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Kinetic analysis of deactivation of immobilized α‐chymotrypsin by water‐miscible organic solvent in kyotorphin synthesis
Author(s) -
Levitsky Vladislav Yu.,
Lozano Pedro,
Iborra José L.
Publication year - 1999
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/(sici)1097-0290(19991020)65:2<170::aid-bit6>3.0.co;2-q
Subject(s) - chemistry , covalent bond , solvent , immobilized enzyme , nucleophile , substrate (aquarium) , adsorption , selectivity , catalysis , derivative (finance) , organic chemistry , enzyme , oceanography , financial economics , economics , geology
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. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 65: 170–175, 1999.