CN110283800B - Glucose oxidation enzyme mutant, double enzyme coexpression vectors and its application - Google Patents

Abstract

The invention discloses one kind by carrying out rite-directed mutagenesis to L-GLOD, obtain a kind of glucose oxidation enzyme mutant that enzyme activity significantly improves, co-express the mutant enzyme and catalase building recombinant strain, using Pidolidone salt as substrate, α-ketoglutaric acid is prepared under normal temperature and pressure conditions.Simple process of the present invention has the characteristics that high conversion, easily separated purifying, working condition is mild, environmental pollution is small, has preferable industrial applications prospect.

Description

Glucose oxidation enzyme mutant, double enzyme coexpression vectors and its application

Technical field

The present invention relates to a kind of glucose oxidation enzyme mutant, double enzyme coexpression vectors and its applications, belong to bioengineering Technical field.

Background technique

α-ketoglutaric acid (α-KG) is a kind of important organic acid, is played an important role in organic cellular metabolic, It is also the important precursor of synthesis various saccharides, amino acid and protein etc., in food, medicine, feed, chemical industry and makeup It has broad application prospects in the industries such as product.In recent years, α-KG is widely used in sport nutrition beverage and health care product, α-KG warp After human consumption absorbs, excessive ammonia be can be incorporated on α-KG to reduce the problem of ammonia poisoning is brought in vivo, simultaneously participate in body The metabolic processes such as interior nitrogen metabolism.In addition, α-KG also can be used as physique reinforcing agent, in conjunction with ornithine after, can improve growth hormone, The level of insulin, and can inhibit the decomposition of muscle fibre, play reparation muscle damage under the premise of reducing protein depletion Effect, health value with higher.

Currently, the production method of α-KG includes chemical synthesis, microbe fermentation method and biological catalysis.Traditional α-KG is raw It produces and uses chemical synthesis, but the harmful reagents such as strong acid and strong base used in chemical synthesis process, cyanide, be not only easy to draw Environmental pollution is played, more limits it in the application of the industries such as food, cosmetics and medicine.Microbe fermentation method fermentation period is long, And the by-products such as pyruvic acid, fumaric acid are more in tunning, will increase difficulty and expense that subsequent α-KG is extracted, not yet realize Industrial mass production.Production by Enzymes α-KG has the advantages such as reaction time is short, conversion ratio is high, and ox expects clear wait by mutagenic obtained The streptomycete mutant strain of high yield dglutamic oxidase, converts 24 h in optimal conditions, and α-KG yield is expected up to 38.1 g/L(oxen Clearly, bioengineering journal, 2014,8:1318-22 are waited)；Using the method for genetic engineering, by glucose oxidation enzyme gene The heterogenous expression in Escherichia coli, by enzyme law catalysis, 24 h generate 104.7 g/L(Niu PQ, et al. J of α-KG yield Biotechnol, 2014,179:56-62).Catalysed in vitro is carried out using purifying enzyme not only to need by cumbersome protein purification Step, and a large amount of expensive catalase auxiliary catalysis of external source addition are needed in reaction process, considerably increase industrial cost. Catalysed in vitro is carried out compared to separation enzyme, whole-cell biocatalyst is easier to prepare, and catalytic performance is more stable, not vulnerable to the external world The influence of the factors such as temperature, pH；External source addition confactor, and the generation without poisonous and harmful product, ring are not required in conversion process Border is friendly, has industrial applications prospect.

Dglutamic oxidase (L-glutamate oxidase, LGOX) is a kind of The flavoprotein enzyme of prothetic group, the substrate alloisomerism selectivity with height, high catalytic efficiency, reaction condition is mild, can be Oxidation Pidolidone generates hydrogen peroxide, ammonia and α-ketoglutaric acid under conditions of not adding exogenous confactor.The study found that The enzyme is primarily present in streptomycete, but enzymatic productivity difference is larger.It excavates and identification has high activity or substrate specificity Dglutamic oxidase, and the enzymatic system for meeting physiological Study and production application requirement is obtained, it will be mentioned for industrial production α-KG For advantage.

Summary of the invention

It is an object of the invention to utilize genetic engineering means to microbe-derived L-GLOD and peroxidating Hydrogen enzyme carries out coexpression optimization, has constructed the excellent coexpression recombinant bacterial strain of catalytic performance, and establish height using the bacterial strain The resting cell method of effect production α-KG, as shown in Figure 1.

It is an object of the present invention to provide the glucose oxidation enzyme mutants that a kind of enzyme activity improves, and are that will correspond to The 280th amino acids of SEQ ID NO:1 are sported the amino acid sequence of threonine T by serine S；Or the glutamic acid oxygen Changing enzyme mutant to be further comprises sporting leucine by histidine H in the 533rd amino acids corresponding to SEQ ID NO:1 The amino acid sequence of the double-site mutant of L.In one embodiment, the glucose oxidation enzyme mutant includes such as SEQ ID Amino acid sequence shown in NO:5, the coding nucleotide sequence of the glucose oxidation enzyme mutant can be SEQ ID NO:6 Shown in nucleotide sequence；In another embodiment, the glucose oxidation enzyme mutant includes such as SEQ ID NO:7 institute The amino acid sequence shown, the coding nucleotide sequence of the glucose oxidation enzyme mutant can be shown in SEQ ID NO:8 Nucleotide sequence.Dglutamic oxidase mutant code nucleotide sequence of the present invention can be from SEQ ID NO:2 Shown in wild type L-GLOD coding nucleotide sequence, such as the coding nucleotide sequence of the mutant can be with It is that nucleotide sequence shown in SEQ ID NO:2 is mutated or the nucleotide of specific site is substituted and obtains.

