 18.5 g/L, sorbitol 91 g/L; The components of the fermentation medium are: glucose 100 g/L, corn steep liquor 15 g/L, ammonium sulfate 20 g/L, magnesium sulfate 1 g/L, potassium dihydrogen phosphate 0.5 g/L, hydrogen phosphate Dipotassium 0.1 g/L, sodium citrate 2 g/L, calcium carbonate 2 g/L, adjust the medium to pH 7.0. In this embodiment, there is no special limitation on the acid-base solution and the concentration used to adjust the pH of the culture medium. In this embodiment, the acid solution is 50% acetic acid, and the alkali solution is 50% ammonia water.

Example 4 Establishment of Whole Cell Transformation Process for Preparation of α-Ketoglutarate

This example is used to provide a method for whole cell transformation to produce α-KG. Based on the co-expression recombinant Corynebacterium glutamicum constructed in the above-mentioned Example 2 as the whole cell catalyst, a biological preparation process of α-KG was established. The production method of α-KG is specifically to use a fermenter to establish a 5 L whole-cell catalytic system, add 270 g/L of sodium glutamate monohydrate (MSG) substrate at a final concentration, and add 10 g/L of the above-mentioned embodiment For the whole-cell sludge prepared in 3, control the fermenter rotation speed to 400 r/min, set the dissolved oxygen to not less than 25%, the catalytic reaction temperature to 35°C, and the catalytic reaction time to 40 h. (HPLC) Quantitative analysis of catalytic reaction solution components.

The α-KG production method established in this example does not require the addition of expensive catalase from an external source, and the entire catalytic reaction process does not need to control the pH value of the reaction system, which can greatly save production costs and simplify the process flow.

The HPLC analysis described in this example, the specific method is: take 1 mL of the whole-cell catalytic reaction solution, centrifuge at 12000×g for 5 min, take the supernatant, and filter it with a filter membrane with a pore size of 0.02 μm. The components of the reaction solution were determined by high-performance liquid chromatography (Agilent1200, USA), and the chromatographic conditions were as follows: the analytical column was a Bio-Rad Aminex HPX-87H chromatographic column, the mobile phase was 5 mMH ₂ SO ₄ , and the flow rate was 0.6 mL/min. The temperature is 30°C, and the detector is an ultraviolet detector with a detection wavelength of 210 nm.

Through liquid chromatography detection and analysis, the co-expression strain obtained based on pXMJ19-tac-lgox-katE showed the highest α-KG production capacity, and the content of α-KG in the whole-cell catalytic reaction liquid could reach 200.0 g/L, and the bottom The molar conversion rate is more than 95%, and there are no complex components in the reaction solution, which helps to simplify the downstream separation and purification process, and has a good technical application prospect.

Example 5 Construction and Activity Analysis of Glutamate Oxidase Mutants

This example is used to provide a method for constructing glutamate oxidase mutants with improved enzyme activity. By analyzing the 3D structure of glutamate oxidase and comparing it with homologous sequences, it was determined that the 280th serine S and the 533rd histidine H of the amino acid sequence of SEQ ID NO: 1 were the target mutation sites. Using site-directed mutagenesis technology, design point mutation primers according to the amino acid site to be mutated, and obtain the glutamic acid oxidase mutant sequence by PCR.

The pXMJ19-tac-lgox-katE plasmid was selected as a template, and the upstream fragment of lgox-S280T was amplified by primer pair S280T-5F and LGOX-DN, and the downstream fragment of lgox-S280T was amplified by primer pair LGOX-UP and S280T-3R. The upstream and downstream fragments were mixed as a template, and based on the principle of fusion PCR, the LGOX-UP/LGOX-DN primer pair was used to amplify the lgox-S280T fragment, which is glutamic acid oxidase containing the S280T single mutation site. The glutamic acid oxidase mutant sequence was connected with the pET21b vector to construct the pET21b-lgox-S280T recombinant plasmid. The lgox-S280T fragment was selected as a template, and the upstream and downstream fragments of lgox-S280T H533L were amplified by using the LGOX-UP/H533L-3R primer pair and the H533L-5F/LGOX-DN primer pair respectively. The upstream and downstream fragments were mixed as a template, and based on the principle of fusion PCR, the LGOX-UP/LGOX-DN primer pair was used to amplify the lgox-S280T H533L fragment, which is glutamate oxidase containing the S280TH533L double mutation site. The glutamic acid oxidase mutant sequence was connected to the pET21b vector to construct the pET21b-lgox-S280TH533L recombinant plasmid. The above-mentioned recombinant plasmids were transformed into E. coli BL21 (DE3) competent cells, and after plate resistance screening, transformants were picked and sequenced for verification. The successfully constructed E. coli BL21/pET21b-lgox-S280T, E. coli BL21/pET21b-lgox-S280TH533L recombinant engineering bacteria and the recombinant engineering bacteria containing the unmutated glutamate oxidase gene were inoculated in 5 mL ampicillin containing In LB medium containing penicillin, after culturing overnight at 37°C and 200 r/min, retransfer to 100 mL LB medium containing ampicillin according to 1% inoculation amount, and when the cell density reaches OD ₆₀₀ value of 0.6 , adding isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 0.4 mM, induced expression at 16°C, 200 r/min for 10 h, and collected by centrifugation to obtain wet bacteria Mud, washed with sterile water and resuspended bacteria. Adjust the concentration of the obtained resuspended bacteria to an OD ₆₀₀ value of about 10, and use an ultrasonic cell disruptor to disrupt the cells, set the ultrasonic power to 200 W, sonicate for 2 s with an interval of 1 s, and sonicate for 10 min. After the sonication process, centrifuge at 8000×g for 10 min at 4°C, and the obtained supernatant can be used for subsequent protein expression analysis and Ni-NTA purification.

The Ni-NTA described in this example was purified using standard laboratory protocols. The specific method is: the filler used for purification is nickel, and the whole purification process is carried out in a refrigerator at 4°C. After injecting 1-2 mL filler into the chromatography column, wash the column with 3-5 times column volume of deionized water, and then add 5 times column volume of lysis buffer (20 mM Na ₂ HPO ₄ , 200 mM NaCl, pH 7.0 ) Equilibrate the column so that the filler is under the same buffer system as the target protein. Add the sample to a well-balanced chromatographic column, turn it upside down to make the target protein fully contact with the filler, collect the effluent after the column liquid is separated, and repeat the above steps 2 to 3 times. Wash with 10-15 times column volume of impurity buffer (20 mM Na ₂ HPO ₄ , 200 mM NaCl, 50 mM imidazole, pH 7.0) to remove non-specifically adsorbed impurities, and use Coomassie brilliant blue solution to detect the effluent , until no significant protein was detected. Elute the target protein with elution buffer solution (20 mM Na ₂ HPO ₄ , 200 mM NaCl, 500 mM imidazole, pH 7.0), collect the effluent, and use Coomassie brilliant blue detection solution to detect the effluent until no obvious protein is detected . Concentrate the collected target protein with a 30 kDa Amicon ultrafiltration tube, centrifuge at 5000×g for 10 min at 4°C, and dilute the protein solution with preservation buffer (20 mMNa ₂ HPO ₄ , pH 7.0) until the concentration of imidazole less than 10 mmol/L.

