Aspergillus niger所產的葡萄糖氧化酶（Glucose oxidase， GOX）是屬於一種醣化蛋白質，在食品工業及醫療上皆有廣泛的應用。利用嗜甲基酵母菌Pichia pastoris可進行大量表現此酵素。在菌體中表現顫菌血紅蛋白(Vitreoscilla Hemoglobin， VHb )，已證明可來提升菌體之氧氣利用率，而提高菌體的生產速率，因此本研究將VHb與GOX融合在AOX1啟動子調控下，利用甲醇當誘導劑在P. pastoris GS115中表現可分泌至胞外之VGOX融合蛋白。在YPF(Yeast 1%， Peptone 2%， Fructose 2%)培養基中添加1% Casamino acid會大幅提升VGOX的表現量。在搖瓶培養時，添加了Casamino acid之VGOX活性可由1.5 U/ml增加至19.3U/ml提高約13倍。VGOX經過Immobilized Metal Affinity Chromatography （IMAC）純化過後，比活性可達到328.6 U/mg，相較於GOX 的237.9 U/mg提升約1.4倍，主要原因可能是融合之VHb可供氧給GOX而加速其反應速率。而Casamino acid添加對提升VGOX活性之效應，主要可能是防止菌體本身所分泌之蛋白分解酶分解VGOX及提供足夠的胺基酸用於VGOX之表現。

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）.

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