Study On The Expression Of Aspergillus Niger Glucose Oxidase In Pichia Pastoris And Its High-efficiency Fermentation

Glucose oxidase is a group of enzymes that could catalyzeβ-D-glucose to produce glucono-δ-lactone,and has broad application prospects in the production of food,medicine,and biosensors.In this study,the coding sequence of glucose oxidase was cloned from Aspergillus niger,and the constitutive and inducible expression of glucose oxidase were achieved in Pichia pastoris,and its enzymatic properties and fermentation conditions were analyzed.The results showed that the coding gene of Aspergillus niger glucose oxidase was1818 bp,encoding 605 amino acids with a signal peptide composed of 21 amino acids.The enzyme activity of glucose oxidase in the fermentation supernatant of Pichia pastoris constitutively expressing glucose oxidase can reach 1.2 U/mL.The purified glucose oxidase by nickel ion affinity chromatography showed a molecular weight of about 100kDa was obtained.After removing glycosylation of glucose oxidase by endoglycosidase H(Endo H),its molecular weight was about 70 kDa,which is similar to its estimated molecular weight.The study of its enzymatic properties showed that the optimal reaction pH of the enzyme was 6.0,the optimal reaction temperature was 35°C,and the specific activity was 60.9 U/mg.In order to further increase the expression level of glucose oxidase in Pichia pastoris,an inducible expression of Pichia pastoris strain was constructed and a highly expressing strain was obtained through geneticin screening.The high-density fermentation of this strain(200 g wet cells/L and 300 g wet cells/L)showed that the optimal methanol addition amount was 2.5%,and the protein concentration in the fermentation broth was about 1.9 g/L and 2.0 g/L by the 7th day of fermentation.On this basis,we further investigated the effect of gene copy number on protein expression.We used Quantitative Real-time PCR technique to construct plasmids p MD18-T-GAPDH and pPIC9K-GOX respectively,using the highly conserved gene of glyceraldehyde-3-phosphate dehydrogenase(GAPDH)in Pichia pastoris as the internal reference gene,and constructed GAPDH and GOX double standard curves by RT-PCR,and their correlation coefficients(R~2)were 0.999 and 0.996,and the amplification efficiencies were 108.802%and 88.328%,respectively,indicating that the double standard curves had good reproducibility.Then the whole genome containing GOX was extracted from the recombinant yeast for RT-PCR,and the copy number of GOX in recombinant yeast was calculated by the double standard curve.The results showed that the four strains screened by0.25、0.5、1、2、4、8、16 mg/mL concentrations of geneticin plates contained 1-2,2-3,3-4and 13-14 copies of glucose oxidase genes,respectively.The fermentation culture of these four strains revealed that the protein expression was positive correlation to the number of copies of glucose oxidase gene integrated into the whole genome of Pichia pastoris.We then performed directed evolution of glucose oxidase,constructed a random mutation library by error-prone PCR and established a high-throughput liquid screening system,and used this screening system to screen out two recombinant strains with significantly improved thermal stability,thus verifying the usefulness of this system.This study provides an experimental basis for the scale-up production and research of Aspergillus niger glucose oxidase and the subsequent directed evolution of glucose oxidase.