甲醇酵母Pichia pastoris高水平表达有活性的辣根过氧化物酶  

High level Expression of Active Horseradish Peroxidase in Methyltrophic Yeast

作　　者：蒋太交[1] 吉鑫松[1] 章如安[1] 黄艳红[1] 吴祥甫[1] 袁中一[1] 

机构地区：[1]中国科学院上海生物化学研究所,上海200031

出　　处：《生物化学与生物物理进展》1999年第6期584-587,共4页Progress In Biochemistry and Biophysics

摘　　要：表达有活性的辣根过氧化物酶（ＨＲＰ） 不仅可以深入揭示ＨＲＰ 结构与功能及其生理作用规律， 而且为ＨＲＰ的广泛需要提供新的来源． 为了在甲醇酵母Ｐ． ｐａｓｔｏｒｉｓ 中成功表达， 将编码ＨＲＰＣ成熟肽的ｃＤＮＡ 构建到ｐＰＩＣ９ 上， 再转化到Ｐ． ｐａｓｔｏｒｉｓ 中， 筛选到了分泌表达非糖基化ＨＲＰ 和高糖基化ＨＲＰ（ 分子质量超过１００ ｋｕ） 两种主要产物的重组细胞株． 优化表达条件， 目标产物在摇瓶发酵液中高效表达， 可达４～６ ｇ／Ｌ． 并且直接从发酵液中可获得具有活性的高糖基化ＨＲＰ， 每毫升发酵液中酶活力约有２ Ｕ， 经初步的纯化ＨＲＰ具有最大吸收峰４０３ ｎｍ ．Expression of active horseradish( Armoracia rusticana) peroxidase (HRP) in microbes is necessary not only to gain an insight into its mechanisms, structure and function, and physiological role in plants, but also to supply large quantities of this useful enzyme. Exploiting new approach to express HRP in methyltrophic yeast, Pichia pastoris , the cDNA coding for mature HRP isozyme C was subcloned into the vector pPIC9. After transformation, the recombinant P.pastoris strain which secreted nonglycosylated HRP(about 30 ku) and hyperglycosylated HRP (about 100 ku) was obtained . Through optimization of the growth conditions, the hyperglycosylated HRP as the sole product was secreted into fermentation broth and reached a level of approximately 4～6 mg per ml of culture medium. Remarkably, a certain activity of 2 24 U/ml was determined in the fermentation broth and showed the maximum absorbance at 403 nm.

关 键 词：高糖基化 甲醇酵母 发酵 基因表达 HRT 

分 类 号：TQ925.9[轻工技术与工程—发酵工程]