CN115975966A - Fusion protein for improving catalytic efficiency of glucose oxidase, method and application - Google Patents
Fusion protein for improving catalytic efficiency of glucose oxidase, method and application Download PDFInfo
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Abstract
The invention discloses a fusion protein for improving the catalytic efficiency of glucose oxidase, which comprises glucose oxidase, vitreoscilla hemoglobin and a section of flexible linked polypeptide. The vitreoscilla hemoglobin and the glucose oxidase are constructed into the fusion protein, and the fusion protein is favorable for improving the catalytic efficiency of the glucose oxidase by utilizing the oxygen transfer capacity of the vitreoscilla hemoglobin. Under the same condition, compared with the pure glucose oxidase, the catalytic efficiency of the fusion protein is obviously improved, and the application of the enzyme in industrial production is facilitated.
Description
Technical Field
The invention belongs to the technical field of biochemical engineering, and particularly relates to a fusion protein for improving the catalytic efficiency of glucose oxidase, a method and application thereof.
Background
Glucose Oxidase (GOD) is an important industrial enzyme, and can catalyze glucose to generate gluconic acid and hydrogen peroxide by taking oxygen as an electron acceptor. In view of their unique substrate consumption (oxygen) and product formation (acid) in catalytic reactions and the concomitant electron transport processes, GOD is widely used in numerous industries, such as food, feed, pharmaceutical and medical.
At present, commercial GOD is mainly Aspergillus niger GOD with better stability. Taking a bulk organic acid product, gluconic Acid (GA) as an example, the main production mode is deep fermentation of Aspergillus niger for producing GOD. GOD-catalyzed reactions are a number of aerobic processes, and oxygen transfer is an important limiting factor in the ability of GOD to function. Research shows that the optimization of the process for improving oxygen transmission and concentration can greatly enhance the yield of the GA, but the economic benefit of the GA is restricted by the increase of aerodynamic cost caused by actual production. How to effectively enhance the oxygen supply capacity in the GOD catalytic reaction process is an important research direction for cost reduction and efficiency improvement.
Vitreoscilla hemoglobin (VHb) is a bacterial hemoglobin from Vitreoscilla C1. VHb has strong oxygen transport capacity and has certain terminal oxidase activity and peroxidase activity. VHb has become a very practical tool and is widely applied to the overexpression in microorganisms, animals and plants to promote the cell growth or improve the product production. The VHb heterologous expression can improve the synthesis of metabolites such as microbial alcohols, antibiotics, organic acids, polysaccharides and the like, and can also improve the secretory expression of enzymes such as lipase 2, xylanase, coenzyme Q10, L-asparaginase and the like.
Therefore, VHb and GOD are constructed into fusion protein, and the utilization of oxygen transfer capability of VHb is helpful to improve catalytic efficiency of GOD. The invention provides a fusion protein method of VHb and GOD, which can obviously improve the catalytic efficiency of GOD.
Through searching, no published documents related to the patent application of the invention are found.
Disclosure of Invention
The invention aims to overcome the problems in the prior art and provides a fusion protein for improving the catalytic efficiency of glucose oxidase, a method and application thereof.
The technical scheme adopted by the invention for solving the technical problem is as follows:
a fusion protein for improving the catalytic efficiency of glucose oxidase is prepared from glucose oxidase, vitreoscilla hemoglobin and a segment of flexible linked polypeptide.
Further, the glucose oxidase, the vitreoscilla hemoglobin and a section of flexible link polypeptide need to construct fusion protein according to a certain link sequence, wherein the certain link sequence is that the two ends of the flexible link polypeptide are respectively linked with the C end of an amino acid sequence of the vitreoscilla hemoglobin and the N end of an amino acid sequence of the glucose oxidase.
Further, the flexible chain polypeptide is composed of polypeptide rich in glycine and serine, the total content of the glycine and the serine in the flexible chain polypeptide is not less than 60%, and the number of amino acids in the flexible chain polypeptide is 4-15.
Further, the amino acid sequence of the glucose oxidase is shown as SEQ ID No.1, the amino acid sequence of the vitreoscilla hemoglobin is shown as SEQ ID No.2, the amino acid sequence of the flexible linking polypeptide is shown as SEQ ID No.3, and the amino acid sequence of the fusion protein is shown as SEQ ID No. 4.
A method for improving the catalytic efficiency of glucose oxidase by using the fusion protein, wherein the method improves the catalytic efficiency of glucose oxidase by using the fusion protein.
The nucleotide sequence of the encoding gene of the fusion protein is shown as SEQ ID NO.5.
The recombinant expression vector containing the encoding gene of the fusion protein is preferably an Aspergillus niger expression vector.
A recombinant strain comprising a gene encoding a fusion protein as described above.
Further, the recombinant strain is recombinant aspergillus niger, which may be a bacterial or fungal cell, preferably a yeast or filamentous fungal cell, more preferably aspergillus niger.
The fusion protein is applied to improving the catalytic efficiency of glucose oxidase.
The beneficial effects obtained by the invention are as follows:
1. the vitreoscilla hemoglobin and the glucose oxidase are constructed into the fusion protein, and the fusion protein is favorable for improving the catalytic efficiency of the glucose oxidase by utilizing the oxygen transfer capacity of the vitreoscilla hemoglobin. Through enzyme kinetic parameter determination, compared with a parent glucose oxidase shown in SEQ ID NO.1, the catalytic efficiency (Kcat/Km) of the fusion protein is improved by more than one time, and the application of the enzyme in industrial production is facilitated.
2. The fusion protein can be applied to 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 the condition of dissolved oxygen limitation, compared with the parent glucose oxidase shown in SEQ ID NO.1, the fusion protein disclosed by the invention keeps higher catalytic efficiency, and is beneficial to reducing aerodynamic cost in production in the application of the enzyme in industrial production.
Drawings
FIG. 1 is a plasmid map of starting vector pLH503 in the present invention;
FIG. 2 shows the electrophoretic image of plasmid pLH1437 of the present invention, which is verified by double digestion (xho I/xba I,4110bp/8304 bp); wherein M is DNASMarker, and 1-3 are double enzyme digestion verification plasmids;
FIG. 3 is a map of plasmid pLH1437 of the present invention;
FIG. 4 is a map of plasmid pLH1540 in the present invention;
FIG. 5 is a SDS-PAGE and Western Blot of the fusion protein and GOD of the present invention; wherein, M is Marker,1 is GOD, and 2 is fusion protein.
FIG. 6 is a graph showing the determination of kinetic parameters of the fusion protein and GOD in the present invention.
Detailed Description
The present invention will be further described in detail with reference to examples for better understanding, but the scope of the present invention is not limited to the examples.
The raw materials used in the invention are all conventional commercial products unless otherwise specified, the methods used in the invention are all conventional in the field, and the quality of each substance used in the invention is conventional quality.
A fusion protein for improving the catalytic efficiency of glucose oxidase is prepared from glucose oxidase, vitreoscilla hemoglobin and a segment of flexible linked polypeptide.
