甘露聚糖酶基因在毕赤酵母中的表达及酶学性质研究

Secreted Expression of M annanase Gene in Pichia pastoris and Anylysis of Enzymic Properties

采用PCR的方法,以枯草芽孢杆菌Bacillus subtilis基因组DNA为模板,克隆出甘露聚糖酶MAN的成熟肽编码序列,将其插入巴斯德毕赤酵母Pichia pastoris表达载体pPIC9K中,并位于α-因子信号肽序列的下游,获得重组质粒pPIC9K-MAN。重组质粒线性化后用聚乙二醇法导入毕赤酵母Pichia pastoris菌株GS115中,经大量筛选,获得高效分泌表达甘露聚糖酶的毕赤酵母工程菌株MAN22。将此菌株在5 L发酵罐中进行高密度发酵,测定酶活最高达1102IU／ml,同时对重组甘露聚糖酶的性质进行了初步研究。

A PCR method was used to amplify the sequence encoding the mature peptide of β-mannanase of Bacillus subtilis. The gene was inserted into the Pichia pastoris vector pPIC9K, downstream of α-factor signal peptide sequence. The resultant recombinant plasmid pPIC9K-MAN was lineared by BglII digestion and introduced into the host Pichia pastoris GS115 by PEG method. After screen, the recombinant P. pastor/s strain MAN22 was obtained and fermented in large scale 5 L fermenter. The recombinant mannanase activity could reach to 1102IU/ml. The properties of the recombinant mannanase were characterized.