title: Sorbitol counteracts temperature- and chemical-induced denaturation of a recombinant α-amylase from alkaliphilic Bacillus sp. TS-23.
abstract: Enzymes are highly complex systems with a substantial degree of structural variability in their folded state. In the presence of cosolvents, fluctuations among vast numbers of folded and unfolded conformations occur via many different pathways; alternatively, certain conformations can be stabilized or destabilized. To understand the contribution of osmolytes to the stabilization of structural changes and enzymatic activity of a truncated Bacillus sp. TS-23 α-amylase (BACΔNC), we monitored amylolytic activity, circular dichroism, and fluorescence as a function of osmolytes. In the presence of trimethylamine N-oxide (TMAO) and sorbitol, BACΔNC activity was retained significantly at elevated temperatures. As compared to the control, the secondary structures of this enzyme were essentially conserved upon the addition of these two kinds of osmolytes. Fluorescence results revealed that the temperature-induced conformational change of BACΔNC was prevented by TMAO and sorbitol. However, glycerol did not provide profound protection against thermal denaturation of the enzyme. Sorbitol was further found to counteract guanidine hydrochloride- and SDS-induced denaturation of BACΔNC. Thus, some well-known naturally occurring osmolytes make a dominant contribution to the stabilization of BACΔNC.
authors.affiliation: Department of Applied Chemistry, National Chiayi University, 300 Syuefu Road, Chiayi County 60004, Taiwan.
authors.forename: Meng-Chun
authors.initials: MC
authors.lastname: Chi
authors.forename: Tai-Jung
authors.initials: TJ
authors.lastname: Wu
authors.forename: Hsing-Ling
authors.initials: HL
authors.lastname: Chen
authors.forename: Huei-Fen
authors.initials: HF
authors.lastname: Lo
authors.forename: Long-Liu
authors.initials: LL
authors.lastname: Lin
mesh_terms: D001407:Bacillus / Q000201:enzymology*; D002942:Circular Dichroism; D004795:Enzyme Stability / Q000187:drug effects; D019791:Guanidine / Q000037:antagonists & inhibitors / Q000494:pharmacology; D006358:Hot Temperature; D008744:Methylamines / Q000494:pharmacology; D009994:Osmolar Concentration; D011489:Protein Denaturation / Q000187:drug effects*; D017433:Protein Structure, Secondary / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013012:Sorbitol / Q000494:pharmacology*; D013050:Spectrometry, Fluorescence; D000516:alpha-Amylases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D008744:Methylamines; D011994:Recombinant Proteins; D012967:Sodium Dodecyl Sulfate; D013012:Sorbitol; D000516:alpha-Amylases; C005855:trimethyloxamine; D019791:Guanidine