CN115975966A - A fusion protein, method and application for improving the catalytic efficiency of glucose oxidase - Google Patents

Abstract

The invention discloses a fusion protein for improving the catalytic efficiency of glucose oxidase, and the fusion protein comprises glucose oxidase, Vitella hyaline hemoglobin and a flexible link polypeptide. In the present invention, the fusion protein is constructed by constructing the hyaline hemoglobin and the glucose oxidase, and the fusion protein utilizes the oxygen transfer ability of the hyaline hemoglobin to help improve the catalytic efficiency of the glucose oxidase. Under the same conditions, compared with the simple glucose oxidase, the catalytic efficiency of the fusion protein is significantly improved, which is conducive to the application of the enzyme in industrial production.

Description

A fusion protein, method and application for improving the catalytic efficiency of glucose oxidase

technical field

The invention belongs to the field of biochemical technology, in particular to a fusion protein, method and application for improving the catalytic efficiency of glucose oxidase.

Background technique

Glucose oxidase (GOD) is a very important industrial enzyme that catalyzes the production of gluconic acid and hydrogen peroxide from glucose using oxygen as the electron acceptor. Due to the unique substrate consumption (oxygen) and product generation (acid) of its catalytic reaction and the accompanying electron transfer process, GOD is widely used in many industries such as food, feed, pharmaceutical and medical.

At present, the commercial GOD is still based on the stable Aspergillus niger GOD. Taking gluconic acid (GA), a bulk organic acid product, as an example, its main production method is submerged fermentation of GOD-producing Aspergillus niger. GOD catalyzed reaction is a massive aerobic process, and oxygen transfer is an important limiting factor for GOD to exert its ability. Studies have shown that process optimization to increase oxygen transfer and concentration can greatly enhance the yield of GA, but the actual production will also lead to increased aerodynamic costs, which restricts the economic benefits of GA. How to effectively enhance the oxygen supply capacity in the catalytic reaction of GOD is an important research direction for cost reduction and efficiency increase.

Vibrella hemoglobin (VHb) is a bacterial hemoglobin derived from Vibrella C1. VHb has strong oxygen transport ability, and has certain terminal oxidase activity and peroxidase activity. VHb has become a very practical tool and is widely used in microorganisms, animals and plants for overexpression to promote cell growth or improve product production. Heterologous expression of VHb can increase the synthesis of metabolites such as microbial alcohols, antibiotics, organic acids, and polysaccharides, and can also increase the secretion and expression of enzymes, such as lipase 2, xylanase, coenzyme Q10, and L-asparaginase, etc. .

Therefore, constructing VHb and GOD as a fusion protein and utilizing the oxygen transfer ability of VHb can help improve the catalytic efficiency of GOD. The invention provides a fusion protein method of VHb and GOD, which can significantly improve the catalytic efficiency of GOD.

Through searching, no published documents related to the patent application of the present invention have been found.

Contents of the invention

The purpose of the present invention is to overcome the problems existing in the prior art, and provide a fusion protein, method and application for improving the catalytic efficiency of glucose oxidase.

The technical scheme that the present invention solves technical problem adopts is:

A fusion protein that improves the catalytic efficiency of glucose oxidase, the fusion protein includes glucose oxidase, Vitiligo hyaline hemoglobin and a flexible link polypeptide.

Further, the glucose oxidase, Vitreum hemoglobin and a flexible link polypeptide need to construct a fusion protein according to a certain link sequence, and the certain link sequence is the amino acid sequence C of the flexible link polypeptide linked to Vitreum hemoglobin respectively. terminal and the amino acid sequence N-terminal of glucose oxidase.

Further, the flexible link polypeptide is composed of glycine and serine-rich polypeptides, the total content of glycine and serine in the flexible link polypeptide is not less than 60%, and the flexible link polypeptide contains 4 to 15 amino acids.

Further, the amino acid sequence of the glucose oxidase is shown in SEQ ID NO.1, the amino acid sequence of the Vitella hyaline hemoglobin is shown in SEQ ID NO.2, and the amino acid sequence of the flexible link polypeptide is shown in SEQ ID NO .3, the amino acid sequence of the fusion protein is shown in SEQ ID NO.4.

A method for improving the catalytic efficiency of glucose oxidase by using the above-mentioned fusion protein, said method improving the catalytic efficiency of glucose oxidase through the fusion protein.

A kind of coding gene of above-mentioned fusion protein, its nucleotide sequence is as shown in SEQ ID NO.5.

A recombinant expression vector comprising the gene encoding the fusion protein as described above, preferably the recombinant expression vector is an Aspergillus niger expression vector.

A recombinant strain comprising the gene encoding the fusion protein as described above.

Further, the recombinant strain is recombinant Aspergillus niger, and the recombinant strain may be bacteria or fungal cells, preferably yeast or filamentous fungal cells, more preferably Aspergillus niger.

The application of the above-mentioned fusion protein in improving the catalytic efficiency of glucose oxidase.

The beneficial effects that the present invention obtains are:

1. In the present invention, a fusion protein is constructed by constructing Vitreum hyaline hemoglobin and glucose oxidase, and the fusion protein helps to improve the catalytic efficiency of glucose oxidase by using the oxygen transfer ability of Vitreum hyaline hemoglobin. According to the determination of enzyme kinetic parameters, compared with the parent glucose oxidase shown in SEQ ID NO.1, the catalytic efficiency (Kcat/Km) of the fusion protein is more than doubled, which is conducive to the application of the enzyme in industrial production.

2. The fusion protein of the present invention can be applied in the fields of food, chemical industry, medicine, agriculture or feed, and can be applied to the production of feed additives, food additives, sodium gluconate or calcium gluconate. Under dissolved oxygen limitation conditions, the fusion protein of the present invention retains a higher catalytic efficiency compared with the parent glucose oxidase shown in SEQ ID NO. Aerodynamic costs.

Description of drawings

Fig. 1 is the plasmid map of starting vector pLH503 in the present invention;

Fig. 2 is the verification electrophoresis figure of the double enzyme digestion (xho I/xba I, 4110bp/8304bp) of plasmid pLH1437 in the present invention; Wherein M is DNAMarker, and 1-3 is double enzyme digestion verification plasmid;

Fig. 3 is the map of plasmid pLH1437 in the present invention;

Fig. 4 is the collection of maps of plasmid pLH1540 among the present invention;

Fig. 5 is fusion protein and GOD SDS-PAGE and Western Blot figure in the present invention; Wherein, M is Marker, 1 is GOD, 2 is fusion protein.

Fig. 6 is a diagram of determination of kinetic parameters of the fusion protein and GOD in the present invention.

Detailed ways

In order to better understand the present invention, the present invention will be further described in detail below in conjunction with the examples, but the protection scope of the present invention is not limited to the range indicated by the examples.

The raw materials used in the present invention, if no special instructions, are conventional commercially available products, the methods used in the present invention, if no special instructions, are conventional methods in this area, and the quality of each material used in the present invention is Normal use quality.

A fusion protein that improves the catalytic efficiency of glucose oxidase, the fusion protein includes glucose oxidase, Vitiligo hyaline hemoglobin and a flexible link polypeptide.

