Improved Thermostability And High Expression Of Glucose Oxidase From Aspergillus Niger

Glucose oxidase（EC 1.1.3.4,GOD）is a flavin protein,which can catalyze the oxidation ofβ-D-glucose to gluconolactone and H₂O₂.GOD has been applied in many fields,such as medical diagnosis,food,feed industry,textile and so on.In nature,many fungi,bacteria,animals and plants can produce GOD.Penicillium and Aspergillus Niger are the most common strains which are used to produce GOD in industry.At present,there are still some problems in the application of GOD,such as poor stability and low catalytic efficiency.When Penicillium and Aspergillus Niger are used to fermentation,the low yield and fermentation by-products limit the large-scale production of GOD.In this study,Pichia pastoris was used to construct genetic engineering bacteria for producing GOD,and the thermostability and catalytic efficiency of GOD were improved by genetic engineering methods.Finally,gene doses were tried to increased to improve the yield of GOD.（1）The glucose oxidase genes were heterologously expressed in P.pastoris.Firstly,the GOD genes from Penicillium（oppn GOD）and Aspergillus niger（opan GOD）were selected and inserted into p PICZαA expression vector respectively.Then,they were transformed into P.pastoris KM71H and P.pastoris X33 and induced to express.By shake flask analysis,the specific enzyme activities of GOD from Aspergillus niger and Penicillium expressed in P.pastoris KM71H were 2.20-fold and 1.25-fold higher than those of P.pastoris X33,respectively When expressed in P.pastoris KM71H or P.pastoris X33,the specific enzyme activity of the A.niger GOD was 4.85-fold or 2.60-fold higher than that of the Penicillium GOD,respectively.The recombinant strain KM71H-p PICZαA-opan GOD was selected for3-L fermentor fermentation.It was found that the strain did not use methanol during induction fermentation,which affected the yield of GOD.To solve the problem,the expression vector and host strain were replaced with p PIC9K and P.pastoris GS115,and the GOD gene from A.niger was selected for subsequent expression.The specific enzyme activity was similar to that of the recombinant strain KM71H-p PICZαA-opan GOD by shake flask analysis.After amplification fermentation,there is no phenomenon of not using methanol.After 120 days of fermentation,the enzyme activity and protein content were 5.99-fold and 14.77-fold higher than those in shake flask respectively.（2）Enhanced of thermostability and catalytic efficiency of glucose oxidase.Firstly,four primary mutants were designed on the basis of wild GOD by bioinformatics analysis and sequence alignment,and then the mutant GODm was constructed by combinatorial design,and the enzyme properties were expressed,purified and studied by P.pastoris.The enzyme properties showed that the specific enzyme activity of godm was 2.10-fold higher than that of the wild type,and the value of(k_cat/K_m)was increased by 1.45-fold.The enzyme activity of GODm remained 37.5%after treated at 55℃for 3 h,and the t_1/2of GODm at 55℃and65℃was 2.28-fold and 3.36-fold higher than that of the wild type.After analyzing the protein tertiary structure of mutant GODm,it was found that the mutation site T30V formed a new hydrogen bond with FAD and enhanced the hydrophobic interaction,D70K and D315K optimized the surface electrostatic interaction,and A162T may improve the transfer efficiency of the electron transfer chain,thus a mutant with thermostability and catalytic efficiency improved simultaneously was obtained.（3）High level expression of glucose oxidase.Three endogenous signal peptides COL1P+SP（pro）、O1-α-factor、α-factor（DSGRG）which are from Saccharomyces cerevisiae were selected,after construction and expression,it was found that the yield of GOD of the strains with signal peptide replacement were significantly lower than that of the original strain,and most of GOD remained in the cell,indicating that the replacement of signal peptide in this study did not solve the problem of low yield of GOD.Then the method of increasing the gene doses was tried to improve the yield of GOD,in this research,Crispr-cas9 was used to delete the gene of the antibiotic which has been integrated into the genome of P.pastoris,and then integrating GOD gene again into the gene of pastoris genome,integrating 1,2 and 3times of GOD gene respectively.After transformation and screening,it was found that the activity of the strain integrating 3 times of GOD gene was 4.19-fold higher than that of the strains integrating 1 time of GOD gene,which improved the yield of GOD.