CN108070532A - A kind of method for producing glucose oxidase - Google Patents

Abstract

The invention discloses a kind of methods for producing glucose oxidase.Present invention firstly provides a kind of recombinant bacteriums, are that the encoding gene of the encoding gene of glucose oxidase and molecular chaperones is imported out obtaining for bacterium germination.The molecular chaperones concretely disulfide bond isomerase and/or tuberculosis immunity globulin.The present invention also protects a kind of method for producing glucose oxidase, includes the following steps：The recombinant bacterium is cultivated, obtains glucose oxidase.The present invention also protects application of the molecular chaperones in microbial expression glucose oxidase is promoted.The present invention is significant to the secreting, expressing and industrialized production for enhancing glucose oxidase.

Description

A kind of method for producing glucose oxidase

Technical field

The invention belongs to biological technical fields, and in particular to a kind of method for producing glucose oxidase.

Background technology

Glucose oxidase (Glucose oxidase, GOD) is a kind of oxidases mainly generated by mould, can be with By the glycoxidative generation glucuronic acid of grape, while by the use of molecular oxygen as electron acceptor, hydrogen peroxide is generated, therefore extensively should For industries such as food, medicine, feed and industrial productions.

Domestic production glucose oxidase level is relatively low at present, and enzyme preparation purity is generally relatively low, still needs to largely rely on every year Import, the market price remain high.

Pichia pastoris (Pichia pastoris) expression system is to start a kind of fast-developing methanol after 1980 Auxotype yeast expression system has nearly 500 kinds of foreign proteins successful expression within the system at present.It not only overcomes protokaryon Expression system background proteins are more, the defects of being unable to secreting, expressing foreign protein and being unable to the albumen of expression structure complexity, also make up Higher eukaryotic expression system is complicated for operation, expression is low and industrialization production is of high cost deficiency, while there is other ferment The incomparable superior part of female expression system, such as successfully avoids saccharomyces cerevisiae

(Saccharomyces cerevisiae) the defects of protein secretion expression efficiency is low, expression plasmid is easy to be lost.

The content of the invention

The object of the present invention is to provide a kind of methods for producing glucose oxidase.

Present invention firstly provides a kind of recombinant bacterium (restructuring fungus beetles), are by the coding of glucose oxidase (GOD albumen) The encoding gene of gene and molecular chaperones imports out what bacterium germination obtained.

The present invention also provides a kind of recombinant bacterium (recombinant bacterium second), are to confer to out bacterium germination glucose oxidase activity and molecule What Chaperone Activity obtained.

The molecular chaperones concretely disulfide bond isomerase (PDI albumen) and tuberculosis immunity globulin (BiP albumen).

The molecular chaperones concretely disulfide bond isomerase (PDI albumen).

The molecular chaperones concretely tuberculosis immunity globulin (BiP albumen).

The glucose oxidase is following (a1) or (a2)：

(a1) protein shown in the sequence 1 of sequence table；

(a2) amino acid sequence of sequence 1 by the substitution of one or several amino acid residues and/or missing and/or is added Add and there is its derivative protein of identical function.

The encoding gene of the glucose oxidase is following (a3), (a4), (a5) or (a6)：

(a3) DNA molecular of the code area as shown in the 1st to 1749 nucleotide of sequence 2 of sequence table；

(a4) DNA molecular of the code area as shown in the sequence 2 of sequence table；

(a5) the DNA sequence dna hybridization limited under strict conditions with (a3) or (a4) and the DNA of coding glucose oxidase Molecule；

(a6) DNA sequence dna limited with (a3) or (a4) at least with 70%, at least with 75%, at least with 80%, At least with 85%, at least with 90%, at least with 95%, at least with 96%, at least with 97%, at least with 98% Or at least there is more than 99% homology and encode the DNA molecular of glucose oxidase.

The disulfide bond isomerase is following (b1) or (b2)：

(b1) protein shown in the sequence 3 of sequence table；

(b2) amino acid sequence of sequence 3 by the substitution of one or several amino acid residues and/or missing and/or is added Add and there is its derivative protein of identical function.

The encoding gene of the disulfide bond isomerase is following (b3), (b4), (b5) or (b6)：

(b3) DNA molecular of the code area as shown in the 1st to 1551 nucleotide of sequence 4 of sequence table；

(b4) DNA molecular of the code area as shown in the sequence 4 of sequence table；

(b5) the DNA sequence dna hybridization limited under strict conditions with (b3) or (b4) and the DNA of coding disulfide bond isomerase Molecule；

(b6) DNA sequence dna limited with (b3) or (b4) at least with 70%, at least with 75%, at least with 80%, At least with 85%, at least with 90%, at least with 95%, at least with 96%, at least with 97%, at least with 98% Or at least there is more than 99% homology and encode the DNA molecular of disulfide bond isomerase.

The tuberculosis immunity globulin is following (c1) or (c2)：

(c1) protein shown in the sequence 5 of sequence table；

(c2) amino acid sequence of sequence 5 by the substitution of one or several amino acid residues and/or missing and/or is added Add and there is its derivative protein of identical function.

The encoding gene of the tuberculosis immunity globulin is following (c3), (c4), (c5) or (c6)：

(c3) DNA molecular of the code area as shown in the 1st to 2034 nucleotide of sequence 6 of sequence table；

(c4) DNA molecular of the code area as shown in the sequence 6 of sequence table；

(c5) DNA sequence dna limited under strict conditions with (c3) or (c4) hybridizes and encodes tuberculosis immunity globulin DNA molecular；

(c6) DNA sequence dna limited with (c3) or (c4) at least with 70%, at least with 75%, at least with 80%, At least with 85%, at least with 90%, at least with 95%, at least with 96%, at least with 97%, at least with 98% Or at least there is more than 99% homology and encode the DNA molecular of tuberculosis immunity globulin.

Above-mentioned stringent condition can be with 0.1 × SSPE (or 0.1 × SSC), and the solution of 0.1%SDS is miscellaneous in DNA or RNA It hands over and hybridizes in experiment at 65 DEG C and wash film.

The recombinant bacterium is concretely by the encoding gene of glucose oxidase, the encoding gene and knot of disulfide bond isomerase The encoding gene of core immunoglobulin imports out what bacterium germination obtained.The encoding gene of glucose oxidase, disulfide bond isomerase The encoding gene of encoding gene and tuberculosis immunity globulin can import out bacterium germination by same expression vector, can also be by With two expression vectors, (i.e. two of which gene shares an expression vector, and the 3rd gene is by another expression vector；Tool Body can say that the encoding gene of disulfide bond isomerase and the encoding gene of tuberculosis immunity globulin can share an expression vector, The encoding gene of glucose oxidase is by another expression vector) bacterium germination is imported out, it can also be (i.e. each by three plasmids Gene is respectively by an expression vector) import out bacterium germination.The construction method of the recombinant bacterium is specific as follows：By recombinant plasmid first and Recombinant plasmid second imports out bacterium germination, obtains recombinant bacterium；The recombinant plasmid first is to be inserted into the encoding gene of glucose oxidase The recombinant expression carrier that the multiple cloning sites of expression vector first obtain；The recombinant plasmid second is by the coding of disulfide bond isomerase The recombinant expression carrier that the multiple cloning sites of the encoding gene of gene and tuberculosis immunity globulin insertion expression vector second obtain.Institute State expression vector first concretely pPIC9K carriers.The recombinant plasmid first concretely recombinant plasmid pPIC9K-GOD.Restructuring The construction method of plasmid pPIC9K-GOD：Sequence table is inserted between I restriction enzyme site of EcoR I and Not of pPIC9K carriers Double chain DNA molecule shown in sequence 2.The expression vector second concretely pPICZ α A carriers.The recombinant plasmid second specifically may be used For recombinant plasmid pPICZ-BiP-PDI.Recombinant plasmid pPICZ-BiP-PDI is the circular plasmids shown in the sequence 7 of sequence table.

