High Level Expression Of Glucose Oxidase And Its Thermostability Modification

Glucose oxidase(EC 1.1.3.4,GOD)is an aerobic dehydrogenase,it catalyzes the oxidation of ?-D-glucose to gluconic acid using molecular oxygen as an electron acceptor,with the production concomitant of hydrogen peroxide.GOD is widely used in the food,medicine,chemistry,feed and other fields,which is used as food and feed additives,preservatives,and glucose biosensors.However,low productivity and poor thermostability of the GOD reported currently limit these large-scale GOD industrial applications.GOD from P.notatum F4 is selected as a research object in this study.The recombinant GOD strain is modified by genetic engineering means to improve the secretion and expression of GOD.At the same time,GOD molecule is reformed by recombination technology in vitro to increase its thermostability.The main results are listed as follows:1.By co-expressing SEC61 complex and UPR transcriptional activator HAC1 from P.pastoris,foldase PDI and ERO1 from S.cerevisiae,the effects on the extracellular enzyme concentration of heterologous GOD were investigated.Compared to wild-type strain,PP-G-SEC61 had a significantly increase of GOD enzymes,resulting in a 3.24-fold increase.PP-G-PDI and PP-G-ERO1 produced larger total quantities of GOD enzymes,resulting in a 1.95-fold and 1.32-fold greater enzyme yield in shake flasks.Similarly,PP-G-HAC1 enhanced the expression and secretion of GOD effectively,extracellular enzyme activity results in a 1.38-fold increase.2.The three-dimensional structure model of GODp from P.notatum F4 was constructed based on homology modeling and molecular dynamics simulation.In the present study,we performed computer-aided design with key amino acid site-directed saturation mutagenesis to improve the thermostability of GOD.The key factors affecting the thermostability of GODm were analysed at the protein structure level,and two mutants with significantly improved thermostabilities,S100 A and D408 W,were obtained.The half-lives of S100 A and D408 W were 5.13-and 4.41-fold greater,respectively,than that of the wild-type enzyme at 55°C.Their protein denaturing temperatures were enhanced by about 4.4°C and 1.2°C,respectively,compared with the wild-type enzyme.This study,which has good theory and application prospects,started with the most outstanding problems of GOD applications,improving the thermostability and expression efficiency of GOD,and then reducing its cost of production and use effectively.This new type GOD will be further expanded its applications in food and feed industry.