In one embodiment, the wild type L-GLOD derives from luxuriant source streptomycete (Streptomyces mobaraensis).

In one embodiment, the amino acid sequence of the glucose oxidation enzyme mutant can also include 1-10 its Substitution, missing or the insertion of his amino acid residue, but still keep L-GLOD activity.

The present invention also provides the recombinant bacterial strains for expressing above-mentioned glucose oxidation enzyme mutant, are by the coding of the mutant Obtained by nucleotide sequence or vector introduction host strain containing the coding nucleotide sequence.The recombinant bacterial strain can be used for Produce α-ketoglutaric acid.

Second object of the present invention is to provide a kind of double enzyme coexpression vectors for producing α-KG, the coexpression vector It is in a plasmid while to import the encoding gene of L-GLOD and catalase.

According to the present invention, the L-GLOD derives from streptomyces, and the catalase derives from intestines bar Pseudomonas.According to the present invention, the carrier for double enzymes coexpressions is not particularly limited, can for can in bacterial strain table Up to the various expression vectors commonly used in the art of target gene, such as E. coli-C. glutamicum shuttle expression plasmid PXMJ19 or pDXW serial carrier or pBL1 serial carrier or colibacillus expression plasmid pET serial carrier or pBAD system It lists body or bacillus subtilis expression plasmid pHT01 or pMA5 carrier etc., the expression carries in a preferred embodiment Body is pXMJ19 plasmid.In one embodiment, the L-GLOD contains the amino as shown in SEQ ID NO:1 Acid sequence, coding nucleotide sequence is as shown in SEQ ID NO:2.In another embodiment, the Pidolidone oxidation Enzyme is the glucose oxidation enzyme mutant as described in the first purpose, the amino acid of the mutant such as SEQ ID NO:5 or SEQ Shown in ID NO:7, coding nucleotide sequence is as shown in SEQ ID NO:6 or SEQ ID NO:8.

In one embodiment, catalase contains as shown in SEQ ID NO:3 in double enzyme coexpression vectors Amino acid sequence, coding nucleotide sequence is as shown in SEQ ID NO:4.

The present invention provides the double enzyme coexpression systems of a variety of simple substance grains, express Pidolidone oxidation simultaneously in a plasmid The encoding gene of enzyme and the encoding gene of catalase.In one embodiment, double enzyme coexpression vectors are to use It is single to start subpattern for the encoding gene of the encoding gene and hydrogen peroxide of a promoter and the L-GLOD Expressing in series is carried out, described two encoding genes can be connected with random order；In another embodiment, double enzymes are total to table It up to carrier is added respectively before the encoding gene of L-GLOD and catalase respectively using double-promoter mode Promoter.The promoter can be tac strong promoter or trc strong promoter.In one embodiment, it is preferred to single starting Subpattern, and promoter is tac strong promoter.

In embodiments of the invention, in singly starting subpattern, conservative can be increased between two encoding genes RBS correlated series are split；Or RBS correlation is increased separately in double-promoter mode, between promoter and encoding gene Sequence.

In a specific embodiment, using single tac strong promoter by L-GLOD encoding gene and mistake Hydrogen oxide encoding gene carry out expressing in series, wherein between L-GLOD encoding gene and hydrogen peroxide enzyme coding gene by Conservative RBS correlated series are divided；In another embodiment, using double-promoter mode respectively in Pidolidone Tac strong promoter and conservative RBS correlated series are added before aoxidizing enzyme coding gene and hydrogen peroxide enzyme coding gene；Another In a specific embodiment, added before L-GLOD encoding gene using double-promoter mode tac strong promoter and Conservative RBS correlated series add trc strong promoter and conservative RBS correlated series before catalase gene.

Promoter and RBS correlated series of the present invention for coexpression system building are as follows:

Third object of the present invention is to provide a kind of double enzymes coexpression recombinant bacterial strain for producing α-KG, the recombinant bacterium Strain contains double enzyme coexpression vectors as described in the second purpose.In one embodiment, the recombinant bacterial strain be will be described double The conversion of enzyme coexpression vector imports obtained by host strain.Host strain of the invention is can to express the dglutamic oxidase With the bacterial strain of catalase, the host strain can be selected from Corynebacterium, Escherichia or bacillus, such as For Corynebacterium glutamicum (Corynebacterium glutamicum), Escherichia coli (Escherichia coli) or withered grass bud Spore bacillus (Bacillus subtilis)；Preferably Corynebacterium glutamicum (Corynebacterium glutamicum), it is more excellent It is selected as Corynebacterium glutamicum ATCC 13032.

Fourth object of the present invention is to provide a kind of whole-cell catalytic bacterium mud, and the bacterium mud includes described in third purpose Double enzymes co-express recombinant bacterial strain；Specifically, the recombinant bacterial strain is prepared by high density liquid submerged fermentation.

In one embodiment, the whole-cell catalytic bacterium mud is prepared with the following method:

(1) coexpression recombinant bacterial strain described in third purpose is inoculated in seed culture medium culture；

(2) seed liquor is then inoculated in fermentation medium fermentation, optionally addition isopropyl-beta D-thio galactopyranosyl Glucosides (IPTG) induces destination protein expression, collects somatic cells, obtains the whole-cell catalytic bacterium mud.

