| Glucose oxidase (β-D-glucose:oxygen 1-oxidoreductase; EC 1.1.2.3.4) turns β-D-glucose into gluconic acid, using molecular oxygen as electron acceptor with a production of hydrogen peroxide at the same time. Due to its wide applications in pharmaceutical, food, chemical, beverage, biotechnology, clinical chemistry and other industries, microbial glucose oxidase is receiving more and more attention. New applications of glucose oxidase in biosensors have increased the demand in recent years. Applications of glucose oxidase in many industries and as analytical enzymes are having an increasing impact on bioprocessing.In this research, GOD gene from A. niger Z-25 is successfully expressed in Pichia yeast GS115, with a flask enzyme activity of 17.35 U·mL-1.However, the limit of expression of GOD from A. niger was found, and it is not likely to raise the enzyme activity just by selecting Mut+ kind of strain, nor by raise the copy number of the genes. Also, after many researchers trying to modify GOD in a rational way, new methods need to be found and bring into reality.As a result, irrational methods are being applied for further development of GOD activity. And the process is three folds as followed.Firstly, single-gene shuffling of GOD-A, expressed in yeast, after screen, 7d flask fermentation shows activity of 32 U · mL-1,3L fermentation show activity of 767 U·mL-1. Known as Generation I.Secondly, bring in GOD-P, creating chimeric gene with GOD-A and GOD-P, expressed in yeast, after screen,7d flask fermentation shows activity of 41 U·mL-1,3L fermentation show activity of 927 U·mL-1. Known as Generation Ⅱ, P. pastoris-GS115-pPIC9K-GOD(SFL-A+P).Last, modification of strains, using gTME, bring mutation into TATA box, raising the protein concentration, receiving strain P. pastoris-GS115-pPIC9K-GOD(SFL)-13-pPICZaA-△a-TBP,3d flask fermentation shows activity of 24 U·mL-1,33% higher than control of 18 U·mL-1.The result shows the mutants have rather good genetical stability, enzyme activity remain stable after generation.The qualitatively measure of copy number of gene imply that modified strain has no difference compared with original ones containing gene copy number of 7-12.As for thermal stability, one strain showing remained activity at 2.56% was screened out,38.38% higher than original strain. |