Enzyme activity was determined by enzymatic reaction using pure enzyme obtained after Ni-NTA purification. The total volume of the enzymatic reaction is 1 mL, including 600 μL 100 mM sodium glutamate substrate, 300 μL ddH ₂ O and 100 μL enzyme solution of appropriate concentration, mix well and react at 37°C for 30 min, in boiling water Boil in a bath for 10 min to terminate the reaction. After the reaction is over, take 20 μL of the enzymatic reaction solution, add 400 μL of 2 mM 2,4-dinitrophenylhydrazine solution, incubate at 37°C for 20 min, then add 1 mL of 1 M sodium hydroxide solution to terminate reaction. After diluting the chromogenic solution 3-10 times, measure the absorbance (detection wavelength 390nm), and calculate the sample concentration by drawing a standard curve. Enzyme activity is defined as the amount of enzyme required to generate 1 μmol of α-KG per minute is 1 U. The results showed that, as shown in Figure 3, compared with the wild-type enzyme, the specific enzyme activity of the glutamate oxidase mutant was significantly improved, and the amino acid change at the 280-position serine key site was increased from 120 U/mg before mutation to The amino acid changes at 185 U/mg, 280 serine and 533 histidine key sites increased from 120 U/mg before mutation to 215 U/mg, indicating that the catalytic properties of the two mutants were improved to a certain extent.

Example 6 Application of Glutamate Oxidase Mutant in Biotransformation Production of α-KG

This example is used to provide an application of a glutamate oxidase mutant in the production of α-KG. Using the site-directed mutagenesis technique, using the pXMJ19-tac-lgox-katE plasmid constructed in Example 1 above as a template, and using S280T-5F/S280T-3R and H533L-5F/H533L-3R as primers, respectively, to obtain both S280T and H533L The recombinant expression plasmid of the mutation site is named pXMJ19-tac-lgoxS280TH533L-katE. The mutant plasmid was transformed into Corynebacterium glutamicum, and after plate resistance screening, a pXMJ19-tac-lgoxS280TH533L-katE co-expression strain was obtained, and α-KG was produced according to the whole cell transformation process established in Example 4. The specific method is: use a fermenter to establish a 5 L whole-cell catalytic system, add 270 g/L final concentration of sodium glutamate monohydrate (MSG) substrate, add 10 g/L whole-cell sludge, and control the speed of the fermenter 400 r/min, set dissolved oxygen not lower than 25%, catalytic reaction temperature 35°C, catalytic reaction time 32 h. After the reaction, the components of the catalytic reaction solution were quantitatively analyzed by liquid chromatography (HPLC).

As shown in Fig. 4, through liquid chromatography detection and analysis, the components in the whole cell transformation liquid are relatively single, and there are few residues of substrates and impurities. The co-expression strain obtained based on the glutamic acid oxidase mutant showed better α-KG production capacity. After 32 h of whole-cell transformation reaction, the content of α-KG in the transformation solution could reach 205.0 g/L, and the bottom The molar conversion rate can reach 97.5%. Compared with the recombinant engineering bacteria containing the unmutated glutamic acid oxidase gene, it has better advantages in terms of whole-cell catalysis time, product yield and molar conversion rate, which can greatly save production. The method has low cost and simplified technological process, and has good industrial application prospect.

The embodiments of the present invention have been described above. However, the present invention is not limited to the above-mentioned embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

sequence listing

<110> Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences

<120> Glutamate oxidase mutant, dual-enzyme co-expression vector and application thereof

<130> CPCN19111019

<141> 2019-08-22

<160> 29

<170> SIPOSequenceListing 1.0

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<213> Streptomyces mobaraensis

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Met Ala Val Pro Ala Lys Ser Thr Ala Asp Trp Asp Thr Cys Leu Glu

