The Directed Evolution Of Glucose Oxidase From Aspergillus Niger Expressed In Pichia Pastoris

Glucose oxidase(GOD;β-D-glucose: oxygen 1-oxidoreductase, EC 1.1.3.4)is a flavoprotein that catalyzes the oxidation of D-glucose by molecular oxygen to glucono-δ-lactone, which subsequently hydrolyzes spontaneously to gluconic acid. GOD activity has been identified from various sources, notably enzymes from Aspergillus niger and Penicillium amagasakiense are of considerable importance. Besides being used in food industry for glucose removal and gluconic acid production, the most frequent application of GOD has been in biosensors, where it is used for the quantitative determination of glucose in the body and in fermentation fluids.Here we describe the cloning and expression of the GOD gene A. niger in Pichia Pastoric, and the directed evolution of GOD toward higher catalytic activity. Through two round of error-prone PCR, several improved variants from about 3000 mutant colonies were obtained by 96-well plate screening procedure. The mutant sites were identified by gene sequencing and catalytic properties of the mutant enzymes were analyzed using a stopped-flow system. Compared with wild type, D401N/A574V and A137S exhibited nearly 3-4 folds higher specific activity. The thermostability analyses of mutants and wild type were determined by incubating enzyme solution at 40℃, 50℃, 60℃, 70℃, respectively, and taking samples at intervals for assay at room temperature. The results showed that the mutant enzymes had similar thermostability to that of wild type. Storage stability of the enzyme at room temperature and 4℃was also measured periodically using a procedure similar to the thermostability assay. After incubated 40 days at 4℃, the activity of wild type GOD dropped to 65％, while the activity of D401N/A574V remained 65％, 80％and 78％of their original activity, respectively. Regarding the storage stability at room temperature, wild type GOD and A137S are very much alike, while D401N/A574V is more stable.In order to understand the mechanism behind higher activity of the mutant GOD, the structure genes of the mutant GOD were sequenced and five activating amino acid substitutions, H57R, S74G, A137S, D401N, and A574Vwere identified. The H57R, S74G and A137S variants were located far away from the center of the catalytic pocket. The variant D401N was located at theα-helix H10, which was the part of substrate-binding domain. The variant A574V was located at theα-helix H13, which was located at FAD-binding domain.