title: Effect of enzyme dehydration on alcalase-catalyzed dipeptide synthesis in near-anhydrous organic media.
abstract: The effect of enzyme dehydration by molecular sieves on the coupling of phenylalanine amide and the carbamoylmethyl ester of N-protected phenylalanine in near-anhydrous tetrahydrofuran was investigated. This coupling was catalyzed by Alcalase covalently immobilized onto macroporous acrylic beads (Cov); these immobilized enzymes were hydrated prior to use. The dehydration kinetics of Cov by molecular sieve powder were determined by incubating Cov with different amounts of molecular sieve powder for different periods of time (0-80 h). Subsequently, the remaining coupling activity of Cov was measured. Dehydration-induced inactivation of Cov by molecular sieve powder was found to occur in three phases: (1) an initial, rapid, major dehydration-induced inactivation that takes place during the first activity measurement, (2) a phase of first-order inactivation, and (3) a plateau phase in activity. These dehydration kinetics were incorporated into a previously found reaction kinetics model. The resulting model was then used to fit progress curve data of the coupling in the presence of different amounts of molecular sieve powder. Upon establishment of parameter values, the model was used to predict independent data sets and found to work well.
authors.affiliation: Bioprocess Engineering, Wageningen University, 6700 EV, Wageningen, The Netherlands. petra.vossenberg@wur.nl
authors.forename: Petra
authors.initials: P
authors.lastname: Vossenberg
authors.forename: Rik
authors.initials: R
authors.lastname: Beeftink
authors.forename: Martien Cohen
authors.initials: MC
authors.lastname: Stuart
authors.forename: Hans
authors.initials: H
authors.lastname: Tramper
mesh_terms: D055162:Biocatalysis; D003681:Dehydration; D004151:Dipeptides / Q000096:biosynthesis*; D005663:Furans / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D013381:Subtilisins / Q000378:metabolism*
chemical_list: D004151:Dipeptides; D005663:Furans; C018674:tetrahydrofuran; D013381:Subtilisins
keywords: dipeptide synthesis; enzyme dehydration; enzyme kinetics; parameter estimation; progress curve analysis