Preferably, the glucose oxidase, the vitreoscilla hemoglobin and the flexible linking polypeptide are required to construct fusion protein according to a certain linking sequence, and the certain linking sequence is that the two ends of the flexible linking polypeptide are respectively linked with the C end of the amino acid sequence of the vitreoscilla hemoglobin and the N end of the amino acid sequence of the glucose oxidase.
Preferably, the flexible chain polypeptide is composed of polypeptide rich in glycine and serine, the total content of glycine and serine in the flexible chain polypeptide is not less than 60%, and the number of amino acids in the flexible chain polypeptide is 4-15.
Preferably, the amino acid sequence of the glucose oxidase is shown as SEQ ID NO.1, the amino acid sequence of the vitreoscilla hemoglobin is shown as SEQ ID NO.2, the amino acid sequence of the flexible linking polypeptide is shown as SEQ ID NO.3, and the amino acid sequence of the fusion protein is shown as SEQ ID NO. 4.
A method for improving the catalytic efficiency of glucose oxidase by using the fusion protein, wherein the method improves the catalytic efficiency of glucose oxidase by using the fusion protein.
The nucleotide sequence of the encoding gene of the fusion protein is shown as 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 a gene encoding a fusion protein as described above.
Preferably, the recombinant strain is recombinant aspergillus niger, which may be a bacterial or fungal cell, preferably a yeast or filamentous fungal cell, more preferably aspergillus niger.
The fusion protein is applied to improving the catalytic efficiency of glucose oxidase.
Specifically, the preparation and detection are as follows:
example 1: construction of fusion protein expression plasmid pLH1437
The starting vector is pLH503 (figure 1, the specific nucleotide sequence of the starting vector pLH503 is shown in SEQ ID NO.6, and the construction method of the plasmid pLH503 is that based on pLH454 (Appl Microbiol Biotechnol.2019,103 (19): 8105-8114. Doi). The vitreoscilla hemoglobin encoding gene nucleotide sequence SEQ ID No.8 and the glucose oxidase encoding gene nucleotide sequence SEQ ID No.9 were synthesized by Jin Weizhi. The C end of the constructed fusion protein contains a 6 × His tag.
The specific construction process is as follows: taking the synthesized vitreoscilla hemoglobin gene fragment as a template, designing an overlap primer, introducing flexible linking polypeptide (GS linker), amplifying VHB (438 bp) by using a primer VHB-f/VHB-r, wherein the nucleotide sequence of the primer is SEQ ID NO.7; taking a nucleotide sequence SEQ ID NO.8 of a glucose oxidase coding gene as a template, and amplifying GOD (1767 bp) by using a primer v6478-F/v 6478-R; the two gene fragments were ligated together (2238 bp) by overlap PCR to obtain the fusion protein-encoding gene fragment SEQ ID NO.5. The ligated fragments were ligated to the starting vector pLH503 which had been digested simultaneously with Sac I and Kpn I using the Novozan C113-Clon express-MultiS One Step Cloning Kit, the ligation products were transformed into competent cells of Escherichia coli JM109, which were then spread on LB dishes containing 100 to 300. Mu.g/mL kanamycin, incubated overnight at 37 ℃ to select single clones, and the plasmid p1437 (FIG. 3) was obtained after double digestion verification (FIG. 2).
TABLE 1 primers used in the present invention
Example 2: construction of fusion protein expression Strain
Agrobacterium containing plasmid pLH1437 and Aspergillus niger host strain ATCC1015 are co-cultured in IM plate, and the co-culture is transferred to CM plate containing 100-500. Mu.M cefotaxime, 100-500. Mu.g/mL ampicillin, 100-500. Mu.g/mL streptomycin and 100-500. Mu.g/mL hygromycin B and cultured at 28-37 deg.c until monoclone is formed. Selecting a single clone to be transferred to a PDA plate containing hygromycin B, screening hygromycin B resistant transformants, extracting a genome for verification, and obtaining a fusion protein overexpression strain S2250.
The IM medium is: 15-20 g of agar powder, adding water to 905.7mL, sterilizing at 121 ℃ for 20-50 min, heating by microwave until the agar is completely dissolved, adding: 0% to 0.2% K buffer,0% to 5% MN buffer,0% to 0.5% CaCl 2 ·2H 2 O，0％～2％0.01％FeSO 4 ，0％～1％IM Trace elements，0％～1％20％NH 4 NO 3 0% to 2% of glycerol, 0% to 6% of 1M MES,0% to 1% of 20% of glucose.
Preparation of required reagents in the IM medium:
1) Kbuffer: 1 to 5M K 2 HPO 4 And 1-5M KH 2 PO 4 Mixing to adjust the pH value to 3-7, and sterilizing at 121 ℃ for 20min.
2)MN buffer：0％～0.6％MgSO 4 ·7H 2 O, 0-0.3% of NaCl, dissolving in deionized water, and sterilizing at 121 deg.C for 20min.
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 And O, adding deionized water for dissolving, and sterilizing at 121 ℃ for 20min.
The CM medium was:
15-20 g of agar powder, adding water to 897mL, and sterilizing at 121 ℃ for 20min. After the agar is completely dissolved, adding the following components: 0% -5% ASP + N,0% -5% glucose, 0% -0.5% The 4 0% to 0.5% by weight of CM Trace elements,0% to 5% by weight of 10% casein hydrolysate, 0% to 10% by weight of yeast extract.
Preparation of required reagents in the CM medium:
1)ASP+N：0％～0.5％KCl(350mM)，0％～1.5％KH 2 PO 4 (550mM)，0％～5％NaNO 3 (3.5M), adding deionized water to dissolve, pH 5.5 (5M KOH), and sterilizing at 121 ℃ 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% of EDTA, adding deionized water for dissolution, and sterilizing at 121 ℃ for 20min.
The PDA culture medium is: 100-500 g of potato is cut into small pieces, 500-3000 mL of water is added for boiling for 20-40 min, and the clear liquid is filtered by double-layer gauze. Then 10-50 g of glucose is added to be completely dissolved, and water is added to be constant volume to 1-3L. Solid culture of 1.5% (W/V) agar powder. Autoclaving at 121 deg.C for 20min.
Example 3: construction of GOD expressing strains
Referring to example 1, plasmid pLH1540 (fig. 4) was obtained by using pLH503 as starting vector and obtaining plasmid overexpressing GOD. Referring to example 2, a strain overexpressing GOD was obtained, resulting in strain S2637.
Example 4: separation and purification of fusion protein and GOD
Inoculating S2250 and S2637 spores into seed culture medium, performing shake culture at 28 deg.C for one day, transferring into fermentation medium, filtering the fermentation broth with gauze after 7 days, replacing the fermentation broth with lysine Buffer, performing protein purification to obtain fusion protein and GOD, and testing with SDS-PAGE and Western Blot. The results of protein gel electrophoresis analysis show that the molecular weights of the single over-expressed glucose oxidase and the fusion protein are respectively 60kDa and 75kDa (figure 5), and the results show that the fusion protein is expressed.
