用毕赤酵母表达理氏木霉的β-葡萄糖苷酶基因  

Using Pichia pastoris to Express of β-glucosidase Gene from Trichoderma reesei

作　　者：林海鹏[1] 沈锦城[2] 尹慧祥[1] 张泽华[1] 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

出　　处：《生物技术》2012年第3期31-33,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项目(IT-BB120503)资助

摘　　要：目的:为提高β-葡萄糖苷酶的产量,用毕赤酵母取代理氏木霉用于生产,以弥补理氏木霉在大规模生产中的缺陷。方法:用套叠PCR法从理氏木霉基因组中扩增β-葡萄糖苷酶基因(bglⅠ)。用T4DNA连接酶和限制性DNA内酶将bglⅠ重组于P.pastoris表达载体pPIC9K的多克隆位点,获得含bglⅠ的重组表达载体pPIC9K-bglⅠ。通过电转法将其pPIC9K-bglⅠ载体转化于P.pastoris基因组,筛选高G418抗性以及高表达bglⅠ酶的重组子作为工程菌。结果:用BMGY-BMMY培养基体系,在摇瓶中发酵48 h,表达BglⅠ30 mg/L,在P.pastoris中表达的BglⅠ能水解对硝基苯-β-D-葡萄糖苷具有β-葡萄糖苷酶活性。其酶活力为56 U/L发酵液。结论:通过这种方法,可以成功地用毕赤酵母表达理氏木霉的β-葡萄糖苷酶基因。Objective:Construction of recombinant P.pastoris replaced of Trichoderma reesei to produce of β-glucosidase to increase the yield of the enzyme.Method:β-glucosidase gene(bgl) was amplified from Trichoderma reesei.After sequence,the DNA segment was inserted directly into pPIC9K to generate P.pastoris expression plasmid of pPIC9K-bglⅠ which was then transformed into the P.pastoris of GS115 by electroporation method.A strain on higher anti-G418 was used as engineering strain.Result:With the medium system of BMGY-BMMY to ferment the engineering strain for 48 h,30 mg/L of recombinant β-glucosidase is secreted into the fermentation liquid,which can hydrolyze 4'-Nitrophenyl-beta-D-glucopyranoside to show of β-glucosidase activity in the degree of 56 U/L fermentation liquid.Conclusion:The β-glucosidase gene from Trichoderma reesei was successfully expressed by recombinant P.pastoris.

关 键 词：Β-葡萄糖苷酶 毕赤酵母 套叠PCR 基因表达 酶活力 

分 类 号：Q786[生物学—分子生物学]