热稳定α-半乳糖苷酶基因在毕赤酵母中的分泌表达及酶学性质研究

Secreted Expression of a Thermostable α-galactosidase Gene in Pichia psatoris and Analysis of Enzymic Properties

克隆前期筛得分枝犁头霉Absidia ramosa WL511的热稳定α-半乳糖苷酶cDNA基因aga(GenBank No.DQ234280),将aga基因插入表达载体pPICZαA并电转化整合到毕赤酵母P.pastoris GS115的染色体上。30℃、甲醇流加量0.5%(V/V)时,酵母发酵上清液中酶活达32U/ml。纯化后酶的比活力为137U/mg,SDS-PAGE显示单一条带,凝胶过滤和SDS-PAGE估算其分子量分别为348kDa和87kDa,该酶为四聚体结构,糖基化导致重组蛋白的分子量比原酶大6kDa。该酶等电点为5.2,最适反应温度73℃,最适pH6.8,60℃以下及pH5.5～9.0范围内活性稳定。75℃时保温2小时保留54%的酶活,85℃时酶活完全消失。以对硝基苯酚-α-D-半乳糖苷为底物,该酶Km值为0.42mmol/Lol/L;Vmax为413U/mg;kcat为64531/min。

DNA sequence（GenBank No. DQ234280）of the α-galactosidase from Absidia ramose WL511 was cloned and inserted into the Pichia pastoris expression vector pPICZαA,and integrated into the genome of P. pastoris GS115 cells by electricity pulse. The culture supernatant showed 32U/ml of enzymatic activity at 30℃ with 0.5%（V/V）methanol added. The specific activity of purified recombinant enzyme at the grade of electrophoresis pure was 137 U/ mg. The α-galactosidase expressed by P.pastoris was glycosyled protein with a 6kDa larger-molecular mass than native enzyme, and a tetramer with the molecular weight of 348 kDa estimated by gel filtration and the subunit molecular weight of 87 kDa by SDS-PAGE. The pI value, optimal pH and temperature of the enzyme are 5.2,6.8 and 73 ℃. With pNPG as the substrate, the Km, Vmax, and kcat are 0.42mmol/Lol/L, 413U/mg and 64 531/min, respectively. This enzyme showed a stable activity at pH5.5~9.0 and below 60℃, still remained 54% of activity for 2 h at 75 ℃, and completely inactivated at 85℃.the secret-expressed α-galactosidase from A. ramose WL511 have better enzymatic properties of high thermostability, high specific activity and wide pH range.