In a specific embodiment, the whole-cell catalytic bacterium mud the preparation method is as follows:

The coexpression recombinant bacterial strain is inoculated in containing 100 mL seed culture mediums (2.5 g/L of yeast powder, albumen Peptone 5 g/L, NaCl 5 g/L, 18.5 g/L of brain heart infusion, 91 g/L of sorbierite) 500 mL triangular flasks in, in 30 °C Shaken cultivation 15-18 h；According to the switching of 5% inoculum concentration in being already equipped with 5 L fermentation mediums (glucose 100 g/L of sugar, corn pulp 15 g/L, 20 g/L of ammonium sulfate, 1 g/L of magnesium sulfate, 0.5 g/L of potassium dihydrogen phosphate, 0.1 g/L of dipotassium hydrogen phosphate, lemon Sour 2 g/L of sodium, 2 g/L of calcium carbonate；PH 7.0) fermentor in, set 30 ~ 32 °C of cultivation temperature, control culture pH value be 7.0, culture beforehand control revolving speed is 300 r/min, when dissolved oxygen is down to 20 % hereinafter, setting revolving speed and dissolved oxygen are coupled.To thallus Concentration OD₆₀₀When growing to about 25 ~ 30, the isopropyl-beta D-thio galactopyranoside (IPTG) of final concentration of 0.4 mM is added Destination protein expression is induced, setting inducing temperature is 25 ~ 30 °C, and control culture pH value is 7.0, about 24 h of induction time, thallus The ultimate density OD of growth₆₀₀Up to 150.0 or so.After the completion of entire Induction Process, by above-mentioned fermentation culture medium carry out from The heart collects somatic cells to get whole-cell catalytic bacterium mud.

Of the invention the 5th is designed to provide above-mentioned glucose oxidation enzyme mutant, double enzyme coexpression vectors, recombination The application of bacterial strain or whole-cell catalytic bacterium mud in preparation α-ketoglutaric acid.

The sixth object of the present invention also provides a kind of method of whole-cell catalytic preparation α-ketoglutaric acid, including culture third Double enzymes described in purpose co-express recombinant bacterial strain.

In one embodiment, it is the addition sodium glutamate monohydrate in water phase fermentation system, above-mentioned recombination is added Bacterial strain or whole-cell catalytic bacterium mud containing the recombinant bacterial strain are resuspended, and carry out catalysis reaction.Optionally, in catalysis reaction Isopropyl-beta D-thio galactopyranoside (IPTG) induction destination protein expression is added.As a preferred embodiment, The present invention prepares α-ketoglutaric acid using whole-cell catalytic method, without adding exogenous enzyme catalyst.

In a particular embodiment, in the fermentation system preferred sodium glutamate monohydrate (monosodium glutamate) it is final concentration of 270 g/L.In one embodiment, the concentration of whole-cell catalytic bacterium mud described in fermentation system is 10 ~ 20 g/L, for example, 12 g/L,15 g/L,18 g/L.In one embodiment, control revolving speed is 400 r/min, and setting dissolved oxygen is 25 ~ 40%, instead Answering temperature is 35 °C, and the catalysis reaction time is 24 ~ 48 h.

Term " corresponding to " has the normally understood meaning of those of ordinary skill in the art in the present invention.Specifically, " right Ying Yu " indicates designated position of the two sequences after homology or sequence identity compare, in a sequence and another sequence Corresponding position.Thus, for example, just " sporting threonine by serine S corresponding to the 280th amino acids of SEQ ID NO:1 For T ", if one end of the amino acid sequence shown in SEQ ID NO:1 add 6 × His label, gained mutant in The 280th corresponding to amino acid sequence shown in SEQ ID NO:1 may be the 286th in the amino acid sequence of mutant. Those of ordinary skill in the art can using it is known in the art it is any measurement sequence homology or the phase same sex method measurement or Compare the homology or the phase same sex of sequence, including but not limited to computer molecular biology (Computational Molecular Biology), Lesk, A.M. are compiled, Oxford University Press, New York, and 1988;Biological computation: informatics and genome item Mesh (Biocomputing:Informatics and Genome Projects), Smith, D.W. are compiled, academic press, New York, 1993;The computer of sequence data analyzes (Computer Analysis of Sequence Data), and first Divide, Griffin, A.M. and Griffin, H.G. is compiled, Humana Press, New Jersey, is remembered in 1994 equal documents The method of load.

Beneficial effects of the present invention

The present invention successfully constructs the recombinant strain of a kind of efficiently coexpression dglutamic oxidase and catalase, And provide a kind of α-ketoglutaric acid biological preparation method for being not required to external source addition catalase, this method bottom with higher Object conversion ratio and by-product is less, preparation method is simple and convenient, and working condition is mild, environmental pollution is small, has good technology Application prospect.The catalytic performance for the L-GLOD that the present invention uses is excellent, being capable of efficient catalysis substrate Pidolidone α-ketoglutaric acid is generated, the catalase that the present invention uses can be fast by the hydrogen peroxide byproduct generated in catalysis reaction Prompt drop solution, so that α-ketoglutaric acid be promoted to produce.The aliment security level glutamic acid microbial rod that the present invention can preferably generally acknowledge Bacterium has going through for many decades as traditional key industry fermentative microorganism, for producing monosodium glutamate as production bacterial strain, the bacterial strain History, therefore using bacterium production α-ketoglutaric acid it is possible to prevente effectively from food safety hazards.