The seed culture medium is as follows: 5-200 g/L glucose, 5-200 g/L corn dry powder, sterilizing for 20min at 115 ℃.
The fermentation medium is as follows: maltose 10-150 g/L, soybean powder 10-60 g/L, sodium citrate 10-70 g/L, (NH 4) 2 SO 4 2～15g/L、NaH 2 PO 4 1～5g/L、MgSO 4 1-5 g/L, tween-80-1-5 mL/L, 1-5 g/L arginine, sterilizing at 115 ℃ for 20min. The protein purification method comprises the following steps: concentrating, dialyzing and desalting the fermentation liquid obtained in the fermentation medium, and passing through a Ni NTA Beads 6FF packed column; collecting the eluent; desalting, dialyzing and dissolving in pH5.1 sodium acetate buffer solution;
reagents used for protein purification:
1)Lysis Buffer：10～50mM NaH 2 PO 4 100 to 300mM NaCl and 10 to 50mM imidazole; adjusting the pH to 8.0 by using NaOH solution;
2) Wash Buffer: 10-50mM NaH2PO4, 100-300 mM NaCl, 5-20 mM imidazole; adjusting the pH to 8.0 by using NaOH solution;
3)Elution Buffer：10～50mM NaH 2 PO 4 100 to 300mM NaCl and 10 to 250mM imidazole; adjusting the pH to 8.0 by using NaOH solution;
example 5: fusion protein and GOD enzyme activity assay
The enzyme activity of the purified pure protein is measured, and the Michaelis equation kinetic fitting is carried out, as shown in FIG. 6, the result shows that the catalytic efficiency Kcat/Km of the fusion protein is 45.3, which is 2 times of GOD, and the catalytic efficiency of the fusion protein is improved by 100% compared with GOD.
Reagents used for determining enzyme activity:
working solution I (o-dianisidine): a: dissolving 0.1-0.3 g of o-dianisidine in 10mL of methanol to obtain a stock solution of o-dianisidine, storing at 4 ℃, shaking up before use, and suggesting to be prepared for use. B: 10-50 g of anhydrous sodium acetate is accurately weighed, dissolved in 500mL of double distilled water, and the pH value is gradually adjusted to about 5.1-7 by glacial acetic acid. C: taking 0.1-0.5 mL of A to be dissolved in 10-20 mL of buffer solution B as working solution I.
Working solution II (glucose solution): 10-30 g of anhydrous glucose is accurately weighed, and the volume is fixed to 100mL by using double distilled water.
Working solution III (horseradish peroxidase): the horseradish peroxidase is diluted to 10-100U/mL of enzyme activity (prepared at present).
The enzyme activity determination steps are as follows:
1) Adding 1-2.5 mL of working solution I, 100-300 mu L of working solution II and 100-300 mu L of working solution III into a 5mL EP tube, and preheating for 1-10 min at 37 ℃.
2) Respectively adding 100-300 mu L of enzyme solution sample, simultaneously adding the corresponding inactivated enzyme solution as a control, and reacting for 3-5 min at 37 ℃.
3) Then, 2mL of a 2mol/L sulfuric acid solution was added to terminate the reaction.
4) 1mL of the reaction solution was taken out of the cuvette, and the absorbance at a wavelength of 500 to 600nm was measured by zeroing the absorbance of the inactivated enzyme solution.
5) A commercial GOD standard (purchased from Solarbio, cat #: g8030 Prepared with buffers to form enzyme solutions with a gradient of 0.4, 0.6, 0.8, 1.0, 2.0 and 4.0U/mL, and numbered 1-6 in sequence. Meanwhile, 500 mu L of No. 1-6 enzyme solution is used for inactivation, and a standard curve is obtained.
6) And (3) bringing the OD value of the diluted enzyme solution after the o-dianisidine-catalase reaction into the standard yeast, and multiplying the obtained result by the dilution times to obtain the enzyme activity of the enzyme solution to be detected.
The related sequences of the invention are as follows:
SEQ ID NO.1: amino acid sequence of glucose oxidase (589 aa)