Preferably, the glucose oxidase, C. hyaline hemoglobin and a flexible link polypeptide need to construct a fusion protein according to a certain link sequence, and the certain link sequence is the amino acid sequence of the two ends of the flexible link polypeptide respectively linked to C. hyaline hemoglobin. C-terminal and amino acid sequence N-terminal of glucose oxidase.

Preferably, the flexible link polypeptide is composed of a glycine- and serine-rich polypeptide, the total content of glycine and serine in the flexible link polypeptide is not less than 60%, and the flexible link polypeptide contains 4 to 15 amino acids.

Preferably, the amino acid sequence of the glucose oxidase is shown in SEQ ID NO.1, the amino acid sequence of the Vitella hyaline hemoglobin is shown in SEQ ID NO.2, and the amino acid sequence of the flexible link polypeptide is shown in SEQ ID Shown in NO.3, the amino acid sequence of described fusion protein is shown in SEQ ID NO.4.

A method for improving the catalytic efficiency of glucose oxidase by using the above-mentioned fusion protein, said method improving the catalytic efficiency of glucose oxidase through the fusion protein.

A kind of coding gene of above-mentioned fusion protein, its nucleotide sequence is as shown in SEQ ID NO.5.

A recombinant expression vector comprising the gene encoding the fusion protein as described above, preferably the recombinant expression vector is an Aspergillus niger expression vector.

A recombinant strain comprising the gene encoding the fusion protein as described above.

Preferably, the recombinant strain is recombinant Aspergillus niger, and the recombinant strain may be bacteria or fungal cells, preferably yeast or filamentous fungal cells, more preferably Aspergillus niger.

The application of the above-mentioned fusion protein in improving the catalytic efficiency of glucose oxidase.

Specifically, the relevant preparation and detection are as follows:

Example 1: Construction of Fusion Protein Expression Plasmid pLH1437

The departure vector is pLH503 (Fig. 1, see SEQ ID NO.6 for the specific nucleotide sequence of the departure vector pLH503, and the construction method of the plasmid pLH503 is: pLH454 (Appl Microbiol Biotechnol.2019, 103(19): 8105-8114.doi:10.1007/s00253-019-10054-3.) as the basis, use SpeⅠ/EcoRI double enzyme digestion to cut off PgpdA in pLH454, and use primer pair 503-F/503-R to amplify the artificially synthesized PglaA -MCS fragment (the nucleotide sequence is SEQ ID NO.7), the PglaA-MCS fragment is inserted into the pLH454 of SpeI/EcoRI double enzyme by one-step cloning method, thereby obtaining pLH503.), the pLH503 vector contains promoter PglaA and terminator TtrpC, which can be used to control the expression of genes encoding fusion proteins. The nucleotide sequence SEQ ID NO.8 of the hemoglobin coding gene of Vitiligo hyaline and the nucleotide sequence SEQ ID NO.9 of the glucose oxidase coding gene were fully synthesized by Jinweizhi Company. The C-terminus of the constructed fusion protein contains 6×His tag.

The specific construction process is as follows: using the synthetic hemoglobin gene fragment of Vitella hyaline as a template, design overlap primers to introduce the flexible link polypeptide (GS linker), use primers vhb-f/vhb-r to amplify VHB (438bp), and its nucleotide sequence It is SEQ ID NO.7; using the nucleotide sequence SEQ ID NO.8 of the glucose oxidase coding gene as a template, using primers v6478-F/v6478-R to amplify GOD (1767bp); using overlap PCR, the two gene fragments Connect together (2238bp), obtain fusion protein encoding gene fragment SEQ ID NO.5. Novazym C113-ClonExpress-MultiS One StepCloning Kit kit was used to connect the ligated fragments to the departure vector pLH503 after Sac I and Kpn I double digestion, and the ligated products were transformed into Escherichia coli JM109 competent cells, and homogenized Spread on LB petri dishes containing 100-300 μg/mL kanamycin, culture overnight at 37°C, pick a single clone, and verify by double enzyme digestion (Figure 2) to obtain plasmid pLH1437 (Figure 3).

Table 1 primers used in the present invention

Embodiment 2: Construction of fusion protein expression strain

Co-cultivate Agrobacterium containing plasmid pLH1437 and Aspergillus niger host strain ATCC1015 on an IM plate, and then transfer the co-culture to cells containing 100-500 μM cefotaxime, 100-500 μg/mL ampicillin, 100-500 μg/mL streptomycin 100-500 μg/mL hygromycin B on a CM plate, culture at 28-37°C until a single colony is formed. Pick a single clone and transfer it to a PDA plate containing hygromycin B, screen hygromycin B-resistant transformants, extract genome verification, and obtain fusion protein overexpression strain S2250.

IM medium: 15-20g agar powder, add water to 905.7mL, sterilize at 121°C for 20-50min, heat in microwave until the agar is completely dissolved, add: 0%-0.2% K buffer, 0%-5% MN buffer, 0%～0.5% 1% CaCl ₂ 2H ₂ O, 0%～2% 0.01% FeSO ₄ , 0%～1% IM Trace elements, 0%～1% 20% NH ₄ NO ₃ , 0%～2% 50% glycerol, 0%-6% 1M MES, 0%-1% 20% glucose.

The preparation of required reagent in the described IM medium:

1) Kbuffer: mix 1-5M K ₂ HPO ₄ with 1-5M KH ₂ PO ₄ to make the pH 3-7, and sterilize at 121°C for 20 minutes.

2) MN buffer: 0%-0.6% MgSO ₄ ·7H ₂ O, 0%-0.3% NaCl, dissolved in deionized water, and sterilized at 121°C for 20 minutes.

3) IM Trace elements: 0%～0.05% ZnSO ₄ ·7H ₂ O, 0%～0.05% CuSO ₄ ·5H ₂ O, 0%～0.05% H ₃ BO ₃ , 0%～0.05% MnSO ₄ ·H ₂ O, 0%-0.05% Na ₂ MoO ₄ ·2H ₂ O, add deionized water to dissolve, and sterilize at 121°C for 20min.

CM medium is:

15-20g agar powder, add water to 897mL, sterilize at 121°C for 20min. After the agar is completely dissolved by microwave heating, add: 0%~5% ASP+N, 0%~5% 50% glucose, 0%~0.5% 1M MgSO ₄ , 0%~0.5% CM Traceelements, 0%~5% 10% casein hydrolyzate, 0% ~ 10% 10% yeast extract.

The preparation of required reagent in the described CM medium:

1) ASP+N: 0%-0.5% KCl (350mM), 0%-1.5% KH ₂ PO ₄ (550mM), 0%-5% NaNO ₃ (3.5M), dissolved in deionized water, pH 5.5 ( 5M KOH), sterilized at 121°C for 20min.

2) CM Trace elements: 0%～0.5% ZnSO ₄ 7H ₂ O, 0%～0.3% H ₃ BO ₃ , 0%～0.1% MnCl ₂ 4H ₂ O, 0%～0.1% FeSO ₄ 7H ₂ O, 0%～0.05% CoCl ₂ 6H ₂ O, 0%～0.05% CuSO ₄ 5H ₂ O, 0%～0.04% Na ₂ MoO ₄ 2H ₂ O, 0%～1% EDTA, add deionized Dissolve in water and sterilize at 121°C for 20 minutes.

