title: Characterization and solvent engineering of wheat β-amylase for enhancing its activity and stability.
abstract: The kinetic and thermodynamic parameters of wheat β-amylase (WBA) were characterized and various additives were evaluated for enhancing its activity and thermostability. WBA activity was examined by neocuproine method using soluble starch as substrate. The Michaelis constant (K(m)) and molecular activity (k(cat)) were determined to be 1.0±0.1% (w/v) and 94±3s(-1), respectively, at pH 5.4 and at 25°C. The optimum reaction temperature (T(opt)) for WBA activity was 55°C and the temperature (T(50)) at which it loses half of the activity after 30-min incubation was 50±1°C. Modifications of the solvent with 182mM glycine and 0.18% (w/v) gelatin have increased the T(50) by 5°C. Glycerol, ethylene glycol, dimethylformamide (DMF) and dimethyl sulfoxide have also slightly enhanced the thermostability plausibly through weakening the water structure and decreasing the water shell around the WBA protein. Ethanol and DMF activated WBA by up to 24% at 25°C probably by inducing favorable conformation for the active site or changing the substrate structure by weakening the hydrogen bonding. Its half-life in the inactivation at 55°C was improved from 23 to 48min by 182mM glycine. The thermodynamic parameters indicate that WBA is thermo-labile and sufficient stabilization was achieved through solvent modification with additives and that the heat inactivation of WBA is entropic-driven. It is suggested that WBA could be applied more widely in starch-saccharification industries with employing suitable additives.
authors.affiliation: Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Tadessa
authors.initials: T
authors.lastname: Daba
authors.forename: Kenji
authors.initials: K
authors.lastname: Kojima
authors.forename: Kuniyo
authors.initials: K
authors.lastname: Inouye
mesh_terms: D001709:Biotechnology / Q000379:methods; D004795:Enzyme Stability / Q000187:drug effects; D006358:Hot Temperature; D006868:Hydrolysis; D007700:Kinetics; D012997:Solvents / Q000737:chemistry / Q000494:pharmacology; D013213:Starch / Q000378:metabolism; D013816:Thermodynamics; D014908:Triticum / Q000201:enzymology*; D001614:beta-Amylase / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*
chemical_list: D012997:Solvents; D013213:Starch; D001614:beta-Amylase