CN115369049B - Genetically engineered bacterium secreting glucose oxidase, construction method and application thereof - Google Patents
Genetically engineered bacterium secreting glucose oxidase, construction method and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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- C—CHEMISTRY; METALLURGY
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
- C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
- C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
- C07K14/395—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Saccharomyces
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
- C12Y101/03004—Glucose oxidase (1.1.3.4)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2800/00—Nucleic acids vectors
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
Abstract
The invention relates to a genetically engineered bacterium secreting glucose oxidase. The engineering bacteria are constructed by taking pichia pastoris as a host cell, introducing a gene for coding Glucose Oxidase (GOX) or a gene for coding Glucose Oxidase (GOX) optimized by a codon, and strengthening and expressing a ubiquitination transport gene of the pichia pastoris, and are genetic engineering bacteria for efficiently secreting the glucose oxidase. The invention also relates to a construction method of the genetically engineered bacterium secreting glucose oxidase. The method enhances the secretion expression level of the glucose oxidase by over-expressing the ubiquitination gene related to the secretion expression, has simple operation, can be applied to the construction of high-yield glucose oxidase strains and the industrialized preparation of the glucose oxidase, and has good market application prospect.
Description
Technical Field
The invention belongs to the technical field of gene recombination, and relates to a genetically engineered bacterium secreting glucose oxidase, and a construction method and application thereof.
Background
In 1928, muller first discovered glucose oxidase from the cell-free extract of Aspergillus niger. The glucose oxidase is fully applied in the seventies of the 20 th century at home and abroad.
Glucose Oxidase (GOX) can be fixed on the surface of an electrode to detect glucose, and is often used for detecting blood sugar in the field of medicine, and is rapid and convenient. Can be used in food field to remove oxygen and protect food from oxidation by oxygen in air, and also can remove glucose in food to avoid Maillard reaction.
In the wine industry, GOX is used for reducing the concentration of alcohol, and can also inhibit the growth of acetic acid bacteria and lactic acid bacteria which cause the deterioration of alcohol, so that the addition of preservative in alcohol can be reduced. Glucose oxidase and catalase can be co-immobilized and can be used for preparing sodium gluconate.
The glucose oxidase is fully applied to the feed for raising animals and is put into chicken feed, so that the survival rate and the feeding rate of broilers are improved. GOX in pig feed can reduce sow perinatal syndrome and improve weaned pig daily gain. GOX eliminates appetite fading in cow and sheep at perinatal period, and promotes digestion and absorption of feed.
The content of glucose oxidase in the animal and plant bodies is low, and huge demands are difficult to meet through an extraction means. Therefore, the glucose oxidase with high purity is produced by adopting a genetic engineering means, separated, extracted and purified, and the existing industrial strains are generally Aspergillus niger and Penicillium.
The glucose oxidase is researched by using a genetic engineering means, and the structure of the enzyme is modified by genes, such as error-prone PCR (polymerase chain reaction) and DNA (deoxyribonucleic acid) shuffle achieves a certain effect, and although theoretical means and technical support are provided for GOX production, the structure is still not satisfactory.
In the existing method for enhancing the secretion efficiency of heterologous proteins by carrying out over-expression enhancement on the secretion pathway of pichia pastoris proteins, the interaction mechanism of related protein secretion pathway factors and the foreign proteins is unclear, and the over-expression of which genes can be judged only by trial and error has the promotion effect on the enzyme activity, so that the operation is complex, the workload is extremely large, and the cost is high.
The heterologous protein adopts a strong promoter in Pichia pastoris, so that the pressure of a transportation path is over high easily, a large amount of target protein cannot be secreted out of cells smoothly, and the method for enhancing the transportation path has extremely important application value.
Thus, there is a problem in that it is necessary to construct a strain producing glucose oxidase at low cost, rapidly and efficiently.
Disclosure of Invention
The invention aims to solve the problem of providing a genetically engineered bacterium for secreting glucose oxidase, which can strengthen the secretion of the glucose oxidase by strengthening a protein transportation and secretion way and has higher enzyme activity.