1 5 10 15

Val Ala Arg Ala Leu Leu Val Val Asp Glu His Asp Arg Pro Leu Val

20 25 30

Pro Glu Tyr Lys Lys Ile Leu Asp Asp Gly Leu Pro Arg Thr Gly Lys

35 40 45

Lys Ala Gly Arg Lys Val Leu Val Val Gly Ala Gly Pro Ala Gly Leu

50 55 60

Val Ala Ala Trp Leu Leu Lys Arg Ala Gly His His Val Thr Leu Leu

65 70 75 80

Glu Ala Asn Gly Asn Arg Val Gly Gly Arg Ile Lys Thr Phe Arg Lys

85 90 95

Gly Gly His Glu His Ala Val Gln Pro Phe Ala Asp Pro Arg Gln Tyr

100 105 110

Ala Glu Ala Gly Ala Met Arg Ile Pro Gly Ser His Pro Leu Val Met

115 120 125

Ser Leu Ile Asp Gly Leu Gly Val Lys Arg Arg Pro Phe Tyr Leu Val

130 135 140

Asp Val Asp Gly Gln Gly Lys Pro Val Asn His Ala Trp Leu His Val

145 150 155 160

Asn Gly Val Arg Val Arg Arg Ala Asp Tyr Val Lys Asp Pro Arg Lys

165 170 175

Val Asn Arg Ser Phe Gly Val Pro Arg Glu Leu Trp Asp Thr Pro Ser

180 185 190

Ser Val Ile Leu Arg Arg Val Leu Asp Pro Val Arg Asp Glu Phe Ser

195 200 205

Thr Ala Gly Ala Asp Gly Lys Arg Val Asp Lys Pro Met Pro Glu Arg

210 215 220

Val Lys Gly Trp Ala Arg Val Ile Gln Lys Tyr Gly Asp Trp Ser Met

225 230 235 240

Tyr Arg Phe Leu Thr Glu Glu Ala Gly Phe Asp Glu Arg Thr Leu Asp

245 250 255

Leu Val Gly Thr Leu Glu Asn Leu Thr Ser Arg Leu Pro Leu Ser Phe

260 265 270

Val His Ser Phe Ile Ser Gln Ser Leu Ile Ser Pro Asp Thr Ala Phe

275 280 285

Trp Glu Leu Val Gly Gly Thr Ala Ser Leu Pro Asp Ala Leu Leu Lys

290 295 300

Lys Val Asp Asp Val Leu Arg Leu Asp Arg Arg Ala Thr Arg Ile Glu

305 310 315 320

Tyr Trp Ser Pro Asp Arg Thr Gly Ala Asp Arg Ala Thr His Val Arg

325 330 335

Glu Gly Gly Pro His Val Trp Ile Asp Thr Val Ser Glu Gly Arg Asp

340 345 350

Gly Lys Val Val Arg Glu Gln Phe Thr Gly Asp Leu Ala Ile Val Thr

355 360 365

Val Pro Phe Thr Gly Leu Arg His Val Gln Val Ser Pro Leu Met Ser

370 375 380

Tyr Gly Lys Arg Arg Ala Val Thr Glu Leu His Tyr Asp Ser Ala Thr

385 390 395 400

Lys Val Leu Leu Glu Phe Ser Arg Arg Trp Trp Glu Phe Thr Glu Glu

405 410 415

Asp Trp Lys Arg Glu Leu Glu Asp Val Arg Pro Gly Leu Tyr Ala Ala

420 425 430

Tyr Arg Asp Gly Lys Ala Pro Ala Asp Gly Ser Leu Leu Gly Thr His

435 440 445

Pro Ser Val Pro His Gly His Ile Ser Gln Ala Gln Arg Ala His Tyr

450 455 460

Ala Ala Asn Tyr Trp Glu Gly Arg Asp Gln Pro Glu Ala Ala His Ile

465 470 475 480

Val Gly Gly Gly Ser Val Ser Asp Asn Pro Asn Arg Phe Met Phe Asn

485 490 495

Pro Ser His Pro Val Pro Gly Ser Glu Gly Gly Val Val Leu Ala Val

500 505 510

Tyr Cys Trp Ala Asp Asp Ala Ser Arg Trp Asp Ser Leu Asp Asp Glu

515 520 525

Ala Arg Tyr Pro His Ala Leu Cys Gly Leu Gln Gln Val Tyr Gly Gln

530 535 540

Arg Val Glu Val Phe Tyr Thr Gly Ala Gly Arg Thr Gln Ser Trp Leu

545 550 555 560

Arg Asp Pro Tyr Ala Tyr Gly Glu Ala Ser Val Leu Leu Pro Gly Gln

565 570 575

His Thr Glu Leu Leu Gly Ala Ile Arg Glu Pro Glu Gly Pro Leu His

580 585 590

Phe Ala Gly Asp His Thr Ser Val Lys Pro Ser Trp Ile Glu Gly Ala

595 600 605

Val Glu Ser Gly Val Arg Ala Ala Leu Glu Ala His Leu Ala

610 615 620

<210> 2

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<400> 2

atggcggttc ctgcgaaaag caccgcggat tgggatacct gtctggaagt ggcgcgcgcg 60

ttattatagtgg tggatgaaca tgatcgtccg ctggtgccgg aatataagaa gattctggat 120

gatggcctgc ctcgcaccgg taaaaaagcg ggccgcaaag tgttagttgt tggtgcgggt 180

cctgcaggtt tagttgcggc gtggctgtta aaacgtgcgg gtcatcatgt gactctgctg 240

gaagcgaacg gcaatcgtgt tggcggccgc attaaaacct ttcgcaaagg cggccatgaa 300

catgcggttc agccgtttgc agatccgcgt cagtatgcag aagcaggcgc gatgcgtatt 360

cctggcagcc atccgttagt gatgagcctg attgatggcc tgggtgttaa acgccgcccg 420

ttttatctgg tggatgtgga tggccaaggc aaaccggtta accatgcgtg gctgcatgtg 480

aatggtgttc gcgttcgccg tgcggattat gtgaaagatc cgcgcaaagt gaaccgcagc 540

tttggtgtgc cgcgtgaatt atgggatacc ccgagcagcg ttaattctgcg ccgcgtttta 600

gatcctgtgc gcgatgaatt ttcaaccgcg ggcgcggatg gtaaacgcgt ggataaaccg 660

atgccggaac gcgttaaagg ttgggcgcgc gtgattcaga aatatggcga ctggagcatg 720

tatcgctttc tgaccgaaga agcgggcttt gatgaacgca ccctggattt agttggcacc 780

ctggaaaact taaccagccg cctgccgtta agctttgtgc atagctttat tagccagagc 840

ctgatttcac cggataccgc gttttgggaa ctggttggtg gcaccgcgag cttacctgat 900

gcgctgctga aaaaagtgga tgatgtgctg cgcttagatc gtcgtgcgac ccgcattgaa 960

tattggagcc cggatcgtac tggtgcggat cgtgcgaccc atgttcgtga aggtggtccg 1020

catgtgtgga ttgataccgt gagcgaaggc cgcgatggca aagttgtgcg cgaacagttt 1080

actggcgatc tggcgattgt gaccgtgccg tttaccggtc tgcgccatgt tcaagtgagc 1140

ccgctgatga gctatggtaa acgtcgcgcg gtgaccgaac tgcattatga tagcgcgacc 1200

aaagtgctgc tggaatttag ccgccgctgg tgggaattta ccgaagaaga ttggaaacgc 1260

gaactggaag atgttcgccc gggcttatat gcagcgtatc gcgatggtaa agcgcctgcg 1320

gatggtagct tattaggcac ccatccgtca gttccgcatg gccatattag ccaagcgcag 1380

cgtgcacatt atgcggcgaa ctattgggaa ggccgcgatc aacctgaagc ggcgcatatt 1440

gttggtggtg gcagcgttag cgataacccg aaccgcttta tgtttaaccc gagccatccg 1500

