重组绿僵菌酸性海藻糖酶在毕赤酵母中的表达、纯化及特性分析
Expression, purification, and characterization of recombinant Metarhizium anisopliae acid trehalase in Pichia pastoris.
作者信息
Liu Yingchun, Wang Zhongkang, Yin Youping, Cao Yueqing, Zhao Hua, Xia Yuxian
机构信息
Genetic Engineering Research Center, Bioengineering College, Chongqing University, Chongqing 400030, PR China.
出版信息
Protein Expr Purif. 2007 Jul;54(1):66-72. doi: 10.1016/j.pep.2007.02.016. Epub 2007 Mar 1.
The mature peptide of Metarhizium anisopliae acid trehalase (ATM1) (EC3.2.1.28) was successfully expressed in Pichia pastoris at high levels under the control of AOX1 promoter. The recombinant ATM1 (reATM1) was secreted into culture medium. After 48-h 0.5% methanol induction, the activity of reATM1 in the culture supernatant reached the peak, 5.35 U/mg. Enzyme with a histidine sequence appended to the C terminus was still active and was purified using metal-chelate affinity chromatography. The yield of purified reATM1 was 2.5 mg from 1L supernatant. The purified reATM1 exhibited a molecular mass of approximately 170 kDa on SDS-PAGE. The optimum temperature and pH of reATM1 were 30 degrees C and 6.0, respectively, and the K(m) and V(max) values for reATM1 were 2.6 mM and 0.305 mmol/min/mg, respectively. Studies showed that the enzymatic properties of reATM1 were similar to those of the native ATM1.
绿僵菌酸性海藻糖酶（ATM1）（EC3.2.1.28）的成熟肽在AOX1启动子的控制下于毕赤酵母中成功实现了高水平表达。重组ATM1（reATM1）分泌到培养基中。经48小时0.5%甲醇诱导后，培养上清液中reATM1的活性达到峰值，为5.35 U/mg。C末端附加有组氨酸序列的酶仍具有活性，并通过金属螯合亲和层析进行纯化。从1L上清液中获得的纯化reATM1产量为2.5 mg。纯化后的reATM1在SDS-PAGE上显示分子量约为170 kDa。reATM1的最适温度和pH分别为30℃和6.0，其K(m)和V(max)值分别为2.6 mM和0.305 mmol/min/mg。研究表明，reATM1的酶学性质与天然ATM1相似。
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