【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A₆₀₀=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。更多还原

【Abstract】 The gene of endo-l,4-β-D-glucanaseⅡ (eg Ⅱ) was amplified from the Trichoderma reesei chromosome by cross-over 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 600 nm,while secreted eg Ⅱ was 90 mg/L.The secreted product showed the hydrolase activity on carboxymethylcellulose.更多还原