Expression of an exoinulinase gene from Aspergillus ficuum in Escherichia coli and its characterization.

Xiao-Ming Chen, Xue-Ming Xu, Zheng-Yu Jin, Han-Qing Chen

Index: Carbohydr. Polym. 92(2) , 1984-90, (2013)

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Abstract

An exoinulinase gene from Aspergillus ficuum JNSP5-06 was overexpressed in Escherichia coli. Two exons of the exoinulinase gene were amplified separately, joined together by an overlap PCR, and expressed in E. coli. The molecular weight of the recombinant exoinulinase was estimated to be 63 kDa. The K(m) and V(max) values for inulin were (7.1±0.2) mM and (1000.0±0.1) μmol/(min mg protein), respectively. The K(m) and V(max) values for sucrose were (347.6±25.9)mM and (12,037.0±801.9) μmol/(min mg protein), respectively. The optimum temperature and pH with inulin as the substrate were 60°C and 4.0, respectively. The optimum temperature and pH with sucrose as the substrate were 55 °C and 5.0, respectively. Its activity was increased by Mn(2+), completely inhibited by Cu(2+), and strongly inhibited by Al(3+), Ag(+), Fe(3+), Fe(2+), Ni(2+), Zn(2+), and Mg(2+). The product of hydrolysis of inulin by the recombinant exoinulinase was fructose.Copyright © 2012 Elsevier Ltd. All rights reserved.