Study On Recombinant Expression And Properties Of Glucose Oxidase In Non-spore Aspergillus Niger

Glucose oxidase?EC 1.1.3.4,GOD?is a FAD-dependent oxidoreductase that catalyzes the oxidation of?-D-glucose into glucolactone and H₂O₂.GOD is widely used in the fodder,graziery,foodstuff,medical science and biotechnology.At present,production volume of GOD enzyme what can be applied to foodstuff is in low level,fermentation by-product is too much and it's unfavorable to purify glucose oxidase,these problems have limited the fermentation product of GOD of food grade.In this work,one of four GOD genes from Aspergillus niger CBS513.88 was choosed,using codon optimization and signal peptide replace to modify GOD CDS,and improve the fermenting enzyme activity.Non-spore A.niger strain SH2 and HL1 which were low-background of secreted proteins without kusA,pyrG genes were used as hosts to expression the GOD gene and the auxotroph marker is pyr G.The specific contents of this study are as follows:?1?Recombinant expression of GOD in host SH2:According to NCBI database,there are 4genes related to GOD expression among Aspergillus niger CBS513.88.Choose the gene goxC though using literature search,BLAST and building a phylogenetic tree by blasting the amino acid sequences,then gox C gene was expressed in A.niger SH2.Screen the highest enzyme activity of transformant SH2-goxC5 by method of o-dianisidine with hydrogen peroxide enzyme,enzyme activity achieved 563.8 U/mL at 168 h in shake fermentation.In the 50L tank fermentation enzyme activity achieved 1128U/mL at 179 h,which increasing more than2 times comparing with shake fermentation.?2?Recombinant expression of GOD in host HL1:To explore the influence of knocking out prtT and a series of amylase of A.niger in utilising fermentation medium.Use the host HL1which had gene prt T to build efficient expression of recombinant strains of GOD.then and screened the highest enzyme activity 635.1 U/mL of transformant HL1-goxC 9.By using 0%⁵%glucose concentrations of shake fermentation and testing,found that 0%glucose concentrations of shaker at 72h reached the highest enzyme activity of 658.3 U/mL,1%glucose concentrations of shaker at 120h reached the second highest enzyme activity of 599.3U/mL,and with the increase of glucose concentration in the culture medium,the number of hours of enzyme activity to maxmum number also increased.?3?Study on enzymatic properties of GOD:The fermentation liquor was purified by affinity chromatography.The purified protein produced an approximately 80 kDa protein and a protein band?approximately 65 kDa?was produced after the deglycosylation,suggesting that the recombinant GOD is a glycoprotein.The optimum temperature and pH of the recombinant GOD was 40°C and5.5.Meanwhile,at the concentration of 10mmol/L,Ni⁺,Ca²⁺,K⁺,Zn²⁺,Mn²⁺and Mg²⁺increase the activity of GOD.EDTA and Fe³⁺had little effect on GOD activity.SDS or Na⁺decreased GOD activity.At the concentration of 100mmol/L,SDS,Ni⁺,K⁺and Mn²⁺increase the activity of GOD.EDTA and Na⁺significantly decreased activity of GOD.Ca²⁺,Zn²⁺,Fe³⁺and Mg²⁺had little effect on GOD activity.