title: Effects of salt and ligand concentrations on the thermal unfolding and refolding of halophilic starch-binding domain from Kocuria varians α-amylase.
abstract: The starch binding domain of α-amlylase from moderate halophile was expressed in E. coli with His tag (His- SBD12) and characterized for its halophilic properties. His-SBD12 was stable up to 35°C and showed binding activity, although at reduced level, to amylose even in the absence of NaCl. Both NaCl and specific ligands exhibited insignificant influence on the secondary structure of His-SBD12, but showed significant stabilization effects against thermal unfolding concentration-dependently, showing its halophilic properties. NaCl increased thermal stability of His-SBD12 by 4°C at 0.2 M and 15°C at 2 M, and enhanced refolding rate by ~7-fold at 0.2 M and ~170-fold at 2 M. Its specific ligands, β- cyclodextrin (at 3 mM) and maltose (at 470 mM), also stabilized the protein by 11° C, most likely reflecting affinity difference between these two ligands. However, they showed marginal effects on refolding rate. These observations suggest that although binding of NaCl and specific ligands to the native structure can explain their stabilization effects on His- SBD12, it is not a sole factor for modulating their effects on folding of His-SBD12.
authors.affiliation: Biochemistry and Applied Biosciences, The United Graduate School of Agricultural Sciences, Kagoshima University, Korimoto, Kagoshima, Japan.
authors.forename: Rui
authors.initials: R
authors.lastname: Yamaguchi
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Arakawa
authors.forename: Hiroko
authors.initials: H
authors.lastname: Tokunaga
authors.forename: Matsujiro
authors.initials: M
authors.lastname: Ishibashi
authors.forename: Masao
authors.initials: M
authors.lastname: Tokunaga
mesh_terms: D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D004795:Enzyme Stability / Q000187:drug effects; D007700:Kinetics; D008024:Ligands; D008320:Maltose / Q000378:metabolism / Q000494:pharmacology; D008835:Micrococcaceae / Q000201:enzymology* / Q000502:physiology; D011485:Protein Binding; D058849:Protein Refolding / Q000187:drug effects*; D017434:Protein Structure, Tertiary; D055049:Salt Tolerance / Q000187:drug effects; D012965:Sodium Chloride / Q000378:metabolism / Q000494:pharmacology*; D013213:Starch / Q000378:metabolism* / Q000494:pharmacology; D013696:Temperature; D000516:alpha-Amylases / Q000737:chemistry* / Q000378:metabolism*; D047392:beta-Cyclodextrins / Q000378:metabolism / Q000494:pharmacology
chemical_list: D008024:Ligands; D047392:beta-Cyclodextrins; D012965:Sodium Chloride; D008320:Maltose; D013213:Starch; D000516:alpha-Amylases