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

作　　者：张多多 郑菲[2,3] 罗会颖[2] 李中媛 罗学刚[1] 

机构地区：[1]工业发酵微生物教育部重点实验室天津市工业微生物重点实验室天津科技大学生物工程学院,天津300457 [2]中国农业科学院饲料研究所,北京100081 [3]北京林业大学生物科学与生物技术学院,北京100083

出　　处：《生物技术通报》2017年第6期207-213,共7页Biotechnology Bulletin

基　　金：国家杰出青年科学基金(31225026)

摘　　要：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.

关 键 词：嗜热真菌 Α-半乳糖苷酶 热稳定性 异源表达 

分 类 号：Q78[生物学—分子生物学]