Abstract
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. Zn2+, Fe2+, and Mg2+ stimulated the activity of the purified enzyme, whereas Co2+, Cu2+, and Ni2+ inhibited enzyme activity. The K m and V max values for inulin hydrolysis were 1.72 mM and 21.69 μmol min−1 mg−1 protein, respectively. The same parameters toward sucrose were 41.09 mM and 78.7 μmol min−1 mg−1 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.
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Acknowledgments
This work was financially supported by the National Natural Science Foundation of China (Grant 21376203), the Natural Science Foundation of Jiangsu Province (Grant BK2010290), the Promotion Program of Achievements in Scientific Research for Industrial Production of Higher Education of Jiangsu Province (Grant JHB2011-54), Qinglan Project of Higher Education of Jiangsu Province, Overseas Research & Training Program for University Prominent Young & Middle-aged Teachers and Presidents of Jiangsu Province, and Jiangsu Agricultural Key Technology Research and Development Program (Grant BE2012394).
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Jian Gao and You-Yong Xu contributed equally to this work.
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Gao, J., Xu, YY., Yang, HM. et al. Gene Cloning, Expression, and Characterization of an Exo-inulinase from Paenibacillus polymyxa ZJ-9. Appl Biochem Biotechnol 173, 1419–1430 (2014). https://doi.org/10.1007/s12010-014-0950-y
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DOI: https://doi.org/10.1007/s12010-014-0950-y