EFFECT OF POLYOLS ON ALPHA-CHYMOTRYPSIN THERMOSTABILITY - A MECHANISTIC ANALYSIS OF THE ENZYME STABILIZATION

被引:81
作者
LOZANO, P
COMBES, D
IBORRA, JL
机构
[1] UNIV MURCIA,FAC QUIM,DEPT BIOQUIM & BIOL MOLEC B & IMMUNOL,E-30001 MURCIA,SPAIN
[2] INST NATL SCI APPL,DEPT GEN BIOCHIM & ALIMENTAIRE,CNRS,UA 544,F-31077 TOULOUSE,FRANCE
关键词
ALPHA-CHYMOTRYPSIN; ENZYME STABILITY; POLYOL; ENZYME DEACTIVATION; MECHANISM; SERIES-TYPE; WATER ACTIVITY;
D O I
10.1016/0168-1656(94)90186-4
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
The influence of the synthetic substrate (N-acetyl-L-tyrosine ethyl ester) and the different polyols (ethylene glycol, glycerol, erythritol, xylitol and sorbitol) on the thermostability of alpha-chymotrypsin at 60 degrees C have been studied. The results obtained showed an important stabilizing effect in the presence of both additives. In order to describe the kinetics of enzyme stabilization, the experimental results were analyzed by a four-parameters deactivation model with excellent agreement. In all cases, alpha-chymotrypsin exhibited non-first-order deactivation kinetics, corresponding to a two-step unimolecular mechanism, where the main protective effect of polyols was observed in the first-step of the deactivation profile. Thus, the presence of polyols increased the level of activity stabilization (alpha(1)), and dereased the first-order deactivation rate constant (k(1)). Additionally, the experimental results were analyzed as a function of both, the change in the standard free energy of denaturation (Delta(Delta G degrees)), and a protective effect, defined as the ratio of alpha-chymotrypsin half-lives (with and without polyols), showing in both cases a clear stabilizing effect of these polyhydroxylic cosolvents for the enzyme. The overall protective effect of polyols was also simultaneously related to their concentration and their water-activity depressing power.
引用
收藏
页码:9 / 18
页数:10
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