The recombinant bacterium concretely leads the encoding gene of the encoding gene of glucose oxidase and disulfide bond isomerase Enter out what bacterium germination obtained.The encoding gene of glucose oxidase and the encoding gene of disulfide bond isomerase can be by same tables Go out bacterium germination up to vector introduction, can also be imported out by with two expression vectors (i.e. each gene is respectively by an expression vector) Bacterium germination.The construction method of the recombinant bacterium is specific as follows：Recombinant plasmid first and recombinant plasmid second are imported into out bacterium germination, recombinated Bacterium；The recombinant plasmid first is the weight for obtaining the multiple cloning sites of the encoding gene insertion expression vector first of glucose oxidase Group expression vector；The recombinant plasmid second is that the encoding gene of disulfide bond isomerase is inserted into the multiple cloning sites of expression vector second Obtained recombinant expression carrier.The expression vector first concretely pPIC9K carriers.The recombinant plasmid first concretely weighs Group plasmid pPIC9K-GOD.The construction method of recombinant plasmid pPIC9K-GOD：In the I digestion position of EcoR I and Not of pPIC9K carriers The double chain DNA molecule shown in the sequence 2 of sequence table is inserted between point.The expression vector second concretely pPICZ α A carriers. The recombinant plasmid second concretely recombinant plasmid pPICZ-PDI.The construction method of recombinant plasmid pPICZ-PDI：In pPICZ α A The double chain DNA molecule shown in the sequence 4 of sequence table is inserted between I restriction enzyme site of BstB I and Not of carrier.

The recombinant bacterium is concretely by the encoding gene of glucose oxidase and the encoding gene of tuberculosis immunity globulin Import out what bacterium germination obtained.The encoding gene of glucose oxidase and the encoding gene of tuberculosis immunity globulin can be by same A expression vector imports out bacterium germination, can also be led by with two expression vectors (i.e. each gene is respectively by an expression vector) Enter out bacterium germination.The construction method of the recombinant bacterium is specific as follows：Recombinant plasmid first and recombinant plasmid second are imported into out bacterium germination, obtained Recombinant bacterium；The recombinant plasmid first is to obtain the multiple cloning sites of the encoding gene insertion expression vector first of glucose oxidase Recombinant expression carrier；The recombinant plasmid second is more grams that the encoding gene of tuberculosis immunity globulin is inserted into expression vector second The recombinant expression carrier that grand site obtains.The expression vector first concretely pPIC9K carriers.The recombinant plasmid first is specific Can be restructuring plasmid pPIC9K-GOD.The construction method of recombinant plasmid pPIC9K-GOD：In the EcoR I of pPIC9K carriers and Not I The double chain DNA molecule shown in the sequence 2 of sequence table is inserted between restriction enzyme site.The expression vector second concretely pPICZ α A carriers.The recombinant plasmid second concretely recombinant plasmid pPICZ-BiP.The construction method of recombinant plasmid pPICZ-BiP： The double chain DNA molecule shown in the sequence 6 of sequence table is inserted between I restriction enzyme site of BstB I and Not of pPICZ α A carriers.

It can be any commonly employed host strain in expression foreign protein to go out bacterium germination described in any of the above.It sets out described in any of the above Bacterium concretely yeast, more specifically can be Pichia pastoris, such as Pichia pastoris X33.

The present invention also protects application of the recombinant bacterium in glucose oxidase is prepared described in any of the above.

The present invention also protects a kind of method for producing glucose oxidase, includes the following steps：The recombinant bacterium is cultivated, is obtained To glucose oxidase.

The method of the production glucose oxidase specifically comprises the following steps：Using restructuring described in BSM medium cultures Bacterium.When concretely 30 DEG C of the condition of the culture, 500rpm shaken cultivations 12-132 are small.

The method of the production glucose oxidase specifically comprises the following steps：

(1) recombinant bacterium is inoculated in BMGY culture mediums, cultivated to OD_600nmIt is worth for 10；

(2) system that 10 parts by volume steps 2 obtain is seeded to 90 parts by volume BSM culture mediums, cultivated.

In step (1), concretely 30 DEG C of condition, the 220rpm shaken cultivations of the culture.

In step (2), when concretely 30 DEG C of the condition of the culture, 500rpm shaken cultivations 12-132 are small.

The method of the production glucose oxidase specifically comprises the following steps：By recombinant bacterium X33/pPIC9K-GOD/ PPICZ-BiP-PDI inoculation BSM culture mediums (initial OD=1-2), 30 DEG C of cultures；Incubation is by one He of stage that carries out successively Stage two forms；

Stage one：It is 5.0 by adding in 28% concentrated ammonia liquor control system pH, by adjusting speed of agitator in incubation It is more than 20% with throughput control system dissolved oxygen；When the glycerol depletion in BSM culture mediums rises so as to cause dissolved oxygen, start Using the rate flow feeding liquid first that adds in 18.15mL in every liter of system per hour, (feed supplement liquid first is is 1.2% containing volume fraction The aqueous solution of PTM1 and 50g/100ml glycerine), OD_600nmTo stop feed supplement during 150-200, continue 30 points of culture after stopping feed supplement When clock -2 is small；

Stage two：It is 6.0 by adding in 28% concentrated ammonia liquor control system pH, by adjusting speed of agitator in incubation It is more than 10% with throughput control system dissolved oxygen；In incubation, added with the rate stream for adding in 5g in every liter of system per hour Feed supplement liquid second (methanol that it is 1.2%PTM1 containing volume fraction that feed supplement liquid second, which is), while to add per hour in every kilogram of system The rate stream for entering 50g adds sorbierite.

The method of the production glucose oxidase specifically comprises the following steps：By recombinant bacterium X33/pPIC9K-GOD/ PPICZ-BiP-PDI inoculation BSM culture mediums (initial OD=1-2), when 30 DEG C of cultures 150 are small；Incubation is by successively carrying out Stage one and stage two form (continuous timing)；

Stage one：It is 5.0 by adding in 28% concentrated ammonia liquor control system pH, by adjusting speed of agitator in incubation It is more than 20% with throughput control system dissolved oxygen；19th starts to add in the speed of 18.15mL in every liter of system per hour when small Rate flow feeding liquid first (feed supplement liquid first is to contain the aqueous solution that volume fraction is 1.2%PTM1 and 50g/100ml glycerine), the 23rd Hour start stop feed supplement, stop feed supplement after continue culture 1 it is small when；

Stage two：It is 6.0 by adding in 28% concentrated ammonia liquor control system pH, by adjusting speed of agitator in incubation It is more than 10% with throughput control system dissolved oxygen；In incubation, the 24th starts to add in per hour in every liter of system when small The rate flow feeding liquid second (methanol that it is 1.2%PTM1 containing volume fraction that feed supplement liquid second, which is) of 5g, while with every kilogram of body The rate stream for adding in 50g in system per hour adds sorbierite.

The present invention also protects application of the molecular chaperones in microbial expression glucose oxidase is promoted.The molecular chaperones Concretely disulfide bond isomerase (PDI albumen) and tuberculosis immunity globulin (BiP albumen).The molecular chaperones concretely two Sulfide linkage isomerase (PDI albumen).Concretely tuberculosis immunity globulin (BiP albumen) described microorganism is specific for the molecular chaperones It can be yeast, more specifically can be Pichia pastoris, such as Pichia pastoris X33.The microorganism that sets out is concretely in Pichia pastoris The recombinant bacterium that the encoding gene of glucose oxidase obtains is imported in X33.

The method that the present invention also protects a kind of expression of raising glucose oxidase in microorganism, including walking as follows Suddenly：The encoding gene of molecular chaperones is imported in the microorganism that sets out, the level of glucose oxidase is expressed so as to improve it.It is described The microorganism that sets out is the microorganism of expression glucose oxidase.The microorganism concretely yeast more specifically can be to finish red ferment Mother, such as Pichia pastoris X33.The microorganism that sets out concretely imports the volume of glucose oxidase in Pichia pastoris X33 The recombinant bacterium that code gene obtains.

The method that the present invention also protects a kind of expression of raising glucose oxidase in microorganism, including walking as follows Suddenly：Increase the level of molecular chaperones in microorganism, so as to improve the level of microbial expression glucose oxidase.The microorganism To express the microorganism of glucose oxidase.The microorganism concretely yeast can be more specifically Pichia pastoris, such as finish red Yeast X33.What the encoding gene that the microorganism that sets out concretely imports glucose oxidase in Pichia pastoris X33 obtained Recombinant bacterium.

The present invention is significant to the secreting, expressing and industrialized production for enhancing glucose oxidase.

Description of the drawings

Fig. 1 is the electrophoretogram of the supernatant obtained different sample times.

Fig. 2 is the glucose oxidase enzyme activity of the supernatant obtained different sample times.

Specific embodiment

Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments Method is conventional method unless otherwise specified.Test material used in following embodiments is certainly unless otherwise specified What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even Average.

