高密度发酵P.pastoris诱导表达葡聚糖外切酶  

Inducible Expresstion of CellobiohydrolaseⅡ by AOXI Promoter in P.pastoris Using High-density Cell Culture

作　　者：杨穗珊[1,2] 易国辉[1] 屠发志[1] 张添元[3] 罗进贤[3] 张爱联[1] 

机构地区：[1]中国热带农业科学院热带生物技术研究所农业部热带作物生物技术重点开放实验室,海南海口571101 [2]海南大学农学院,海南海口570228 [3]中山大学基因工程教育部重点实验室/生物化学系,广东广州510275

出　　处：《生物技术》2010年第6期64-66,共3页Biotechnology

基　　金：中央级公益性科研院所基本科研业务费专项资金资助项目(ITBBZX2008-4-2);国家"863"项目(No.2007AA021307)资助

摘　　要：目的:应用P.pastoris的pAOX1表达系统表达CBHⅡ酶。方法:PCR法扩增木霉的cbhⅡ基因。将其克隆进P.pastoris表达载体pPIC9k,电转法将其cbhⅡ基因重组于P.pastoris基因组,筛选高G418抗性的克隆为工程菌。重组CBHⅡ酶的生产是在50 L生物反应器中进行。连续24h补加甘油-PTM4增殖细胞,然后用甲醇诱导表达64h。结果:放罐时生物量为A600=180,重组CBHⅡ产量为80mg/L。表达产物具有酶解羧甲基纤维素的活性。结论:实现应用pAOX1表达系统在生物反应器中高密度发酵P.pastoris诱导表达CBHⅡ。该研究为重组CBHⅡ的规模化生产打下基础。Objective：Using P.pastoris to express of CBHⅡ protein.Method： Cellobiohydrolase Ⅱ（cbhⅡ） gene was amplified by PCR technique and cloned directly into the P.pastoris vector pPIC9K resulted in the expression plasmid pPIC9K-cbhⅡ which was then transformed into P.pastoris GS115 by electroporation method.A recombinant with G418 high resistant characteristic was selected as engineering strain.The fermentation was carried out in a 50 L bioreactor.Result：The cells were first grown in glycerol-PTM4 trace salts for 24 h and then induction by methanol for 64 h.The biomass growth was 180 as measured by absorption of 600nm,while secreted CBHⅡ was 80 mg/L.The secreted product showed the enzyme activity on hydrolyzing carboxymethylcellulose.Conclusion： cbhⅡ gene under the control of pAOX1 expresses successfully in P.pastoris by the way of high-density cell culture.This work has established the base for large amount production of CBHⅡ enzyme with the pAOX1 expression system of P.pastoris.

关 键 词：葡聚糖外切酶 P.PASTORIS 高密度发酵 

分 类 号：Q786[生物学—分子生物学]