title: Gene cloning, expression, and characterization of an exo-inulinase from Paenibacillus polymyxa ZJ-9.
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. Zn(2+), Fe(2+), and Mg(2+) stimulated the activity of the purified enzyme, whereas Co(2+), Cu(2+), and Ni(2+) 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.
authors.affiliation: School of Chemical and Biological Engineering, Yancheng Institute of Technology, Yancheng, 224051, People's Republic of China, gaojian@ycit.edu.cn.
authors.forename: Jian
authors.initials: J
authors.lastname: Gao
authors.forename: You-Yong
authors.initials: YY
authors.lastname: Xu
authors.forename: Hong-Mei
authors.initials: HM
authors.lastname: Yang
authors.forename: Hong
authors.initials: H
authors.lastname: Xu
authors.forename: Feng
authors.initials: F
authors.lastname: Xue
authors.forename: Sha
authors.initials: S
authors.lastname: Li
authors.forename: Xiao-Hai
authors.initials: XH
authors.lastname: Feng
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001709:Biotechnology; D002072:Butylene Glycols / Q000378:metabolism; D003001:Cloning, Molecular; D005285:Fermentation; D005798:Genes, Bacterial; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007444:Inulin / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D056507:Paenibacillus / Q000201:enzymology* / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012988:Soil Microbiology; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D002072:Butylene Glycols; D011994:Recombinant Proteins; C026978:2,3-butylene glycol; D007444:Inulin; D006026:Glycoside Hydrolases; C023086:inulinase