PPIC9K carriers：Invitrogen, catalog number V17520；It is located at multiple cloning sites in pPIC9K carriers (MCS) promoter of upstream is AOX1 promoters, has signal peptide in AOX1 promoters downstream and MCS upstreams, signal peptide carries Beginning codon ATG, therefore the foreign gene being inserted at MCS can not have initiation codon, as long as codeword triplet phase therewith Symbol, without frameshit, signal peptide can voluntarily be cut off during protein expression processing is ripe.Pichia pastoris (Pichia Pastoris) X33, abbreviation Pichia pastoris X33：Invitrogen, article No. C18000.PPICZ α A carriers：Invitrogen is public Department, article No. V19020.

Glucose oxidase (Glucose oxidase) abbreviation GOD albumen.Tuberculosis immunity globulin (Binding Immunoglobulin protein) abbreviation BiP albumen.Disulfide bond isomerase (Protein disulfide isomerase) Abbreviation PDI albumen.

BMGY culture mediums (1L)：Yeast extract 10g, peptone 20g, YNB 13.4g, 400 μ g of biotin, glycerine 10g, Surplus is pH 6.0, the kaliumphosphate buffer of 100mM.

BSM culture mediums (1L)：85%H₃PO₄Aqueous solution 26.7mL, CaSO₄ 0.93g、K₂SO₄ 18.2g、MgSO₄·7H₂O PTM1 4.35mL after 14.9g, KOH 4.13g, glycerine 40g, filtration sterilization, surplus are water.

PTM1(1L)：CuSO₄·5H₂O 6g、NaI 0.08g、MnSO₄·H₂O 3g、NaMoO₄·2H₂O 0.2g、H₃BO₃ 0.02g、CoCl₂ 0.5g、ZnCl₂ 20g、FeSO₄·7H₂O 65g, biotin 0.2g, 98%H₂SO₄Aqueous solution 5mL, surplus For water.

Embodiment 1, structure recombinant bacterium

First, recombinant bacterium X33/pPIC9K-GOD is built

1st, construction recombination plasmid pPIC9K-GOD.According to sequencing result, structure is carried out to restructuring plasmid pPIC9K-GOD and is retouched It states as follows：The double-stranded DNA shown in the sequence 2 of sequence table is inserted between I restriction enzyme site of EcoR I and Not of pPIC9K carriers Molecule.DNA molecular shown in the sequence 2 of sequence table is GOD genes, the GOD albumen shown in the sequence 1 of polynucleotide.External source Albumen extracellular expression.

2nd, recombinant plasmid pPIC9K-GOD restriction enzyme BglII are linearized, are then introduced into Pichia pastoris X33, Recombinant bacterium is obtained, is named as X33/pPIC9K-GOD.

2nd, recombinant bacterium X33/pPIC9K-GOD/pPICZ-PDI is built

1st, construction recombination plasmid pPICZ-PDI.According to sequencing result, structure description is carried out to recombinant plasmid pPICZ-PDI It is as follows：The double-stranded DNA point shown in the sequence 4 of sequence table is inserted between I restriction enzyme site of BstB I and Not of pPICZ α A carriers Son.DNA molecular shown in the sequence 4 of sequence table is PDI genes, the PDI albumen shown in the sequence 3 of polynucleotide.Due to inserting Angle of striking is between BstB I and Not I, so eliminating the nucleotide of coding for alpha-mating signal peptides in pPICZ α A carriers, outside Source protein intracellular expression.

2nd, recombinant plasmid pPICZ-PDI restriction enzyme Sac I are linearized, are then introduced into X33/pPIC9K-GOD, Recombinant bacterium is obtained, is named as X33/pPIC9K-GOD/pPICZ-PDI.

3rd, recombinant bacterium X33/pPIC9K-GOD/pPICZ-BiP is built

1st, construction recombination plasmid pPICZ-BiP.According to sequencing result, structure description is carried out to recombinant plasmid pPICZ-BiP It is as follows：The double-stranded DNA point shown in the sequence 6 of sequence table is inserted between I restriction enzyme site of BstB I and Not of pPICZ α A carriers Son.DNA molecular shown in the sequence 6 of sequence table is BiP genes, the BiP albumen shown in the sequence 5 of polynucleotide.

2nd, recombinant plasmid pPICZ-BiP restriction enzyme Sac I are linearized, are then introduced into X33/pPIC9K-GOD, Recombinant bacterium is obtained, is named as X33/pPIC9K-GOD/pPICZ-BiP.

4th, recombinant bacterium X33/pPIC9K-GOD/pPICZ-BiP-PDI is built

1st, using recombinant plasmid pPICZ-BiP as the carrier that sets out, construction recombination plasmid pPICZ-BiP-PDI.Through sequence verification, Recombinant plasmid pPICZ-BiP-PDI is the circular plasmids shown in the sequence 7 of sequence table.In the sequence 7 of sequence table, 938-2974 Position nucleotide is BiP genes, and 4342-5895 nucleotide are PDI genes.

2nd, recombinant plasmid pPICZ-BiP-PDI restriction enzyme Sac I are linearized, is then introduced into X33/pPIC9K- GOD obtains recombinant bacterium, is named as X33/pPIC9K-GOD/pPICZ-BiP-PDI.

5th, recombinant bacterium X33/pPIC9K and recombinant bacterium X33/pPIC9K/pPICZ is built

PPIC9K carriers restriction enzyme BglII is linearized, Pichia pastoris X33 is then introduced into, obtains recombinant bacterium, It is named as X33/pPIC9K.

PPICZ α A carrier restriction enzymes Sac I is linearized, X33/pPIC9K is then introduced into, obtains recombinant bacterium, It is named as X33/pPIC9K/pPICZ.

The ability of embodiment 2, detection recombinant bacterium production glucose oxidase

Test strain is：Recombinant bacterium X33/pPIC9K-GOD, recombinant bacterium X33/pPIC9K-GOD/pPICZ-PDI, recombinant bacterium X33/pPIC9K-GOD/pPICZ-BiP, recombinant bacterium X33/pPIC9K-GOD/pPICZ-BiP-PDI, recombinant bacterium X33/pPIC9K, Recombinant bacterium X33/pPIC9K/pPICZ or Pichia pastoris X33.

1st, take test strain, 30 DEG C, 220rpm shaken cultivations for 24 hours, obtain seed liquor.

2nd, the seed liquor that 1 parts by volume step 1 obtains is inoculated in 99 parts by volume BMGY culture mediums, 30 DEG C, 220rpm vibrations It cultivates to OD_600nmIt is worth for 10.

3rd, the system that 10 parts by volume steps 2 obtain is seeded to 90 parts by volume BSM culture mediums, 30 DEG C, 500rpm vibration trainings It supports.In incubation, it is every 12 it is small when sample, 4 DEG C, 10000rpm centrifugation 10min, collect supernatant.Supernatant is carried out poly- third Alkene acyl ammonia gel electrophoresis.Detect the glucose oxidase enzyme activity of supernatant.Detect GOD protein concentrations (the GOD albumen in supernatant Extracellular expression, PDI albumen and the equal intracellular expression of BiP albumen, so GOD protein concentrations are in terms of the total protein concentration in supernatant).

Glucose oxidase enzyme activity determination method is as follows：

1. taking supernatant, diluted with the PBS buffer solution of pH5.0,50mM, obtain dilution.

2. prepare reaction system (3.1mL)：Dilution, 100 μ L HRP solution and the 2.9mL 1. obtained by 100 μ L steps Dianisidine-glucose solution composition.HRP concentration in HRP solution is 300U/mL.Dianisidine-glucose solution Solvent is pH5.0,50mM acetate buffer solution；Solute and its concentration are as follows：2mM dianisidines, 10g/100ml glucose. PH4.5,50mM acetate buffer solution：Solvent is water, and solute is acetic acid and sodium acetate；The concentration of sodium acetate is 50mM, with acetic acid tune PH value is 4.5.

3. 2. reaction system that step obtains, 37 DEG C stand reaction 1min.

Using UV1800 at 500nm wavelength, the preceding light absorption value with after reaction of reaction is measured, the light absorption value after reaction-anti- Light absorption value=△ G before answering.Enzyme activity=(△ G × V_Always)/(ε×L)；V_Always=3.1ml, ε=7.5 × 10³M^-1·cm^-1；

L is the optical path of cuvette, L=0.1cm in this experiment.