LPHYIRSNGIEASLLTDPKDVSGRTVDYIIAGGGLTGLTTAARLTENPNISVLVIESGSYESDRGPIIEDLNAYGDIFGSSVDHAYETVELATNNQTALIRSGNGLGGSTLVNGGTWTRPHKAQVDSWETVFGNEGWNWDNVAAYSLQAERARAPNAKQIAAGHYFNASCHGTNGTVHAGPRDTGDDYSPIVKALMSAVEDRGVPTKKDFGCGDPHGVSMFPNTLHEDQVRSDAAREWLLPNYQRPNLQVLTGQYVGKVLLSQNGTTPRAVGVEFGTHKGNTHNVYAKHEVLLAAGSAVSPTILEYSGIGMKSILEPLGIDTVVDLPVGLNLQDQTTATVRSRITSAGAGQGQAAWFATFNETFGDYAEKAHELLNTKLEQWAEEAVARGGFHNTTALLIQYENYRDWIVNHNVAYSELFLDTAGVASFDVWDLLPFTRGYVHILDKDPYLHHFAYDPQYFLNELDLLGQAAATQLARNISNSGAMQTYFAGETIPGDNLAYDADLSAWTEYIPYHFRPNYHGVGTCSMMPKEMGGVVDNAARVYGVQGLRVIDGSIPPTQMSSHVMTVFYAMALKIADAILEDYASMQ
SEQ ID NO.2: amino acid sequence of Vitreoscilla hemoglobin (146 aa)
MLDQQTINIIKATVPVLKEHGVTITTTFYKNLFAKHPEVRPLFDMGRQESLEQPKALAMTVLAAAQNIENLPAILPAVKKIAVKHCQAGVAAAHYPIVGQELLGAIKEVLGDAATDDILDAWGKAYGVIADVFIQVEADLYAQAVE
SEQ ID NO.3: flexible linker polypeptide amino acid sequence (4 aa)
GSSG
SEQ ID NO.4: fusion protein amino acid sequence (739 aa)
MLDQQTINIIKATVPVLKEHGVTITTTFYKNLFAKHPEVRPLFDMGRQESLEQPKALAMTVLAAAQNIENLPAILPAVKKIAVKHCQAGVAAAHYPIVGQELLGAIKEVLGDAATDDILDAWGKAYGVIADVFIQVEADLYAQAVEGSSGLPHYIRSNGIEASLLTDPKDVSGRTVDYIIAGGGLTGLTTAARLTENPNISVLVIESGSYESDRGPIIEDLNAYGDIFGSSVDHAYETVELATNNQTALIRSGNGLGGSTLVNGGTWTRPHKAQVDSWETVFGNEGWNWDNVAAYSLQAERARAPNAKQIAAGHYFNASCHGTNGTVHAGPRDTGDDYSPIVKALMSAVEDRGVPTKKDFGCGDPHGVSMFPNTLHEDQVRSDAAREWLLPNYQRPNLQVLTGQYVGKVLLSQNGTTPRAVGVEFGTHKGNTHNVYAKHEVLLAAGSAVSPTILEYSGIGMKSILEPLGIDTVVDLPVGLNLQDQTTATVRSRITSAGAGQGQAAWFATFNETFGDYAEKAHELLNTKLEQWAEEAVARGGFHNTTALLIQYENYRDWIVNHNVAYSELFLDTAGVASFDVWDLLPFTRGYVHILDKDPYLHHFAYDPQYFLNELDLLGQAAATQLARNISNSGAMQTYFAGETIPGDNLAYDADLSAWTEYIPYHFRPNYHGVGTCSMMPKEMGGVVDNAARVYGVQGLRVIDGSIPPTQMSSHVMTVFYAMALKIADAILEDYASMQ
SEQ ID No.5: fusion protein nucleotide sequence
atgctggatcagcagaccatcaacatcatcaaggccaccgtccccgtcctgaaggagcacggtgtcactattaccaccaccttctacaagaacctgttcgccaagcaccccgaggtccgccctttgtttgatatgggccgccaggagtccctggagcagcctaaagctctggctatgaccgtcctggctgctgctcaaaatatcgagaacctgcccgctattctgcccgccgtcaaaaagatcgccgtcaagcactgccaggccggcgttgccgctgctcattatcctattgtcggtcaggagctgctgggcgccattaaggaagtcctgggcgatgccgccaccgatgatatcctggatgcctggggcaaggcctacggcgttattgccgatgtctttatccaggtcgaggccgatctgtacgcccaggccgttgaaGGTTCCagcggcCTGCCACACTACATCAGGAGCAATGGCATTGAAGCCAGCCTCCTGACTGACCCCAAGGATGTCTCCGGCCGCACGGTCGACTACATCATCGCTGGTGGAGGTCTGACTGGACTCACCACCGCTGCTCGTCTGACGGAGAACCCCAACATCAGTGTGCTCGTCATCGAAAGTGGCTCCTACGAGTCGGACAGAGGTCCTATCATTGAGGACCTGAACGCCTACGGCGACATTTTTGGCAGCAGTGTAGACCACGCCTACGAGACCGTTGAGCTCGCTACCAACAATCAAACCGCGCTGATCCGCTCCGGAAATGGTCTCGGTGGCTCTACTCTAGTGAATGGTGGCACCTGGACTCGCCCCCACAAGGCACAGGTTGATTCTTGGGAGACTGTCTTTGGAAATGAGGGCTGGAACTGGGACAATGTGGCCGCCTACTCCCTCCAGGCTGAGCGTGCTCGCGCACCAAATGCCAAACAGATCGCTGCTGGCCACTACTTCAACGCATCCTGTCATGGTACCAATGGTACTGTCCATGCCGGACCCCGTGACACCGGCGATGACTATTCCCCCATCGTCAAGGCTCTCATGAGCGCTGTCGAAGACCGGGGCGTTCCCACCAAGAAGGACTTCGGATGCGGTGACCCTCATGGTGTGTCCATGTTCCCCAACACCTTGCACGAAGACCAAGTTCGCTCCGATGCCGCTCGCGAATGGCTCCTTCCCAACTACCAACGTCCCAACCTGCAAGTCCTGACCGGACAATATGTTGGTAAGGTGCTCCTTAGCCAGAACGGCACCACCCCTCGTGCCGTCGGCGTGGAATTCGGCACCCACAAGGGCAACACCCACAACGTTTACGCTAAGCACGAGGTCCTCCTGGCTGCTGGCTCGGCTGTCTCTCCCACCATCCTCGAATATTCCGGTATCGGAATGAAGTCCATCCTGGAACCCCTTGGTATCGACACCGTCGTTGACCTGCCCGTCGGCCTGAACCTGCAGGACCAGACCACCGCTACCGTCCGCTCCCGCATCACCTCTGCTGGTGCCGGACAGGGACAGGCCGCTTGGTTCGCCACCTTCAACGAGACCTTTGGTGACTATGCCGAAAAGGCACACGAGCTGCTCAACACCAAGCTGGAGCAGTGGGCCGAAGAGGCCGTCGCCCGTGGCGGATTCCACAACACCACCGCCTTGCTCATCCAGTACGAGAACTATCGCGACTGGATTGTCAATCACAACGTCGCGTACTCGGAACTCTTCCTCGACACTGCCGGAGTGGCCAGCTTCGATGTGTGGGACCTTCTGCCCTTCACGAGAGGATACGTCCACATCCTCGACAAGGACCCCTACCTCCACCACTTTGCCTACGACCCTCAGTACTTCCTCAACGAGCTCGACCTGCTCGGTCAGGCTGCCGCTACTCAGCTGGCCCGTAACATCTCCAACTCCGGTGCTATGCAGACCTACTTCGCTGGCGAGACTATCCCCGGTGATAACCTCGCGTATGATGCCGATTTGAGCGCCTGGACTGAGTACATCCCGTACCACTTCCGTCCTAACTACCATGGCGTGGGTACTTGCTCCATGATGCCGAAGGAGATGGGCGGTGTTGTCGATAATGCTGCCCGTGTGTACGGTGTGCAGGGACTGCGTGTCATTGATGGTTCTATTCCCCCTACGCAGATGTCGTCCCATGTCATGACTGTGTTCTACGCCATGGCGTTGAAGATTGCGGATGCTATTTTGGAGGACTACGCTTCTATGCAGcaccaccaccaccaccactaa
SEQ ID NO.6: pLH503 nucleotide sequence