The invention also provides a construction method of the genetically engineered bacterium for secreting glucose oxidase, which can strengthen a protein transportation and secretion way, so that secretion of the glucose oxidase is enhanced, enzyme activity of the glucose oxidase is improved, and an industrial strain capable of being used for fermenting and synthesizing the glucose oxidase is obtained.
To this end, the first aspect of the present invention provides a genetically engineered bacterium that secretes glucose oxidase, which is a recombinant pichia pastoris that contains a ubiquitinated transporter gene and a gene encoding Glucose Oxidase (GOX) or a codon optimized gene encoding Glucose Oxidase (GOX).
According to the invention, the ubiquitinated transporter gene includes an endogenous pichia ubiquitinated transporter gene, and optionally an exogenous ubiquitinated transporter gene.
In some embodiments of the invention, the exogenous ubiquitinated transporter gene is derived from saccharomyces cerevisiae.
In another embodiment of the invention, the gene encoding Glucose Oxidase (GOX) is derived from Penicillium sp (Penicillium Amagasakiense), genebank accession number AAD01493.1.
According to the invention, the ubiquitinated transporter gene is preferably an endogenous pichia ubiquitinated transporter gene.
In some embodiments of the invention, the endogenous pichia ubiquitination transport gene comprises one or more of the SEC12, SEC13, SEC23, SEC24 and SEC31 genes.
Specifically, the Genebank accession number of the SEC12 gene is AF216960.1;
and/or the Genebank accession number of SEC13 gene is AAB01155.2;
and/or the Genebank accession number of SEC23 gene is CAY67406.1;
and/or the Genebank accession number of SEC24 gene is CAY71741.1;
and/or, the Genebank accession number of the SEC31 gene is CAY68066.1.
According to the invention, the genetically engineered bacterium consists of a ubiquitination transport gene and a Glucose Oxidase (GOX) gene or a codon-optimized gene encoding Glucose Oxidase (GOX) over-expressed in Pichia pastoris GS115 through a vector plasmid.
In some embodiments of the invention, the gene encoding Glucose Oxidase (GOX) or the codon optimized gene encoding Glucose Oxidase (GOX) is integrated into Pichia pastoris by PPIC9K plasmid.
In another embodiment of the present invention, the ubiquitinated transporter gene is integrated into pichia pastoris via PGAPZB plasmid.
In the invention, the pichia pastoris is pichia pastoris GS115.
The second aspect of the present invention provides a method for constructing a genetically engineered bacterium secreting glucose oxidase according to the first aspect of the present invention, comprising:
step A, integrating a gene encoding Glucose Oxidase (GOX) or a gene encoding Glucose Oxidase (GOX) subjected to codon optimization into pichia pastoris through a vector plasmid I to obtain pichia pastoris containing the gene encoding Glucose Oxidase (GOX) or the gene encoding Glucose Oxidase (GOX) subjected to codon optimization;
and B, integrating the ubiquitination transport gene into pichia pastoris containing Glucose Oxidase (GOX) gene or gene which codes for Glucose Oxidase (GOX) through codon optimization through a II-th vector plasmid to obtain genetically engineered bacteria secreting glucose oxidase.
In some embodiments of the invention, the I-th vector plasmid is a PPIC9K plasmid.
In other embodiments of the invention, the vector ii plasmid is a PGAPZB plasmid.
In a third aspect, the invention provides the use of a genetically engineered bacterium according to the first aspect of the invention or a genetically engineered bacterium constructed by a method according to the second aspect of the invention in the fermentative production of Glucose Oxidase (GOX).
In some embodiments of the invention, the application comprises inoculating genetically engineered bacteria that secrete glucose oxidase into a fermentation medium for fermentation culture to obtain glucose oxidase.
In some specific embodiments of the invention, the fermentation culture conditions are: the fermentation temperature is 28 ℃, the inoculation amount is 8%, the rotating speed is 500rpm, the ventilation amount is 1.5vvm, the pH value is 5.5, the culture medium is BMGY culture medium, methanol is added after dissolved oxygen rises, the concentration of the methanol is controlled at 5g/L, the fermentation time is more than or equal to 240h, and the enzyme activity of the glucose oxidase is more than or equal to 1638.96U/mL.
