The Study of Glucose Oxidase Fuses with Vitreoscilla Hemoglobin(VHb-GOX) Expression in Methylotrophic Yeast Pichia pastoris

• Main Authors: Chen-hsuan Lee, 李承軒
• Other Authors: C. K. Lee
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/ne552d

碩士 === 國立臺灣科技大學 === 化學工程系 === 94 === The glucose oxidase（GOX）from Aspergillus niger is a glycoprotein. it is widely use in food industry and diagnosis kit for glucose because it catalyzes the glucose oxidation with O2. The methylotrophic yeast Pichia pastoris can been used to extracellularly express GOX. Vitreoscilla Hemoglobin（VHb）from Vitreoscilla sp. has been demonstrated that it can enhance oxygen uptake and cell growth when expressed intracellularly in most microorganisms. In this study, VHb was fused with GOX（VGOX） in order to take advantage of O2 carrying ability of VHb so that GOX activity enhancement can be expected. The extracellular expression of fusion protein VGOX in P. pastoris GS115 was under to control of methanol induced AOX1 promoter. Casamino acid（CA）was found to be the crucial nutrition factor that determined the expressed GOX activity in the culture of VGOX expressing strain GS115 / καVG. The activity of GOX increased from 1.5 U/ml to 19.3 U/ml when 1 % CA was employed in YPF medium. Maintaining culture at neutral pH to prevent expressed VGOX from degradating by usually expressed acidic protease of GS115 was found to be one of the reasons that a higher GOX activity could be obtained in the culture supplemented with CA. The specific activity of IMAC purified VGOX is 328.6 U/mg. It is about 1.4 fold higher than that of GOX（237.9 U/mg）.