| Summary: | 为提高南极假丝酵母脂肪酶b(CAndIdA AnTArCTICAlIPASE b,CAlb)的表达量,根据毕赤酵母(PICHIA PASTOrIS)密码子偏好性设计合成CAlb基因,插入表达载体PPIC9k构建重组质粒PPIC9k-CAlb.重组质粒转化毕赤酵母gS115,经过g418抗性筛选得到多拷贝转化子.摇瓶发酵120 H后上清液酶活力达到46 u/Ml,通过金属螯合层析纯化发酵液将CAlb纯化了5.23倍,比活力达到856.7 u/Mg,去糖基化实验显示重组CAlb比野生型CAlb大5 ku.实验考察了不同反应温度、PH值和金属离子对重组CAlb活性和稳定性的影响,发现重组CAlb最适反应温度和PH分别为30℃和6.5,在PH 5.0--8.0之间以及40℃以下有较好稳定性,金属离子(10 MMOl/l)CA2+,zn2+,Mn2+有助于CAlb酶活力的提高,而Cu2+,Ag+,fE3+强烈抑制酶催化反应. To improve expression efficiency of recombinant Candida antarctica lipase B(CALB) in methylotrophic yeast Pichia pastoris GS115,the DNA sequence encoding CALB was designed and synthesized based on the codon bias of P.pastoris.The new CALB gene was cloned into pPIC9K,yielding pPIC9K-CALB,and transformed into P.pastoris GS115.In shake-flask cultivation,the enzyme activity was 46 U/mL after induction by methanol for 120 h.The CALB was purified by metal chelate chromatography by 5.23 folds.Deglycosylation showed the recombinant CALB was 5 ku bigger than wild type CALB.The optimal pH and temperature of this recombinant enzyme were pH 6.5 and 30°C,respectively.CALB was relatively stable at temperatures below 40 ℃ within a pH range from 5.0 to 8.0.Ca2+,Zn2+,Mn2+ conduced to enzyme activity,while Cu2+,Ag+,Fe3+ inhibited it. 广东省教育部产学研结合项目(2008B090500218)
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