青霉菌聚半乳糖醛酸酶基因在毕赤酵母中的表达及酶学性质研究

Cloning,Expression and Characterization of a Polygalacturonase from Penicillium sp.in Pichia pastoris

筛选得到一株能分解果胶的青霉菌(Penicillium sp.),使用简并引物PCR和TAIL-PCR方法从该菌中克隆了一个聚半乳糖醛酸酶基因pgp1。pgp1基因全长1 225 bp,包含2个内含子,其cDNA全长1 104 bp,编码367个氨基酸和一个终止密码子,前18个氨基酸为信号肽序列。将pgp1基因连接pPIC9载体,在巴斯德毕赤酵母表达系统中进行了异源表达。在3L发酵罐水平,培养基中聚半乳糖醛酸酶活力达到700 U/mL。酶学性质测定表明,重组酶蛋白PGP1的最适pH为5.0,在pH4.0-6.0下处理1 h后,剩余酶活力超过90%;最适温度为38℃,以聚半乳糖醛酸为底物,PGP1的Km=(1.172±0.169)mg/mL,Vmax=(0.061±0.002)mg/min/mL。

Polygalacturonase as a kind of pectinase that can hydrolyze pectin,have been widely used in food,feed,textile and paper industry.A Penicillium sp.FJ2,which produced polygalacturonase was selected in this research.In addition,a polygalacturonase gene pgp1 was cloned using degenerated PCR and TAIL-PCR.Length of the polygalacturonase pgp1 gene and its cDNA are 1 225 bp and 1 104 bp,respectively.The cDNA of pgp1 encode 367 amino acids and a termination codon,first 18-residue amino acids is signal peptide.This gene was cloned into a expression vector pPIC9 and overexpressed in Pichia pastoris GS115 with the maximum polygalacturonase activity of 700 U/mL.The recombinant PGP1 exhibited activity towards polygalacturonic acid was optimally active at pH5.0 and 38℃,and remained 90% relative activity at pH4.0-6.0.The Km and Vmax values for polygalacturonic acid were（1.172±0.169）mg/mL and（0.061±0.002）mg/min/mL,respectively.