1 enzyme activity unit refers under the conditions of 37 DEG C, pH5.0, and 1 μm of ol glucose is converted in 1min and generates 1 μm of ol Gluconic acid and H₂O₂Required enzyme amount.

The electrophoretogram for the supernatant that different sample times obtain is shown in Fig. 1.In Fig. 1, A X33/pPIC9K-GOD；B is X33/ PPIC9K-GOD/pPICZ-BiP, C X33/pPIC9K-GOD/pPICZ-PDI, D X33/pPIC9K-GOD/pPICZ-BiP- PDI.GOD albumen is marked with regard to arrow in Fig. 1.

The glucose oxidase enzyme activity for the supernatant that different sample times obtain see Fig. 2 (five times repetition experiment be averaged Value).Cultivate 132 it is small when the obtained glucose oxidase enzyme activity of supernatant be shown in Table 1 (average value of five repetition experiments).

Table 1

The result shows that expressing PDI albumen, expression BiP albumen while expressing PDI albumen and BiP albumen, can increase Recombinant bacterium produces the ability of GOD albumen, wherein expressing PDI albumen and the effect of BiP albumen is especially prominent simultaneously.

Embodiment 3 mass produces glucose oxidase using recombinant bacterium by fermentation tank

It is (initial that recombinant bacterium X33/pPIC9K-GOD/pPICZ-BiP-PDI is seeded to the fermentation tank equipped with BSM culture mediums OD_600nm=1-2), 30 DEG C of cultures.The stage two is carried out after the completion of stage one, stage one and stage two are carried out continuously.

Stage one：It is 5.0 by adding in 28% concentrated ammonia liquor control system pH, by adjusting speed of agitator in incubation It is more than 20% with throughput control system dissolved oxygen；When the glycerol depletion in BSM culture mediums rises so as to cause dissolved oxygen, start Using the rate flow feeding liquid first that adds in 18.15mL in every liter of system per hour, (feed supplement liquid first is is 1.2% containing volume fraction The aqueous solution of PTM1 and 50g/100ml glycerine), OD_600nmTo stop feed supplement during 150-200, continue 30 points of culture after stopping feed supplement When clock -2 is small.

Stage two：It is 6.0 by adding in 28% concentrated ammonia liquor control system pH, by adjusting speed of agitator in incubation It is more than 10% with throughput control system dissolved oxygen；In incubation, added with the rate stream for adding in 5g in every liter of system per hour Feed supplement liquid second (methanol that it is 1.2%PTM1 containing volume fraction that feed supplement liquid second, which is), while to add per hour in every kilogram of system The rate stream for entering 50g adds sorbierite.

A specific process is as follows：Recombinant bacterium X33/pPIC9K-GOD/pPICZ-BiP-PDI is seeded to equipped with 3L The 5L specification fermentation tank (initial ODs of BSM culture mediums_600nm=1)；19th starts flow feeding liquid first when small, the 23rd starts to stop when small Only feed supplement, the 24th starts flow feeding liquid second and sorbierite when small.Cultivate 150 it is small when after, the glucose oxidase enzyme of supernatant Work reaches 957U/mL (Enzyme activity assay method is with embodiment 2).

Sequence table

<110>Fujian Li Duoli bio tech ltd

<120>A kind of method for producing glucose oxidase

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Ser Asn Gly Ile Glu Ala Ser Leu Leu Thr Asp Pro Lys Asp Val Ser

1 5 10 15

Gly Arg Thr Val Asp Tyr Ile Ile Ala Gly Gly Gly Leu Thr Gly Leu

20 25 30

Thr Thr Ala Ala Arg Leu Thr Glu Asn Pro Asn Ile Ser Val Leu Val

35 40 45

Ile Glu Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp

50 55 60

Leu Asn Ala Tyr Gly Asp Ile Phe Gly Ser Ser Val Asp His Ala Tyr

65 70 75 80

Glu Thr Val Glu Leu Ala Thr Asn Asn Gln Thr Ala Leu Ile Arg Ser

85 90 95

Gly Asn Gly Leu Gly Gly Ser Thr Leu Val Asn Gly Gly Thr Trp Thr

100 105 110

Arg Pro His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn

115 120 125

Glu Gly Trp Asn Trp Asp Asn Val Ala Ala Tyr Ser Leu Gln Ala Glu

130 135 140

Arg Ala Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe

145 150 155 160

Asn Ala Ser Cys His Gly Val Asn Gly Thr Val His Ala Gly Pro Arg

165 170 175

Asp Thr Gly Asp Asp Tyr Ser Pro Ile Val Lys Ala Leu Met Ser Ala

180 185 190

Val Glu Asp Arg Gly Val Pro Thr Lys Lys Asp Phe Gly Cys Gly Asp

195 200 205

Pro His Gly Val Ser Met Phe Pro Asn Thr Leu His Glu Asp Gln Val

210 215 220

Arg Ser Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro

225 230 235 240

Asn Leu Gln Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser

245 250 255

Gln Asn Gly Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His

260 265 270

Lys Gly Asn Thr His Asn Val Tyr Ala Lys His Glu Val Leu Leu Ala

275 280 285

Ala Gly Ser Ala Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly

290 295 300

Met Lys Ser Ile Leu Glu Pro Leu Gly Ile Asp Thr Val Val Asp Leu

305 310 315 320

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

325 330 335

Arg Ile Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala

340 345 350

Thr Phe Asn Glu Thr Phe Gly Asp Tyr Ser Glu Lys Ala His Glu Leu

355 360 365

Leu Asn Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly

370 375 380

Gly Phe His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg

385 390 395 400

Asp Trp Ile Val Asn His Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp

405 410 415

Thr Ala Gly Val Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr

420 425 430

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

435 440 445

Ala Tyr Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln

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Ala Ala Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met

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Gln Thr Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr

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Asp Ala Asp Leu Ser Ala Trp Thr Glu Tyr Ile Pro Tyr His Phe Arg

