title: Stabilization of invertase by molecular engineering.
abstract: Extracellular invertase (EC 3.2.1.26) of Saccharomyces cerevisiae was stabilized against thermal denaturation by intermolecular and intramolecular crosslinking of the surface nucleophilic functional groups with diisocyanate homobifunctional reagents (O==C==N(CH(2))(n)N==C==O) of various lengths (n = 4, 6, 8). Crosslinking with 1,4-diisocyanatobutane (n = 4) proved most effective in enhancing thermostability. Stability was improved dramatically by crosslinking 0.5 mg/mL of protein with 30 mumol/mL of the reagent. Molecular engineering by crosslinking reduced the first-order thermal denaturation constant at 60 degrees C from 1.567 min(-1) (for the native enzyme) to 0.437 min(-1) (for the stabilized enzyme). Similarly, the best crosslinking treatment increased the activation energy for denaturation from 391 kJ mol(-1) (for the native protein) to 466 kJ mol(-1) (for the stabilized enzyme). Crosslinking was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis.
authors.affiliation: School of Engineering, Massey University, Palmerston North, New Zealand.
authors.forename: Pattamawadee
authors.initials: P
authors.lastname: Tananchai
authors.forename: Yusuf
authors.initials: Y
authors.lastname: Chisti
mesh_terms: D002616:Chemical Engineering; D003432:Cross-Linking Reagents / Q000737:chemistry* / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability / Q000187:drug effects; D006358:Hot Temperature; D011489:Protein Denaturation / Q000187:drug effects; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D043324:beta-Fructofuranosidase / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D003432:Cross-Linking Reagents; D043324:beta-Fructofuranosidase