gttcctggta gcgaaggtgg tgttgtgctg gcggtttatt gctgggcgga tgatgcaagc 1560

cgttgggata gcctggatga tgaagcgcgc tatccgcatg cactgtgtgg tctgcaacag 1620

gtttatggcc agcgcgtgga agtgttttat accggcgcgg gtcgtactca atcatggctg 1680

cgtgatccgt atgcgtatgg cgaagcgagc gttttattac ctggccagca taccgaatta 1740

ctgggcgcga ttcgcgaacc tgaaggccct ttacattttg cgggcgatca tacctcagtg 1800

aaaccgagct ggattgaagg tgcggtggaa agcggtgttc gtgcggcgtt agaagcgcat 1860

ttagcgtaa 1869

<210> 3

<211> 753

<212> PRT

<213> Escherichia coli

<400> 3

Met Ser Gln His Asn Glu Lys Asn Pro His Gln His Gln Ser Pro Leu

1 5 10 15

His Asp Ser Ser Glu Ala Lys Pro Gly Met Asp Ser Leu Ala Pro Glu

20 25 30

Asp Gly Ser His Arg Pro Ala Ala Glu Pro Thr Pro Pro Gly Ala Gln

35 40 45

Pro Thr Ala Pro Gly Ser Leu Lys Ala Pro Asp Thr Arg Asn Glu Lys

50 55 60

Leu Asn Ser Leu Glu Asp Val Arg Lys Gly Ser Glu Asn Tyr Ala Leu

65 70 75 80

Thr Thr Asn Gln Gly Val Arg Ile Ala Asp Asp Gln Asn Ser Leu Arg

85 90 95

Ala Gly Ser Arg Gly Pro Thr Leu Leu Glu Asp Phe Ile Leu Arg Glu

100 105 110

Lys Ile Thr His Phe Asp His Glu Arg Ile Pro Glu Arg Ile Val His

115 120 125

Ala Arg Gly Ser Ala Ala His Gly Tyr Phe Gln Pro Tyr Lys Ser Leu

130 135 140

Ser Asp Ile Thr Lys Ala Asp Phe Leu Ser Asp Pro Asn Lys Ile Thr

145 150 155 160

Pro Val Phe Val Arg Phe Ser Thr Val Gln Gly Gly Ala Gly Ser Ala

165 170 175

Asp Thr Val Arg Asp Ile Arg Gly Phe Ala Thr Lys Phe Tyr Thr Glu

180 185 190

Glu Gly Ile Phe Asp Leu Val Gly Asn Asn Thr Pro Ile Phe Phe Ile

195 200 205

Gln Asp Ala His Lys Phe Pro Asp Phe Val His Ala Val Lys Pro Glu

210 215 220

Pro His Trp Ala Ile Pro Gln Gly Gln Ser Ala His Asp Thr Phe Trp

225 230 235 240

Asp Tyr Val Ser Leu Gln Pro Glu Thr Leu His Asn Val Met Trp Ala

245 250 255

Met Ser Asp Arg Gly Ile Pro Arg Ser Tyr Arg Thr Met Glu Gly Phe

260 265 270

Gly Ile His Thr Phe Arg Leu Ile Asn Ala Glu Gly Lys Ala Thr Phe

275 280 285

Val Arg Phe His Trp Lys Pro Leu Ala Gly Lys Ala Ser Leu Val Trp

290 295 300

Asp Glu Ala Gln Lys Leu Thr Gly Arg Asp Pro Asp Phe His Arg Arg

305 310 315 320

Glu Leu Trp Glu Ala Ile Glu Ala Gly Asp Phe Pro Glu Tyr Glu Leu

325 330 335

Gly Phe Gln Leu Ile Pro Glu Glu Asp Glu Phe Lys Phe Asp Phe Asp

340 345 350

Leu Leu Asp Pro Thr Lys Leu Ile Pro Glu Glu Leu Val Pro Val Gln

355 360 365

Arg Val Gly Lys Met Val Leu Asn Arg Asn Pro Asp Asn Phe Phe Ala

370 375 380

Glu Asn Glu Gln Ala Ala Phe His Pro Gly His Ile Val Pro Gly Leu

385 390 395 400

Asp Phe Thr Asn Asp Pro Leu Leu Gln Gly Arg Leu Phe Ser Tyr Thr

405 410 415

Asp Thr Gln Ile Ser Arg Leu Gly Gly Pro Asn Phe His Glu Ile Pro

420 425 430

Ile Asn Arg Pro Thr Cys Pro Tyr His Asn Phe Gln Arg Asp Gly Met

435 440 445

His Arg Met Gly Ile Asp Thr Asn Pro Ala Asn Tyr Glu Pro Asn Ser

450 455 460

Ile Asn Asp Asn Trp Pro Arg Glu Thr Pro Pro Gly Pro Lys Arg Gly

465 470 475 480

Gly Phe Glu Ser Tyr Gln Glu Arg Val Glu Gly Asn Lys Val Arg Glu

485 490 495

Arg Ser Pro Ser Phe Gly Glu Tyr Tyr Ser His Pro Arg Leu Phe Trp

500 505 510

Leu Ser Gln Thr Pro Phe Glu Gln Arg His Ile Val Asp Gly Phe Ser

515 520 525

Phe Glu Leu Ser Lys Val Val Arg Pro Tyr Ile Arg Glu Arg Val Val

530 535 540

Asp Gln Leu Ala His Ile Asp Leu Thr Leu Ala Gln Ala Val Ala Lys

545 550 555 560

Asn Leu Gly Ile Glu Leu Thr Asp Asp Gln Leu Asn Ile Thr Pro Pro

565 570 575

Pro Asp Val Asn Gly Leu Lys Lys Asp Pro Ser Leu Ser Leu Tyr Ala

580 585 590

Ile Pro Asp Gly Asp Val Lys Gly Arg Val Val Ala Ile Leu Leu Asn

595 600 605

Asp Glu Val Arg Ser Ala Asp Leu Leu Ala Ile Leu Lys Ala Leu Lys

610 615 620

Ala Lys Gly Val His Ala Lys Leu Leu Tyr Ser Arg Met Gly Glu Val

625 630 635 640

Thr Ala Asp Asp Gly Thr Val Leu Pro Ile Ala Ala Thr Phe Ala Gly

645 650 655

Ala Pro Ser Leu Thr Val Asp Ala Val Ile Val Pro Cys Gly Asn Ile

660 665 670

Ala Asp Ile Ala Asp Asn Gly Asp Ala Asn Tyr Tyr Leu Met Glu Ala

675 680 685

Tyr Lys His Leu Lys Pro Ile Ala Leu Ala Gly Asp Ala Arg Lys Phe

690 695 700

Lys Ala Thr Ile Lys Ile Ala Asp Gln Gly Glu Glu Gly Ile Val Glu

705 710 715 720

Ala Asp Ser Ala Asp Gly Ser Phe Met Asp Glu Leu Leu Thr Leu Met

725 730 735

Ala Ala His Arg Val Trp Ser Arg Ile Pro Lys Ile Asp Lys Ile Pro

740 745 750

Ala

<210> 4

<211> 2262

<212>DNA

<213> Escherichia coli

<400> 4

atgtcgcaac ataacgaaaa gaacccacat cagcaccagt caccactaca cgattccagc 60

gaagcgaaac cggggatgga ctcactggca cctgaggacg gctctcatcg tccagcggct 120

gaaccaacac cgccaggtgc acaacctacc gccccaggga gcctgaaagc ccctgatacg 180

cgtaacgaaa aacttaattc tctggaagac gtacgcaaag gcagtgaaaa ttatgcgctg 240

accactaatc agggcgtgcg catcgccgac gatcaaaact cactgcgtgc cggtagccgt 300

ggtccaacgc tgctggaaga ttttattctg cgcgagaaaa tcacccactt tgaccatgag 360

cgcattccgg aacgtattgt tcatgcacgc ggatcagccg ctcacggtta tttccagcca 420

tataaaagct taagcgatat taccaaagcg gatttcctct cagatccgaa caaaatcacc 480