The invention has the beneficial effects that:
(1) The genetic engineering bacteria for secreting glucose oxidase provided by the invention can strengthen the secretion of the glucose oxidase by strengthening the protein transportation and secretion pathway, and the secreted glucose oxidase has higher enzyme activity.
(2) The construction method of the genetically engineered bacterium for secreting glucose oxidase solves the problem that the transfer and secretion channels of glucose oxidase gene expression in pichia pastoris are limited by the intensified expression of pichia pastoris transfer genes.
(3) The invention only needs to express two genes, such as GOX gene and SEC12 gene, has simpler operation and lower application cost, improves the extracellular enzyme activity of glucose oxidase, and can be applied to the construction and application of high-yield glucose oxidase strains.
Drawings
The invention is described in further detail below with reference to the accompanying drawings:
FIG. 1 shows the results of the effect of over-expressed transport genes on biomass of recombinant Pichia strains.
FIG. 2 shows the results of the effect of over-expression of a transport gene on heterologous expression of glucose oxidase by recombinant Pichia strains.
FIG. 3 shows the change in the fermentation biomass and enzyme activity of PPG at 5L.
FIG. 4 shows the change in fermentation biomass and enzyme activity of PPG-SEC12 at 5L.
Detailed Description
In order that the invention may be readily understood, the invention will be described in detail below with reference to the accompanying drawings. Before the present invention is described in detail, it is to be understood that this invention is not limited to particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
Unless defined otherwise, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.
I terminology
The term "genetically engineered bacterium" as used herein refers to a fungus, such as pichia pastoris, that produces a desired protein by introducing a gene of interest into a host organism (i.e., a host cell or a fungus body) for expression. The core technology of genetic engineering is a recombinant technology of DNA, and thus, in the present invention, genetically engineered bacteria are also referred to as recombinant microorganisms.
The term "recombinant" as used herein refers to a transgenic organism constructed by using genetic material of a donor organism or an artificially synthesized gene, cutting the gene by in vitro or ex vivo restriction enzymes, then ligating the gene with a suitable vector to form a recombinant DNA molecule, and introducing the recombinant DNA molecule into a recipient cell or a recipient organism, wherein the organism can exhibit a property of another organism according to a blueprint designed in advance by human.
II. Embodiment
As described above, the prior art has been developed using genetic engineering techniques to improve the yield of glucose oxidase by modifying the structure of the enzyme, and has the disadvantages of complex operation, high workload and high cost; meanwhile, the existing genetically engineered bacteria for secreting glucose oxidase have the problem that the transportation and secretion channels of glucose oxidase gene expression in pichia pastoris are limited. In view of this, the present inventors have made a great deal of research on the construction of genetically engineered bacteria that secrete glucose oxidase, and have succeeded in constructing a strain that produces glucose oxidase at low cost, rapidly and efficiently by enhancing expression of a pichia pastoris ubiquitination transport gene, solving the problem of limited transport and secretion pathways for expression of glucose oxidase genes in pichia pastoris.
Therefore, the invention provides a new way for synthesizing glucose oxidase, which enhances the secretion of the glucose oxidase by enhancing the protein transportation and secretion way, improves the enzyme activity of the glucose oxidase and realizes the efficient synthesis of the glucose oxidase.
In order to achieve the technical scheme, the invention firstly provides a genetically engineered bacterium capable of secreting glucose oxidase, which expresses a gene of a glucose oxidase synthesis path in original or modified fungal cells, and prepares a host capable of synthesizing glucose oxidase; based on the host capable of synthesizing the glucose oxidase, the invention strengthens the ubiquitination transport gene, thereby constructing and obtaining the strain capable of rapidly and efficiently producing the glucose oxidase.
In an embodiment of the first aspect of the present invention, the present invention provides a genetically engineered bacterium that secretes glucose oxidase, which is a recombinant pichia pastoris that contains a ubiquitinated transporter gene and a gene encoding Glucose Oxidase (GOX) or a codon optimized gene encoding Glucose Oxidase (GOX).
According to the invention, the ubiquitinated transporter gene includes an endogenous pichia ubiquitinated transporter gene, and optionally an exogenous ubiquitinated transporter gene.