500 505 510

Pro Asn Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met

515 520 525

Gly Gly Val Val Asp Asn Ala Ala Arg Val Tyr Gly Val Gln Gly Leu

530 535 540

Arg Val Ile Asp Gly Ser Ile Pro Pro Thr Gln Met Ser Ser His Val

545 550 555 560

Met Thr Val Phe Tyr Ala Met Ala Leu Lys Ile Ser Asp Ala Ile Leu

565 570 575

Glu Asp Tyr Ala Ser Met Gln

580

<210> 2

<211> 1752

<212> DNA

<213>Artificial sequence

<400> 2

agcaatggca ttgaagccag cctcctgact gatcccaagg atgtctccgg ccgcacggtc 60

gactacatca tcgctggtgg aggtctgact ggactcacca ccgctgctcg tctgacggag 120

aaccccaaca tcagtgtgct cgtcatcgaa agtggctcct acgagtcgga cagaggtcct 180

atcattgagg acctgaacgc ctacggcgac atctttggca gcagtgtaga ccacgcctac 240

gagaccgtgg agctcgctac caacaatcaa accgcgctga tccgctccgg aaatggtctc 300

ggtggctcta ctctagtgaa tggtggcacc tggactcgcc cccacaaggc acaggttgac 360

tcttgggaga ctgtctttgg aaatgagggc tggaactggg acaatgtggc cgcctactcc 420

ctccaggctg agcgtgctcg cgcaccaaat gccaaacaga tcgctgctgg ccactacttc 480

aacgcatcct gccatggtgt taatggtact gtccatgccg gaccccgcga caccggcgat 540

gactattctc ccatcgtcaa ggctctcatg agcgctgtcg aagaccgggg cgttcccacc 600

aagaaagact tcggatgcgg tgacccccat ggtgtgtcca tgttccccaa caccttgcac 660

gaagaccaag tgcgctccga tgccgctcgc gaatggctac ttcccaacta ccaacgtccc 720

aacctgcaag tcctgaccgg acagtatgtt ggtaaggtgc tccttagcca gaacggcacc 780

acccctcgtg ccgttggcgt ggaattcggc acccacaagg gcaacaccca caacgtttac 840

gctaagcacg aggtcctcct ggccgcgggc tccgctgtct ctcccacaat cctcgaatat 900

tccggtatcg gaatgaagtc catcctggag ccccttggta tcgacaccgt cgttgacctg 960

cccgtcggct tgaacctgca ggaccagacc accgctaccg tccgctcccg catcacctct 1020

gctggtgcag gacagggaca ggccgcttgg ttcgccacct tcaacgagac ctttggtgac 1080

tattccgaaa aggcacacga gctgctcaac accaagctgg agcagtgggc cgaagaggcc 1140

gtcgcccgtg gcggattcca caacaccacc gccttgctca tccagtacga gaactaccgc 1200

gactggattg tcaaccacaa cgtcgcgtac tcggaactct tcctcgacac tgccggagta 1260

gccagcttcg atgtgtggga ccttctgccc ttcacccgag gatacgttca catcctcgac 1320

aaggacccct accttcacca cttcgcctac gaccctcagt acttcctcaa cgagctggac 1380

ctgctcggtc aggctgccgc tactcaactg gcccgcaaca tctccaactc cggtgccatg 1440

cagacctact tcgctgggga gactatcccc ggtgataacc tcgcgtatga tgccgatttg 1500

agcgcctgga ctgagtacat cccgtaccac ttccgtccta actaccatgg cgtgggtact 1560

tgctccatga tgccgaagga gatgggcggt gttgttgata atgctgcccg tgtgtatggt 1620

gtgcagggac tgcgtgtcat tgatggttct attcctccta cgcaaatgtc gtcccatgtc 1680

atgacggtgt tctatgccat ggcgctaaaa atttcggatg ctatcttgga agattatgct 1740

tccatgcagt ga 1752

<210> 3

<211> 517

<212> PRT

<213>Artificial sequence

<400> 3

Met Gln Phe Asn Trp Asn Ile Lys Thr Val Ala Ser Ile Leu Ser Ala

1 5 10 15

Leu Thr Leu Ala Gln Ala Ser Asp Gln Glu Ala Ile Ala Pro Glu Asp

20 25 30

Ser His Val Val Lys Leu Thr Glu Ala Thr Phe Glu Ser Phe Ile Thr

35 40 45

Ser Asn Pro His Val Leu Ala Glu Phe Phe Ala Pro Trp Cys Gly His

50 55 60

Cys Lys Lys Leu Gly Pro Glu Leu Val Ser Ala Ala Glu Ile Leu Lys

65 70 75 80

Asp Asn Glu Gln Val Lys Ile Ala Gln Ile Asp Cys Thr Glu Glu Lys

85 90 95

Glu Leu Cys Gln Gly Tyr Glu Ile Lys Gly Tyr Pro Thr Leu Lys Val

100 105 110

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

115 120 125

Ser Gln Ser Ile Val Ser Tyr Met Leu Lys Gln Ser Leu Pro Pro Val

130 135 140

Ser Glu Ile Asn Ala Thr Lys Asp Leu Asp Asp Thr Ile Ala Glu Ala

145 150 155 160

Lys Glu Pro Val Ile Val Gln Val Leu Pro Glu Asp Ala Ser Asn Leu

165 170 175

Glu Ser Asn Thr Thr Phe Tyr Gly Val Ala Gly Thr Leu Arg Glu Lys

180 185 190

Phe Thr Phe Val Ser Thr Lys Ser Thr Asp Tyr Ala Lys Lys Tyr Thr

195 200 205

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

210 215 220

Ser Val Tyr Ser Gly Glu Glu Leu Asp Glu Thr His Leu Val His Trp

225 230 235 240

Ile Asp Ile Glu Ser Lys Pro Leu Phe Gly Asp Ile Asp Gly Ser Thr

245 250 255

Phe Lys Ser Tyr Ala Glu Ala Asn Ile Pro Leu Ala Tyr Tyr Phe Tyr

260 265 270

Glu Asn Glu Glu Gln Arg Ala Ala Ala Ala Asp Ile Ile Lys Pro Phe

275 280 285

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

290 295 300

Lys Phe Gly Lys His Ala Lys Asn Leu Asn Met Asp Glu Glu Lys Leu

305 310 315 320

Pro Leu Phe Val Ile His Asp Leu Val Ser Asn Lys Lys Phe Gly Val

325 330 335

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

340 345 350

Lys Phe Ile Ala Gly Glu Ala Glu Pro Ile Val Lys Ser Glu Pro Ile

355 360 365

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

370 375 380

Asp Glu Val Val Phe Asp Glu Ser Lys Asp Val Leu Val Lys Tyr Tyr

385 390 395 400

Ala Pro Trp Cys Gly His Cys Lys Arg Met Ala Pro Ala Tyr Glu Glu

405 410 415

Leu Ala Thr Leu Tyr Ala Asn Asp Glu Asp Ala Ser Ser Lys Val Val

420 425 430

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

435 440 445

Gln Gly Tyr Pro Thr Leu Ile Leu Tyr Pro Ala Gly Asp Lys Ser Asn

450 455 460

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

465 470 475 480

Val Lys Glu Arg Gly Thr His Lys Val Asp Ala Leu Ala Leu Arg Pro

485 490 495

Val Glu Glu Glu Lys Glu Ala Glu Glu Glu Ala Glu Ser Glu Ala Asp

500 505 510

Ala His Asp Glu Leu

515

<210> 4

<211> 1554

<212> DNA

<213>Artificial sequence

<400> 4

atgcaattca actggaatat taaaactgtg gcaagtattt tgtccgctct cacactagca 60

caagcaagtg atcaggaggc tattgctcca gaggactctc atgtcgtcaa attgactgaa 120

gccacttttg agtctttcat caccagtaat cctcacgttt tggcagagtt ttttgcccct 180

tggtgtggtc actgtaagaa gttgggccct gaacttgttt ctgctgccga gatcttaaag 240

gacaatgagc aggttaagat tgctcaaatt gattgtacgg aggagaagga attatgtcaa 300

ggctacgaaa ttaaagggta tcctactttg aaggtgttcc atggtgaggt tgaggtccca 360

agtgactatc aaggtcaaag acagagccaa agcattgtca gctatatgct aaagcagagt 420

ttaccccctg tcagtgaaat caatgcaacc aaagatttag acgacacaat cgccgaggca 480

aaagagcccg tgattgtgca agtactaccg gaagatgcat ccaacttgga atctaacacc 540

acattttacg gagttgccgg tactctcaga gagaaattca cttttgtctc cactaagtct 600

actgattatg ccaaaaaata cactagcgac tcgactcctg cctatttgct tgtcagacct 660

ggcgaggaac ctagtgttta ctctggtgag gagttagatg agactcattt ggtgcactgg 720

attgatattg agtccaaacc tctatttgga gacattgacg gatccacctt caaatcatat 780

gctgaagcta acatcccttt agcctactat ttctatgaga acgaagaaca acgtgctgct 840

gctgccgata ttattaaacc ttttgctaaa gagcaacgtg gcaaaattaa ctttgttggc 900

ttagatgccg ttaaattcgg taagcatgcc aagaacttaa acatggatga agagaaactc 960

cctctatttg tcattcatga tttggtgagc aacaagaagt ttggagttcc tcaagaccaa 1020

gaattgacga acaaagatgt gaccgagctg attgagaaat tcatcgcagg agaggcagaa 1080

