Pronucleus expression and characterization of thermotoga lettingae TMO pullulanase TMP in recombinant escherichia coli

被引：0

作者：

Wei T. ^{[1
]}

Chi L. ^{[1
]}

Yu X. ^{[1
]}

Zhang J. ^{[1
]}

Liu K. ^{[1
]}

Mao D. ^{[1
]}

机构：

[1] School of Food and Biological Engineering, Zhengzhou University of Light Industry, Zhengzhou

来源：

Mao, Duobin | 1600年 / Chinese Institute of Food Science and Technology卷 / 16期

关键词：

Cloning and expression; Enzymatic property; Pullulanase; Site-directed mutant;

D O I：

10.16429/j.1009-7848.2016.08.003

中图分类号：

学科分类号：

摘要：

A pullulanase gene (Tlet_l262) from Thermotoga lettingae TMO was amplified by PCR method, cloned into the vector pET15b and functionally overexpressed in Escherichia coli. The recombinant enzyme was purified to homogeneity after heat treatment at 80℃ and Ni-NTA affinity. The purified recombinant protein had a molecular weight of about 97 ku by SDS-PAGE. The enzyme displayed optimal activity at 90℃ and pH 5.4. It retained over 80% of enzymatic activity after incubation in reaction mixture at 90℃ for 150 min. TMP was found to have high acid tolerance and maintained about 50% of activity even after 20 h of treatment at 90℃ and pH 5.0. The enzyme was found to have high tolerance to metal ions, SDS, urea, Tween 80 and Trtion-XlOO. The enzymatic activity was activated to 18% and 28% by the addition of DTT (0.5 or 5 mmol/L). Three site-directed mutants C501S, C649S and C704S were constructed using the overlap PCR method. As compared with the wild-type pullulanase TMP, the catalytic efficiency (kcat/Km) to pullulan and starch of C649S increased to about 37.8% and 40.8% through the analysis of catalytic dynamic, which demonstrated that C649 was critical residues in the catalytic site for enzymatic activity of pullulanase TMP. © 2016, Editorial Office of Journal of CIFST. All right reserved.

引用

页码：16 / 22

页数：6

共 6 条

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