The PDA medium is: 100-500g of potatoes, cut into small pieces, add 500-3000mL of water to boil for 20-40min, filter with double-layer gauze to obtain clear liquid. Then add 10-50g of glucose to dissolve completely, add water to make up to 1-3L. Solid culture 1.5% (W/V) agar powder. Autoclave at 121°C for 20 minutes.

Embodiment 3: the construction of GOD expression bacterial strain

Referring to Example 1, using pLH503 as the starting vector, a plasmid for overexpressing GOD was obtained, and plasmid pLH1540 was obtained ( FIG. 4 ). Referring to Example 2, a strain overexpressing GOD was obtained, and strain S2637 was obtained.

Embodiment 4: separation and purification of fusion protein and GOD

S2250 and S2637 spores were inoculated into the seed medium, and after shaking culture at 28°C for one day, they were transferred to the fermentation medium. After 7 days, the fermentation broth was filtered with gauze, the fermentation broth was replaced with Lysis Buffer, and the protein was purified to obtain Fusion protein and GOD were tested by SDS-PAGE and Western Blot. The results of protein gel electrophoresis analysis showed that the molecular weights of the single overexpressed glucose oxidase and the fusion protein were 60kDa and 75kDa respectively (Figure 5), and there were obvious protein bands. The results indicated that the fusion protein had been expressed.

The seed medium is: 5-200g/L glucose, 5-200g/L corn dry powder, sterilized at 115°C for 20min.

The fermentation medium is: maltose 10-150g/L, fermented soybean powder 10-60g/L, sodium citrate 10-70g/L, (NH4) ₂ SO ₄ 2-15g/L, NaH ₂ PO ₄ 1-5g /L, MgSO ₄ 1~5g/L, Tween-80 1~5mL/L, arginine 1~5g/L, sterilized at 115°C for 20min. The protein purification method is as follows: the fermentation broth obtained in the fermentation medium is concentrated, dialyzed and desalted through NiNTA Beads 6FF packed column; the eluate is collected; the desalted dialyzed solution is dissolved in pH5.1 sodium acetate buffer;

Reagents used for protein purification:

1) Lysis Buffer: 10-50mM NaH ₂ PO ₄ , 100-300mM Nacl, 10-50mM imidazole; use NaOH solution to adjust the pH to 8.0;

2) Wash Buffer: 10-50mM NaH2PO4, 100-300mM Nacl, 5-20mM imidazole; use NaOH solution to adjust the pH to 8.0;

3) Elution Buffer: 10-50mM NaH ₂ PO ₄ , 100-300mM Nacl, 10-250mM imidazole; use NaOH solution to adjust the pH to 8.0;

Embodiment 5: fusion protein and GOD enzyme activity assay

The enzymatic activity of the purified protein was measured, and Michaelis-Menten equation kinetic fitting was carried out, as shown in Figure 6. The results showed that the catalytic efficiency Kcat/Km of the fusion protein was 45.3, which was twice that of GOD. The catalytic efficiency of the protein was increased by 100% compared with GOD.

Reagents used to measure enzyme activity:

Working solution I (o-dianisidine): A: Dissolve 0.1-0.3g of o-dianisidine in 10mL of methanol to form a stock solution of o-dianisidine. Store at 4°C and shake well before use. It is recommended to prepare and use immediately. B: Accurately weigh 10-50g of anhydrous sodium acetate, dissolve it in 500mL double-distilled water, and gradually adjust the pH value to about 5.1-7 with glacial acetic acid. C: Dissolve 0.1-0.5mL of A in 10-20mL of buffer B as working solution I.

Working solution II (glucose solution): Accurately weigh 10-30g of anhydrous glucose, and dilute to 100mL with double distilled water.

Working solution III (horseradish peroxidase): Dilute horseradish peroxidase to an enzyme activity of 10-100U/mL (preparation for use now).

The enzyme activity assay steps are as follows:

1) Add 1 to 2.5 mL of working solution I, 100 to 300 μL of working solution II and 100 to 300 μL of working solution III into a 5 mL EP tube, and preheat at 37°C for 1 to 10 minutes.

2) Add 100-300 μL of enzyme solution samples, and at the same time add corresponding inactivated enzyme solution as a control, and react at 37°C for 3-5 minutes.

3) Then add 2 mL of 2 mol/L sulfuric acid solution to terminate the reaction.

4) Take 1 mL of the reaction solution in a cuvette, adjust to zero the absorbance of the inactivated enzyme solution, and measure the absorbance at a wavelength of 500-600 nm.

5) The commercial GOD standard (purchased from Solarbio, product number: G8030) was prepared with buffer solution into enzyme solutions with gradients of 0.4, 0.6, 0.8, 1.0, 2.0 and 4.0 U/mL, and numbered 1 to 6 in sequence. At the same time, take 500 μL of No. 1-6 enzyme solution to inactivate, and obtain the standard curve.

6) Bring the OD value of the diluted enzyme solution after the dianisidine-catalase reaction into the above-mentioned standard song, and multiply the obtained result by the dilution factor to obtain the enzyme activity of the enzyme solution to be tested.

The relevant sequences of the present invention are as follows:

SEQ ID NO.1: Amino acid sequence (589aa) of glucose oxidase

LPHYIRSNGIEASLLTDPKDVSGRTVDYIIAGGGLTGLTTAARLTENPNISVLVIESGSYESDRGPIIEDLNAYGDIFGSSVDHAYETVELATNNQTALIRSGNGLGGSTLVNGGTWTRPHKAQVDSWETVFGNEGWNWDNVAAYSLQAERARAPNAKQIAAGHYFNASCHGTNGTVHAGPRDTGDDYSPIVKALMS AVEDRGVPTKKDFGCGDPHGVSMFPNTLHEDQVRSDAAREWLLPNYQRPNLQVLTGQYVGKVLLSQNGTTPRAVGVEFGTHKGNTHNVYAKHEVLLAAGSAVSPTILEYSGIGMKSILEPLGIDTVVDLPVGLNLQDQTTATVRSRITSAGAGQGQAAWFATFNETFGDYAEKAHELLNTKLE QWAEEAVARGGFHNTTALLIQYENYRDWIVNHNVAYSELFLDTAGVASFDVWDLLPFTRGYVHILDKDPYLHHFAYDPQYFLNELDLLGQAAATQLARNISNSGAMQTYFAGETIPGDNLAYDADLSAWTEYIPYHFRPNYHGVGTCSMMPKEMGGVVDNAARVYGVQGLRVIDGSIPPTQMS SHVMTVFYAMALKIDAILEDYASMQ

SEQ ID NO.2: Amino acid sequence (146aa) of C. hyaline hemoglobin

MLDQQTINIIKATVPVLKEHGVTITTTFYKNLFAKHPEVRPLFDMGRQESLEQPKALAMTVLAAAQNIENLPAILPAVKKIAVKHCQAGVAAAHYPIVGQELLGAIKEVLGDAATDDILDAWGKAYGVIADVFIQVEADLYAQAVE