ccaattgtga aatcagagcc aattccagaa attcaagaag agaaagtctt caagctagtc 1140

ggaaaggccc acgatgaagt tgtcttcgat gaatctaaag atgttctagt caagtactac 1200

gccccttggt gtggtcactg taagagaatg gctcctgctt atgaggaatt ggctactctt 1260

tacgccaatg atgaggatgc ctcttcaaag gttgtgattg caaaacttga tcacactttg 1320

aacgatgtcg acaacgttga tattcaaggt tatcctactt tgatccttta tccagctggt 1380

gataaatcca atcctcaact gtatgatgga tctcgtgacc tagaatcatt ggctgagttt 1440

gtaaaggaga gaggaaccca caaagtggat gccctagcac tcagaccagt cgaggaagaa 1500

aaggaagctg aagaagaagc tgaaagtgag gcagacgctc acgacgagct ttaa 1554

<210> 5

<211> 678

<212> PRT

<213>Artificial sequence

<400> 5

Met Leu Ser Leu Lys Pro Ser Trp Leu Thr Leu Ala Ala Leu Met Tyr

1 5 10 15

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

20 25 30

Asp Val Glu Ser Tyr Gly Thr Val Ile Gly Ile Asp Leu Gly Thr Thr

35 40 45

Tyr Ser Cys Val Gly Val Met Lys Ser Gly Arg Val Glu Ile Leu Ala

50 55 60

Asn Asp Gln Gly Asn Arg Ile Thr Pro Ser Tyr Val Ser Phe Thr Glu

65 70 75 80

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

85 90 95

Pro Lys Asn Thr Ile Phe Asp Ile Lys Arg Leu Ile Gly Met Lys Tyr

100 105 110

Asp Ala Pro Glu Val Gln Arg Asp Leu Lys Arg Leu Pro Tyr Thr Val

115 120 125

Lys Ser Lys Asn Gly Gln Pro Val Val Ser Val Glu Tyr Lys Gly Glu

130 135 140

Glu Lys Ser Phe Thr Pro Glu Glu Ile Ser Ala Met Val Leu Gly Lys

145 150 155 160

Met Lys Leu Ile Ala Glu Asp Tyr Leu Gly Lys Lys Val Thr His Ala

165 170 175

Val Val Thr Val Pro Ala Tyr Phe Asn Asp Ala Gln Arg Gln Ala Thr

180 185 190

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

195 200 205

Glu Pro Thr Ala Ala Ala Leu Ala Tyr Gly Leu Asp Lys Thr Gly Glu

210 215 220

Glu Arg Gln Ile Ile Val Tyr Asp Leu Gly Gly Gly Thr Phe Asp Val

225 230 235 240

Ser Leu Leu Ser Ile Glu Gly Gly Ala Phe Glu Val Leu Ala Thr Ala

245 250 255

Gly Asp Thr His Leu Gly Gly Glu Asp Phe Asp Tyr Arg Val Val Arg

260 265 270

His Phe Val Lys Ile Phe Lys Lys Lys His Asn Ile Asp Ile Ser Asn

275 280 285

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

290 295 300

Arg Thr Leu Ser Ser Gln Met Thr Thr Arg Ile Glu Ile Asp Ser Phe

305 310 315 320

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

325 330 335

Glu Ile Asn Ile Glu Leu Phe Lys Lys Thr Leu Lys Pro Val Glu Gln

340 345 350

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

355 360 365

Leu Val Gly Gly Ser Thr Arg Ile Pro Lys Val Gln Gln Leu Leu Glu

370 375 380

Asp Tyr Phe Asp Gly Lys Lys Ala Ser Lys Gly Ile Asn Pro Asp Glu

385 390 395 400

Ala Val Ala Tyr Gly Ala Ala Val Gln Ala Gly Val Leu Ser Gly Glu

405 410 415

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

420 425 430

Gly Ile Glu Thr Thr Gly Gly Val Met Thr Thr Leu Ile Asn Arg Asn

435 440 445

Thr Ala Ile Pro Thr Lys Lys Ser Gln Ile Phe Ser Thr Ala Ala Asp

450 455 460

Asn Gln Pro Thr Val Leu Ile Gln Val Tyr Glu Gly Glu Arg Ala Leu

465 470 475 480

Ala Lys Asp Asn Asn Leu Leu Gly Lys Phe Glu Leu Thr Gly Ile Pro

485 490 495

Pro Ala Pro Arg Gly Thr Pro Gln Val Glu Val Thr Phe Val Leu Asp

500 505 510

Ala Asn Gly Ile Leu Lys Val Ser Ala Thr Asp Lys Gly Thr Gly Lys

515 520 525

Ser Glu Ser Ile Thr Ile Asn Asn Asp Arg Gly Arg Leu Ser Lys Glu

530 535 540

Glu Val Asp Arg Met Val Glu Glu Ala Glu Lys Tyr Ala Ala Glu Asp

545 550 555 560

Ala Ala Leu Arg Glu Lys Ile Glu Ala Arg Asn Ala Leu Glu Asn Tyr

565 570 575

Ala His Ser Leu Arg Asn Gln Val Thr Asp Asp Ser Glu Thr Gly Leu

580 585 590

Gly Ser Lys Leu Asp Glu Asp Asp Lys Glu Thr Leu Thr Asp Ala Ile

595 600 605

Lys Asp Thr Leu Glu Phe Leu Glu Asp Asn Phe Asp Thr Ala Thr Lys

610 615 620

Glu Glu Leu Asp Glu Gln Arg Glu Lys Leu Ser Lys Ile Ala Tyr Pro

625 630 635 640

Ile Thr Ser Lys Leu Tyr Gly Ala Pro Glu Gly Gly Thr Pro Pro Gly

645 650 655

Gly Gln Gly Phe Asp Asp Asp Asp Gly Asp Phe Asp Tyr Asp Tyr Asp

660 665 670

Tyr Asp His Asp Glu Leu

675

<210> 6

<211> 2037

<212> DNA

<213>Artificial sequence

<400> 6

atgctgtcgt taaaaccatc ttggctgact ttggcggcat taatgtatgc catgctattg 60

gtcgtagtgc catttgctaa acctgttaga gctgacgatg tcgaatctta tggaacagtg 120

attggtatcg atttgggtac cacgtactct tgtgtcggtg tgatgaagtc gggtcgtgta 180

gaaattcttg ctaatgacca aggtaacaga atcactcctt cctacgttag tttcactgaa 240

gacgagagac tggttggtga tgctgctaag aacttagctg cttctaaccc aaaaaacacc 300

atctttgata ttaagagatt gatcggtatg aagtatgatg ccccagaggt ccaaagagac 360

ttgaagcgtc ttccttacac tgtcaagagc aagaacggcc aacctgtcgt ttctgtcgag 420

tacaagggtg aggagaagtc tttcactcct gaggagattt ccgccatggt cttgggtaag 480

atgaagttga tcgctgagga ctacttagga aagaaagtca ctcatgctgt cgttaccgtt 540

ccagcctact tcaacgacgc tcaacgtcaa gccactaagg atgccggtct catcgccggt 600

ttgactgttc tgagaattgt gaacgagcct accgccgctg cccttgctta cggtttggac 660

aagactggtg aggaaagaca gatcatcgtc tacgacttgg gtggaggaac cttcgatgtt 720

tctctgcttt ctattgaggg tggtgctttc gaggttcttg ctaccgccgg tgacacccac 780

ttgggtggtg aggactttga ctacagagtt gttcgccact tcgttaagat tttcaagaag 840

aagcataaca ttgacatcag caacaatgat aaggctttag gtaagctgaa gagagaggtc 900

gaaaaggcca agcgtacttt gtcttcccag atgactacca gaattgagat tgactctttc 960

gtcgacggta tcgacttctc tgagcaactg tctagagcta agtttgagga gatcaacatt 1020

gaattattca agaagacact gaaaccagtt gaacaagtcc tcaaagacgc tggtgtcaag 1080

aaatctgaaa ttgatgacat tgtcttggtt ggtggttcta ccagaattcc aaaggttcaa 1140

caattattgg aggattactt tgacggaaag aaggcttcta agggaattaa cccagatgaa 1200

gctgtcgcat acggtgctgc tgttcaggct ggtgttttgt ctggtgagga aggtgtcgat 1260

gacatcgtct tgcttgatgt gaacccccta actctgggta tcgagactac tggtggcgtt 1320

atgactacct taatcaacag aaacactgct atcccaacta agaaatctca aattttctcc 1380

actgctgctg acaaccagcc aactgtgttg attcaagttt atgagggtga gagagccttg 1440

gctaaggaca acaacttgct tggtaaattc gagctgactg gtattccacc agctccaaga 1500

ggtactcctc aagttgaggt tacttttgtt ttagacgcta acggaatttt gaaggtctct 1560

gccaccgata agggaactgg aaaatccgag tccatcacca tcaacaatga tcgtggtaga 1620

ttgtccaagg aggaggttga ccgtatggtt gaagaggccg agaagtacgc cgctgaggat 1680

gctgcactaa gagaaaagat tgaggctaga aacgctctgg agaactacgc tcattccctt 1740

aggaaccaag ttactgatga ctctgaaacc gggcttggtt ctaaattgga cgaggacgac 1800

aaagagacat tgacagatgc catcaaagat accctagagt tcttggaaga caacttcgac 1860

accgcaacca aggaagaatt agacgaacaa agagaaaagc tttccaagat tgcttaccca 1920

atcacttcta agctatacgg tgctccagag ggtggtactc cacctggtgg tcaaggtttt 1980

gacgatgatg atggagactt tgactacgac tatgactatg atcatgatga gttgtag 2037

<210> 7

<211> 8221

<212> DNA

<213>Artificial sequence

