杂合木聚糖酶的设计及在毕赤酵母中的表达

Design of heterozygous xylanase and the expression in Pichia pastoris

人工设计合成木聚糖酶,为木聚糖酶分子改造研究提供新思路。采用合成生物学思路,以黑曲霉XZ-3S木聚糖酶XynZF-2基因序列为基础,将N端48个氨基酸替换成EvXyn11 N端的34个氨基酸;引入芳香族氨基酸P9Y和H14F;在C端引入二硫键Cys38-Cys191;α-螺旋及cord区域分别引入疏水性氨基酸K164M、G166A、N160I、V111A以及G109A,设计杂合酶XynZL。基因全合成后,构建毕赤酵母重组表达质粒pPIC9K-ZL,将其转化酵母菌GS115,对工程菌GS115-XynZL进行单因素发酵条件优化及重组酶酶学性质测定。最佳发酵培养基为土豆培养基,最佳诱导温度为28℃,最佳种龄为20 h,最佳诱导时间为168 h,最佳诱导起始pH值为6.5,甲醇诱导最佳体积分数为15 mL/L。重组酶酶学性质显示:最佳反应温度为55℃,最适pH值为5.0,杂合酶XynZL在毕赤酵母中表达后具有特定性质和功能,其设计方法拓宽了木聚糖酶分子改造研究的思路。

In order to provide a new idea for molecular modification of xylanase, heterozygous xylanase was designed and synthetized in this study. Based on the xylanase XynZF-2 gene sequence of Aspergillus niger XZ-3S, 48 N-terminal amino acids were replaced by 34 N-terminal amino acids of EvXyn11. Heterozygous enzyme Xyn-ZL was designed by introducing aromatic amino acids P9Y and H14F, and disulfide bond Cys38-Cys191 at the C-terminal, hydrophobic amino acids K164M, G166A, N160I, and V111A at α-helix and G109A at cord area. After gene synthesis, the recombinant expression plasmid pPIC9K-ZL was constructed and transformed into P. pastoris GS115 to obtain the recombinant strain GS115-Xyn-ZL. It was found that the optimal fermentation condition was as follows: using potato medium as the medium, inoculated for 20 h before induction at 28 ℃ for 168 h at pH 6.5, and the optimal concentration of methanol for induction was 15 mL/L. The optimal reaction temperature and pH of the recombinant enzyme was 55 ℃ and pH 5.0, respectively. It was concluded that the XynZL expressed in P. pastoris exhibited specific properties and functions, which broadens the idea of modifying xylanase molecularly.