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

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 Mn²⁺, completely inhibited by Cu²⁺, and strongly inhibited by Al³⁺, Ag⁺, Fe³⁺, Fe²⁺, Ni²⁺, Zn²⁺, and Mg²⁺. The product of hydrolysis of inulin by the recombinant exoinulinase was fructose.

Introduction

Inulin is a linear polymer of β-2,1-linked d-fructofuranose molecules terminated by a d-glucose residue at the reducing end (Vandamme & Derycke, 1983). It occurs as a reserve carbohydrate in the roots and tubers of plants such as Jerusalem artichoke, chicory, and dahlia. In recent years, inulin has received increasing attention as a raw material for fructose syrup production, ethanol fermentation, and inulo-oligosaccharide production.

Microbial inulinases can be divided into two types: endo-inulinase (2,1-β-d-fructan fructanohydrolase; EC 3.2.1.7) and exo-inulinase (β-d-fructan fructohydrolase; EC 3.2.1.80) according to the mode of action on inulin. Endoinulinase are specific for inulin and hydrolyze the internal β-2,1-fructofuranosidic linkages to yield inulotriose, inulotetraose, and inulopentaose as the main products. In contrast, exoinulinase split off terminal fructose units successively from the nonreducing end of the inulin molecule, and also hydrolyze sucrose and raffinose.

In recent years, exoinulinases have been attracting worldwide research interests due to their great potential industrial application in the preparation of high fructose syrups from inulin. However, the yields of exoinulinase from naturally isolated microorganisms are often low and not satisfied with the requirements for the large-scale industrial production. An efficient method for improving the yields of exoinulinase is the usage of recombinant DNA technology. A number of bacterial exoinulinases have cloned and overexpressed using recombinant DNA technology (Kwon et al., 2003, Moriyama et al., 2003, Tsujimoto et al., 2003, Wang et al., 2011, Zhang et al., 2004, Zhang et al., 2012, Zhang et al., 2009).

In our laboratory, we isolated a filamentous fungus, Aspergillus ficuum JNSP5-06, from soil samples. It can produce endoinulinase as well as exoinulinase (Chen et al., 2009). Our previous study indicated that endoinulinase-encoding gene from A. ficuum JNSP5-06 was expressed in Escherichia coli (Chen, Xu, Jin, & Chen, 2012). However, to the best of our knowledge, no study on the expression of an exoinulinase (exo I) gene from A. ficuum in E. coli has been reported. In this study, the expression of this exoinulinase gene from A. ficuum JNSP5-06 in E. coli and some properties of the recombinant exoinulinase are investigated.