Kinetic analysis of deactivation of immobilized alpha-chymotrypsin by water-miscible organic solvent in kyotorphin synthesis

Two different immobilized chymotrypsin derivatives were used to synthesize kyotorphin, using N-benzoyl-L-tyrosine ethyl ester and L-arginine ethyl est er as substrates, in water-DMF media. The first was adsorbed onto Celite pa rticles and the second was multipoint covalently attached into polyacrylami de gel. In all cases, the conversion of the carboxyl substrate was carried out in first-order reaction conditions. For the adsorbed enzyme, the reacti on kinetics deviated from first-order likely due to a fast irreversible ina ctivation of enzyme during the reaction time even at low DMF concentration (15-20% v/v). The covalent attachment of enzyme resulted in elimination of irreversible activity loss by organic solvent up to 60% (v/v) of DMF. The c atalytic activity of the covalent derivative was conserved as appropriate f or performing a synthetic reaction up to 60% v/v of DMF tin comparison to 3 0% v/v for the adsorbed derivative), showing a clear improvement in its sta bility against reversible denaturation by this solvent. The selectivity of the synthetic reaction was slightly enhanced (from 40-50%) with the increas e in DMF concentration to 80% v/v, but it was significantly improved (to 80 %) when L-argininamide was used as nucleophile. (C) 1999 John Wiley & Sons, Inc.