SEQ ID NO.3: Flexible link polypeptide amino acid sequence (4aa)

GSSG

SEQ ID NO.4: Fusion protein amino acid sequence (739aa)

MLDQQTINIIKATVPVLKEHGVTITTTFYKNLFAKHPEVRPLFDMGRQESLEQPKALAMTVLAAAQNIENLPAILPAVKKIAVKHCQAGVAAAHYPIVGQELLGAIKEVLGDAATDDILDAWGKAYGVIADVFIQVEADLYAQAVEGSSGLPHYIRSNGIEASLLTDPKDVSGRTVDYIIAGGGLTG LTTAARLTENPNISVLVIESGSYESDRGPIIEDLNAYGDIFGSSVDHAYETVELATNNQTALIRSGNGLGGSTLVNGGTWTRPHKAQVDSWETVFGNEGWNWDNVAAYSLQAERARAPNAKQIAAGHYFNASCHGTNGTVHAGPRDTGDDYSPIVKALMSAVEDRGVPTKKDFGCGDPHGVSMFPNTLHEDQVRSDAA REWLLPNYQRPNLQVLTGQYVGKVLLSQNGTTPRAVGVEFGTHKGNTHNVYAKHEVLLAAGSAVSPTILEYSGIGMKSILEPLGIDTVVDLPVGLNLQDQTTATVRSRITSAGAGQGQAAWFATFNETFGDYAEKAHELLNTKLEQWAEEAVARGGFHNTTALLIQYENYRDWIVNHNVAYS ELFLDTAGVASFDVWDLLPFTRGYVHILDKDPYLHHFAYDPQYFLNELDLLGQAAATQLARNISNSGAMQTYFAGETIPGDNLAYDADLSAWTEYIPYHFRPNYHGVGTCSMMPKEMGGVVDNAARVYGVQGLRVIDGSIPPTQMSSHVMTVFYAMALKIADAILEDYASMQ

SEQ ID NO.5: fusion protein nucleotide sequence

atgctggatcagcagaccatcaacatcatcaaggccaccgtccccgtcctgaaggagcacggtgtcactattattaccaccaccttctacaagaacctgttcgccaagcaccccgaggtccgccctttgtttgatatgggccgccaggaggtccctggagcagcctaaagctctggctatgaccgtcctggctgctgctcaaa atatcgagaacctgcccgctattctgcccgccgtcaaaaagatcgccgtcaagcactgccaggccggcgttgccgctgctcattatcctattgtcggtcaggagctgctgggcgccattaaggaagtcctgggcgatgccgccaccgatgatatcctggatgcctggggcaaggcctacggcgtattgccgat gtctttatccaggtcgaggccgatctgtacgcccaggccgttgaaGGTTCCagcggcCTGCCACACTACATCAGGAGCAATGGCATTGAAGCCAGCCTCCTGACTGACCCCAAGGATGTCTCCGGCCGCACGGTCGACTACATCATCGCTGGTGGAGGTCTGACTGGACTCACCACCGCTGCTCGTCTGACGGAGAACCCCAACATCA GTGTGCTCGTCATCGAAAGTGGCTCCTACGAGTCGGACAGAGGTCCTATCATTGAGGACCTGAACGCCTACGGCGACATTTTTGGCAGCAGTGTAGACCACGCCTACGAGACCGTTGAGCTCGCTACCAACAATCAAACCGCGCTGATCCGCTCCGGAAATGGTCTCGGTGGCTCTACTCTAGTGAATGGTGGCACCTGGACTCGCCCCACAAGGCAC AGGTTGATTCTTGGGAGACTGTCTTTGGAAATGAGGGCTGGAACTGGGACAATGTGGCCGCCTACTCCCTCCAGGCTGAGCGTGCTCGCGCACCAAATGCCAAACAGATCGCTGCTGGCCACTACTTCAACGCATCCTGTCATGGTACCAATGGTACTGTCCATGCCGGACCCCGTGACACCGGCGATGACTATTCCCCCATCGTCAAGGCTC ATGAGCGCTGTCGAAGACCGGGGCGTTCCCACCAAGAAGGACTTCGGATGCGGTGACCTCATGGTGTGTCCATGTTCCCAACACCTTGCACGAAGACCAAGTTCGCTCCGATGCCGCTCGCGAATGGCTCCTTCCAACTACCAACGTCCCAACCTGCAAGTCCTGACCGGACAATATGTTGGTAAGGTGCTCCTTAGCCAGAACGGCACCACCTCGT GCCGTCGGCGTGGAATTCGGCACCCCAAGGGCAACACCCACAACGTTTACGCTAAGCACGAGGTCCTCCTGGCTGCTGGCTCGGCTGTCTCTCCCCACCATCCTCGAATATTCCGGTATCGGAATGAAGTCCATCCTGGAACCCTTGGTATCGACACCGTCGTTGACCTGCCCGTCGGCCTGAACCTGCAGGACCAGACCACCGCTACCGTCCGCTC CCGCATCACCCTCTGCTGGTGCCGGACAGGGACAGGCCGCTTGGTTCGCCACCTTCAACGAGACCTTTGGTGACTATGCCGAAAAGGCACACGAGCTGCTCAACACCAAGCTGGAGCAGTGGGCCGAAGAGGCCGTCGCCCGTGGCGGATTCCACAACACCACCGCCTTGCTCATCCAGTACGAGAACTATCGCGACTGGATTGTCAATCCAACGTC GCGTACTCGGAACTTCTTCCTCGACACTGCCGGAGTGGCCAGCTTCGATGTGTGGGACCTTCTGCCCTTCACGAGAGGATACGTCCACATCCTCGACAAGGACCCCTACCTCCACCACTTTGCCTACGACCCTCAGTACTTCCTCAACGAGCTCGACCTGCTCGGTCAGGCTGCCGCTACTCAGCTGGCCCGTAACATCTCCAACTCCGGTGCTATGCAGACC TACTTCGCTGGCGAGACTATCCCCGGTGATAACCTCGCGTATGATGCCGATTTGAGCGCCTGGACTGAGTACATCCCGTACCACTTCCGTCCTAACTACCATGGCGTGGGTACTTGCTCCATGATGCCGAAGGAGATGGGCGGTGTTGTCGATAATGCTGCCCGTGTGTACGGTGTGCAGGGACTGCGTGTCATTGATGGTTCTATTCCCCCTACGCAG ATGTCGTCCCATGTCATGACTGTGTTCTACGCCATGGCGTTGAAGATTGCGGATGCTATTTTGGAGGACTACGCTTCTATGCAGcaccaccaccaccaccactaa