Detailed description of the invention

Fig. 1 is to produce α-ketoglutaric acid schematic diagram based on whole-cell catalytic.

Fig. 2 is the coexpression full cell total soluble protein SDS-PAGE electrophoresis of recombinant bacterial strain.

Fig. 3 is the analysis of dglutamic oxidase mutant activity.

Fig. 4 is α-ketoglutaric acid content analysis detection figure in whole-cell catalytic liquid.

Specific embodiment

Following implementation further illustrates the contents of the present invention, but should not be construed as limiting the invention.Implement below In example, bacillus coli DH 5 alpha and Corynebacterium glutamicum are commercially available, and bacillus coli DH 5 alpha is for all genes in the present invention Clone, Corynebacterium glutamicum produces α-ketoglutaric acid for expression of gene protein and resting cell in the present invention.Large intestine bar Bacterium DH5 α competent cell is conventionally prepared with Corynebacterium glutamicum competent cell.Tool is not specified in the following example The test method of concrete conditions in the establishment of a specific crime carries out according to normal conditions, such as condition described in " molecular cloning: laboratory manual ", or presses Condition proposed by the manufacturer of biological reagent is answered in photograph.

The building of embodiment 1 dglutamic oxidase and catalase coexpression vector

It will be understood by those skilled in the art that Corynebacterium glutamicum and luxuriant source streptomycete etc. be when expressing protein, All it is presented with different degrees of codon-bias.It is excellent by codon is carried out from the glucose oxidation enzyme gene of luxuriant source streptomycete Change, target protein can be made more effectively to express in Corynebacterium glutamicum expression system, the method for the codon optimization is Known to one of skill in the art, details are not described herein.

The present invention will from luxuriant source streptomycete (S. mobaraensisDSM40903 glucose oxidation enzyme gene) according to The codon usage frequency of Corynebacterium glutamicum optimizes, and nucleotide sequence is as shown in SEQ ID No:2, and commission Suzhou gold is only Intelligence Biotechnology Co., Ltd carries out gene chemical synthesis, and using Lgox-5F and Lgox-3R as primer pair, is risen by PCR amplification The dglutamic oxidase gene order of conservative RBS correlated series is added in beginning codon front end.With rbs-katE-5F and rbs- KatE-3R is primer pair, with Escherichia coli (E. coliMG1655) genome is template, and it is close to obtain starting by PCR amplification The catalase gene sequence of conservative RBS correlated series is added in numeral front end.Using above-mentioned two sections of gene orders as template, And using Lgox-5F and rbs-katE-3R as primer, the glutamic acid for adding RBS sequence respectively is obtained by bridging PCR amplification Oxidizing ferment and catalase fusion segment (rbs-lgox-rbs-katE).Using pXMJ19-5F and pXMJ19-3R as primer pair, Plasmid pXMJ19 is template, obtains the pXMJ19 plasmid backbone containing AarI restriction enzyme site by PCR.Using based on Golden Fusion segment rbs-lgox-rbs-katE and pXMJ19 plasmid backbone is connected into and is singly opened by the segment assembly method of Gate clone Mover expressing in series plasmid.Above-mentioned obtained recombinant plasmid is named as pXMJ19-tac-lgox-katE, and send Suzhou Jin Weizhi Biotechnology Co., Ltd carries out sequencing confirmation.

Selection pXMJ19-tac-lgox-katE plasmid is template, utilizes pXMJ19-lk-5F and pXMJ19-lk-3R primer It is right, the pXMJ19-tac-lgox-katE plasmid backbone containing AarI restriction enzyme site is obtained by PCR.By tac-fusionF and Tac-fusionR primer forms the double chain DNA fragment containing cohesive end, i.e. tac promoter sequence after merging after being annealed； The double chain DNA fragment containing cohesive end is formed after merging after trc-fusionF and trc-fusionR primer is annealed, That is trc promoter sequence.By plasmid backbone after AarI inscribe enzymatic treatment, respectively with the tac promoter sequence of above-mentioned acquisition It is attached with trc promoter sequence, obtains two kinds of dual promoter expression plasmid pXMJ19-tac-lgox-tac-katE respectively And pXMJ19-tac-lgox-trc-katE, send Suzhou Jin Weizhi Biotechnology Co., Ltd be sequenced really obtained plasmid Recognize.

The primer sequence in above-mentioned case study on implementation are as follows:

In the present embodiment, pcr amplification reaction system are as follows: 10 μ L 5 × HF Phusion buffer, 2.5 μ L 2.5 10 μM of Primer of mM 1,2.5 μ L of dNTP, 2.5 10 μM of μ L Primer 2,0.5 μ L Template, 0.5 μ L DMSO, 0.5 μ L Phusion archaeal dna polymerase supplement ddH₂O to 50 μ L.PCR reaction condition are as follows: 98 °C of initial denaturations 1 Min, 98 °C of 10 s of denaturation, 60 °C of 20 s of annealing, 72 °C of 1 min of extension, 35 recycle；72 °C of 8 min of extension, 16 °C of preservations.