ccagtatttg tacgtttctc taccgttcag ggtggtgctg gctctgctga taccgtgcgt 540

gatatccgtg gctttgccac caagttctat accgaagagg gtatttttga cctcgttggc 600

aataacacgc caatcttctt tatccaggat gcgcataaat tccccgattt tgttcatgcg 660

gtaaaaccag aaccgcactg ggcaattcca caagggcaaa gtgcccacga tactttctgg 720

gattatgttt ctctgcaacc tgaaactctg cacaacgtga tgtgggcgat gtcggatcgc 780

ggcatccccc gcagttaccg caccatggaa ggcttcggta ttcacacctt ccgcctgatt 840

aatgccgaag ggaaggcaac gtttgtacgt ttccactgga aaccactggc aggtaaagcc 900

tcactcgttt gggatgaagc acaaaaactc accggacgtg acccggactt ccaccgccgc 960

gagttgtggg aagccattga agcaggcgat tttccggaat acgaactggg cttccagttg 1020

attcctgaag aagatgaatt caagttcgac ttcgatcttc tcgatccaac caaacttatc 1080

ccggaagaac tggtgcccgt tcagcgtgtc ggcaaaatgg tgctcaatcg caacccggat 1140

aacttctttg ctgaaaacga acaggcggct ttccatcctg ggcatatcgt gccgggactg 1200

gacttcacca acgatccgct gttgcaggga cgtttgttct cctataccga tacacaaatc 1260

agtcgtcttg gtgggccgaa tttccatgag attccgatta accgtccgac ctgcccttac 1320

cataatttcc agcgtgacgg catgcatcgc atggggatcg acactaaccc ggcgaattac 1380

gaaccgaact cgattaacga taactggccg cgcgaaacac cgccggggcc gaaacgcggc 1440

ggttttgaat cataccagga gcgcgtggaa ggcaataaag ttcgcgagcg cagcccatcg 1500

tttggcgaat attattccca tccgcgtctg ttctggctaa gtcagacgcc atttgagcag 1560

cgccatattg tcgatggttt cagttttgag ttaagcaaag tcgttcgtcc gtatattcgt 1620

gagcgcgttg ttgaccagct ggcgcatatt gatctcactc tggcccaggc ggtggcgaaa 1680

aatctcggta tcgaactgac tgacgaccag ctgaatatca ccccacctcc ggacgtcaac 1740

ggtctgaaaa aggatccatc cttaagtttg tacgccattc ctgacggtga tgtgaaaggt 1800

cgcgtggtag cgattttact taatgatgaa gtgagatcgg cagaccttct ggccattctc 1860

aaggcgctga aggccaaagg cgttcatgcc aaactgctct actcccgaat gggtgaagtg 1920

actgcggatg acggtacggt gttgcctata gccgctacct ttgccggtgc accttcgctg 1980

acggtcgatg cggtcattgt cccttgcggc aatatcgcgg atatcgctga caacggcgat 2040

gccaactact acctgatgga agcctacaaa caccttaaac cgattgcgct ggcgggtgac 2100

gcgcgcaagt ttaaagcaac aatcaagatc gctgaccagg gtgaagaagg gattgtggaa 2160

gctgacagcg ctgacggtag ttttatggat gaactgctaa cgctgatggc agcacaccgc 2220

gtgtggtcac gcattcctaa gattgacaaa attcctgcct ga 2262

<210> 5

<211>622

<212> PRT

<213> Artificial sequence

<400> 5

Met Ala Val Pro Ala Lys Ser Thr Ala Asp Trp Asp Thr Cys Leu Glu

1 5 10 15

Val Ala Arg Ala Leu Leu Val Val Asp Glu His Asp Arg Pro Leu Val

20 25 30

Pro Glu Tyr Lys Lys Ile Leu Asp Asp Gly Leu Pro Arg Thr Gly Lys

35 40 45

Lys Ala Gly Arg Lys Val Leu Val Val Gly Ala Gly Pro Ala Gly Leu

50 55 60

Val Ala Ala Trp Leu Leu Lys Arg Ala Gly His His Val Thr Leu Leu

65 70 75 80

Glu Ala Asn Gly Asn Arg Val Gly Gly Arg Ile Lys Thr Phe Arg Lys

85 90 95

Gly Gly His Glu His Ala Val Gln Pro Phe Ala Asp Pro Arg Gln Tyr

100 105 110

Ala Glu Ala Gly Ala Met Arg Ile Pro Gly Ser His Pro Leu Val Met

115 120 125

Ser Leu Ile Asp Gly Leu Gly Val Lys Arg Arg Pro Phe Tyr Leu Val

130 135 140

Asp Val Asp Gly Gln Gly Lys Pro Val Asn His Ala Trp Leu His Val

145 150 155 160

Asn Gly Val Arg Val Arg Arg Ala Asp Tyr Val Lys Asp Pro Arg Lys

165 170 175

Val Asn Arg Ser Phe Gly Val Pro Arg Glu Leu Trp Asp Thr Pro Ser

180 185 190

Ser Val Ile Leu Arg Arg Val Leu Asp Pro Val Arg Asp Glu Phe Ser

195 200 205

Thr Ala Gly Ala Asp Gly Lys Arg Val Asp Lys Pro Met Pro Glu Arg

210 215 220

Val Lys Gly Trp Ala Arg Val Ile Gln Lys Tyr Gly Asp Trp Ser Met

225 230 235 240

Tyr Arg Phe Leu Thr Glu Glu Ala Gly Phe Asp Glu Arg Thr Leu Asp

245 250 255

Leu Val Gly Thr Leu Glu Asn Leu Thr Ser Arg Leu Pro Leu Ser Phe

260 265 270

Val His Ser Phe Ile Ser Gln Thr Leu Ile Ser Pro Asp Thr Ala Phe

275 280 285

Trp Glu Leu Val Gly Gly Thr Ala Ser Leu Pro Asp Ala Leu Leu Lys

290 295 300

Lys Val Asp Asp Val Leu Arg Leu Asp Arg Arg Ala Thr Arg Ile Glu

305 310 315 320

Tyr Trp Ser Pro Asp Arg Thr Gly Ala Asp Arg Ala Thr His Val Arg

325 330 335

Glu Gly Gly Pro His Val Trp Ile Asp Thr Val Ser Glu Gly Arg Asp

340 345 350

Gly Lys Val Val Arg Glu Gln Phe Thr Gly Asp Leu Ala Ile Val Thr

355 360 365

Val Pro Phe Thr Gly Leu Arg His Val Gln Val Ser Pro Leu Met Ser

370 375 380

Tyr Gly Lys Arg Arg Ala Val Thr Glu Leu His Tyr Asp Ser Ala Thr

385 390 395 400

Lys Val Leu Leu Glu Phe Ser Arg Arg Trp Trp Glu Phe Thr Glu Glu

405 410 415

Asp Trp Lys Arg Glu Leu Glu Asp Val Arg Pro Gly Leu Tyr Ala Ala

420 425 430

Tyr Arg Asp Gly Lys Ala Pro Ala Asp Gly Ser Leu Leu Gly Thr His

435 440 445

Pro Ser Val Pro His Gly His Ile Ser Gln Ala Gln Arg Ala His Tyr

450 455 460

Ala Ala Asn Tyr Trp Glu Gly Arg Asp Gln Pro Glu Ala Ala His Ile

465 470 475 480

Val Gly Gly Gly Ser Val Ser Asp Asn Pro Asn Arg Phe Met Phe Asn

485 490 495

Pro Ser His Pro Val Pro Gly Ser Glu Gly Gly Val Val Leu Ala Val

500 505 510

Tyr Cys Trp Ala Asp Asp Ala Ser Arg Trp Asp Ser Leu Asp Asp Glu

515 520 525

Ala Arg Tyr Pro His Ala Leu Cys Gly Leu Gln Gln Val Tyr Gly Gln

