摘要
聚半乳糖醛酸酶属于果胶水解酶类,在食品、饲料、纺织和造纸等工业生产中应用广泛。本研究筛选获得一株能分解果胶的青霉菌Penicilliumsp.FJ2,使用简并引物PCR和TAIL-PCR方法从该菌中克隆得到一个聚半乳糖醛酸酶基因pgp1。pgp1基因全长1225bp,包含2个内含子,其cDNA全长1104bp,编码367个氨基酸和一个终止密码子。将pgp1基因连接pET-30a(+)载体并转化大肠杆菌BL21(DE3),重组蛋白以包涵体形式获得表达。通过尿素溶解和梯度稀释方法对重组蛋白PGP1进行了重折叠复性试验,复性后的PGP1聚半乳糖醛酸酶活力达到12.9U/mL,比活力为5.83U/mg。
Polygalacturonases are a class of pectinase that can hydrolyze pectin, and they are widely used in food, feed, textile and paper industry. A Penicillium sp. FJ2, which produced polygalacturonase, has been screened in this research. In addition, a polygalacturonase gene pgp1 has been 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 encodes 367 amino acids and a termination codon. Pgp1 was cloned into pET-30a（＋） vector and expressed in Escherichia coli BL21（DE3）. The recombinant PGP1 was expressed as inclusion bodies without activity towards polygalacturonic acid. The renaturation of the PGP1 was through with urea resolution and gradient dilution. Polygalacturonase activity of the renatured PGP1 was detected as 12.9 U/mL, and with specific activity of 5.83 U/mg.
出处
《生物技术进展》
2011年第6期426-431,F0003,共7页
Current Biotechnology
基金
中国农业科学院生物技术研究所基本科研业务费专项资金(2008-08)资助
关键词
聚半乳糖醛酸酶
果胶酶
青霉菌
包涵体
复性
polygalacturonase
pectinase
Penicillium
inclusion body
renaturation