In some embodiments of the invention, the exogenous ubiquitinated transporter gene is derived from saccharomyces cerevisiae.
In another embodiment of the present invention, the gene encoding Glucose Oxidase (GOX) is derived from Penicillium citrinum (Penicillium Amagasakiense), which has Genebank accession number AAD01493.1, accession number ATCC 332245.
In some preferred embodiments of the invention, the ubiquitinated transporter is an endogenous pichia ubiquitinated transporter comprising one or more of the SEC12, SEC13, SEC23, SEC24, and SEC31 genes; preferably, the endogenous pichia ubiquitination transport gene is selected from the group consisting of SEC12, SEC13, SEC23, SEC24 and SEC31 genes; further preferably, the endogenous pichia ubiquitination transport gene is a SEC12 gene.
In the invention, the nucleotide sequence of the SEC12 gene is shown as Genebank: AF216960.1; the nucleotide sequence of the SEC13 gene is shown as Genebank AAB01155.2; the nucleotide sequence of the SEC23 gene is shown as genebank:CAY 67406.1; the nucleotide sequence of the SEC24 gene is shown as genebank:CAY 71741.1; the nucleotide sequence of the SEC31 gene is shown as genebank:CAY 68066.1.
According to the invention, the genetically engineered bacterium consists of a ubiquitination transport gene and a Glucose Oxidase (GOX) gene or a codon-optimized gene encoding Glucose Oxidase (GOX) over-expressed in Pichia pastoris GS115 through a vector plasmid.
In some embodiments of the invention, the gene encoding Glucose Oxidase (GOX) or the codon optimized gene encoding Glucose Oxidase (GOX) is integrated into Pichia pastoris by PPIC9K plasmid.
In another embodiment of the present invention, the ubiquitinated transporter gene is integrated into pichia pastoris via PGAPZB plasmid.
In some particularly preferred embodiments of the present invention, the genetically engineered bacterium is composed of a codon optimized gene encoding Glucose Oxidase (GOX) integrated into and overexpressed in Pichia pastoris via PPIC9K plasmid, and a gene encoding Glucose Oxidase (GOX) integrated into and overexpressed in Pichia pastoris via PGAPZB plasmid.
In the invention, the nucleotide sequence of the gene encoding Glucose Oxidase (GOX) subjected to codon optimization is shown in SEQ No. 1.
In the invention, the pichia is pichia GS115, and the strain deposit number is ATCC20864 (Bai Oribo Bo).
In a second aspect, the present invention provides a method for constructing a genetically engineered bacterium for secretion of glucose oxidase according to the first aspect of the present invention, which can be understood as a method for enhancing secretion of glucose oxidase by enhancing a protein transport secretion pathway, comprising:
step A, integrating a gene encoding Glucose Oxidase (GOX) or a gene encoding Glucose Oxidase (GOX) subjected to codon optimization into pichia pastoris through a vector plasmid I (PPIC 9K plasmid) to obtain pichia pastoris containing the gene encoding Glucose Oxidase (GOX) or the gene encoding Glucose Oxidase (GOX) subjected to codon optimization;
and B, integrating the ubiquitination transport gene into pichia pastoris containing Glucose Oxidase (GOX) genes or genes encoding the Glucose Oxidase (GOX) through a II-th vector plasmid (PGAPZB plasmid), and obtaining genetically engineered bacteria secreting the glucose oxidase.
In some specific embodiments of the present invention, a method for enhancing glucose oxidase secretion by enhancing expression of a pichia pastoris transport gene and thereby constructing a genetically engineered bacterium that efficiently secretes glucose oxidase comprises the steps of:
(1) The PPIC9K plasmid is adopted to transform a gene which codes Glucose Oxidase (GOX) and is subjected to codon optimization into pichia pastoris GS115, so that pichia pastoris GS115 containing the gene of Glucose Oxidase (GOX) is obtained;
(2) The genetic recombination technology is adopted to clone the ubiquitination transport gene of the pichia pastoris onto a common carrier PGAPZB of the pichia pastoris, and the ubiquitination transport gene is co-expressed in pichia pastoris GS115 containing a Glucose Oxidase (GOX) gene.