<400> 7

agatctaaca tccaaagacg aaaggttgaa tgaaaccttt ttgccatccg acatccacag 60

gtccattctc acacataagt gccaaacgca acaggagggg atacactagc agcagaccgt 120

tgcaaacgca ggacctccac tcctcttctc ctcaacaccc acttttgcca tcgaaaaacc 180

agcccagtta ttgggcttga ttggagctcg ctcattccaa ttccttctat taggctacta 240

acaccatgac tttattagcc tgtctatcct ggcccccctg gcgaggttca tgtttgttta 300

tttccgaatg caacaagctc cgcattacac ccgaacatca ctccagatga gggctttctg 360

agtgtggggt caaatagttt catgttcccc aaatggccca aaactgacag tttaaacgct 420

gtcttggaac ctaatatgac aaaagcgtga tctcatccaa gatgaactaa gtttggttcg 480

ttgaaatgct aacggccagt tggtcaaaaa gaaacttcca aaagtcggca taccgtttgt 540

cttgtttggt attgattgac gaatgctcaa aaataatctc attaatgctt agcgcagtct 600

ctctatcgct tctgaacccc ggtgcacctg tgccgaaacg caaatgggga aacacccgct 660

ttttggatga ttatgcattg tctccacatt gtatgcttcc aagattctgg tgggaatact 720

gctgatagcc taacgttcat gatcaaaatt taactgttct aacccctact tgacagcaat 780

atataaacag aaggaagctg ccctgtctta aacctttttt tttatcatca ttattagctt 840

actttcataa ttgcgactgg ttccaattga caagcttttg attttaacga cttttaacga 900

caacttgaga agatcaaaaa acaactaatt attcgaaatg ctgtcgttaa aaccatcttg 960

gctgactttg gcggcattaa tgtatgccat gctattggtc gtagtgccat ttgctaaacc 1020

tgttagagct gacgatgtcg aatcttatgg aacagtgatt ggtatcgatt tgggtaccac 1080

gtactcttgt gtcggtgtga tgaagtcggg tcgtgtagaa attcttgcta atgaccaagg 1140

taacagaatc actccttcct acgttagttt cactgaagac gagagactgg ttggtgatgc 1200

tgctaagaac ttagctgctt ctaacccaaa aaacaccatc tttgatatta agagattgat 1260

cggtatgaag tatgatgccc cagaggtcca aagagacttg aagcgtcttc cttacactgt 1320

caagagcaag aacggccaac ctgtcgtttc tgtcgagtac aagggtgagg agaagtcttt 1380

cactcctgag gagatttccg ccatggtctt gggtaagatg aagttgatcg ctgaggacta 1440

cttaggaaag aaagtcactc atgctgtcgt taccgttcca gcctacttca acgacgctca 1500

acgtcaagcc actaaggatg ccggtctcat cgccggtttg actgttctga gaattgtgaa 1560

cgagcctacc gccgctgccc ttgcttacgg tttggacaag actggtgagg aaagacagat 1620

catcgtctac gacttgggtg gaggaacctt cgatgtttct ctgctttcta ttgagggtgg 1680

tgctttcgag gttcttgcta ccgccggtga cacccacttg ggtggtgagg actttgacta 1740

cagagttgtt cgccacttcg ttaagatttt caagaagaag cataacattg acatcagcaa 1800

caatgataag gctttaggta agctgaagag agaggtcgaa aaggccaagc gtactttgtc 1860

ttcccagatg actaccagaa ttgagattga ctctttcgtc gacggtatcg acttctctga 1920

gcaactgtct agagctaagt ttgaggagat caacattgaa ttattcaaga agacactgaa 1980

accagttgaa caagtcctca aagacgctgg tgtcaagaaa tctgaaattg atgacattgt 2040

cttggttggt ggttctacca gaattccaaa ggttcaacaa ttattggagg attactttga 2100

cggaaagaag gcttctaagg gaattaaccc agatgaagct gtcgcatacg gtgctgctgt 2160

tcaggctggt gttttgtctg gtgaggaagg tgtcgatgac atcgtcttgc ttgatgtgaa 2220

ccccctaact ctgggtatcg agactactgg tggcgttatg actaccttaa tcaacagaaa 2280

cactgctatc ccaactaaga aatctcaaat tttctccact gctgctgaca accagccaac 2340

tgtgttgatt caagtttatg agggtgagag agccttggct aaggacaaca acttgcttgg 2400

taaattcgag ctgactggta ttccaccagc tccaagaggt actcctcaag ttgaggttac 2460

ttttgtttta gacgctaacg gaattttgaa ggtctctgcc accgataagg gaactggaaa 2520

atccgagtcc atcaccatca acaatgatcg tggtagattg tccaaggagg aggttgaccg 2580

tatggttgaa gaggccgaga agtacgccgc tgaggatgct gcactaagag aaaagattga 2640

ggctagaaac gctctggaga actacgctca ttcccttagg aaccaagtta ctgatgactc 2700

tgaaaccggg cttggttcta aattggacga ggacgacaaa gagacattga cagatgccat 2760

caaagatacc ctagagttct tggaagacaa cttcgacacc gcaaccaagg aagaattaga 2820

cgaacaaaga gaaaagcttt ccaagattgc ttacccaatc acttctaagc tatacggtgc 2880

tccagagggt ggtactccac ctggtggtca aggttttgac gatgatgatg gagactttga 2940

ctacgactat gactatgatc atgatgagtt gtaggcggcc gccagcttac gtagaacaaa 3000

aactcatctc agaagaggat ctgaatagcg ccgtcgacca tcatcatcat catcattgag 3060

tttgtagcct tagacatgac tgttcctcag ttcaagttgg gcacttacga gaagaccggt 3120

cttgctagat tctaatcaag aggatgtcag aatgccattt gcctgagaga tgcaggcttc 3180

atttttgata cttttttatt tgtaacctat atagtatagg attttttttg tcattttgtt 3240

tcttctcgta cgagcttgct cctgatcagc ctatctcgca gctgatgaat atcttgtggt 3300

aggggtttgg gaaaatcatt cgagtttgat gtttttcttg gtatttccca ctcctcttca 3360

gagtacagaa gattaagtga gaccttcgtt tgtgcggatc cagatctaac atccaaagac 3420

gaaaggttga atgaaacctt tttgccatcc gacatccaca ggtccattct cacacataag 3480

tgccaaacgc aacaggaggg gatacactag cagcagaccg ttgcaaacgc aggacctcca 3540

ctcctcttct cctcaacacc cacttttgcc atcgaaaaac cagcccagtt attgggcttg 3600

attggagctc gctcattcca attccttcta ttaggctact aacaccatga ctttattagc 3660

ctgtctatcc tggcccccct ggcgaggttc atgtttgttt atttccgaat gcaacaagct 3720

ccgcattaca cccgaacatc actccagatg agggctttct gagtgtgggg tcaaatagtt 3780

tcatgttccc caaatggccc aaaactgaca gtttaaacgc tgtcttggaa cctaatatga 3840

caaaagcgtg atctcatcca agatgaacta agtttggttc gttgaaatgc taacggccag 3900

ttggtcaaaa agaaacttcc aaaagtcggc ataccgtttg tcttgtttgg tattgattga 3960

cgaatgctca aaaataatct cattaatgct tagcgcagtc tctctatcgc ttctgaaccc 4020

cggtgcacct gtgccgaaac gcaaatgggg aaacacccgc tttttggatg attatgcatt 4080

gtctccacat tgtatgcttc caagattctg gtgggaatac tgctgatagc ctaacgttca 4140

tgatcaaaat ttaactgttc taacccctac ttgacagcaa tatataaaca gaaggaagct 4200

gccctgtctt aaaccttttt ttttatcatc attattagct tactttcata attgcgactg 4260

gttccaattg acaagctttt gattttaacg acttttaacg acaacttgag aagatcaaaa 4320

aacaactaat tattcgaaac gatgcaattc aactggaata ttaaaactgt ggcaagtatt 4380

ttgtccgctc tcacactagc acaagcaagt gatcaggagg ctattgctcc agaggactct 4440

catgtcgtca aattgactga agccactttt gagtctttca tcaccagtaa tcctcacgtt 4500

ttggcagagt tttttgcccc ttggtgtggt cactgtaaga agttgggccc tgaacttgtt 4560

tctgctgccg agatcttaaa ggacaatgag caggttaaga ttgctcaaat tgattgtacg 4620

gaggagaagg aattatgtca aggctacgaa attaaagggt atcctacttt gaaggtgttc 4680

catggtgagg ttgaggtccc aagtgactat caaggtcaaa gacagagcca aagcattgtc 4740

agctatatgc taaagcagag tttaccccct gtcagtgaaa tcaatgcaac caaagattta 4800

gacgacacaa tcgccgaggc aaaagagccc gtgattgtgc aagtactacc ggaagatgca 4860

tccaacttgg aatctaacac cacattttac ggagttgccg gtactctcag agagaaattc 4920

acttttgtct ccactaagtc tactgattat gccaaaaaat acactagcga ctcgactcct 4980

gcctatttgc ttgtcagacc tggcgaggaa cctagtgttt actctggtga ggagttagat 5040

gagactcatt tggtgcactg gattgatatt gagtccaaac ctctatttgg agacattgac 5100

ggatccacct tcaaatcata tgctgaagct aacatccctt tagcctacta tttctatgag 5160

aacgaagaac aacgtgctgc tgctgccgat attattaaac cttttgctaa agagcaacgt 5220

ggcaaaatta actttgttgg cttagatgcc gttaaattcg gtaagcatgc caagaactta 5280

aacatggatg aagagaaact ccctctattt gtcattcatg atttggtgag caacaagaag 5340