530 535 540

Arg Val Glu Val Phe Tyr Thr Gly Ala Gly Arg Thr Gln Ser Trp Leu

545 550 555 560

Arg Asp Pro Tyr Ala Tyr Gly Glu Ala Ser Val Leu Leu Pro Gly Gln

565 570 575

His Thr Glu Leu Leu Gly Ala Ile Arg Glu Pro Glu Gly Pro Leu His

580 585 590

Phe Ala Gly Asp His Thr Ser Val Lys Pro Ser Trp Ile Glu Gly Ala

595 600 605

Val Glu Ser Gly Val Arg Ala Ala Leu Glu Ala His Leu Ala

610 615 620

<210> 6

<211> 1869

<212>DNA

<213> Artificial sequence

<400> 6

atggcggttc ctgcgaaaag caccgcggat tgggatacct gtctggaagt ggcgcgcgcg 60

ttattatagtgg tggatgaaca tgatcgtccg ctggtgccgg aatataagaa gattctggat 120

gatggcctgc ctcgcaccgg taaaaaagcg ggccgcaaag tgttagttgt tggtgcgggt 180

cctgcaggtt tagttgcggc gtggctgtta aaacgtgcgg gtcatcatgt gactctgctg 240

gaagcgaacg gcaatcgtgt tggcggccgc attaaaacct ttcgcaaagg cggccatgaa 300

catgcggttc agccgtttgc agatccgcgt cagtatgcag aagcaggcgc gatgcgtatt 360

cctggcagcc atccgttagt gatgagcctg attgatggcc tgggtgttaa acgccgcccg 420

ttttatctgg tggatgtgga tggccaaggc aaaccggtta accatgcgtg gctgcatgtg 480

aatggtgttc gcgttcgccg tgcggattat gtgaaagatc cgcgcaaagt gaaccgcagc 540

tttggtgtgc cgcgtgaatt atgggatacc ccgagcagcg ttaattctgcg ccgcgtttta 600

gatcctgtgc gcgatgaatt ttcaaccgcg ggcgcggatg gtaaacgcgt ggataaaccg 660

atgccggaac gcgttaaagg ttgggcgcgc gtgattcaga aatatggcga ctggagcatg 720

tatcgctttc tgaccgaaga agcgggcttt gatgaacgca ccctggattt agttggcacc 780

ctggaaaact taaccagccg cctgccgtta agctttgtgc atagctttat tagccagacc 840

ctgatttcac cggataccgc gttttgggaa ctggttggtg gcaccgcgag cttacctgat 900

gcgctgctga aaaaagtgga tgatgtgctg cgcttagatc gtcgtgcgac ccgcattgaa 960

tattggagcc cggatcgtac tggtgcggat cgtgcgaccc atgttcgtga aggtggtccg 1020

catgtgtgga ttgataccgt gagcgaaggc cgcgatggca aagttgtgcg cgaacagttt 1080

actggcgatc tggcgattgt gaccgtgccg tttaccggtc tgcgccatgt tcaagtgagc 1140

ccgctgatga gctatggtaa acgtcgcgcg gtgaccgaac tgcattatga tagcgcgacc 1200

aaagtgctgc tggaatttag ccgccgctgg tgggaattta ccgaagaaga ttggaaacgc 1260

gaactggaag atgttcgccc gggcttatat gcagcgtatc gcgatggtaa agcgcctgcg 1320

gatggtagct tattaggcac ccatccgtca gttccgcatg gccatattag ccaagcgcag 1380

cgtgcacatt atgcggcgaa ctattgggaa ggccgcgatc aacctgaagc ggcgcatatt 1440

gttggtggtg gcagcgttag cgataacccg aaccgcttta tgtttaaccc gagccatccg 1500

gttcctggta gcgaaggtgg tgttgtgctg gcggtttatt gctgggcgga tgatgcaagc 1560

cgttgggata gcctggatga tgaagcgcgc tatccgcatg cactgtgtgg tctgcaacag 1620

gtttatggcc agcgcgtgga agtgttttat accggcgcgg gtcgtactca atcatggctg 1680

cgtgatccgt atgcgtatgg cgaagcgagc gttttattac ctggccagca taccgaatta 1740

ctgggcgcga ttcgcgaacc tgaaggccct ttacattttg cgggcgatca tacctcagtg 1800

aaaccgagct ggattgaagg tgcggtggaa agcggtgttc gtgcggcgtt agaagcgcat 1860

ttagcgtaa 1869

<210> 7

<211>622

<212> PRT

<213> Artificial sequence

<400> 7

Met Ala Val Pro Ala Lys Ser Thr Ala Asp Trp Asp Thr Cys Leu Glu

1 5 10 15

Val Ala Arg Ala Leu Leu Val Val Asp Glu His Asp Arg Pro Leu Val

20 25 30

Pro Glu Tyr Lys Lys Ile Leu Asp Asp Gly Leu Pro Arg Thr Gly Lys

35 40 45

Lys Ala Gly Arg Lys Val Leu Val Val Gly Ala Gly Pro Ala Gly Leu

50 55 60

Val Ala Ala Trp Leu Leu Lys Arg Ala Gly His His Val Thr Leu Leu

65 70 75 80

Glu Ala Asn Gly Asn Arg Val Gly Gly Arg Ile Lys Thr Phe Arg Lys

85 90 95

Gly Gly His Glu His Ala Val Gln Pro Phe Ala Asp Pro Arg Gln Tyr

100 105 110

Ala Glu Ala Gly Ala Met Arg Ile Pro Gly Ser His Pro Leu Val Met

115 120 125

Ser Leu Ile Asp Gly Leu Gly Val Lys Arg Arg Pro Phe Tyr Leu Val

130 135 140

Asp Val Asp Gly Gln Gly Lys Pro Val Asn His Ala Trp Leu His Val

145 150 155 160

Asn Gly Val Arg Val Arg Arg Ala Asp Tyr Val Lys Asp Pro Arg Lys

165 170 175

Val Asn Arg Ser Phe Gly Val Pro Arg Glu Leu Trp Asp Thr Pro Ser

180 185 190

Ser Val Ile Leu Arg Arg Val Leu Asp Pro Val Arg Asp Glu Phe Ser

195 200 205

Thr Ala Gly Ala Asp Gly Lys Arg Val Asp Lys Pro Met Pro Glu Arg

210 215 220

Val Lys Gly Trp Ala Arg Val Ile Gln Lys Tyr Gly Asp Trp Ser Met

225 230 235 240

Tyr Arg Phe Leu Thr Glu Glu Ala Gly Phe Asp Glu Arg Thr Leu Asp

245 250 255

Leu Val Gly Thr Leu Glu Asn Leu Thr Ser Arg Leu Pro Leu Ser Phe

260 265 270

Val His Ser Phe Ile Ser Gln Thr Leu Ile Ser Pro Asp Thr Ala Phe

275 280 285

Trp Glu Leu Val Gly Gly Thr Ala Ser Leu Pro Asp Ala Leu Leu Lys

290 295 300

Lys Val Asp Asp Val Leu Arg Leu Asp Arg Arg Ala Thr Arg Ile Glu

305 310 315 320

Tyr Trp Ser Pro Asp Arg Thr Gly Ala Asp Arg Ala Thr His Val Arg

325 330 335

Glu Gly Gly Pro His Val Trp Ile Asp Thr Val Ser Glu Gly Arg Asp

340 345 350

Gly Lys Val Val Arg Glu Gln Phe Thr Gly Asp Leu Ala Ile Val Thr

355 360 365

Val Pro Phe Thr Gly Leu Arg His Val Gln Val Ser Pro Leu Met Ser

370 375 380

Tyr Gly Lys Arg Arg Ala Val Thr Glu Leu His Tyr Asp Ser Ala Thr

385 390 395 400

Lys Val Leu Leu Glu Phe Ser Arg Arg Trp Trp Glu Phe Thr Glu Glu

405 410 415

