Genetically engineered bacterium for producing glucose oxidase as well as construction and application thereof
A technology of glucose oxidase and genetically engineered bacteria, which is applied in the direction of oxidoreductase, fungi, and microbial-based methods, can solve the problems of high cost, complicated separation and extraction, and achieve the effect of reducing production costs
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Embodiment 1
[0020] Construction and Identification of Embodiment 1 Recombinant Bacteria
[0021] According to the gene of Aspergillus niger CCTCC NO: M2011291 screened and preserved in our laboratory, primers were designed to obtain the GOD gene, and the purified GOD gene and vector pPIC9K were digested with restriction endonucleases SnaB I and Not I, and T4DNA The ligase was ligated overnight at 16°C, and the ligated product was transformed into the host strain JM109 by chemical transformation method, and the transformed bacteria liquid was spread on the LB plate containing kanamycin (30 mg / mL), cultured overnight at 37°C, and GOD-F and GOD-R was used as a primer to carry out colony PCR to identify positive clones, and finally the recombinant expression plasmid pPIC9K-GOD containing GOD gene was obtained, which was verified by double enzyme digestion.
[0022] The recombinant plasmid pPIC9K-GOD was electroporated to transform Pichia pastoris GS115 competent cells to obtain genetically en...
Embodiment 2
[0024] Enzyme activity assay and protein electrophoresis of embodiment 2 recombinant bacteria
[0025] Use the yeast engineered bacteria obtained in Example 1 as the production strain, after activation, cultivate it to OD at 30°C and 200rpm 600 The seeds between 1.6-1.7 are transferred to the basic fermentation medium with 2% inoculum, and cultured at 30°C and 200rpm; when the OD value is 1.2-1.5, the yeast cells are transferred to BMMY medium to induce protein generation.
[0026] Medium: YPD medium (1L) for seed and slant medium: tryptone 20g, yeast extract 10g, glucose 20g; slant medium with agar 20g; basic fermentation medium is BMGY medium (1L): tryptone 20g, yeast extract 10g, glycerin 10mL, YNB 13.4g, 100mM phosphate buffer (pH 6.0); induction medium is BMMY medium (1L): tryptone 20g, yeast extract 10g, methanol 8mL, YNB 13.4 g, 100mM phosphate buffer (pH 6.0);
[0027] A protein band with a molecular weight of about 68kDa was obtained by protein electrophoresis (SDS...
Embodiment 3
[0028] The enzymatic property research of embodiment 3 recombinant protein
[0029] The enzymatic activity of the recombinant GOD glucose oxidase at different temperatures and the thermostability at a gradient temperature of 20-80°C were determined. The results show that the optimal temperature of recombinant GOD is 40°C, and it has good thermal stability at 20-60°C. The recombinant GOD is incubated at 60°C for 3h, and the enzyme activity can still reach 80%. After 3 hours of incubation, more than 98% of the enzyme activity remains, and after exceeding 80°C, the enzyme activity is seriously lost ( figure 2 ).
[0030] The pH mainly affects the activity of the enzyme by changing the spatial structure of the enzyme and affecting the dissociation of the active center group of the enzyme molecule. The activity of the recombinant GOD glucose oxidase was tested at its optimum temperature and in a buffer system with pH 2.0-10.0 to determine its optimum pH. The results showed that...
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