| 南极假丝酵母脂肪酶B在毕赤酵母的表达及酶学性质研究 |
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| 引用本文： | 孙金鹏,钱圣一,敬科举,卢英华.南极假丝酵母脂肪酶B在毕赤酵母的表达及酶学性质研究[J].厦门大学学报(自然科学版),2011,50(4):765-771. |
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| 作者姓名： | 孙金鹏 钱圣一 敬科举 卢英华 |
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| 作者单位： | 厦门大学化学化工学院,福建厦门,361005 |
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| 基金项目： | 广东省教育部产学研结合项目(2008B090500218) |
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| 摘 要： | 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.
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| 关 键 词： | 南极假丝酵母脂肪酶B 毕赤酵母 分泌表达 酶学性质 |
Expression of Candida antarctica lipase B in Pichia pastoris and Studying of Enzyme Properties |
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| SUN Jin-peng,QIAN Sheng-yi,JING Ke-ju,LU Ying-hua.Expression of Candida antarctica lipase B in Pichia pastoris and Studying of Enzyme Properties[J].Journal of Xiamen University(Natural Science),2011,50(4):765-771. |
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| Authors: | SUN Jin-peng QIAN Sheng-yi JING Ke-ju LU Ying-hua |
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| Institution: | SUN Jin-peng,QIAN Sheng-yi,JING Ke-ju,LU Ying-hua(College of Chemistry and Chemical Engineering,Xiamen Univeristy,Xiamen 361005,China) |
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| Abstract: | 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 showe... |
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| Keywords: | Candida antarctica lipase B Pichia pastoris secretory expression enzyme properties |
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