阿魏酸酯酶O42807在毕赤酵母GS115中的表达

研究阿魏酸酯酶O42807基因(fae)在巴斯德毕赤酵母GS115中的表达及重组阿魏酸酯酶的酶学特性.化学合成fae基因序列,构建分泌型重组质粒pPIC9K-fae,经线性化后电转化至毕赤酵母GS115,对筛选出的高活性转化子进行诱导表达.SDS-PAGE分析显示:发酵上清液为单一条带,表观相对分子质量为42 ku,酶活为78.49 nkat·mL^-1,比活力为524.38 nkat·mg^-1,最适反应温度为50 ℃,在40~45 ℃温度范围内较稳定,最适反应p

Expression of Feruloyl Esterase O42807 in Pichia pastoris GS115

This paper studied the expression of the feruloyl estrase O42807 in Pichia pastoris GS115 and the enzymatic properties of the recombinant feruloyl estrase. Based on the amino acids of feruloyl estrase O42807, the gene(fae)of feruloyl estrase was synthesized chemically. The secreted expression vector pPIC9K-fae was constructed by the ligation of the fae gene into the shuttle vector pPIC9K. The plasmid pPIC9K-fae was linearized and then electrotransformed into Pichia pastoris GS115. Afterwards, the recombinant strain with high level of feruloyl estrase activity was obtained through activity screening. The SDS-PAGE result showed a single band in the fermentation supernatant. The molecular weight of the recombinant feruloyl estrase was about 42 ku and the enzyme activity was 78.49 nkat·mL^-1. The specific activity of the recombinant feruloyl estrase was 524.38 nkat·mg^-1. The results also showed that the optimal reaction temperature was 50 ℃, and stable from 40 ℃ to 45 ℃. And the optimal pH was from 5.0 to 5.5, stable at pH 6.0. Furthermore, the enzymatic activity was slightly enhanced by K⁺, Ca²⁺, Na⁺, whereas it was slightly inhibited by Fe²⁺, Zn²⁺, and strongly inhibited by Mn²⁺, Cu²⁺.