Asp Trp Lys Arg Glu Leu Glu Asp Val Arg Pro Gly Leu Tyr Ala Ala

420 425 430

Tyr Arg Asp Gly Lys Ala Pro Ala Asp Gly Ser Leu Leu Gly Thr His

435 440 445

Pro Ser Val Pro His Gly His Ile Ser Gln Ala Gln Arg Ala His Tyr

450 455 460

Ala Ala Asn Tyr Trp Glu Gly Arg Asp Gln Pro Glu Ala Ala His Ile

465 470 475 480

Val Gly Gly Gly Ser Val Ser Asp Asn Pro Asn Arg Phe Met Phe Asn

485 490 495

Pro Ser His Pro Val Pro Gly Ser Glu Gly Gly Val Val Leu Ala Val

500 505 510

Tyr Cys Trp Ala Asp Asp Ala Ser Arg Trp Asp Ser Leu Asp Asp Glu

515 520 525

Ala Arg Tyr Pro Leu Ala Leu Cys Gly Leu Gln Gln Val Tyr Gly Gln

530 535 540

Arg Val Glu Val Phe Tyr Thr Gly Ala Gly Arg Thr Gln Ser Trp Leu

545 550 555 560

Arg Asp Pro Tyr Ala Tyr Gly Glu Ala Ser Val Leu Leu Pro Gly Gln

565 570 575

His Thr Glu Leu Leu Gly Ala Ile Arg Glu Pro Glu Gly Pro Leu His

580 585 590

Phe Ala Gly Asp His Thr Ser Val Lys Pro Ser Trp Ile Glu Gly Ala

595 600 605

Val Glu Ser Gly Val Arg Ala Ala Leu Glu Ala His Leu Ala

610 615 620

<210> 8

<211> 1869

<212>DNA

<213> Artificial sequence

<400> 8

atggcggttc ctgcgaaaag caccgcggat tgggatacct gtctggaagt ggcgcgcgcg 60

ttattatagtgg tggatgaaca tgatcgtccg ctggtgccgg aatataagaa gattctggat 120

gatggcctgc ctcgcaccgg taaaaaagcg ggccgcaaag tgttagttgt tggtgcgggt 180

cctgcaggtt tagttgcggc gtggctgtta aaacgtgcgg gtcatcatgt gactctgctg 240

gaagcgaacg gcaatcgtgt tggcggccgc attaaaacct ttcgcaaagg cggccatgaa 300

catgcggttc agccgtttgc agatccgcgt cagtatgcag aagcaggcgc gatgcgtatt 360

cctggcagcc atccgttagt gatgagcctg attgatggcc tgggtgttaa acgccgcccg 420

ttttatctgg tggatgtgga tggccaaggc aaaccggtta accatgcgtg gctgcatgtg 480

aatggtgttc gcgttcgccg tgcggattat gtgaaagatc cgcgcaaagt gaaccgcagc 540

tttggtgtgc cgcgtgaatt atgggatacc ccgagcagcg ttaattctgcg ccgcgtttta 600

gatcctgtgc gcgatgaatt ttcaaccgcg ggcgcggatg gtaaacgcgt ggataaaccg 660

atgccggaac gcgttaaagg ttgggcgcgc gtgattcaga aatatggcga ctggagcatg 720

tatcgctttc tgaccgaaga agcgggcttt gatgaacgca ccctggattt agttggcacc 780

ctggaaaact taaccagccg cctgccgtta agctttgtgc atagctttat tagccagacc 840

ctgatttcac cggataccgc gttttgggaa ctggttggtg gcaccgcgag cttacctgat 900

gcgctgctga aaaaagtgga tgatgtgctg cgcttagatc gtcgtgcgac ccgcattgaa 960

tattggagcc cggatcgtac tggtgcggat cgtgcgaccc atgttcgtga aggtggtccg 1020

catgtgtgga ttgataccgt gagcgaaggc cgcgatggca aagttgtgcg cgaacagttt 1080

actggcgatc tggcgattgt gaccgtgccg tttaccggtc tgcgccatgt tcaagtgagc 1140

ccgctgatga gctatggtaa acgtcgcgcg gtgaccgaac tgcattatga tagcgcgacc 1200

aaagtgctgc tggaatttag ccgccgctgg tgggaattta ccgaagaaga ttggaaacgc 1260

gaactggaag atgttcgccc gggcttatat gcagcgtatc gcgatggtaa agcgcctgcg 1320

gatggtagct tattaggcac ccatccgtca gttccgcatg gccatattag ccaagcgcag 1380

cgtgcacatt atgcggcgaa ctattgggaa ggccgcgatc aacctgaagc ggcgcatatt 1440

gttggtggtg gcagcgttag cgataacccg aaccgcttta tgtttaaccc gagccatccg 1500

gttcctggta gcgaaggtgg tgttgtgctg gcggtttatt gctgggcgga tgatgcaagc 1560

cgttgggata gcctggatga tgaagcgcgc tatccgcttg cactgtgtgg tctgcaacag 1620

gtttatggcc agcgcgtgga agtgttttat accggcgcgg gtcgtactca atcatggctg 1680

cgtgatccgt atgcgtatgg cgaagcgagc gttttattac ctggccagca taccgaatta 1740

ctgggcgcga ttcgcgaacc tgaaggccct ttacattttg cgggcgatca tacctcagtg 1800

aaaccgagct ggattgaagg tgcggtggaa agcggtgttc gtgcggcgtt agaagcgcat 1860

ttagcgtaa 1869

<210> 9

<211> 81

<212>DNA

<213> Artificial sequence

<400> 9

aactgttgac aattaatcat cggctcgtat aatgtgtgga attgtgagcg gataacaatt 60

tcacacagga aacagaatta a 81

<210> 10

<211> 66

<212>DNA

<213> Artificial sequence

<400> 10

tgaaatgagc tgttgacaat taatcatccg gctcgtataa tgtgtggaat tgtgagcgga 60

taacaa 66

<210> 11

<211> 21

<212>DNA

<213> Artificial sequence

<400> 11

ctttaagaag gagatataca t 21

<210> 12

<211> 64

<212>DNA

<213> Artificial sequence

<400> 12

cactacgcac ctgcaaaata agctttaaga aggagatata catatggcgg ttcctgcgaa 60

aagc 64

<210> 13

<211> 64

<212>DNA

<213> Artificial sequence

<400> 13

cttttcgtta tgttgcgaca tatgtatatc tccttcttaa agttacgcta aatgcgcttc 60

taac 64

<210> 14

<211> 64

<212>DNA

<213> Artificial sequence

<400> 14

gttagaagcg catttagcgt aactttaaga aggagatata catatgtcgc aacataacga 60

aaag 64

<210> 15

<211> 47

<212>DNA

<213> Artificial sequence

<400> 15

cactacgcac ctgcaaaaat gctcaggcag gaattttgtc aatctta 47

<210> 16

<211> 41

<212>DNA

<213> Artificial sequence

<400> 16

cactacgcac ctgcaaaagc atgcctgcag gtcgactcta g 41

<210> 17

<211> 42

<212>DNA

<213> Artificial sequence

<400> 17

cactacgcac ctgcaaaact taattaattc tgtttcctgt gt 42

<210> 18

<211> 48

<212>DNA

<213> Artificial sequence

<400> 18

cactacgcac ctgcaaaact atgccaagtg tgcttcgagt gctgcacg 48

<210> 19

<211> 60

<212>DNA

<213> Artificial sequence

<400> 19

cactacgcac ctgcaaaact ttaagaagga gatatacata tgtcgcaaca taacgaaaag 60

<210> 20

<211> 85

<212>DNA

<213> Artificial sequence

<400> 20

