嗜热子囊菌JCM12803的α-半乳糖苷酶基因tcgal27A在毕赤酵母中的表达

Expression of α-Galactosidases from Thermoascus crustaceus JCM12803 in Pichia pastoris

从嗜热子囊菌(Thermoascus crustaceus)JCM12803克隆α-半乳糖苷酶基因,并对其酶学性质进行系统的研究,旨为获得工业应用性质优良的α-半乳糖苷酶。通过RT-PCR方法从T.crustaceus JCM12803中克隆α-半乳糖苷酶基因序列,并对其酶学性质进行了系统的分析。结果显示,tcgal27A属于糖苷水解酶27家族,基因全长1 918 bp,含有4个内含子,c DNA全长1 419 bp,编码472个氨基酸,预测tcgal27A N端含有24个氨基酸为其可能的信号肽。将TCGal27A在毕赤酵母中成功地进行了表达,所获得的重组TCGal27A具有高比活(336.5 U/mg)及宽泛的底物特异性,对蜜二糖(14.4 U/mg)、棉子糖(9.1 U/mg)、角豆胶(3.6 U/mg)、魔芋粉(1.6 U/mg)、瓜尔豆胶(1.3 U/mg)、水苏糖(0.7 U/mg)都有着不同程度的降解作用。以pNPG为底物时的Km值为1.6mol/m L,Vmax为536.8μmo L/(min·mg)。TCGal27A的最适作用pH为4.5,最适温度为65℃,50℃处理1 h之后酶活力还能保持86.0%。这些结果表明耐高温TCGal27A性质优良,可添加到饲料中,在消化和提高豆粕蛋白能量利用率方面有良好的潜在应用价值。

This work is to clone the α-galactosidase gene from Thermoascus crustaceus JCM12803 and systematically study its enzymaticproperties for obtaining high-quality α-galactosidase in industry. The gene sequence of α-galactosidase was cloned from T. crustaceus JCM12803 by RT-PCR and its enzymatic properties were systematically analyzed. As results revealed,the full-length of tcgal27A belonging to glycosidehydrolase 27 was 1 918 bp,contained 4 introns,the c DNA of tcgal27 was 1 419 bp,and encoded 472 amino acids. Signal P analysis indicated24 residues in the N-terminal of tcgal27 might be signal peptides. Recombinant TCGal27A successfully expressed in Pichia pastoris had a highspecific activity（336.5 U/mg）,and the broad substrate specificity,i.e.,presenting different degrees of degradation to melibiose（14.4 U/mg）,raffinose（9.1 U/mg）,gum tragon（3.6 U/mg）,konjaku flour（1.6 U/mg）,guar gum（1.3 U/mg）,stachyose（0.7 U/mg）. Usingp NPG as the substrate,the Km and Vmax of TCGal27A were determined to be 1.6 mol/m L and 536.8 μmo L/（min·mg）,respectively. Likemost fungal a-galactosidases,TCGal27A had an optimal acidic pH 4.5. Purified recombinant TCGal27A was thermophilic,exhibiting themaximum activity at 65℃,and the enzyme remained 86% of initial activity at 50℃ for 1 h. All above results imply that heat-tolerant proteinTCGal27A with excellent enzymatic properties can be additive for feedstuff,and will be solid potential applicable value in digesting andimproving the energy utilization ratio of soybean meal protein.