In the present embodiment, Golden Gate assembly system are as follows: 1.5 μ L 10 × T4 Ligase buffer, 1 μ L T4 Ligase, 1 μ L AarI restriction endonuclease, 0.5 μ L 100 × BSA, 0.2 μ L Oligo, 200 ng genetic fragments or Plasmid backbone supplements ddH₂O to 15 μ L.Golden Gate assembly program are as follows: 37 °C, 3 min；22 °C, 4 min；Circulation 30 It is secondary；22 °C, 20 min；50 °C, 2 min；80 °C, 2 min；16 °C, 5 min.After reaction, reaction solution is directly converted big Enterobacteria DH5 α competence, after plate resistance screening, picking transformant is verified.

In the present embodiment, digestion system are as follows: 5 μ 10 × FastDigest of L buffer, 2 μ L AarI restriction endonucleases； 0.5 100 × BSA of μ L, 200 ng DNA fragmentations supplement ddH₂O to 50 μ L.Endonuclease reaction condition is 37 °C, the digestion time For 2 h.

The expression vector pXMJ19 that the present embodiment is chosen is inducible expression carrier, and the plasmid itself contains strong promoter Tac correlated series (including manipulation sequence lacO), when the inducers such as IPTG or lactose are added, can promote aporepressor to leave behaviour Vertical sequence is expressed to initial gene.

2 dglutamic oxidase of embodiment and catalase coexpression recombinant bacterial strain building and expression analysis

Extract co-expression plasmid pXMJ19-tac-lgox-katE, the pXMJ19-tac- obtained in above-described embodiment 1 Lgox-tac-katE and pXMJ19-tac-lgox-trc-katE is transferred to Corynebacterium glutamicum using electrotransformation, described Specific method for transformation are as follows: take 100 μ L-Glu rod bacterium competence cells, be separately added into the above-mentioned coexpression matter of about 200 ng Grain, is transferred to after mixing well and has been pre-chilled in 2 mm electricity revolving cups and has placed on ice 20 min, and adjusting electroporation voltage is 2.1 KV carries out electric shock, 46 °C of 6 min of water-bath heat shock is placed in, at 32 ° in 1 mL LBHIS fluid nutrient medium is added immediately after the completion C, after 150 rpm shaking tables recovery culture, 2 h, the LBHIS solid medium containing 15 μ g/mL ampicillins, 32 °C of mistakes are coated with Night culture, it is to be generated grow single colonie after verified.

The recombination Corynebacterium glutamicum containing different co-expression plasmids of above-mentioned acquisition is subjected to protein induced detection of expression, Analyze dglutamic oxidase and catalase expression.Method particularly includes: it picks them separately monoclonal and is inoculated in containing 100 In the shaking flask of mL LBHIS fluid nutrient medium, it is incubated overnight in 32 °C, 200 r/min shaking tables.It then, will according to 2% inoculum concentration Bacterium is transferred again in 100 mL LBHIS fluid nutrient mediums overnight, is cultivated in 32 °C, 200 r/min shaking tables to cell concentration OD₆₀₀To 1.0 or so, final concentration of 0.4 mM IPTG induction destination protein expression is added, cultivation temperature is adjusted to 28 °C, continues 12 h of Fiber differentiation.After the completion of inducing, after taking 1 mL bacterium solution to carry out ultrasonication processing, detected altogether followed by SDS-PAGE Express the expression of double enzymes in bacterial strain.

As shown in Fig. 2, protein expression analysis is found, pXMJ19-tac-lgox-tac-katE co-expresses peroxidating in bacterial strain Hydrogenase expression amount is apparently higher than dglutamic oxidase, shows that tac promoter is added before hydrogen peroxide enzyme coding gene to be facilitated Gene expression；PXMJ19-tac-lgox-trc-katE is co-expressed in bacterial strain, and catalase expression quantity is but decreased obviously, The trc promoter is prompted not to be suitable for double enzyme coexpression systems；PXMJ19-tac-lgox-katE co-expresses bacterial strain Glutamic Acid Oxidizing ferment and catalase have relatively high expression, and the two protein concentration is substantially similar.

3 high density fermentation culture of embodiment prepares whole-cell catalytic bacterium mud

The present embodiment is used to provide a kind of high density fermentation culture and bacterium mud preparation method for co-expressing recombinant bacterial strain, described The method of fermented and cultured may comprise steps of: the coexpression recombinant bacterial strain obtained in above-described embodiment 2 being seeded to and has been contained Have in the seed culture medium of chlorampenicol resistant, 16 h of shake culture in 32 °C, 200 r/min shaking tables, obtains coexpression recombination Bacterial strain seed liquor；Seed liquor is inoculated according to 5% inoculum concentration in the fermentor for having been loaded with 5 L fermentation mediums, setting culture temperature 32 °C, 300 r/min of speed of agitator of degree, control culture pH value is 7.0, and when dissolved oxygen is down to 20% or less, revolving speed and dissolved oxygen is arranged Coupling.Monitor cell concentration OD₆₀₀, to thalli growth to OD₆₀₀When about 25, final concentration of 0.4 mM IPTG inducer is added Destination protein expression is activated, sets about 28 °C of cultivation temperature, control culture pH value is 7.0, about 24 h of induction time, final thallus Concentration OD₆₀₀Up to 150.0 or so；After aforesaid liquid fermentation culture medium is centrifuged 10 min under the conditions of 8000 × g of revolving speed, Fall fermented liquid supernatant, collects somatic cells to get recombination Corynebacterium glutamicum bacterium mud, it is spare that 4 °C of refrigerators can be placed in.