tttggagttc ctcaagacca agaattgacg aacaaagatg tgaccgagct gattgagaaa 5400

ttcatcgcag gagaggcaga accaattgtg aaatcagagc caattccaga aattcaagaa 5460

gagaaagtct tcaagctagt cggaaaggcc cacgatgaag ttgtcttcga tgaatctaaa 5520

gatgttctag tcaagtacta cgccccttgg tgtggtcact gtaagagaat ggctcctgct 5580

tatgaggaat tggctactct ttacgccaat gatgaggatg cctcttcaaa ggttgtgatt 5640

gcaaaacttg atcacacttt gaacgatgtc gacaacgttg atattcaagg ttatcctact 5700

ttgatccttt atccagctgg tgataaatcc aatcctcaac tgtatgatgg atctcgtgac 5760

ctagaatcat tggctgagtt tgtaaaggag agaggaaccc acaaagtgga tgccctagca 5820

ctcagaccag tcgaggaaga aaaggaagct gaagaagaag ctgaaagtga ggcagacgct 5880

cacgacgagc tttagcggcc gcgaacaaaa actcatctca gaagaggatc tgaatagcgc 5940

cgtcgaccat catcatcatc atcattgagt ttgtagcctt agacatgact gttcctcagt 6000

tcaagttggg cacttacgag aagaccggtc ttgctagatt ctaatcaaga ggatgtcaga 6060

atgccatttg cctgagagat gcaggcttca tttttgatac ttttttattt gtaacctata 6120

tagtatagga ttttttttgt cattttgttt cttctcgtac gagcttgctc ctgatcagcc 6180

tatctcgcag ctgatgaata tcttgtggta ggggtttggg aaaatcattc gagtttgatg 6240

tttttcttgg tatttcccac tcctcttcag agtacagaag attaagtgag accttcgttt 6300

gtgcggatcc cccacacacc atagcttcaa aatgtttcta ctcctttttt actcttccag 6360

attttctcgg actccgcgca tcgccgtacc acttcaaaac acccaagcac agcatactaa 6420

attttccctc tttcttcctc tagggtgtcg ttaattaccc gtactaaagg tttggaaaag 6480

aaaaaagaga ccgcctcgtt tctttttctt cgtcgaaaaa ggcaataaaa atttttatca 6540

cgtttctttt tcttgaaatt ttttttttta gtttttttct ctttcagtga cctccattga 6600

tatttaagtt aataaacggt cttcaatttc tcaagtttca gtttcatttt tcttgttcta 6660

ttacaacttt ttttacttct tgttcattag aaagaaagca tagcaatcta atctaagggg 6720

cggtgttgac aattaatcat cggcatagta tatcggcata gtataatacg acaaggtgag 6780

gaactaaacc atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc gcgacgtcgc 6840

cggagcggtc gagttctgga ccgaccggct cgggttctcc cgggacttcg tggaggacga 6900

cttcgccggt gtggtccggg acgacgtgac cctgttcatc agcgcggtcc aggaccaggt 6960

ggtgccggac aacaccctgg cctgggtgtg ggtgcgcggc ctggacgagc tgtacgccga 7020

gtggtcggag gtcgtgtcca cgaacttccg ggacgcctcc gggccggcca tgaccgagat 7080

cggcgagcag ccgtgggggc gggagttcgc cctgcgcgac ccggccggca actgcgtgca 7140

cttcgtggcc gaggagcagg actgacacgt ccgacggcgg cccacgggtc ccaggcctcg 7200

gagatccgtc ccccttttcc tttgtcgata tcatgtaatt agttatgtca cgcttacatt 7260

cacgccctcc ccccacatcc gctctaaccg aaaaggaagg agttagacaa cctgaagtct 7320

aggtccctat ttattttttt atagttatgt tagtattaag aacgttattt atatttcaaa 7380

tttttctttt ttttctgtac agacgcgtgt acgcatgtaa cattatactg aaaaccttgc 7440

ttgagaaggt tttgggacgc tcgaaggctt taatttgcaa gctggagacc aacatgtgag 7500

caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata 7560

ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc 7620

cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg 7680

ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc 7740

tttctcaatg ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg 7800

gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc 7860

ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga 7920

ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg 7980

gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa 8040

aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg 8100

tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt 8160

ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat 8220

c 8221

Claims (10)

1. a kind of recombinant bacterium is that the encoding gene of the encoding gene of glucose oxidase and molecular chaperones is imported out obtaining for bacterium germination It arrives.

2. a kind of recombinant bacterium, it is to confer to out bacterium germination glucose oxidase activity and chaperone activity obtains.

3. recombinant bacterium as claimed in claim 1 or 2, it is characterised in that：

The glucose oxidase is following (a1) or (a2)：

(a1) protein shown in the sequence 1 of sequence table；

(a2) by the amino acid sequence of sequence 1 by the substitution and/or missing and/or addition of one or several amino acid residues and Its derivative protein with identical function.

4. recombinant bacterium as claimed in claim 3, it is characterised in that：

The encoding gene of the glucose oxidase is following (a3), (a4), (a5) or (a6)：

(a3) DNA molecular of the code area as shown in the 1st to 1749 nucleotide of sequence 2 of sequence table；

(a4) DNA molecular of the code area as shown in the sequence 2 of sequence table；

(a5) the DNA sequence dna hybridization limited under strict conditions with (a3) or (a4) and the DNA molecular of coding glucose oxidase；

(a6) DNA sequence dna limited with (a3) or (a4) at least with 70%, at least with 75%, at least with 80%, at least With 85%, at least with 90%, at least with 95%, at least with 96%, at least with 97%, at least with 98% or extremely There is more than 99% homology less and encode the DNA molecular of glucose oxidase.

5. the recombinant bacterium as described in any in Claims 1-4, it is characterised in that：The molecular chaperones is disulfide bond isomerase And/or tuberculosis immunity globulin.

6. recombinant bacterium as claimed in claim 5, it is characterised in that：

The disulfide bond isomerase is following (b1) or (b2)：

(b1) protein shown in the sequence 3 of sequence table；

(b2) by the amino acid sequence of sequence 3 by the substitution and/or missing and/or addition of one or several amino acid residues and Its derivative protein with identical function；

The tuberculosis immunity globulin is following (c1) or (c2)：

(c1) protein shown in the sequence 5 of sequence table；

(c2) by the amino acid sequence of sequence 5 by the substitution and/or missing and/or addition of one or several amino acid residues and Its derivative protein with identical function.

7. application of any recombinant bacterium in glucose oxidase is prepared in claim 1 to 6.

8. a kind of method for producing glucose oxidase, includes the following steps：Cultivate any restructuring in claim 1 to 6 Bacterium obtains glucose oxidase.

9. application of the molecular chaperones in microbial expression glucose oxidase is promoted.

10. a kind of method of expression of raising glucose oxidase in microorganism, includes the following steps：In microorganism It imports the encoding gene of molecular chaperones or increases the level of molecular chaperones in microorganism, so as to improve microbial expression glucose The level of oxidizing ferment.