SEQ ID NO.6: pLH503 nucleotide sequence

CTAGACTAGTCCTCTCGTATGCAGAGGAAATCTCCCCTGATCTTCCGAACTGGTCGTACCTGGCGACCTATGACTATGGCACCCCAGTTCTGGGGACCTTCCACGGAAGTGACCTGCTGCAGGTGTTCTATGGGATCAAGCCAAACTATGCAGCTAGTTCTAGCCACACGTACTATCTGAGCTTTGTGTATACGCTGGATCCGAACTCCAACCGGG GGGAGTACATTGAGTGGCCGCAGTGGAAGGAATCGCGGCAGTTGATGAATTTCGGAGCGAACGACGCCAGTCTCTCTTACGGATGATTTCCGCAACGGGACATATGAGTTCATCCTGCAGAATACCGCGGCGTTCCACATCTGATGCCATTGGCGGAGGGGTCCGGACGGTCAGGAACTTAGCCTTATGAGATGAATGATGGACGTGTCT GGCCTCGGAAAAGGATATATGGGGATCATAATAGTACTAGCCATATTAATGAAGGGCATATACCACGCGTTGGACCTGCGTTATAGCTTCCCGTTAGTTATAGTACCATCGTTATACCAGCCAATCAAGTCACCGACCGGGGACGGCGAATCCCCGGTATAGTACCATCGTTATACCAGCCAATCAAGTCACCGCACGACCGGGGACGGC GAATCCCCGGGTATAGTACCATCGTTATACCAGCCAATCAAGTCACCGCACGACCGGGGACGGCGAATCCCCGGGTATAGTACCATCGTTATACCAGCCAATCAAGTCACCACGACCGGGGACGGCGAATCCCCGGGTATAGTACCAGCCAATCAAGTCACCGACCGGGGACGGCGAATCGAATTCAAGCTAGATGC TAAGCGATATTGCATGGCAATATGTGTTGATGCATGTGCTTCTTCCTTCAGCTTCCCTCGTGCAGATGAGGTTTGGCTATAAATTGAAGTGGTTGGTCGGGGTTCCGTGAGGGGCTGAAGTGCTTCCTCCCTTTTAGACGCAACTGAGAGCCTGAGCTTCATCCCCAGCATCATTACACCTCAGCAATGTCGTTCCGATCTCTACTCGCCCTGAGCG GCCTCGTCTGCACAGGGTTGGCAAACGTGATTTCCAAGCGCGAGCTCAGATCTGGTACCGCATGCGGATCCGATCCACTTAACGTTACTGAAATCATCAAACAGCTTGACGAATCTGGATATAAGATCGTTGGTGTCGATGTCAGCTCCGGAGTTGAGACAAATGGTGTTCAGGATCTCGATAAGATACGTTCATTTGTCAAGCAGCAAAGAGTGCCTTCTAGT GATTTAATAGCTCCATGTCAACAAGAATAAAACGCGTTTTCGGGTTTACCCTTCCAGATACAGCTCATCTGCAATGCATTAATGCATTGACTGCAACCTAGTAACGCCTTNCAGGCTCCGGCGAAGAGAAGAATAGCTTAGCAGAGCTATTTTCAAGCATTTTCGGGAGACGAGATCAAGCAGATCAACGGTCGTCAAGAGACCTACGAGACTGAGGAATCCGCTCTTGGCT CCACGCGACTATATATTTGTCTCTAATTGTACTTTGACATGCTCCTCTTCTTACTCTGATAGCTTGACTATGAAAATTCCGTCACCAGCNCCTGGGTTCGCAAAGATAATTGCATGTTTCTAAGCCTTGAACTTCTCAAGCCTACAGGACACACATTCATCGTAGGTATAAACCTCGAAATCANTTCCTACTAAGATGGTATACAATAGTAACCATGCATGGTTGCCTAGTGAAT GCTCCGTAACACCCCAATACGCCGGCCGAAACTTTTTTACAACTCTCCTATGAGTCGTTTACCCAGAATGCACAGGTACACTTGTTTAGAGGTAATCCTTCTTTCTAGACCCGGGGGGCCCTACGTAATAACTTCGTATAATGTATGCTATACGAAGTTATGTCGACGTTAACTGATATTGAAGGAGCATTTTTTGGGCTTGGCTGGAGCTAGTGGAGGT CAACAATGAATGCCTATTTTGGTTTAGTCGTCCAGGCGGTGAGCACAAAATTTGTGTCGTTTGACAAGATGGTTCATTTAGGCAACTGGTCAGATCAGCCCCACTTGTAGCAGTAGCGGCGGCGCTCGAAGTGTGACTCTTATTAGCAGACAGGAACGAGGACATTATTATCATCTGCTGCTTGGTGCACGATAACTTGGTGCGTTTGTCAAGCAAGGTAAGTGGAC GACCCGGTCATACCTTCTTAAGTTCGCCCTTCCTCCCTTTATTTCAGATTCAATCTGACTTACCTATTCTACCCAAGCATCCAAATGAAAAAGCCTGAACTCACCGCGACGTCTGTCGAGAAGTTTCTGATCGAAAAGTTCGACAGCGTCTCCGACCTGATGCAGCTCTCGGAGGGCGAAGAATCTCGTGCTTTCAGCTTCGATGTAGGAGGGCGTGGATATGTC CTGCGGGTAAATAGCTGCGCCGATGGTTTCTACAAAGATCGTTATGTTTATCGGCACTTTGCATCGGCCGCGCTCCCGATTCCGGAAGTGCTTGACATTGGGGAGTTCAGCGAGAGCCTGACCTATTGCATCTCCCGCCGTGCACAGGGTGTCACGTTGCAAGACCTGCCTGAAACCGAACTGCCCGCTGTTTCCAGCCGGTCGCGGAGGCC ATGGATGCGATCGCTGCGGCCGATCTTAGCCAGACGAGCGGGTTCGGCCCATTCGGACCGCAAGGAATCGGTCAATACACTACATGGCGTGATTTCATATGCGCGATTGCTGATCCCCATGTGTATCACTGGCAAACTGTGATGGACGACACCGTCAGTGCGTCCGTCGCAGGCTCTCGATGAGCTGATGCTTTGGGCCGAGGACTGCCCCGA AGTCCGGCACCTCGTGCATGCGGATTTCGGCTCCAACAATGTCCTGACGGACAATGGCCGCATAACAGCGGTCATTGACTGGAGCGAGGCGATGTTCGGGGATTCCCAATACGAGGTCGCCAACATCCTCTTCTGGAGGCCGTGGTTGGCTTGTATGGAGCAGCAGACGCGCTACTTCGAGCGGAGGCATCCGGAGCTTGCAGGATCGCCGCGC CTCCGGGCGTATATGCTCCGCATTGGTCTTGACCAACTCTATCAGAGCTTGGTTGACGGCAATTTCGATGATGCAGCTTGGGCGCAGGGTCGATGCGACGCAATCGTCCGATCCGGAGCCGGGACTGTCGGGCGTACACAAATCGCCCGCAGAAGCGGCCGTCTGGACCGATGGCTGTGTAGAAGTACTCGCCGATAGTGGAAACCG ACGCCCCAGCACTCGTCCGAGGGCAAAGGAATAGAGTAGATGCCGACCGGGAACCAGTTAACGTCGAATAACTTCGTATAATGTATGCTATACGAAGTTATAAGCTTGGCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCAT CCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGCTAGAGCAGCTTGAGCTTGGATCAGATTGTCGTTTCCCGCCTTCAGTTTAAACTATCAGTGTTTGACAGGATATATTGGCGGGTAAACCTAAGAGAAAAGAGCGTTTTATTAGAATAACGGATATTTAAAAGGGCGTGAAAAGGTTTATCGTCGTCCATTTGTATGTGCATGCCAACCACAGGGT TCCCCTCGGGATCAAAGTACTTTGATCCAACCCCTCCGCTGCTATAGTGCAGTCGGCTTCTGACGTTCAGTGCAGCCGTCTTCTGAAAACGACATGTCGCACAAGTCCTAAGTTACGCGACAGGCTGCCGCCCTGCCCTTTTCCTGGCGTTTTTCTTGTCGCGTTTTTAGTCGCATAAAGTAGAATACTTGCGACTAGAACCGGAGACATTACGCCATGAACAAGA GCGCCGCCGCTGGCCTGCTGGGCTATGCCCGCGTCAGCACCGACGACCAGGACTTGACCAACCAACGGGCCGAACTGCACGCGGCCGGCTGCACCAAGCTGTTTTCCGAGAAGATCACCGGCACCAGGCGCGACCGCCCGGAGCTGGCCAGGATGCTTGACCACCTACGCCCTGGCGACGTTGTGACAGTGACCAGGCTAGACCGCCTGGCCC GCAGCACCCGCGACCTACTGGACATTGCCGAGCGCATCCAGGAGGCCGGCGCGGGCCTGCGTAGCCTGGCAGAGCCGTGGGCCGACACCACCACGCCGGCCGGCCGCATGGTGTTGACCGTGTTCGCCGGCATTGCCGAGTTCGAGCGTTCCCTAATCATCGACCGCACCCGGAGCGGGCGCGAGGCCGCCAAGGCCCGAGGCGTGAAGTTTG GCCCCCGCCCTACCCTCACCCCGGCACAGATCGCGCACGCCCGCGAGCTGATCGACCAGGAAGGCCGCACCGTGAAAGAGGCGGCTGCACTGCTTGGCGTGCATCGCTCGACCCTGTACCGCGCACTTGAGCGCAGCGAGGAAGTGACGCCCACCGAGGCCAGGCGGCGCGGTGCCTTCCGTGAGGACGCATTGACCGAGGCCGACGCCCTGGCG GCCGCCGAGAATGAACGCCAAGAGGAACAAGCATGAAACCGCACCAGGACGGCCAGGACGAACCGTTTTTCATTACCGAAGAGATCGAGGCGGAGATGATCGCGGCCGGGTACGTGTTCGAGCCGCCCGCGCACGTCTCAACCGTGCGGCTGCATGAAATCCTGGCCGGTTTGTCTGATGCCAAGCTGGCGGCCTGGCCGGCCAGCTTGGCCG