atagaactgt tgacaattaa tcatcggctc gtataatgtg tggaattgtg agcggataac 60

aatttcacac aggaaacaga attaa 85

<210> 21

<211> 83

<212>DNA

<213> Artificial sequence

<400> 21

aaagttaatt ctgtttcctg tgtgaaattg ttatccgctc acaattccac attatacg 60

agccgatgat taagtcaaca gtt 83

<210> 22

<211> 70

<212>DNA

<213> Artificial sequence

<400> 22

atagtgaaat gagctgttga caattaatca tccggctcgt ataatgtgtg gaattgtgag 60

cggataacaa 70

<210> 23

<211> 70

<212>DNA

<213> Artificial sequence

<400> 23

aaagttgtta tccgctcaca attccacaca ttatacgagc cggatgatta attgtcaaca 60

gctcatttca 70

<210> 24

<211> 35

<212>DNA

<213> Artificial sequence

<400> 24

gctttattag ccagaccctg atttcaccgg atacc 35

<210> 25

<211> 34

<212>DNA

<213> Artificial sequence

<400> 25

aaatcagggt ctggctaata aagctatgca caaa 34

<210> 26

<211> 33

<212>DNA

<213> Artificial sequence

<400> 26

aagcgcgcta tccgcttgca ctgtgtggtc tgc 33

<210> 27

<211> 32

<212>DNA

<213> Artificial sequence

<400> 27

acagtgcaag cggatagcgc gcttcatcat cc 32

<210> 28

<211> 30

<212>DNA

<213> Artificial sequence

<400> 28

catgcatatg gcggttcctg cgaaaagcac 30

<210> 29

<211> 40

<212>DNA

<213> Artificial sequence

<400> 29

ctagctcgag cgctaaatgc gcttctaacg ccgcacgaac 40

Claims (16)

1. a glutamic acid oxidase mutant, is characterized in that, the aminoacid sequence of described glutamic acid oxidase mutant is selected from following group: a) the amino acid sequence corresponding to the 280th amino acid of SEQ ID NO:1 mutated from serine S to threonine T, or b) The amino acid sequence corresponding to the 280th amino acid of SEQ ID NO: 1 is mutated from serine S to threonine T, and the 533rd amino acid corresponding to SEQ ID NO: 1 is mutated from histidine H to leucine Amino acid sequence of L. 2. The coding gene of the glutamate oxidase mutant according to claim 1, wherein the coding gene of the amino acid sequence of the group a) is the nucleotide sequence shown in SEQ ID NO:6. 3. The coding gene of the glutamic acid oxidase mutant according to claim 1, wherein the coding gene of the amino acid sequence of the b) group is the nucleotide sequence shown in SEQ ID NO:8. 4. A recombinant bacterial strain, which is obtained by introducing the vector containing the coding gene of claim 1-the mutant or the coding gene of claim 2 or 3 into the host bacterial strain. 5. Application of the glutamate oxidase mutant according to claim 1, the coding gene according to claim 2 or 3, or the recombinant strain according to claim 4 in the production of α-ketoglutarate. 6. A double-enzyme co-expression vector, wherein the co-expression vector comprises the coding gene of the glutamic acid oxidase mutant and the coding gene of catalase in one plasmid. 7. The dual-enzyme co-expression vector according to claim 6, the coding gene of the glutamic acid oxidase mutant is the nucleotide sequence shown in SEQ ID NO:6 or SEQ ID NO:8. 8. The dual-enzyme co-expression vector according to claim 6 or 7, wherein the gene encoding catalase in the dual-enzyme co-expression vector contains the nucleotide sequence shown in SEQ ID NO:4. 9. The double-enzyme co-expression carrier according to claim 6 or 7, wherein the double-enzyme co-expression carrier adopts a single promoter mode to combine promoter and the coding gene of the glutamic acid oxidase mutant, catalase The coding gene of the glutamate oxidase mutant is expressed in tandem; or the dual promoter mode is used, and the promoters are respectively added before the coding gene of the glutamate oxidase mutant and the coding gene of catalase. 10. The dual-enzyme co-expression vector according to claim 9, wherein the promoter is a strong tac promoter or a strong trc promoter. 11. A dual-enzyme co-expression recombinant strain, containing the dual-enzyme co-expression vector according to any one of claims 6-10. 12. The dual-enzyme co-expression recombinant strain according to claim 11, wherein the recombinant strain is to introduce the dual-enzyme co-expression vector into a host strain, and the host strain is selected from Corynebacterium glutamicum , large intestine Bacillus ( Escherichia coli ) or Bacillus subtilis ( Bacillus subtilis ). 13. The application of the dual-enzyme co-expression vector according to any one of claims 6-10 in the preparation of α-ketoglutarate. 14. The application of the dual-enzyme co-expression recombinant strain according to any one of claims 11-12 in the preparation of α-ketoglutarate. 15. A method for preparing α-ketoglutarate, comprising culturing the dual-enzyme co-expression recombinant strain according to claim 11 or 12. 16. The method for preparing α-ketoglutarate according to claim 15, said method comprising taking glutamate as a substrate, and using said double enzyme co-expression recombinant strain to carry out whole-cell catalytic preparation of α-ketoglutarate Diacid.