Gene Cloning, Expression, and Characterization of an Exo-inulinase from Paenibacillus polymyxa ZJ-9.

Gao, Jian; Xu, You-Yong; Yang, Hong-Mei; Xu, Hong; Xue, Feng; Li, Sha; Feng, Xiao-Hai

An inulinase-producing strain, Paenibacillus polymyxa ZJ-9, was isolated from natural sources to produce R, R-2,3-butanediol via one-step fermentation of raw inulin extracted from Jerusalem artichoke tubers. The inulinase gene from P. polymyxa ZJ-9 was cloned and overexpressed in Escherichia coli BL21 (DE3), and the purified recombinant inulinase was estimated to be approximately 56 kDa by both sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography. This result suggests that the active form of the inulinase is probably a monomer. Terminal hydrolysis fructose units from the inulin indicate that enzymes are exo-inulinase. The purified recombinant enzyme showed maximum activity at 25 °C and pH 6.0, which indicate its extreme suitability for industrial applications. Zn, Fe, and Mg stimulated the activity of the purified enzyme, whereas Co, Cu, and Ni inhibited enzyme activity. The K and V values for inulin hydrolysis were 1.72 mM and 21.69 μmol min mg protein, respectively. The same parameters toward sucrose were 41.09 mM and 78.7 μmol min mg protein, respectively. Considering its substrate specificity and other enzymatic characteristics, we believe that this inulinase gene from P. polymyxa ZJ-9 could be transformed into other special bacterial strains to allow inulin conversion to other biochemicals and bioenergy through one-step fermentation.

PAENIBACILLUS; GENE expression in bacteria; MOLECULAR cloning; INULASE; BACTERIAL enzymes

Applied Biochemistry & Biotechnology, 2014, Vol 173, Issue 6, p1419

0273-2289

Academic Journal

10.1007/s12010-014-0950-y