CTAGACTAGTCCTCTCGTATGCAGAGGAAATCTCCCCTGATCTTCCGAACTGGTCGTACCTGGCGACCTATGACTATGGCACCCCAGTTCTGGGGACCTTCCACGGAAGTGACCTGCTGCAGGTGTTCTATGGGATCAAGCCAAACTATGCAGCTAGTTCTAGCCACACGTACTATCTGAGCTTTGTGTATACGCTGGATCCGAACTCCAACCGGGGGGAGTACATTGAGTGGCCGCAGTGGAAGGAATCGCGGCAGTTGATGAATTTCGGAGCGAACGACGCCAGTCTCCTTACGGATGATTTCCGCAACGGGACATATGAGTTCATCCTGCAGAATACCGCGGCGTTCCACATCTGATGCCATTGGCGGAGGGGTCCGGACGGTCAGGAACTTAGCCTTATGAGATGAATGATGGACGTGTCTGGCCTCGGAAAAGGATATATGGGGATCATAATAGTACTAGCCATATTAATGAAGGGCATATACCACGCGTTGGACCTGCGTTATAGCTTCCCGTTAGTTATAGTACCATCGTTATACCAGCCAATCAAGTCACCACGCACGACCGGGGACGGCGAATCCCCGGGTATAGTACCATCGTTATACCAGCCAATCAAGTCACCACGCACGACCGGGGACGGCGAATCCCCGGGTATAGTACCATCGTTATACCAGCCAATCAAGTCACCACGCACGACCGGGGACGGCGAATCCCCGGGTATAGTACCATCGTTATACCAGCCAATCAAGTCACCACGCACGACCGGGGACGGCGAATCCCCGGGTATAGTACCATCGTTATACCAGCCAATCAAGTCACCACGCACGACCGGGGACGGCGAATCGAATTCAAGCTAGATGCTAAGCGATATTGCATGGCAATATGTGTTGATGCATGTGCTTCTTCCTTCAGCTTCCCCTCGTGCAGATGAGGTTTGGCTATAAATTGAAGTGGTTGGTCGGGGTTCCGTGAGGGGCTGAAGTGCTTCCTCCCTTTTAGACGCAACTGAGAGCCTGAGCTTCATCCCCAGCATCATTACACCTCAGCAATGTCGTTCCGATCTCTACTCGCCCTGAGCGGCCTCGTCTGCACAGGGTTGGCAAACGTGATTTCCAAGCGCGAGCTCAGATCTGGTACCGCATGCGGATCCGATCCACTTAACGTTACTGAAATCATCAAACAGCTTGACGAATCTGGATATAAGATCGTTGGTGTCGATGTCAGCTCCGGAGTTGAGACAAATGGTGTTCAGGATCTCGATAAGATACGTTCATTTGTCCAAGCAGCAAAGAGTGCCTTCTAGTGATTTAATAGCTCCATGTCAACAAGAATAAAACGCGTTTTCGGGTTTACCTCTTCCAGATACAGCTCATCTGCAATGCATTAATGCATTGACTGCAACCTAGTAACGCCTTNCAGGCTCCGGCGAAGAGAAGAATAGCTTAGCAGAGCTATTTTCATTTTCGGGAGACGAGATCAAGCAGATCAACGGTCGTCAAGAGACCTACGAGACTGAGGAATCCGCTCTTGGCTCCACGCGACTATATATTTGTCTCTAATTGTACTTTGACATGCTCCTCTTCTTTACTCTGATAGCTTGACTATGAAAATTCCGTCACCAGCNCCTGGGTTCGCAAAGATAATTGCATGTTTCTTCCTTGAACTCTCAAGCCTACAGGACACACATTCATCGTAGGTATAAACCTCGAAATCANTTCCTACTAAGATGGTATACAATAGTAACCATGCATGGTTGCCTAGTGAATGCTCCGTAACACCCAATACGCCGGCCGAAACTTTTTTACAACTCTCCTATGAGTCGTTTACCCAGAATGCACAGGTACACTTGTTTAGAGGTAATCCTTCTTTCTAGACCCGGGGGGCCCTACGTAATAACTTCGTATAATGTATGCTATACGAAGTTATGTCGACGTTAACTGATATTGAAGGAGCATTTTTTGGGCTTGGCTGGAGCTAGTGGAGGTCAACAATGAATGCCTATTTTGGTTTAGTCGTCCAGGCGGTGAGCACAAAATTTGTGTCGTTTGACAAGATGGTTCATTTAGGCAACTGGTCAGATCAGCCCCACTTGTAGCAGTAGCGGCGGCGCTCGAAGTGTGACTCTTATTAGCAGACAGGAACGAGGACATTATTATCATCTGCTGCTTGGTGCACGATAACTTGGTGCGTTTGTCAAGCAAGGTAAGTGGACGACCCGGTCATACCTTCTTAAGTTCGCCCTTCCTCCCTTTATTTCAGATTCAATCTGACTTACCTATTCTACCCAAGCATCCAAATGAAAAAGCCTGAACTCACCGCGACGTCTGTCGAGAAGTTTCTGATCGAAAAGTTCGACAGCGTCTCCGACCTGATGCAGCTCTCGGAGGGCGAAGAATCTCGTGCTTTCAGCTTCGATGTAGGAGGGCGTGGATATGTCCTGCGGGTAAATAGCTGCGCCGATGGTTTCTACAAAGATCGTTATGTTTATCGGCACTTTGCATCGGCCGCGCTCCCGATTCCGGAAGTGCTTGACATTGGGGAGTTCAGCGAGAGCCTGACCTATTGCATCTCCCGCCGTGCACAGGGTGTCACGTTGCAAGACCTGCCTGAAACCGAACTGCCCGCTGTTCTCCAGCCGGTCGCGGAGGCCATGGATGCGATCGCTGCGGCCGATCTTAGCCAGACGAGCGGGTTCGGCCCATTCGGACCGCAAGGAATCGGTCAATACACTACATGGCGTGATTTCATATGCGCGATTGCTGATCCCCATGTGTATCACTGGCAAACTGTGATGGACGACACCGTCAGTGCGTCCGTCGCGCAGGCTCTCGATGAGCTGATGCTTTGGGCCGAGGACTGCCCCGAAGTCCGGCACCTCGTGCATGCGGATTTCGGCTCCAACAATGTCCTGACGGACAATGGCCGCATAACAGCGGTCATTGACTGGAGCGAGGCGATGTTCGGGGATTCCCAATACGAGGTCGCCAACATCCTCTTCTGGAGGCCGTGGTTGGCTTGTATGGAGCAGCAGACGCGCTACTTCGAGCGGAGGCATCCGGAGCTTGCAGGATCGCCGCGCCTCCGGGCGTATATGCTCCGCATTGGTCTTGACCAACTCTATCAGAGCTTGGTTGACGGCAATTTCGATGATGCAGCTTGGGCGCAGGGTCGATGCGACGCAATCGTCCGATCCGGAGCCGGGACTGTCGGGCGTACACAAATCGCCCGCAGAAGCGCGGCCGTCTGGACCGATGGCTGTGTAGAAGTACTCGCCGATAGTGGAAACCGACGCCCCAGCACTCGTCCGAGGGCAAAGGAATAGAGTAGATGCCGACCGGGAACCAGTTAACGTCGAATAACTTCGTATAATGTATGCTATACGAAGTTATAAGCTTGGCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGCTAGAGCAGCTTGAGCTTGGATCAGATTGTCGTTTCCCGCCTTCAGTTTAAACTATCAGTGTTTGACAGGATATATTGGCGGGTAAACCTAAGAGAAAAGAGCGTTTATTAGAATAACGGATATTTAAAAGGGCGTGAAAAGGTTTATCCGTTCGTCCATTTGTATGTGCATGCCAACCACAGGGTTCCCCTCGGGATCAAAGTACTTTGATCCAACCCCTCCGCTGCTATAGTGCAGTCGGCTTCTGACGTTCAGTGCAGCCGTCTTCTGAAAACGACATGTCGCACAAGTCCTAAGTTACGCGACAGGCTGCCGCCCTGCCCTTTTCCTGGCGTTTTCTTGTCGCGTGTTTTAGTCGCATAAAGTAGAATACTTGCGACTAGAACCGGAGACATTACGCCATGAACAAGAGCGCCGCCGCTGGCCTGCTGGGCTATGCCCGCGTCAGCACCGACGACCAGGACTTGACCAACCAACGGGCCGAACTGCACGCGGCCGGCTGCACCAAGCTGTTTTCCGAGAAGATCACCGGCACCAGGCGCGACCGCCCGGAGCTGGCCAGGATGCTTGACCACCTACGCCCTGGCGACGTTGTGACAGTGACCAGGCTAGACCGCCTGGCCCGCAGCACCCGCGACCTACTGGACATTGCCGAGCGCATCCAGGAGGCCGGCGCGGGCCTGCGTAGCCTGGCAGAGCCGTGGGCCGACACCACCACGCCGGCCGGCCGCATGGTGTTGACCGTGTTCGCCGGCATTGCCGAGTTCGAGCGTTCCCTAATCATCGACCGCACCCGGAGCGGGCGCGAGGCCGCCAAGGCCCGAGGCGTGAAGTTTGGCCCCCGCCCTACCCTCACCCCGGCACAGATCGCGCACGCCCGCGAGCTGATCGACCAGGAAGGCCGCACCGTGAAAGAGGCGGCTGCACTGCTTGGCGTGCATCGCTCGACCCTGTACCGCGCACTTGAGCGCAGCGAGGAAGTGACGCCCACCGAGGCCAGGCGGCGCGGTGCCTTCCGTGAGGACGCATTGACCGAGGCCGACGCCCTGGCGGCCGCCGAGAATGAACGCCAAGAGGAACAAGCATGAAACCGCACCAGGACGGCCAGGACGAACCGTTTTTCATTACCGAAGAGATCGAGGCGGAGATGATCGCGGCCGGGTACGTGTTCGAGCCGCCCGCGCACGTCTCAACCGTGCGGCTGCATGAAATCCTGGCCGGTTTGTCTGATGCCAAGCTGGCGGCCTGGCCGGCCAGCTTGGCCGCTGAAGAAACCGAGCGCCGCCGTCTAAAAAGGTGATGTGTATTTGAGTAAAACAGCTTGCGTCATGCGGTCGCTGCGTATATGATGCGATGAGTAAATAAACAAATACGCAAGGGGAACGCATGAAGGTTATCGCTGTACTTAACCAGAAAGGCGGGTCAGGCAAGACGACCATCGCAACCCATCTAGCCCGCGCCCTGCAACTCGCCGGGGCCGATGTTCTGTTAGTCGATTCCGATCCCCAGGGCAGTGCCCGCGATTGGGCGGCCGTGCGGGAAGATCAACCGCTAACCGTTGTCGGCATCGACCGCCCGACGATTGACCGCGACGTGAAGGCCATCGGCCGGCGCGACTTCGTAGTGATCGACGGAGCGCCCCAGGCGGCGGACTTGGCTGTGTCCGCGATCAAGGCAGCCGACTTCGTGCTGATTCCGGTGCAGCCAAGCCCTTACGACATATGGGCCACCGCCGACCTGGTGGAGCTGGTTAAGCAGCGCATTGAGGTCACGGATGGAAGGCTACAAGCGGCCTTTGTCGTGTCGCGGGCGATCAAAGGCACGCGCATCGGCGGTGAGGTTGCCGAGGCGCTGGCCGGGTACGAGCTGCCCATTCTTGAGTCCCGTATCACGCAGCGCGTGAGCTACCCAGGCACTGCCGCCGCCGGCACAACCGTTCTTGAATCAGAACCCGAGGGCGACGCTGCCCGCGAGGTCCAGGCGCTGGCCGCTGAAATTAAATCAAAACTCATTTGAGTTAATGAGGTAAAGAGAAAATGAGCAAAAGCACAAACACGCTAAGTGCCGGCCGTCCGAGCGCACGCAGCAGCAAGGCTGCAACGTTGGCCAGCCTGGCAGACACGCCAGCCATGAAGCGGGTCAACTTTCAGTTGCCGGCGGAGGATCACACCAAGCTGAAGATGTACGCGGTACGCCAAGGCAAGACCATTACCGAGCTGCTATCTGAATACATCGCGCAGCTACCAGAGTAAATGAGCAAATGAATAAATGAGTAGATGAATTTTAGCGGCTAAAGGAGGCGGCATGGAAAATCAAGAACAACCAGGCACCGACGCCGTGGAATGCCCCATGTGTGGAGGAACGGGCGGTTGGCCAGGCGTAAGCGGCTGGGTTGTCTGCCGGCCCTGCAATGGCACTGGAACCCCCAAGCCCGAGGAATCGGCGTGACGGTCGCAAACCATCCGGCCCGGTACAAATCGGCGCGGCGCTGGGTGATGACCTGGTGGAGAAGTTGAAGGCCGCGCAGGCCGCCCAGCGGCAACGCATCGAGGCAGAAGCACGCCCCGGTGAATCGTGGCAAGCGGCCGCTGATCGAATCCGCAAAGAATCCCGGCAACCGCCGGCAGCCGGTGCGCCGTCGATTAGGAAGCCGCCCAAGGGCGACGAGCAACCAGATTTTTTCGTTCCGATGCTCTATGACGTGGGCACCCGCGATAGTCGCAGCATCATGGACGTGGCCGTTTTCCGTCTGTCGAAGCGTGACCGACGAGCTGGCGAGGTGATCCGCTACGAGCTTCCAGACGGGCACGTAGAGGTTTCCGCAGGGCCGGCCGGCATGGCCAGTGTGTGGGATTACGACCTGGTACTGATGGCGGTTTCCCATCTAACCGAATCCATGAACCGATACCGGGAAGGGAAGGGAGACAAGCCCGGCCGCGTGTTCCGTCCACACGTTGCGGACGTACTCAAGTTCTGCCGGCGAGCCGATGGCGGAAAGCAGAAAGACGACCTGGTAGAAACCTGCATTCGGTTAAACACCACGCACGTTGCCATGCAGCGTACGAAGAAGGCCAAGAACGGCCGCCTGGTGACGGTATCCGAGGGTGAAGCCTTGATTAGCCGCTACAAGATCGTAAAGAGCGAAACCGGGCGGCCGGAGTACATCGAGATCGAGCTAGCTGATTGGATGTACCGCGAGATCACAGAAGGCAAGAACCCGGACGTGCTGACGGTTCACCCCGATTACTTTTTGATCGATCCCGGCATCGGCCGTTTTCTCTACCGCCTGGCACGCCGCGCCGCAGGCAAGGCAGAAGCCAGATGGTTGTTCAAGACGATCTACGAACGCAGTGGCAGCGCCGGAGAGTTCAAGAAGTTCTGTTTCACCGTGCGCAAGCTGATCGGGTCAAATGACCTGCCGGAGTACGATTTGAAGGAGGAGGCGGGGCAGGCTGGCCCGATCCTAGTCATGCGCTACCGCAACCTGATCGAGGGCGAAGCATCCGCCGGTTCCTAATGTACGGAGCAGATGCTAGGGCAAATTGCCCTAGCAGGGGAAAAAGGTCGAAAAGGTCTCTTTCCTGTGGATAGCACGTACATTGGGAACCCAAAGCCGTACATTGGGAACCGGAACCCGTACATTGGGAACCCAAAGCCGTACATTGGGAACCGGTCACACATGTAAGTGACTGATATAAAAGAGAAAAAAGGCGATTTTTCCGCCTAAAACTCTTTAAAACTTATTAAAACTCTTAAAACCCGCCTGGCCTGTGCATAACTGTCTGGCCAGCGCACAGCCGAAGAGCTGCAAAAAGCGCCTACCCTTCGGTCGCTGCGCTCCCTACGCCCCGCCGCTTCGCGTCGGCCTATCGCGGCCGCTGGCCGCTCAAAAATGGCTGGCCTACGGCCAGGCAATCTACCAGGGCGCGGACAAGCCGCGCCGTCGCCACTCGACCGCCGGCGCCCACATCAAGGCACCCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGTAGCGATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGCATTCTAGGTACTAAAACAATTCATCCAGTAAAATATAATATTTTATTTTCTCCCAATCAGGCTTGATCCCCAGTAAGTCAAAAAATAGCTCGACATACTGTTCTTCCCCGATATCCTCCCTGATCGACCGGACGCAGAAGGCAATGTCATACCACTTGTCCGCCCTGCCGCTTCTCCCAAGATCAATAAAGCCACTTACTTTGCCATCTTTCACAAAGATGTTGCTGTCTCCCAGGTCGCCGTGGGAAAAGACAAGTTCCTCTTCGGGCTTTTCCGTCTTTAAAAAATCATACAGCTCGCGCGGATCTTTAAATGGAGTGTCTTCTTCCCAGTTTTCGCAATCCACATCGGCCAGATCGTTATTCAGTAAGTAATCCAATTCGGCTAAGCGGCTGTCTAAGCTATTCGTATAGGGACAATCCGATATGTCGATGGAGTGAAAGAGCCTGATGCACTCCGCATACAGCTCGATAATCTTTTCAGGGCTTTGTTCATCTTCATACTCTTCCGAGCAAAGGACGCCATCGGCCTCACTCATGAGCAGATTGCTCCAGCCATCATGCCGTTCAAAGTGCAGGACCTTTGGAACAGGCAGCTTTCCTTCCAGCCATAGCATCATGTCCTTTTCCCGTTCCACATCATAGGTGGTCCCTTTATACCGGCTGTCCGTCATTTTTAAATATAGGTTTTCATTTTCTCCCACCAGCTTATATACCTTAGCAGGAGACATTCCTTCCGTATCTTTTACGCAGCGGTATTTTTCGATCAGTTTTTTCAATTCCGGTGATATTCTCATTTTAGCCATTTATTATTTCCTTCCTCTTTTCTACAGTATTTAAAGATACCCCAAGAAGCTAATTATAACAAGACGAACTCCAATTCACTGTTCCTTGCATTCTAAAACCTTAAATACCAGAAAACAGCTTTTTCAAAGTTGTTTTCAAAGTTGGCGTATAACATAGTATCGACGGAGCCGATTTTGAAACCGCGGTGATCACAGGCAGCAACGCTCTGTCATCGTTACAATCAACATGCTACCCTCCGCGAGATCATCCGTGTTTCAAACCCGGCAGCTTAGTTGCCGTTCTTCCGAATAGCATCGGTAACATGAGCAAAGTCTGCCGCCTTACAACGGCTCTCCCGCTGACGCCGTCCCGGACTGATGGGCTGCCTGTATCGAGTGGTGATTTTGTGCCGAGCTGCCGGTCGGGGAGCTGTTGGCTGGCTGGTGGCAGGATATATTGTGGTGTAAACAAATTGACGCTTAGACAACTTAATAACACATTGCGGACGTTTTTAATGTACTGAATTAACGCCGAATTAATTCGGGGGATCTGGATTTTAGTACTGGATTTTGGTTTTAGGAATTAGAAATTTTATTGATAGAAGTATTTTACAAATACAAATACATACTAAGGGTTTCTTATATGCTCAACACATGAGCGAAACCCTATAGGAACCCTAATTCCCTTATCTGGGAACTACTCACACATTATTATGGAGAAACTCGAGA
Nucleotide sequence of a fragment of SEQ ID NO.7PglaA-MCS
ACTAGTcctctcgtatgcagaggaaatctcccctgatcttccgaactggtcgtacctggcgacctatgactatggcaccccagttctggggaccttccacggaagtgacctgctgcaggtgttctatgggatcaagccaaactatgcagctagttctagccacacgtactatctgagctttgtgtatacgctggatccgaactccaaccggggggagtacattgagtggccgcagtggaaggaatcgcggcagttgatgaatttcggagcgaacgacgccagtctccttacggatgatttccgcaacgggacatatgagttcatcctgcagaataccgcggcgttccacatctgatgccattggcggaggggtccggacggtcaggaacttagccttatgagatgaatgatggacgtgtctggcctcggaaaaggatatatggggatcataatagtactagccatattaatgaagggcatataccacgcgttggacctgcgttatagcttcccgttagttatagtaccatcgttataccagccaatcaagtcaccacgcacgaccggggacggcgaatccccgggtatagtaccatcgttataccagccaatcaagtcaccacgcacgaccggggacggcgaatccccgggtatagtaccatcgttataccagccaatcaagtcaccacgcacgaccggggacggcgaatccccgggtatagtaccatcgttataccagccaatcaagtcaccacgcacgaccggggacggcgaatccccgggtatagtaccatcgttataccagccaatcaagtcaccacgcacgaccggggacggcgaatcgaattcaagctagatgctaagcgatattgcatggcaatatgtgttgatgcatgtgcttcttccttcagcttcccctcgtgcagatgaggtttggctataaattgaagtggttggtcggggttccgtgaggggctgaagtgcttcctcccttttagacgcaactgagagcctgagcttcatccccagcatcattacacctcagcaatgtcgttccgatctctactcgccctgagcggcctcgtctgcacagggttggcaaacgtgatttccaagcgcGAGCTCAGATCTGGTACCGCATGCGGATCC
SEQ ID NO.8: VHb nucleotide sequence
atgctggatcagcagaccatcaacatcatcaaggccaccgtccccgtcctgaaggagcacggtgtcactattaccaccaccttctacaagaacctgttcgccaagcaccccgaggtccgccctttgtttgatatgggccgccaggagtccctggagcagcctaaagctctggctatgaccgtcctggctgctgctcaaaatatcgagaacctgcccgctattctgcccgccgtcaaaaagatcgccgtcaagcactgccaggccggcgttgccgctgctcattatcctattgtcggtcaggagctgctgggcgccattaaggaagtcctgggcgatgccgccaccgatgatatcctggatgcctggggcaaggcctacggcgttattgccgatgtctttatccaggtcgaggccgatctgtacgcccaggccgttgaa
SEQ ID NO.9: GOD nucleotide sequence
CTGCCACACTACATCAGGAGCAATGGCATTGAAGCCAGCCTCCTGACTGACCCCAAGGATGTCTCCGGCCGCACGGTCGACTACATCATCGCTGGTGGAGGTCTGACTGGACTCACCACCGCTGCTCGTCTGACGGAGAACCCCAACATCAGTGTGCTCGTCATCGAAAGTGGCTCCTACGAGTCGGACAGAGGTCCTATCATTGAGGACCTGAACGCCTACGGCGACATTTTTGGCAGCAGTGTAGACCACGCCTACGAGACCGTTGAGCTCGCTACCAACAATCAAACCGCGCTGATCCGCTCCGGAAATGGTCTCGGTGGCTCTACTCTAGTGAATGGTGGCACCTGGACTCGCCCCCACAAGGCACAGGTTGATTCTTGGGAGACTGTCTTTGGAAATGAGGGCTGGAACTGGGACAATGTGGCCGCCTACTCCCTCCAGGCTGAGCGTGCTCGCGCACCAAATGCCAAACAGATCGCTGCTGGCCACTACTTCAACGCATCCTGTCATGGTACCAATGGTACTGTCCATGCCGGACCCCGTGACACCGGCGATGACTATTCCCCCATCGTCAAGGCTCTCATGAGCGCTGTCGAAGACCGGGGCGTTCCCACCAAGAAGGACTTCGGATGCGGTGACCCTCATGGTGTGTCCATGTTCCCCAACACCTTGCACGAAGACCAAGTTCGCTCCGATGCCGCTCGCGAATGGCTCCTTCCCAACTACCAACGTCCCAACCTGCAAGTCCTGACCGGACAATATGTTGGTAAGGTGCTCCTTAGCCAGAACGGCACCACCCCTCGTGCCGTCGGCGTGGAATTCGGCACCCACAAGGGCAACACCCACAACGTTTACGCTAAGCACGAGGTCCTCCTGGCTGCTGGCTCGGCTGTCTCTCCCACCATCCTCGAATATTCCGGTATCGGAATGAAGTCCATCCTGGAACCCCTTGGTATCGACACCGTCGTTGACCTGCCCGTCGGCCTGAACCTGCAGGACCAGACCACCGCTACCGTCCGCTCCCGCATCACCTCTGCTGGTGCCGGACAGGGACAGGCCGCTTGGTTCGCCACCTTCAACGAGACCTTTGGTGACTATGCCGAAAAGGCACACGAGCTGCTCAACACCAAGCTGGAGCAGTGGGCCGAAGAGGCCGTCGCCCGTGGCGGATTCCACAACACCACCGCCTTGCTCATCCAGTACGAGAACTATCGCGACTGGATTGTCAATCACAACGTCGCGTACTCGGAACTCTTCCTCGACACTGCCGGAGTGGCCAGCTTCGATGTGTGGGACCTTCTGCCCTTCACGAGAGGATACGTCCACATCCTCGACAAGGACCCCTACCTCCACCACTTTGCCTACGACCCTCAGTACTTCCTCAACGAGCTCGACCTGCTCGGTCAGGCTGCCGCTACTCAGCTGGCCCGTAACATCTCCAACTCCGGTGCTATGCAGACCTACTTCGCTGGCGAGACTATCCCCGGTGATAACCTCGCGTATGATGCCGATTTGAGCGCCTGGACTGAGTACATCCCGTACCACTTCCGTCCTAACTACCATGGCGTGGGTACTTGCTCCATGATGCCGAAGGAGATGGGCGGTGTTGTCGATAATGCTGCCCGTGTGTACGGTGTGCAGGGACTGCGTGTCATTGATGGTTCTATTCCCCCTACGCAGATGTCGTCCCATGTCATGACTGTGTTCTACGCCATGGCGTTGAAGATTGCGGATGCTATTTTGGAGGACTACGCTTCTATGCAG
