高密度发酵P.pastoris诱导表达egⅡ基因  

Expression of endo-l,4-β-D-glucanase Ⅱ gene of Trichoderma reesei in P.pastoris by high-density cell culture

作　　者：尹慧祥[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,海口571101 [2]海南大学农学院,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,广州510275

出　　处：《工业微生物》2011年第2期43-46,共4页Industrial Microbiology

基　　金：国家863项目(No.2007AA021307);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)

摘　　要：用套叠PCR法扩增里氏木霉(Trichoderma reesei)的葡聚糖内切酶Ⅱ(egⅡ)基因。扩增基因经EcoR Ⅰ和Not Ⅰ双酶切后克隆进P.pastoris表达载体pPIC9k,获得重组表达质粒pPIC9K-egⅡ。通过电转法将egⅡ基因重组于P.pastoris基因组,筛选高G418抗性的转化子作为工程菌。工程菌的发酵在生物反应器中进行,在50 L的发酵罐中加入20 L发酵液。连续24 h补加甘油-PTM4增殖细胞,然后以甲醇为碳源诱导egⅡ基因表达48 h。放罐时生物量为A_(600)=203,重组EGⅡ产量为90 mg/L。表达产物具有酶解羧甲基纤维素的活性。The gene of endo-1,4-β-D-glucanase Ⅱ （ eg Ⅱ ） was amplified from the Trichoderma reesei chromosome by crossover PCR technique according to the published sequence of eg Ⅱ and cloned directly into the P. pastoris vector pPIC9K resulted in the expression plasmid pPIC9K-eg Ⅱ which was then transformed into P. pastoris GS115 by electroporation method. A recombinant transformant with high G418 resistant characteristic was selected as engineering strain. The fermentation was carried out in a 50 L bioreactor with 20 L modified growth medium recommended by Invitrogen. The cells were first grown in glycerol-PTM4 trace salts for 24 h and then induced by methanol for 48 h. The biomass growth was 203 as measured by absorption of 600nm, while secreted eg 11 was 90 mg/L. The secreted product showed the hydrolase activity on carboxymethylcellulose.

关 键 词：里氏木霉 葡聚糖内切酶 基因表达 P.PASTORIS 高密度发酵 

分 类 号：Q78[生物学—分子生物学]