CTGAAGAAACCGAGCGCCGCCGTCTAAAAAAGGTGATGTGTATTTGAGTAAACAGCTTGCGTCATGCGGTCGCTGCGTATATGATGCGATGAGTAAATAAACAAATACGCAAGGGGAACGCATGAAGGTTATCGCTGTACTTAACCAGAAAAGGCGGGTCAGGCAAGACGACCATCGCAACCCATCTAGCCCGCGCCCTGCAACTCGCCGGGGCCGATGT TCTGTTAGTCGATTCCGATCCCCAGGGCAGTGCCCGCGATTGGGCGGCCGTGCGGGAAGATCAACCGCTAACCGTTGTCGGCATCGACCGCCCGACGATTGACCGCGACGTGAAGGCCATCGGCCGGCGCGACTTCGTAGTGATCGACGGAGCGCCCCAGGCGGCGGACTTGGCTGTGTCCGCGATCAAGGCAGCCGACTTCGTGCTGATTCC GGTGCAGCCAAGCCCTTACGACATATGGGCCACCGCCGACCTGGTGGAGCTGGTTAAGCAGCGCATTGAGGTCACGGATGGAAGGCTACAAGCGGCCTTTGTCGTGTCGCGGGCGATCAAAGGCACGCGCATCGGCGGTGAGGTTGCCGAGGCGCTGGCCGGGTACGAGCTGCCCATTCTTGAGTCCCGTATCACGCAGCGCGTGAG CTACCCAGGCACTGCCGCCGCCGGCACAACCGTTCTTGAATCAGAACCCGAGGGCGACGCTGCCCGCGAGGTCCAGGCGCTGGCCGCTGAAATTAAATCAAAACTCATTTGAGTTAATGAGGTAAAGAGAAAATGAGCAAAAGCACAAACACGCTAAGTGCCGGCCGTCCGAGCGCACGCAGCAGCAAGGCTGCAACGTTGGCCAGCCTGGCAGACACG CCAGCCATGAAGCGGGTCAACTTTCAGTTGCCGGCGGAGGATCACACCAAGCTGAAGATGTACGCGGTACGCCAAGGCAAGACCATTACCGAGCTGCTATCTGAATACATCGCGCAGCTACCAGAGTAAATGAGCAAATGAATAAATGAGTAGATGAATTTTAGCGGCTAAAGGAGGCGGCATGGAAAATCAAGAACCAGGCACCGACGCCGTGGAATGCC CCATGTGTGGAGGAACGGGCGGTTGGCCAGGCGTAAGCGGCTGGGTTGTCTGCCGGCCCTGCAATGGCACTGGAACCCCCAAGCCCGAGGAATCGGCGTGACGGTCGCAAACCATCCGGCCCGGTACAAATCGGCGCGGCGCTGGGTGATGACCTGGTGGAGAAGTTGAAGGCCGCGCAGGCCGCCCAGCGGCAACGCATC GAGGCAGAAGCACGCCCCGGTGAATCGTGGCAAGCGGCCGCTGATCGAATCCGCAAAGAATCCCGGCAACCGCCGGCAGCCGGTGCGCCGTCGATTAGGAAGCCGCCCAAGGGCGACGAGCAACCAGATTTTTTCGTTCCGATGCTCTATGACGTGGGCACCCGCGATAGTCGCAGCATCATGGACGTGGCCGTTTTCCGTCTGTCGAAGCGTGA CCGACGAGCTGGCGAGGTGATCCGCTACGAGCTTCCAGACGGGCACGTAGAGGTTTCCGCAGGGCCGGCCGGCATGGCCAGTGTGTGGGATTACGACCTGGTACTGATGGCGGTTTCCATCTAACCGAATCCATGAACCGATACCGGGAAGGGAAGGGACAAGCCCGGCCGCGTGTTCCGTCCACACGTTGCGGACGTACTCAAGTT CTGCCGGCGAGCCGATGGCGGAAAGCAGAAAGACGACCTGGTAGAAACCTGCATTCGGTTAAACACCACGCACGTTGCCATGCAGCGTACGAAGAAGGCCAAGAACGGCCGCCTGGTGACGGTATCCGAGGGTGAAGCCTTGATTAGCCGCTACAAGATCGTAAAGAGCGAAACCGGGCGGCCGGAGTACATCGAGATCGAGCTAGCTGATTGG ATGTACCGCGAGATCACAGAAGGCAAGAACCCGGACGTGCTGACGGTTCACCCCGATTACTTTTTGATCGATCCCGGCATCGGCCGTTTTTCTCTACCGCCTGGCACGCCGCGCCGCAGGCAAGGCAGAAGCCAGATGGTTGTTCAAGACGATCTACGAACGCAGTGGCAGCGCCGGAGAGTTCAAGTTCTGTTTCACCGTGCGCAAGCTGATCG GGTCAAATGACCTGCCGGAGTACGATTTGAAGGAGGAGGCGGGGCAGGCTGGCCCGATCCTAGTCATGCGCTACCGCAACCTGATCGAGGGCGAAGCATCCGCCGGTTCCTAATGTACGGAGCAGATGCTAGGGCAAATTGCCCTAGCAGGGGAAAAAGGTCGAAAAGGTCTCTTTCCTGTGGATAGCACGTACATTGGGAACCCAAAGCCGT ACATTGGGAACCGGAACCCGTACATTGGGAACCCAAAGCCGTACATTGGGAACCGGTCACACATGTAAGTGACTGATATAAAAGAGAAAAAAGGCGATTTTTCCGCCTAAAACTCTTTAAAAACTTATTAAAAACTCTTAAAACCCGCCTGGCCTGTGCATAACTGTCTGGCCAGCGCACAGCCGAAGAGCTGCAAAAGCGCCTACCCTTCGGTCGCTGCGCT CCCTACGCCCCGCCGCTTCGCGTCGGCCTATCGCGGCCGCTGGCCGCTCAAAAATGGCTGGCCTACGGCCAGGCAATCTACCAGGGCGCGGACAAGCCGCGCCGTCGCCACTCGACCGCCGGCGCCCACATCAAGGCACCCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTT GTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGTAGCGATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCTC TTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTACCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCAAAATC GACGCTCCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAAC CCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCG GAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGCATTCTAGGTACTAAAACAATTCATCCAGTAAAATATAATATTTT ATTTTCTCCCAATCAGGCTTGATCCCCAGTAAGTCAAAAATAGCTCGACATACTGTTCTTCCCCGATATCCTCCCTGATCGACCGGACGCAGAAGGCAATGTCATACCACTTGTCCGCCCTGCCGCTTCTCCCAAGATCAATAAAAGCCACTTACTTTGCCATCTTTCACAAAGATGTTGCTGTCTCCCAGGTCGCCGTGGGAAAAGACAAGTTCCCTTCGGGCTTTT CCGTCTTTAAAAAATCATACAGCTCGCGCGGATCTTTAAATGGAGTGTCTTTCTCCCAGTTTTCGCAATCCACATCGGCCAGATCGTTATTCAGTAAGTAATCCAATTCGGCTAAGCGCTGTCTAAGCTATTCGTATAGGGACAATCCGATATGTCGATGGAGTGAAAGAGCCTGATGCACTCCGCATACAGCTCGATAATCTTTTCAGGGCTTTGTTCATCTTC ATACTCTTTCCGAGCAAAGGACGCCATCGGCCTCACTCATGAGCAGATTGCTCCAGCCATCATGCCGTTCAAAGTGCAGGACCTTTGGAACAGGCAGCTTTCCTTCCAGCCATAGCATCATGTCCTTTTCCCGTTCCACATCATAGGTGGTCCCTTTTATACCGGCTGTCCGTCATTTTAAATAGGTTTTTCATTTTCCCACCAGCTTATATACCTTAGCAGGAGACATT CCTTCCGTATCTTTTACGCAGCGGTATTTTTCGATCAGTTTTTTCAATTCCGGTGATATTTCTCATTTTAGCCATTTATTTTCCTTCCTCTTTTCAGTATTTTAAAGATACCCAAGAAGCTAATTATAACAAGACGAACTTCCAATTCACTGTTCCTTGCATTCTAAAACCTTAAATACCAGAAACAGCTTTTCAAAGTTGTTTTTCAAAGTTGGCGTATAACATAGTATC GACGGAGCCGATTTTGAAACCGCGGTGATCACAGGCAGCAACGCTCTGTCATCGTTACAATCAACATGCTACCCTCCGCGAGATCATCCGTGTTTCAAACCCGGCAGCTTAGTTGCCGTTCTTCCGAATAGCATCGGTAACATGAGCAAAGTCTGCCGCCTTACAACGGCTCTCCCGCTGACGCCGTCCCGGACTGATGGGCTGCCTGTATCGAGTGGTGA TTTTGTGCCGAGCTGCCGGTCGGGGAGCTGTTGGCTGGCTGGTGGCAGGATATTGTGGTGTAAACAAATTGACGCTTAGACAACTTAATAACACATTGCGGACGTTTTTAATGTACTGAATTAACGCCGAATTAATTCGGGGGATCTGGATTTTAGTACTGGATTTTGGTTTTAGGAATTAGAAATTTTATTGATAGAAGTATTTACACA ATACAAATACATACTAAGGGTTTCTTATATGCTCAACACATGAGCGAAACCCTATAGGAACCCTAATTCCCTTATCTGGGAACTACTCACACATTATTATGGAGAAACTCGAGA