(3) Finally screening and identifying to obtain a high secretion expression glucose oxidase strain, wherein the enzyme activity in a 5L fermentation tank is 1638.96U/mL.
Specifically, the invention adopts Gibson self-assembly technology to connect, firstly, primers are used for respectively PCR to obtain genes and vectors, and the genes, the vectors and the Gibbsen enzyme are recovered after gel running and gel recovery according to a:5-a:5 (microliter) of the mixture is added, the temperature is 50 ℃ for 45min, the mixture is transformed into commercial competent cells trans10, colony PCR (polymerase chain reaction) verification sequencing is carried out, and the AVRII linearization plasmid is electrically transformed into PPG competent cells after the sequencing is correct; the strains and plasmids constructed according to the invention are shown in Table 1, and the primers used for the plasmids constructed according to the invention are shown in Table 2.
TABLE 1 construction of strains and plasmids according to the invention
TABLE 2 primers for plasmids constructed according to the invention
The method enhances the secretion expression level of the glucose oxidase by over-expressing the ubiquitination gene related to the secretion expression, is simple to operate, can be applied to the construction of high-yield glucose oxidase strains and the industrialized preparation of the glucose oxidase, and has good market application prospect.
In a third aspect, the invention provides the use of a genetically engineered bacterium according to the first aspect of the invention or a genetically engineered bacterium constructed by a method according to the second aspect of the invention in the fermentative production of Glucose Oxidase (GOX).
In some embodiments of the invention, the application comprises inoculating genetically engineered bacteria that secrete glucose oxidase into a fermentation medium for fermentation culture to obtain glucose oxidase.
In some specific embodiments of the invention, the fermentation culture is performed in a 5L fermenter under the following conditions: the fermentation temperature is 28 ℃, the inoculation amount is 8%, the rotating speed is 500rpm, the ventilation amount is 1.5vvm, the pH is adjusted to 5.5 by 3mol/L phosphoric acid and 28% ammonia water (v/v), the culture medium is BMGY culture medium (glycerol is 40 g/L), methanol is added after dissolved oxygen rises, the concentration of the methanol is controlled to be 5g/L by an FC2002 type methanol detection fed-batch controller, and the enzyme activity is measured after fermentation for 240 hours; preferably, the enzyme activity of the glucose oxidase is more than or equal to 1638.96U/mL.
In the invention, a shake flask culture medium for amplifying biomass of bacteria is a BMGY culture medium, a shake flask culture medium for inducing and synthesizing glucose oxidase is a BMMY culture medium, a seed culture medium for the upper tank is a YPD culture medium, and a fermentation culture medium is a BMGY culture medium.
YPD medium (1L) used in the fermentation process of the present invention: yeast powder 10g, peptone 20g and glucose 20g; BMGY Medium (1L): yeast powder 10g, peptone 20g, glycerin 40g, YNB13.4g,100mM phosphate buffer solution (pH 6.0).
The method for measuring the enzyme activity of the glucose oxidase comprises the following steps:
(1) 0.1mol/L sodium phosphate buffer solution with pH=6, 180g/L glucose solution, 2mol/L sulfuric acid solution, 100U/mL horseradish peroxidase solution, 1g o-dianisidine dissolved and fixed in 100mL methanol to obtain o-dianisidine methanol solution, and 1mL o-dianisidine methanol solution mixed and fixed to 100mL sodium phosphate buffer solution with pH=6 to obtain o-dianisidine buffer solution.
(2) Measuring standard curve, preparing 10 groups of glucose oxidase standard solutions with different concentrations, adding o-dianisidine buffer solution, glucose solution and horseradish peroxidase solution into a 10mL centrifuge tube, finally mixing glucose oxidase standard solutions with accurate volumes of 2.5mL, 0.3mL, 0.1mL and 0.1mL respectively,after the reaction is finished for 3min, the absorbance value OD is measured by an enzyme label instrument after the sulfuric acid solution is reacted 500 The average was taken three times in duplicate and used as a standard curve.