It will be understood by those skilled in the art that the antibiotic being added in seed culture medium and fermentation medium is as sieve Choosing marks the fermented and cultured for co-expressing recombinant bacterial strain, and the concentration of the antibiotic is not particularly limited, and working concentration is 15 μg/L.In the present embodiment, the component of the seed culture medium are as follows: 2.5 g/L of yeast powder, peptone 5 g/L, NaCl 5 g/L, 18.5 g/L of brain heart infusion, 91 g/L of sorbierite；The component of the fermentation medium are as follows: glucose 100 g/L of sugar, 15 g/L of corn pulp, 20 g/L of ammonium sulfate, 1 g/L of magnesium sulfate, 0.5 g/L of potassium dihydrogen phosphate, 0.1 g/ of dipotassium hydrogen phosphate L, 2 g/L of sodium citrate, 2 g/L of calcium carbonate, adjustment culture medium to pH 7.0.In the present embodiment to the tune of medium pH Section, used acid-base solution and concentration are not particularly limited, and the present embodiment uses acid solution for 50% acetic acid, and aqueous slkali is 50% ammonium hydroxide.

Embodiment 4 establishes resting cell technique preparation α-ketoglutaric acid

What the present embodiment constructed in 2 for providing a kind of resting cell production α-KG method, based on the above embodiment is total to Expression recombination Corynebacterium glutamicum is whole-cell catalyst, establishes the biological preparation process of α-KG.The production method of the α-KG 5 L Whole cell catalytic systems specifically are established using fermentor, add the sodium glutamate monohydrate (taste of 270 g/L final concentrations Essence) substrate, the full cell bacterium mud prepared in 10 g/L above-described embodiments 3 is added, controls 400 r/min of fermentor revolving speed, setting Dissolved oxygen is not less than 25%, 35 °C of catalytic reaction temperature, 40 h of reaction time is catalyzed, to after reaction, utilize liquid chromatography (HPLC) quantitative analysis is carried out to catalysis reaction solution component.

α-KG the production method established in the present embodiment does not need the expensive catalase of external source addition, entirely urges Change reaction process without controlling pH value of reaction system, production cost and simplification of flowsheet can be greatlyd save.

The analysis of HPLC described in the present embodiment, method particularly includes: take 1 mL of whole-cell catalytic reaction solution, 12000 × g centrifugation Supernatant is taken after 5 min, is filtered using 0.02 μm of aperture filter membrane.Reaction solution component uses high performance liquid chromatography (Agilent 1200, USA) it measures, chromatographic condition are as follows: analytical column uses Bio-Rad Aminex HPX-87H chromatographic column, and mobile phase is 5 mM H₂SO₄, flow velocity is 0.6 mL/min, and detection temperature is 30 °C, and detector is UV detector detection, 210 nm of Detection wavelength.

It is analyzed through liquid chromatographic detection, the coexpression strains expressed obtained based on pXMJ19-tac-lgox-katE building is gone out Highest α-KG production capacity, up to 200.0 g/L, substrate molar yield reaches the content of α-KG in whole-cell catalytic reaction solution 95% or more, and without complex component in reaction solution, help to simplify downstream separation purifying process, before there is preferable technical application Scape.

5 dglutamic oxidase mutation construction of embodiment and activity analysis

The present embodiment is used to provide a kind of dglutamic oxidase mutation construction method that enzyme activity improves.By analyzing paddy ammonia 3D structure and the homologous sequence of acid oxidase compare, determine the amino acid sequence of SEQ ID NO:1 the 280th serine S and 533rd hyte propylhomoserin H is purpose mutational site.Using site-directed mutagenesis technique, according to amino acid sites to be mutated come design point Mutant primer obtains dglutamic oxidase mutant nucleotide sequence by PCR method.

Selection pXMJ19-tac-lgox-katE plasmid is template, is obtained using S280T-5F and LGOX-DN primer pair amplifies Lgox-S280T fragment upstream is obtained, LGOX-UP and S280T-3R primer pair amplifies is utilized to obtain lgox-S280T segments downstream.It will It is used as template after the mixing of this upstream and downstream segment, is based on fusion DNA vaccine principle, is obtained using LGOX-UP/LGOX-DN primer pair amplifies Lgox-S280T segment, the as dglutamic oxidase containing S280T single mutation site.By the dglutamic oxidase mutant nucleotide sequence It is attached with pET21b carrier, building obtains pET21b-lgox-S280T recombinant plasmid.Selection lgox-S280T segment is mould Plate, is utilized respectively LGOX-UP/H533L-3R primer pair and H533L-5F/LGOX-DN and primer pair, and amplification obtains lgox- The upstream and downstream segment of S280T H533L.It is used as template after this upstream and downstream segment is mixed, is based on fusion DNA vaccine principle, utilizes LGOX-UP/LGOX-DN primer pair amplifies obtain lgox-S280T H533L segment, as contain the bis- mutation positions S280TH533L The dglutamic oxidase of point.The dglutamic oxidase mutant nucleotide sequence and pET21b carrier are attached, building obtains pET21b- Lgox-S280TH533L recombinant plasmid.Above-mentioned recombinant plasmid is converted respectivelyE. coli BL21 (DE3) competent cell, warp After crossing plate resistance screening, picking transformant and sequence verification.It will construct successfulE. coli BL21/pET21b-lgox- S280T、E. coli BL21/pET21b-lgox-S280TH533L recombination engineering with contain unmutated dglutamic oxidase base The recombination engineering of cause is inoculated in respectively in the LB culture medium that 5 mL contain ampicillin, and 37 °C, 200 r/min train overnight It after supporting, is transferred in the LB culture medium that 100 mL contain ampicillin again according to 1% inoculum concentration, reaches OD to cell density₆₀₀ When value is 0.6, the isopropyl-beta D-thio galactopyranoside (IPTG) of final concentration of 0.4 mM is added, in 16 °C, 200 r/ It under min condition of culture after 10 h of inducing expression, is collected by centrifugation and obtains wet bacterium mud, with thallus is resuspended after sterile water washing.It will obtain Resuspension cell concentration adjust to OD₆₀₀Value is 10 or so, carries out clasmatosis, setting ultrasound using ultrasonic cell disruption instrument 200 W of wave power, ultrasound 2 s interval 1 s, 10 min of ultrasound.After to ultrasonic procedure, 8000 × g is centrifuged under the conditions of 4 °C 10 min, obtained supernatant can be used for subsequent protein expression analysis and Ni-NTA purifying.