SEQ ID NO.7PglaA-MCS fragment nucleotide sequence

ACTAGTcctctcgtatgcagaggaaatctcccctgatcttccgaactggtcgtacctggcgacctatgactatggcaccccagttctggggaccttccacggaagtgacctgctgcaggtgttctatgggatcaagccaaactatgcagctagttctagccacacgtactatctgagctttgtgtatacgctggatccga actccaaccggggggagtcattgagtggccgcagtggaaggaatcgcggcagttgatgaatttcggagcgaacgacgccagtctccttacggatgatttccgcaacgggacatatgagttcatcctgcagaataccgcggcgttccacatctgatgccattggcggaggggtccggacggtcaggaacttagccttatgagatga atgatggacgtgtctggcctcggaaaaggatatatggggatcataatagtactagccatattaatgaagggcatataccacgcgttggacctgcgttatagcttcccgttagttatagtaccatcgttataccagccaatcaagtcaccacgcacgaccggggacggcgaatccccgggtatatagtaccatcgttataccagccaat caagtcaccacgcacgaccggggacggcgaatccccgggtatagtaccagccaatcaagtcaccacgcacgaccggggacggcgaatccccgggtatagtaccatcgttataccagccaatcaagtcaccacgcacgaccggggacggcgaatccccgggtatagtaccatcgttataccagccaatcaagtcaccacgcac gaccggggacggcgaatcgaattcaagctagatgctaagcgatattgcatggcaatatgtgttgatgcatgtgcttcttccttcagcttcccctcgtgcagatgaggtttggctataaattgaagtggttggtcggggttccgtgaggggctgaagtgcttcctcccttttagacgcaactgagagcctgagcttcatcc ccagcatcattacacctcagcaatgtcgttccgatctctactcgccctgagcggcctcgtctgcacagggttggcaaacgtgatttccaagcgcGAGCTCAGATCTGGTACCGCATGCGGATCC

