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Stabilization of invertase by molecular engineering
Author(s) -
Tananchai Pattamawadee,
Chisti Yusuf
Publication year - 2009
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1002/btpr.314
Subject(s) - thermostability , denaturation (fissile materials) , chemistry , sodium dodecyl sulfate , invertase , reagent , thermal stability , gel electrophoresis , polyacrylamide gel electrophoresis , enzyme , chromatography , polymer chemistry , nuclear chemistry , organic chemistry , biochemistry
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 (OCN(CH 2 ) n NCO) 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 μmol/mL of the reagent. Molecular engineering by crosslinking reduced the first‐order thermal denaturation constant at 60°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. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010