生物技术通报2017，Vol.33Issue(6)：207-213,7.DOI:10.13560/j.cnki.biotech.bull.1985.2016-1083
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
摘要
Abstract
This work is to clone the α-galactosidase gene from Thermoascus crustaceus JCM12803 and systematically study its enzymatic properties 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 glycoside hydrolase 27 was 1918 bp,contained 4 introns,the cDNA of tcgal27 was 1419 bp,and encoded 472 amino acids. SignalP analysis indicated 24 residues in the N-terminal of tcgal27 might be signal peptides. Recombinant TCGal27A successfully expressed in Pichia pastoris had a high specific 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). Using pNPG as the substrate,the Km and Vmax of TCGal27A were determined to be 1.6 mol/mL and 536.8 μmoL/(min·mg),respectively. Like most fungal a-galactosidases,TCGal27A had an optimal acidic pH 4.5. Purified recombinant TCGal27A was thermophilic,exhibiting the maximum activity at 65℃,and the enzyme remained 86% of initial activity at 50℃ for 1 h. All above results imply that heat-tolerant protein TCGal27A with excellent enzymatic properties can be additive for feedstuff,and will be solid potential applicable value in digesting and improving 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Key words
Thermoascus crustaceus JCM12803/α-galactosidase/thermophilic/heterologous expression引用本文复制引用
张多多,郑菲,罗会颖,李中媛,罗学刚..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[J].生物技术通报,2017,33(6):207-213,7.基金项目
国家杰出青年科学基金(31225026) （31225026）