Although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the embodiments disclosed.
Claims (10)
1. The fusion protein for improving the catalytic efficiency of glucose oxidase is characterized in that: the fusion protein comprises glucose oxidase, vitreoscilla hemoglobin and a section of flexible linked polypeptide.
2. The fusion protein for improving catalytic efficiency of glucose oxidase according to claim 1, characterized in that: the glucose oxidase, the vitreoscilla hemoglobin and a section of flexible linked polypeptide need to construct fusion protein according to a certain linking sequence, and the certain linking sequence is that the two ends of the flexible linked polypeptide are respectively linked with the C end of an amino acid sequence of the vitreoscilla hemoglobin and the N end of an amino acid sequence of the glucose oxidase.
3. The fusion protein for improving catalytic efficiency of glucose oxidase according to claim 1, characterized in that: the flexible linked polypeptide is composed of polypeptide rich in glycine and serine, the total content of glycine and serine in the flexible linked polypeptide is not less than 60%, and the number of amino acids in the flexible linked polypeptide is 4-15.
4. The fusion protein for improving catalytic efficiency of glucose oxidase according to any of claims 1 to 3, characterized in that: the amino acid sequence of the glucose oxidase is shown as SEQ ID No.1, the amino acid sequence of the vitreoscilla hemoglobin is shown as SEQ ID No.2, the amino acid sequence of the flexible link polypeptide is shown as SEQ ID No.3, and the amino acid sequence of the fusion protein is shown as SEQ ID No. 4.
5. A method of increasing the catalytic efficiency of glucose oxidase using the fusion protein of any of claims 1 to 4, characterized in that: the method improves the catalytic efficiency of glucose oxidase by fusion protein.
6. A gene encoding the fusion protein according to any one of claims 1 to 4, wherein: the nucleotide sequence is shown in SEQ ID NO.5.
7. A recombinant expression vector comprising a gene encoding the fusion protein of claim 6.
8. A recombinant strain comprising a gene encoding the fusion protein of claim 6.
9. The recombinant strain of claim 8, wherein: the recombinant strain is recombinant Aspergillus niger.
10. Use of the fusion protein of any one of claims 1 to 4 for increasing the catalytic efficiency of glucose oxidase.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211082946.8A CN115975966A (en) | 2022-09-06 | 2022-09-06 | Fusion protein for improving catalytic efficiency of glucose oxidase, method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211082946.8A CN115975966A (en) | 2022-09-06 | 2022-09-06 | Fusion protein for improving catalytic efficiency of glucose oxidase, method and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115975966A true CN115975966A (en) | 2023-04-18 |
Family
ID=85958703
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211082946.8A Pending CN115975966A (en) | 2022-09-06 | 2022-09-06 | Fusion protein for improving catalytic efficiency of glucose oxidase, method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115975966A (en) |
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2022
- 2022-09-06 CN CN202211082946.8A patent/CN115975966A/en active Pending
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