alpha-chymotrypsin and kyotorphin

Two different immobilized chymotrypsin derivatives were used to synthesize kyotorphin, using N-benzoyl-L-tyrosine ethyl ester and L-arginine ethyl ester as substrates, in water-DMF media. The first was adsorbed onto Celite particles and the second was multipoint covalently attached into polyacrylamide gel. In all cases, the conversion of the carboxyl substrate was carried out in first-order reaction conditions. For the adsorbed enzyme, the reaction kinetics deviated from first-order likely due to a fast irreversible inactivation 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 catalytic activity of the covalent derivative was conserved as appropriate for performing a synthetic reaction up to 60% v/v of DMF (in comparison to 30% v/v for the adsorbed derivative), showing a clear improvement in its stability against reversible denaturation by this solvent. The selectivity of the synthetic reaction was slightly enhanced (from 40-50%) with the increase in DMF concentration to 80% v/v, but it was significantly improved (to 80%) when L-argininamide was used as nucleophile.

Topics: Acrylic Resins; Animals; Cattle; Chymotrypsin; Diatomaceous Earth; Dimethylformamide; Dipeptides; Endorphins; Enzyme Stability; Enzymes, Immobilized; Kinetics; Solvents; Tyrosine

Topics: Amino Acid Sequence; Amino Acids; Analgesics; Carboxypeptidases; Chymotrypsin; Dipeptides; Endorphins; Indicators and Reagents; Molecular Sequence Data; Oligopeptides; Peptides

A kinetic analysis of reaction-rate data obtained during a series of optimization experiments of the alpha-chymotrypsin-catalyzed synthesis of kyotorphin has been performed. The kinetic data have been fitted to a model equation derived from a proposed sequential mechanism, which has been further simplified to a first-order equation as a function of the substrate consumption. Statistical tests performed validate the model, since the fitted constants were statistically significant. In addition, the activation energy of the process has been calculated and resulted to be 32.5 +/- 2.3 kJ/mol which is within the range of other enzymatic reactions.

Topics: Chymotrypsin; Endorphins; Kinetics

Regarding the alpha-chymotrypsin-catalyzed synthesis of Z-Tyr-Arg-NH2, the effect on the reaction yield of the following experimental factors is discussed: DMF/buffer proportions, reaction temperature, and donor/nucleophile ratios. The experimental design shows that relatively better yields are obtained by increasing the reaction temperature and lowering the cosolvent proportion while maintaining a slight excess of nucleophile. The ascensional line reaches an optimal response surface very flat voided of a well-defined maximum.

Topics: Analgesics; Chymotrypsin; Endorphins; Indicators and Reagents; Kinetics; Thermodynamics