(3) And (3) multiplying the absorbance OD500 corresponding to the supernatant by the corresponding enzyme activity concentration corresponding to the standard curve to obtain the corresponding enzyme activity.
III, examples
In order that the invention may be more readily understood, the invention will be described in detail below with reference to the following examples and the accompanying drawings, which are given by way of illustration only and are not limiting of the scope of application of the invention. Reagents or materials used in the present invention may be purchased commercially or prepared by conventional methods unless otherwise specified, and specific experimental methods not mentioned in the following examples are generally performed according to conventional experimental methods.
Example 1: construction of recombinant strains
The glucose oxidase gene from Penicillium Amagasakiense is connected to PPIC9K plasmid after codon optimization, 2.0mg/mL G418 antibiotics are adopted for screening, high-yield strain PPG is obtained as chassis cells, then endogenous genes of SEC12, SEC13, SEC23, SEC24 and SEC31 in Pichia pastoris are respectively connected to plasmid pGAPZB by adopting a gene recombination technology, and the constructed plasmid pGAPZB is linearized to integrate ubiquitinated genes SEC12, SEC13, SEC23, SEC24 and SEC31 into the high-yield strain PPG respectively, so that PPG-SEC12, PPG-SEC13, PPG-SEC23, PPG-SEC24 and PPG-SEC31 recombinant strains are obtained.
Example 2: shake flask fermentation of different recombinant strains
The recombinant strain was cultured at 28℃and 200rpm until OD600 was 1% of the inoculum size and transferred to 50mL of the natural medium BMGY at 28℃and 200rpm, 24h was allowed to incubate at 3000rpm,3min was allowed to centrifuge at 4℃to empty the supernatant, and the whole cell was transferred to 100mL of BMMY medium, and 1mL of methanol was added every 24h to induce glucose oxidase synthesis. As shown in FIG. 1, the growth trend of the PPG-SEC12, PPG-SEC13, PPG-SEC23, PPG-SEC24, PPG-SEC31 strains was substantially the same as that of the control strain PPG, the final fermentation 168h, the biomass OD600 of the PPG, PPG-SEC12, PPG-SEC13, PPG-SEC23, PPG-SEC24 and PPG-SEC31 strains was 37.9, 39.1, 40.9, 38.4, 40.2 and 39.5, respectively, indicating that overexpression of vesicle-related ubiquitination transport factors SEC12, SEC13, SEC23, SEC24, SEC31 did not affect the normal growth of recombinant Pichia cells. As to the trend of the enzyme activity, the enzyme activities of the PPG, PPG-SEC12, PPG-SEC13, PPG-SEC23, PPG-SEC24 and PPG-SEC31 strains were 118.86U/mL, 154.23U/mL, 129.21U/mL, 137.14U/mL, 131.15U/mL and 128.17U/mL, respectively, when the fermentation was carried out for 168 hours, as shown in FIG. 2. The increased enzyme activities of the PPG-SEC12, PPG-SEC13, PPG-SEC23, PPG-SEC24, PPG-SEC31 strains compared to the control strain PPG, by 29.76%, 8.71%, 15.4%, 10.3%, 7.8%, respectively, indicate that overexpression of the vesicle-associated transport factors SEC12, SEC13, SEC23, SEC24, SEC31 increases the enzyme activity of the pichia pastoris producing glucose oxidase, and that SEC12 ubiquitination transport factor is preferred when the vesicle-associated ubiquitination transport factor and glucose oxidase genes are co-expressed.
