摘要
采用RT-PCR法,以草菇Volvariella.volvacea V23总RNA为模板,克隆了包括自身信号肽在内的中性内切葡聚糖酶Ⅰ基因(egⅠ)的cDNA序列。测序结果表明,它全长1167 bp,编码389个氨基酸,推测第1～23位氨基酸为信号肽,第24～389位氨基酸为成熟肽,属于糖苷水解酶第5家族。PCR扩增成熟肽编码基因并将其插入到分泌型表达载体pPIC9K中,重组载体经SalⅠ线性化后电转化毕赤酵母(Pichia pastoris)GS115,筛选得到了1株高产中性内切葡聚糖酶Ⅰ的工程菌株P.pastoris-EGⅠ1。SDS-PAGE检测表明,重组中性内切葡聚糖酶Ⅰ分子质量约为42 ku。在甲醇诱导96h时,酶活达到3 698 U/mL。
An endoglucanase Ⅰ （EG Ⅰ ） cDNA including signal peptide was isolated from V. volvacea V23 by RT-PCR. The full-length cDNA contained an ORF of 1 167 bp which encoded a signal peptide of 23 amino-acids and a mature peptide of 365 amino-acids. Similarity analysis showed that the EG Ⅰ belonged to family 5 of glycosyl hydrolases. The matured peptide cDNA was amplified by PCR and cloned into the secretive expression vector pPIC9K. The recombinant plasmid was introduced into Pichia pastoris GS115 by electroporation after linearized by Sal Ⅰ. After screening, a high yielding recombinant strain P. pastoris-EG Ⅰ 1 was obtained. SDS-PAGE showed that the molecular weight of EG Ⅰ was about 42 ku. With methanol inducing 96 h, the enzyme activity was up to 3 698 U/mL.
出处
《食品与发酵工业》
CAS
CSCD
北大核心
2008年第7期7-11,16,共6页
Food and Fermentation Industries
基金
国家自然科学基金(No．20776061)
关键词
中性内切葡聚糖酶Ⅰ
草菇
毕赤酵母
分泌表达
neutral endoglucanase Ⅰ , Volvariella volvacea, Pichia pastoris, secretive expression