The purifying of Ni-NTA described in the present embodiment uses standard laboratory protocols.Method particularly includes: purifying filler used is Nickel, whole process of purification carry out in 4 °C of refrigerators.After 1-2 mL filler is injected chromatographic column, with 3 ~ 5 times of column volumes go from Sub- water rinses pillar, adds lysis buffer (the 20 mM Na of 5 times of column volumes₂HPO₄, 200 mM NaCl, pH 7.0) and balance Pillar is in filler under buffer system identical with destination protein.Sample is added in the chromatographic column balanced, is turned upside down It comes into full contact with destination protein with filler, after the layering of column liquid, collects efflux, repeat the above steps 2 ~ 3 times.Use 10 ~ 15 Times column volume washes miscellaneous caching liquid (20 mM Na₂HPO₄, 200 mM NaCl, 50 mM imidazoles, pH 7.0) and it is cleaned, it removes The foreign protein of non-specific adsorption detects efflux using Coomassie Brillant Blue solution, until unobvious albumen is detected.With Elution caching liquid (20 mM Na₂HPO₄, 200 mM NaCl, 500 mM imidazoles, pH 7.0) and elution destination protein, collect outflow Liquid detects efflux using Coomassie brilliant blue detection liquid, until unobvious albumen is detected.Destination protein use will be collected into 30 kDa Amicon super filter tubes are concentrated, and 5000 × g is centrifuged 10 min under the conditions of 4 °C, with preservation buffer (20 mM Na₂HPO₄, pH 7.0) and diluted protein liquid, until the concentration of imidazoles is lower than 10 mmol/L.

The pure enzyme obtained after purification using Ni-NTA carries out enzymatic reaction to measure enzyme activity.The total volume of enzymatic reaction is 1 mL, including 600 μ L, 100 mM sodium glutamate substrate, 300 μ L ddH₂O and the appropriate concentration enzyme solution of 100 μ L, after mixing 30 min are reacted under the conditions of 37 °C, and 10 min are boiled in boiling water bath and terminate reaction.To after reaction, take 20 μ L enzymatics anti- Liquid is answered, 400 μ L, 2 mM 2,4-dinitrophenylhydrazine solution is added, after being incubated for 20 min under the conditions of 37 °C, 1 mL is then added 1 M sodium hydroxide solution terminates reaction.Light absorption value measurement (Detection wavelength 390nm) will be carried out after 3 ~ 10 times of developing solution dilutions, led to It crosses drafting standard curve and calculates sample concentration.Enzyme amount needed for enzyme activity is defined as the α-KG for generating 1 μm of ol per minute is 1 U.Knot Fruit shows as shown in figure 3, glucose oxidation enzyme mutant is compared with wild-type enzyme, and specific enzyme activity is significantly improved, 280 The amino acid change of serine critical sites is increased to 185 U/mg by 120 U/mg before being mutated, 280 serines and 533 The amino acid change of histidine critical sites is increased to 215 U/mg by 120 U/mg before being mutated, and illustrates urging for two kinds of mutant Change performance and obtains a degree of raising.

Application of the 6 glucose oxidation enzyme mutant of embodiment in bioconversion production α-KG

The present embodiment is used to provide a kind of application of glucose oxidation enzyme mutant in α-KG production.Utilize rite-directed mutagenesis Technology, using the pXMJ19-tac-lgox-katE plasmid constructed in above-described embodiment 1 as template, respectively with S280T-5F/ S280T-3R and H533L-5F/H533L-3R is primer, obtains the recombinant expression simultaneously containing the mutational site S280T and H533L Plasmid is named as pXMJ19-tac-lgoxS280TH533L-katE.The mutant plasmid is converted into Corynebacterium glutamicum, by flat After plate resistance screening, obtains pXMJ19-tac-lgoxS280TH533L-katE and co-express bacterial strain, according to what is established in embodiment 4 Resting cell technique carries out α-KG production.Method particularly includes: 5 L Whole cell catalytic systems are established using fermentor, are added The full cell bacterium mud of 10 g/L is added in sodium glutamate monohydrate (monosodium glutamate) substrate of 270 g/L final concentrations, controls fermentor revolving speed 400 r/min, setting dissolved oxygen are not less than 25%, 35 °C of catalytic reaction temperature, are catalyzed 32 h of reaction time.To after reaction, benefit Quantitative analysis is carried out to catalysis reaction solution component with liquid chromatography (HPLC).

