工程菌P.pastoris GS115(pPIC9K-lac2)的构建及诱导表达漆酶的研究  

Expression of laccase lac2 gene of Bacillus subtilis in P.pastoris by high-density cell culture

作　　者：沈锦城[1,2] 张泽华[1,2] 杨穗珊[2] 张添元[3] 罗进贤[3] 张爱联[2] 

机构地区：[1]海南大学农学院,海口570228 [2]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,海口571101 [3]中山大学 基因工程教育部重点实验室 生物化学系,广州510275

出　　处：《工业微生物》2013年第1期36-40,共5页Industrial Microbiology

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(ITBB120503)

摘　　要：用PCR法扩增枯草芽孢杆菌的漆酶基因lac2。构建表达质粒pPIC9K-lac2。通过电转法将lac2基因重组于P.pastoris基因组,筛选高G418抗性和高表达漆酶的转化子作为工程菌GS115(pPIC9K-lac2)。在发酵罐中发酵GS115(pPIC9K-lac2)表达重组蛋白。在50 L发酵罐中加入20 L无机盐发酵培养基。在发酵的第一阶段连续24 h补加50%甘油-0.8%PTM4增殖P.pastoris,然后用甲醇-0.8%PTM4诱导49 h。在发酵过程中,通过调节搅拌的频率和通气量,将溶氧维持于20%～30%,用氨水维持pH 5.0.放罐时生物量为A_(600)=266.5,表达漆酶1097.5U/L发酵液。The gene of lac2 was amplified from the Bacillus subtilis chromosome by PCR technique according to the pub- lished sequence of lac2 and cloned directly into the P. pastoris vector pPIC9K, resulted in the expression plasmid pPICgK-/ac2 which was then transformed into P. pastoris GS115 by electroporation method. A recombinant transformant with high G418 resistant characteristics and well expression was selected as engineering strain GSll5 （pPIC9K-lac2）. The fermentation was carried out in a 50 L bioreactor with 20 L inorganic salt fermentation medium. The cells were first grown in 50% glycerol -0.8% PTM4 trace salts for 24 h and then induced by methanol -0.8% PTM4 for 49 h. During the process of the fermentation, the dissolved oxygen was maintained between 20% -30% by adjusting the rates of agitation and aeration, and pH was eontrolled at 5 by NH4OH. At the end of the fermentation, the biomass growth was 266.5 as measured by absorption of 600nm, while expressed lacease was 1097.5U/L fermentation broth.

关 键 词：漆酶 枯草芽孢杆菌 基因表达 P PASTORIS 发酵 高密度发酵 

分 类 号：Q93[生物学—微生物学]