title: Enhanced exo-inulinase activity and stability by fusion of an inulin-binding module.
abstract: In this study, an inulin-binding module from Bacillus macerans was successfully fused to an exo-inulinase from Kluyveromyces marxianus, creating a hybrid functional enzyme. The recombinant exo-inulinase (rINU), the hybrid enzyme (rINUIBM), and the recombinant inulin-binding module (rIBM) were, respectively, heterologously expressed and biochemically characterized. It was found that both the inulinase activity and the catalytic efficiency (k cat/K m(app)) of the rINUIBM were considerably higher than those of rINU. Though the rINU and the rINUIBM shared the same optimum pH of 4.5, the optimum temperature of the rINUIBM (60 °C) was 5 °C higher than that of the rINU. Notably, the fused IBM significantly enhanced both the pH stability and the thermostability of the rINUIBM, suggesting that the rINUIBM obtained would have more extensive potential applications. Furthermore, the fusion of the IBM could substantially improve the inulin-binding capability of the rINUIBM, which was consistent with the determination of the K m(app). This meant that the fused IBM could play a critical role in the recognition of polysaccharides and enhanced the hydrolase activity of the associated inulinase by increasing enzyme-substrate proximity. Besides, the extra supplement of the independent non-catalytic rIBM could also improve the inulinase activity of the rINU. However, this improvement was much better in case of the fusion. Consequently, the IBM could be designated as a multifunctional domain that was responsible for the activity enhancement, the stabilization, and the substrate binding of the rINUIBM. All these features obtained in this study make the rINUIBM become an attractive candidate for an efficient inulin hydrolysis.
authors.affiliation: College of Marine Life Science, Ocean University of China, Yushan Road, No. 5, Qingdao, Shandong, 266003, China.
authors.forename: Shun-Hua
authors.initials: SH
authors.lastname: Zhou
authors.affiliation: College of Marine Life Science, Ocean University of China, Yushan Road, No. 5, Qingdao, Shandong, 266003, China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Liu
authors.affiliation: College of Marine Life Science, Ocean University of China, Yushan Road, No. 5, Qingdao, Shandong, 266003, China.
authors.forename: Yu-Juan
authors.initials: YJ
authors.lastname: Zhao
authors.affiliation: College of Marine Life Science, Ocean University of China, Yushan Road, No. 5, Qingdao, Shandong, 266003, China.
authors.forename: Zhe
authors.initials: Z
authors.lastname: Chi
authors.affiliation: College of Marine Life Science, Ocean University of China, Yushan Road, No. 5, Qingdao, Shandong, 266003, China.
authors.forename: Zhen-Ming
authors.initials: ZM
authors.lastname: Chi
authors.affiliation: College of Marine Life Science, Ocean University of China, Yushan Road, No. 5, Qingdao, Shandong, 266003, China. liugl@ouc.edu.cn.
authors.forename: Guang-Lei
authors.initials: GL
authors.lastname: Liu
mesh_terms: D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007444:Inulin / Q000378:metabolism; D007700:Kinetics; D007716:Kluyveromyces / Q000201:enzymology / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013696:Temperature
chemical_list: D011993:Recombinant Fusion Proteins; D007444:Inulin; D006026:Glycoside Hydrolases; C023086:inulinase
keywords: Exo-inulinase; Inulin-binding capability; Inulin-binding module; Protein engineering; Thermostability; pH stability