| Glucose oxidase(GOD)is an aerobic dehydrogenase that can convert glucose into gluconic acid in the presence of oxygen and at the same time produce hydrogen peroxide.Microbial glucose oxidase is mainly derived from molds such as Aspergillus and Penicillium,and has a large potential for widely application in the fields of medicine,chemical industry,and food.Since wild fungi have low level of glucose oxidase enzyme activity,poor stability and many by-products,they are difficult to separate and purify and can not meet the market’s requirements of glucose oxidase.There is an urgent need to mine high-performance glucose oxidase to achieve efficient secretion expression by using genetic engineering technology.To this end,this research conduct in-depth research on glucose oxidase gene mining,heterologous expression system screening,signal peptide optimization and fermentation technology,and preliminary studies on enzyme separation and purification,enzymatic properties,and application in glucose biosensors are carried out.The main contents are as follows:(1)Mining and heterologous expression of glucose oxidase.BLAST search and analysis the amino acid sequence homology and phylogenetic tree by using the GOD nucleotide sequence derived from Penicillium amagasakiense(AF012277.1)as a template.Screened out a nucleotide sequence derives from A.nomius NRRL13137 that has not been reported to be presumed to encode glucose oxidase and named god.The gene is 1818 bp in length and encodes 605 amino acids.They are expressed heterologously in Escherichia coli and Pichia pastoris expression systems.Among them,the inactive inclusion bodies are expressed in E.coli system.The optimal expression plasmid and host were screened in P.pastoris system.The enzyme activity of the strain P.pastoris SMD1168H/pGAPZαA-god is 6.37 U·mL-1.Follow-up related research is carried out with this expression system.(2)Signal peptide adaptability modification and enzyme production fermentation optimization.In order to improve the secretion expression level of glucose oxidase in Pichia pastoris,the signal peptide apre with the highest secretion expression level of enzyme activity is screened by constructing a signal peptide combinatorial library,and its recombinant strain enzyme activity is 14.92 U·mL-1,which is 2.31-time of the control.The signal peptide recombinant strain was further studied on the shake flask level and the fermentation process on the 15 L fermentor.The results show that the optimal culture conditions are as follows:glycerol content 60 g·L-1,YNB content 0.9%,50 mL/500 mL of the liquid volume,6%of the inoculum volume(volume fraction,V/V),initial pH 6.0,25℃ fermentation culture,the maximum enzyme activity of the shake flask reach 28.90 U·mL-1.while in the 15 L fermenter,the mixed feeding strategy of glycerol and sorbitol is adopted,and the mass volume fraction ratio of the two is 5:1.Under the conditions of 25℃ and pH 6.0,the enzyme activity reach 108.93 U·mL-1 at 90 h via scale-up fermentation,which is 3.77-time higher than that of the shake flask.(3)Purification,enzymatic properties and application of glucose oxidase.In order to investigate the enzymatic performance and application potential of the enzyme,the affinity chromatography is used to purify the recombinant glucose oxidase.The recovery rate is 49.94%and the specific enzyme activity is 97.56 U·mg-1.SDS-PAGE analysis confirm that the 66 kDa band was GOD protein,which is consistent with the theoretical value.Enzymatic characteristics research experiments show that the optimum temperature and pH of glucose oxidase are 35℃and 6.0,respectively,which are stable in the range of 35-45℃,and have a relative enzymatic activity of more than 80%in the range of pH 3.0-9.0;metal ions Mn2+,Mg2+ and Na+ can promote the catalytic activity of the enzyme,while Sn2+and PMSF have significant inhibitory effects on the enzyme;the recombinant glucose oxidase is relatively stable to most surfactants.Through the analysis of enzymatic kinetics experiment,Km is 170 mM.This research also carried out the application research of GOD biosensor.When the temperature is 35℃ and pH 7.0,it can show good detection linearity in the range of glucose concentration 1×10-5-3.5×10-3 M.The detection limit is 1×10-6 M,which can be used for clinically accurate diagnosis of diabetes. |
