title: Cloning, Expression, Characterization, and Mutagenesis of a Thermostable Exoinulinase From Kluyveromyces cicerisporus.
abstract: Inulinase is an enzyme that belongs to glycoside hydrolase family 32. It converts inulin into high-fructose syrups and fructoligosaccharides, both of which are widely used in pharmaceutical and food industries. In this study, the kcINU1 gene (GenBank accession number AF178979) encoding an exoinulinase was cloned from Kluyveromyces cicerisporus CBS4857 and expressed in Pichia pastoris X-33, yielding a maximum of 45.2 ± 0.6 U mL(-1) of inulinase activity of culture supernatant. The expressed inulinase was purified and characterized. The enzyme had an optimum temperature of 55 °C and an optimum pH of 4.5. It had a K m of 0.322 mM and a V max of 4317 μM min(-1) mg(-1) protein when inulin was used as a substrate. It retained nearly 90 % of the maximal activity after pre-incubation at 50 °C for 1 h or at pH ranging from 3.0 to 6.0 at 4 °C for 24 h, demonstrating that KcINU1 was stable at high temperature and low pH. Moreover, we constructed two KcINU1 mutants, Asp30Ala and Glu215Ala, by site-directed mutagenesis and confirmed via zymogram analysis that Asp-30 and Glu-215 of the enzyme were the catalytic active center. The present study has provided important information for understanding the catalytic mechanism of exoinulinase.
authors.affiliation: Natural Products and Glyco-Biotechnology Research Group, Liaoning Provincial Key Laboratory of Carbohydrates, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, CAS, Dalian, 116023, People's Republic of China.|Medical College of Dalian University, Dalian, 116022, People's Republic of China.|University of Chinese Academy of Sciences, Beijing, 100049, People's Republic of China.
authors.forename: Jun-Yan
authors.initials: JY
authors.lastname: Ma
authors.affiliation: Natural Products and Glyco-Biotechnology Research Group, Liaoning Provincial Key Laboratory of Carbohydrates, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, CAS, Dalian, 116023, People's Republic of China.
authors.forename: Hai-Long
authors.initials: HL
authors.lastname: Cao
authors.affiliation: Natural Products and Glyco-Biotechnology Research Group, Liaoning Provincial Key Laboratory of Carbohydrates, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, CAS, Dalian, 116023, People's Republic of China.
authors.forename: Hai-Dong
authors.initials: HD
authors.lastname: Tan
authors.affiliation: Medical College of Dalian University, Dalian, 116022, People's Republic of China.
authors.forename: Xue-Jun
authors.initials: XJ
authors.lastname: Hu
authors.affiliation: Natural Products and Glyco-Biotechnology Research Group, Liaoning Provincial Key Laboratory of Carbohydrates, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, CAS, Dalian, 116023, People's Republic of China.
authors.forename: Wu-Jun
authors.initials: WJ
authors.lastname: Liu
authors.affiliation: Natural Products and Glyco-Biotechnology Research Group, Liaoning Provincial Key Laboratory of Carbohydrates, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, CAS, Dalian, 116023, People's Republic of China. duyg@dicp.ac.cn.|Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 100190, People's Republic of China. duyg@dicp.ac.cn.
authors.forename: Yu-Guang
authors.initials: YG
authors.lastname: Du
authors.affiliation: Natural Products and Glyco-Biotechnology Research Group, Liaoning Provincial Key Laboratory of Carbohydrates, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, CAS, Dalian, 116023, People's Republic of China. yinheng@dicp.ac.cn.
authors.forename: Heng
authors.initials: H
authors.lastname: Yin
mesh_terms: D003001:Cloning, Molecular; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics*; D007716:Kluyveromyces / Q000235:genetics / Q000378:metabolism*; D016296:Mutagenesis; D013696:Temperature
chemical_list: D006026:Glycoside Hydrolases; C023086:inulinase
keywords: Exoinulinase; Pichia pastoris X-33; Site-directed mutagenesis; Zymogram analysis; kcINU1