School of Life Science and Engineering, Harbin Institute of Technology, P.O. 437, No 92, West Da-Zhi Street, Harbin, 150001, Heilongjiang, China.
World J Microbiol Biotechnol. 2013 Nov;29(11):2087-94. doi: 10.1007/s11274-013-1373-6. Epub 2013 May 16.
Trichoderma asperellum parasitizes a large variety of phytopathogenic fungi. The mycoparasitic activity of T. asperellum depends on the secretion of complex mixtures of hydrolytic enzymes able to degrade the host cell wall and proteases which are a group of enzymes capable of degrading proteins from host. In this study, a full-length cDNA clone of aspartic protease gene, TaAsp, from T. asperellum was obtained and sequenced. The 1,185 bp long cDNA sequence was predicted to encode a 395 amino acid polypeptide with molecular mass of 42.3 kDa. The cDNA of TaAsp was inserted into the pPIC9K vector and transformed into yeast Pichia pastoris GS115 for heterologous expression. A clearly visible band with molecular mass about 42 kDa in the SDS-PAGE gel indicated that the transformant harboring the gene TaAsp had been successfully translated in P. pastoris and produced a recombinant protein. Enzyme characterization test showed that the optimum fermentation time for P. pastoris GS115 transformant was 72 h. Enzyme activity of the recombinant aspartic proteinase remained relatively stable at 25-60 °C and pH 3.0-9.0, which indicated its good prospect of application in biocontrol. The optimal pH value and temperature of the enzyme activity were pH 4.0 and 40 °C, and under this condition, with casein as the substrate, the recombinant protease activity was 18.5 U mL(-1). In order to evaluate antagonistic activity of the recombinant protease against pathogenic fungi, five pathogenic fungi, Fusarium oxysporum, Alternaria alternata, Cytospora chrysosperma, Sclerotinia sclerotiorum and Rhizoctonia solani, were applied to the test of in vitro inhibition of their mycelial growth by culture supernatant of P. pastoris GS115 transformant.
木霉asperellum 寄生了各种各样的植物病原真菌。木霉 asperellum 的寄生活性依赖于复杂的水解酶混合物的分泌，这些酶能够降解宿主细胞壁，以及蛋白酶，蛋白酶是一组能够降解宿主蛋白的酶。在这项研究中，从木霉 asperellum 中获得并测序了一个全长 cDNA 克隆的天冬氨酸蛋白酶基因 TaAsp。该 1185bp 长的 cDNA 序列预测编码一个 395 个氨基酸的多肽，分子量为 42.3kDa。将 TaAsp 的 cDNA 插入 pPIC9K 载体，并转化酵母毕赤酵母 GS115 进行异源表达。SDS-PAGE 凝胶中分子量约为 42kDa 的明显条带表明，携带 TaAsp 基因的转化体已在毕赤酵母中成功翻译，并产生了重组蛋白。酶特性测试表明，毕赤酵母 GS115 转化体的最佳发酵时间为 72h。重组天冬氨酸蛋白酶的酶活性在 25-60°C 和 pH3.0-9.0 下相对稳定，这表明其在生物防治中有很好的应用前景。酶活性的最佳 pH 值和温度分别为 pH4.0 和 40°C，在这种条件下，以酪蛋白为底物，重组蛋白酶的活性为 18.5U mL(-1)。为了评价重组蛋白酶对病原真菌的拮抗活性，将五种病原真菌，尖孢镰刀菌、链格孢菌、生尾孢菌、核盘菌和茄腐镰刀菌，应用于毕赤酵母 GS115 转化体培养上清液对其菌丝生长的体外抑制试验。