Example 3: 5L tank fermentation of different recombinant strains
A method for producing glucose oxidase using said strain in a 5L fermenter comprising the steps of: activating recombinant Pichia pastoris strain in a 4mL YPD test tube, inoculating 1% into a 100mL YPD liquid culture medium shake flask (two flasks), mixing with 2.3L BMGY culture medium according to the inoculum size of 8%, maintaining the temperature at 28 ℃, stirring at 500rpm, ventilating at 1.5vvm, regulating pH to 5.5 with 3mol/L phosphoric acid and 28% ammonia water, enriching thallus with 40g/L glycerol in the early stage, adding methanol after dissolved oxygen rises, and controlling the concentration of methanol to 5g/L by an FC2002 type methanol detection fed-batch controller. The change of the PPG at 5L fermentation biomass and the enzyme activity is shown in figure 3, the change of the PPG-SEC12 at 5L fermentation biomass and the enzyme activity is shown in figure 4, and as can be seen from figures 3 and 4, the PPG strain is fermented in a 5L tank for 24 hours, the biomass is 58.5, methanol is induced, the biomass is 291.8, the enzyme activity is 1249.79U/mL, the PPG-SEC12 strain is 58.4, the biomass is 24 hours, the recombinant strain is induced by methanol and heterologously expresses glucose oxidase, the fermentation is 240 hours, the biomass is 289.2, the enzyme activity is 1638.96U/mL, compared with the PPG strain, the biomass is basically unchanged after the SEC12 is overexpressed, and the enzyme activity is increased by 31.1%.
It should be noted that the above-described embodiments are only for illustrating and explaining the present invention, and do not limit the present invention in any way. It is understood that the words which have been used in the embodiments are words of description and illustration, rather than words of limitation. Modifications may be made to the invention as defined in the appended claims, and the invention may be modified without departing from the scope and spirit of the invention. While all such modifications are intended to be included within the scope of the claims, and the invention as described herein is not intended to be limited to the particular embodiments disclosed herein, although the invention is described herein with reference to particular methods, materials and embodiments; rather, the invention extends to all other methods and applications having the same functionality.
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Claims (7)
1. A genetically engineered bacterium that secretes glucose oxidase, which is a recombinant pichia pastoris that expresses a ubiquitination transport gene and a codon optimized gene encoding Glucose Oxidase (GOX);
the nucleotide sequence of the gene for coding Glucose Oxidase (GOX) subjected to codon optimization is shown in SEQ ID NO. 1;
the ubiquitination transport gene is an endogenous pichia ubiquitination transport gene, the endogenous pichia ubiquitination transport gene is a SEC12 gene, and the Genebank accession number is AF216960.1;
the pichia pastoris is pichia pastoris GS115.
2. The genetically engineered bacterium of claim 1, wherein the genetically engineered bacterium consists of a ubiquitinated transporter gene and a codon-optimized gene encoding Glucose Oxidase (GOX) over-expressed in pichia pastoris GS115 via a vector plasmid.
3. Genetically engineered bacterium according to claim 2, wherein the codon-optimized gene encoding Glucose Oxidase (GOX) is integrated into pichia pastoris by PPIC9K plasmid; and/or, the ubiquitinated transporter gene is integrated into pichia pastoris by PGAPZB plasmid.
4. The method for constructing a genetically engineered bacterium which secretes glucose oxidase as claimed in any one of claims 1 to 3, comprising:
step A, integrating a gene encoding Glucose Oxidase (GOX) subjected to codon optimization into pichia pastoris through a vector plasmid I to obtain pichia pastoris containing the gene encoding Glucose Oxidase (GOX) subjected to codon optimization;
and B, integrating the ubiquitination transport gene into pichia pastoris containing a gene which codes for Glucose Oxidase (GOX) through codon optimization through a II vector plasmid to obtain genetically engineered bacteria which secrete the glucose oxidase.
5. The method of claim 4, wherein the vector plasmid I is PPIC 9K; and/or, the II vector plasmid is PGAPZB plasmid.
6. Use of a genetically engineered bacterium according to any one of claims 1-3 or a genetically engineered bacterium constructed according to the method of claim 4 or 5 for the fermentative production of Glucose Oxidase (GOX); the application comprises that genetic engineering bacteria secreting glucose oxidase are inoculated into a fermentation culture medium for fermentation culture, and the glucose oxidase is obtained.
7. The use according to claim 6, wherein the fermentation culture conditions are: the fermentation temperature is 28 ℃, the inoculation amount is 8%, the rotating speed is 500rpm, the ventilation amount is 1.5vvm, the pH value is 5.5, the culture medium is BMGY culture medium, methanol is added after dissolved oxygen rises, the concentration of the methanol is controlled at 5g/L, the fermentation time is more than or equal to 240h, and the enzyme activity of the glucose oxidase is more than or equal to 1638.96U/mL.
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