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Effects of subzero temperatures on the kinetics of protease catalyzed dipeptide synthesis in organic media
Author(s) -
Jönsson Åsa,
Adlercreutz Patrick,
Mattiasson Bo
Publication year - 1995
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/bit.260460506
Subject(s) - chemistry , dipeptide , hydrolysis , nucleophile , substrate (aquarium) , aminolysis , acetonitrile , esterase , organic chemistry , catalysis , protease , medicinal chemistry , enzyme , peptide , biochemistry , oceanography , geology
A depeptide synthesis was drastically influenced by the reaction temperature, in the range from −30° to 25°C. This article shows the kinetic reasons of this effect. α‐Chymotrypsin was immobilized on celite and used in four different water‐miscible solvents containing small amounts of water‐miscible solvents containing small amounts of water. The reaction studied was the aminolysis of N ‐acetyl‐ L ‐phenylalanine ethyl ester (Ac‐PheOEt) with L ‐alaninamide (Ala‐NH 2 ) and water for the acylenzyme complex, the nucleophile was favoured by low reaction temperatures. This effect (quantified as p ‐values) was observed in all four solvents, and it was greatest in acetonitrile and tetrahydrofuran. The esterase and amidase activities of the enzyme were studies using AcPheOEt and N ‐acetyl‐ L ‐phenylalanyl‐ L ‐ananinamide (AcPheAla‐NH 2 ) as substrates. The Michaelis–Menten parameters, K m ,app and V max , were determined for ester hydrolysis and dipeptide hydrolysis. Both K m ,app and V max tended to increase with increasing temperature. Secondary hydrolysis was reduced at subzero temperatures because ester hydrolysis was favoured in relation to depeptide hydrolysis. Depeptide synthesis was thus favored by low temperatures in two ways: first, in the competition between the nucleophile and water for the acyl enzyme; and, second, in the competition between the ester substrate and the peptide substrate for the free enzyme. As a result, in acetonitrile containing 10% water, the maximal yield was 99% at −20%C compared with 84% at 25°C. © 1995 John Wiley & Sons, Inc.