SEQ ID NO.8: VHb nucleotide sequence

atgctggatcagcagaccatcaacatcatcaaggccaccgtccccgtcctgaaggagcacggtgtcactattattaccaccaccttctacaagaacctgttcgccaagcaccccgaggtccgccctttgtttgatatgggccgccaggaggtccctggagcagcctaaagctctggctatgaccgtcctggctgctgctcaaa atatcgagaacctgcccgctattctgcccgccgtcaaaaagatcgccgtcaagcactgccaggccggcgttgccgctgctcattatcctattgtcggtcaggagctgctgggcgccattaaggaagtcctgggcgatgccgccaccgatgatatcctggatgcctggggcaaggcctacggcgtattgccgat gtctttatccaggtcgaggccgatctgtacgcccaggccgttgaa

SEQ ID NO.9: GOD nucleotide sequence

CTGCCACACTACATCAGGAGCAATGGCATTGAAGCCAGCCTCCTGACTGACCCCAAGGATGTCTCCGGCCGCACGGTCGACTACATCATCGCTGGTGGAGGTCTGACTGGACTCCACCACCGCTGCTCGTCTGACGGAGAACCCCAACATCAGTGTGCTCGTCATCGAAAGTGGCTCCTACGAGTCGGACAGAGGTCCTATCATTGAGGACCTGAAC GCCTACGGCGACATTTTTGGCAGCAGTGTAGACCACGCCTACGAGACCGTTGAGCTCGCTACCAACAATCAAACCGCGCTGATCCGCTCCGGAAATGGTCTCGGTGGCTCTACTCTAGTGAATGGTGGCACCTGGACTCGCCCCCACAAGGCACAGGTTGATTCTTGGGAGACTGTCTTTGGAAATGAGGGCTGGAACTGGGACAATGTGG CCGCCTACTCCCTCCAGGCTGAGCGTGCTCGCGCACCAAATGCCAAACAGATCGCTGCTGGCCACTACTTCAACGCATCCTGTCATGGTACCAATGGTACTGTCCATGCCGGACCCCGTGACACCGGCGATGACTATTCCCCCATCGTCAAGGCTCTCATGAGCGCTGTCGAAGACCGGGGCGTTCCCACCAAAGAAGGACTTCGGATGCGGTGACCCTC ATGGTGTGTCCATGTTCCCCAACACCTTGCACGAAGACCAAGTTCGCTCCGATGCCGCTCGCGAATGGCTCCTTCCCAACTACCAACGTCCCAACCTGCAAGTCCTGACCGGACAATATGTTGGTAAGGTGCTCCTTAGCCAGAACGGCACCACCCCTCGTGCCGTCGGCGTGGAATTCGGCACCCCAAGGGCAACACCCCACAACGTTTAACGCTAAGCACGAG GTCCTCCTGGCTGCTGGCTCGGCTGTCTCTCCCCACCATCCTCGAATATTCCGGTATCGGAATGAAGTCCATCCTGGAACCCTTGGTATCGACACCGTCGTTGACCTGCCCGTCGGCCTGAACCTGCAGGACCAGACCACCGCTACCGTCCCGCTCCCGCATCACCTCTGCTGGTGCCGGACAGGGACAGGCCGCTTGGTTCGCCACCTTCAACGAG ACCTTTGGTGACTATGCCGAAAAGGCACACGAGCTGCTCAACACCAAGCTGGAGCAGTGGGCCGAAGAGGCCGTCGCCCGTGGCGGATTCCACAACACCACCGCCTTGCTCATCCAGTACGAGAACTATCGCGACTGGATTGTCAATCACAACGTCGCGTACTCGGAACTTCTTCCTCGACACTGCCGGAGTGGCCAGCTTCGATGTGTGGGACCTTCTG CCCTTCACGAGAGGATACGTCCACATCCTCGACAAGGACCCCTACCTCCACCACTTTGCCTACGACCTCCAGTACTTCCTCAACGAGCTCGACCTGCTCGGTCAGGCTGCCGCTACTCAGCTGGCCCGTAACATCTCCAACTCCGGTGCTATGCAGACCTACTTCGCTGGCGAGACTATCCCCGGTGATAACCTCGCGTATGATGCCGATTTGAGCGCCTGGA CTGAGTACATCCCGTACCACTTCCGTCCTAACTACCATGGCGTGGGTACTTGCTCCATGATGCCGAAGGAGATGGGCGGTGTTGTCGATAATGCTGCCCGTGTGTACGGTGTGCAGGGACTGCGTGTCATTGATGGTTCTATTCCCCCTACGCAGATGTCGTCCCATGTCATGACTGTGTTTCTACGCCATGGCGTTGAAGATTGCGGATGCTATTTT GGAGGACTACGCTTCTATGCAG

Although the embodiments of the present invention are disclosed for the purpose of illustration, those skilled in the art will understand that various alternatives, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims, therefore However, the scope of the present invention is not limited to the content disclosed in the embodiments.

Claims (10)

1. A fusion protein that improves the catalytic efficiency of glucose oxidase, characterized in that: the fusion protein includes glucose oxidase, Vitella hyaline hemoglobin and a flexible link polypeptide. 2. The fusion protein improving the catalytic efficiency of glucose oxidase according to claim 1, characterized in that: said glucose oxidase, Vitella hyaline hemoglobin and a section of flexible link polypeptide need to construct fusion protein according to a certain link sequence, said A certain linking sequence is that the two ends of the flexible linking polypeptide are respectively linked to the C-terminus of the amino acid sequence of C. hyaline hemoglobin and the N-terminus of the amino acid sequence of glucose oxidase. 3. The fusion protein for improving the catalytic efficiency of glucose oxidase according to claim 1, characterized in that: the flexible link polypeptide is composed of polypeptides rich in glycine and serine, and the total content of glycine and serine in the flexible link polypeptide is not low At 60%, the flexible link polypeptide contains 4 to 15 amino acids. 4. The fusion protein improving the catalytic efficiency of glucose oxidase according to any one of claims 1 to 3, characterized in that: the amino acid sequence of the glucose oxidase is as shown in SEQ ID NO.1, and the Vitiligo hyaline The amino acid sequence of hemoglobin is shown in SEQ ID NO.2, the amino acid sequence of the flexible link polypeptide is shown in SEQ ID NO.3, and the amino acid sequence of the fusion protein is shown in SEQ ID NO.4. 5. A method for improving the catalytic efficiency of glucose oxidase by using the fusion protein according to any one of claims 1 to 4, characterized in that: the method uses the fusion protein to improve the catalytic efficiency of glucose oxidase. 6. A gene encoding the fusion protein according to any one of claims 1 to 4, characterized in that: its nucleotide sequence is as shown in SEQ ID NO.5. 7. The recombinant expression vector comprising the coding gene of the fusion protein as claimed in claim 6. 8. A recombinant strain comprising the gene encoding the fusion protein as claimed in claim 6. 9. The recombinant bacterial strain according to claim 8, characterized in that: the recombinant bacterial strain is recombinant Aspergillus niger. 10. The application of the fusion protein according to any one of claims 1 to 4 in improving the catalytic efficiency of glucose oxidase.

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