As shown in figure 4, analyzing through liquid chromatographic detection, ingredient is more single in resting cell liquid, and substrate and impurity are residual It stays less.Go out more preferably α-KG production capacity, In based on the coexpression strains expressed that dglutamic oxidase mutation construction obtains After resting cell reacts 32 h, in conversion fluid the content of α-KG up to 205.0 g/L, substrate molar yield up to 97.5%, With containing unmutated glucose oxidation enzyme gene recombination engineering compared with, the whole-cell catalytic time, Product yields and mole Conversion ratio etc. all has more preferable advantage, can greatly save production cost and simplification of flowsheet, has good industrialization Application prospect.

More than, embodiments of the present invention are illustrated.But the present invention is not limited to above embodiment.It is all Within the spirit and principles in the present invention, any modification, equivalent substitution, improvement and etc. done should be included in guarantor of the invention Within the scope of shield.

Sequence table

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

<120>glucose oxidation enzyme mutant, double enzyme coexpression vectors and its application

<130> CPCN19111019

<141> 2019-08-22

<160> 29

<170> SIPOSequenceListing 1.0

<210> 1

<211> 622

<212> PRT

<213> Streptomyces mobaraensis

<400> 1

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

<211> 1869

<212> DNA

<213> Streptomyces mobaraensis

<400> 2

atggcggttc ctgcgaaaag caccgcggat tgggatacct gtctggaagt ggcgcgcgcg 60

ttattagtgg 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 ttattctgcg 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

ttattagtgg 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 ttattctgcg 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

ttattagtgg 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 ttattctgcg 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 acattatacg 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 kind of glucose oxidation enzyme mutant, which is characterized in that the amino acid sequence of the glucose oxidation enzyme mutant selects From following group:

A) correspond to the amino acid sequence that the 280th amino acids of SEQ ID NO:1 are sported threonine T by serine S, or

B) correspond to the amino acid sequence that the 280th amino acids of SEQ ID NO:1 are sported threonine T by serine S, and The amino acid sequence of leucine L is sported by histidine H in the 533rd amino acids corresponding to SEQ ID NO:1.

2. the encoding gene of glucose oxidation enzyme mutant as described in claim 1, which is characterized in that described a) to organize amino acid The encoding gene of sequence is nucleotide sequence shown in SEQ ID NO:6.

3. the encoding gene of glucose oxidation enzyme mutant as described in claim 1, which is characterized in that described b) to organize amino acid The encoding gene of sequence is nucleotide sequence shown in SEQ ID NO:8.

4. a kind of recombinant bacterial strain, being will volume described in the encoding gene containing mutant described in claim 1 or Claims 2 or 3 Obtained by the vector introduction host strain of code gene.

5. glucose oxidation enzyme mutant described in claim 1, encoding gene described in claim 2 or 3 or right are wanted Application of the recombinant bacterial strain described in asking 4 in α-ketoglutaric acid production.

6. a kind of double enzyme coexpression vectors, the coexpression vector is in a plasmid while comprising described in claim 1 The encoding gene of glucose oxidation enzyme mutant and the encoding gene of catalase.

7. double enzyme coexpression vectors according to claim 6, the encoding gene of the glucose oxidation enzyme mutant be as Nucleotide sequence shown in SEQ ID NO:6 or SEQ ID NO:8.

8. double enzyme coexpression vectors according to claim 6 or 7, the volume of catalase in double enzyme coexpression vectors Code gene contains the nucleotide sequence as shown in SEQ ID NO:4.

9. double enzyme coexpression vectors according to claim 6 or 7, double enzyme coexpression vectors are using single starting subpattern The encoding gene of the encoding gene of promoter and the glucose oxidation enzyme mutant, catalase is subjected to expressing in series； Or double-promoter mode is used, divide before the encoding gene of glucose oxidation enzyme mutant and the encoding gene of catalase It Tian Jia not promoter.

10. double enzyme coexpression vectors according to claim 9, the promoter is that tac strong promoter or trc start by force Son.

11. a kind of double enzymes co-express recombinant bacterial strain, containing such as the described in any item double enzyme coexpression vectors of claim 6-10.

12. double enzymes according to claim 11 co-express recombinant bacterial strain, the recombinant bacterial strain is to co-express double enzymes Vector introduction host strain, the host strain be selected from Corynebacterium glutamicum (Corynebacterium glutamicum), it is big Enterobacteria (Escherichia coli) or bacillus subtilis (Bacillus subtilis).

13. application of the described in any item double enzyme coexpression vectors of claim 6-10 in preparation α-ketoglutaric acid.

14. application of the described in any item double enzyme coexpression recombinant bacterial strains of claim 11-12 in preparation α-ketoglutaric acid.

15. a kind of method for preparing α-ketoglutaric acid co-expresses recombinant bacterium including double enzymes described in culture claim 11 or 12 Strain.

16. the method for preparation α-ketoglutaric acid according to claim 15, the method includes using glutamate as substrate, Whole-cell catalytic, which is carried out, using double enzyme coexpression recombinant bacterial strains prepares α-ketoglutaric acid.