Control of subtilisin substrate specificity by solvent engineering in organic solvents and supercritical fluoroform

被引:69
作者
Chaudhary, AK
Kamat, SV
Beckman, EJ
Nurok, D
Kleyle, RM
Hajdu, P
Russell, AJ
机构
[1] UNIV PITTSBURGH, CTR BIOTECHNOL & BIOENGN, PITTSBURGH, PA 15219 USA
[2] UNIV PITTSBURGH, DEPT CHEM ENGN, PITTSBURGH, PA 15219 USA
[3] INDIANA UNIV PURDUE UNIV, SCH SCI, DEPT CHEM, INDIANAPOLIS, IN 46202 USA
[4] INDIANA UNIV PURDUE UNIV, SCH SCI, DEPT MATH SCI, INDIANAPOLIS, IN 46202 USA
关键词
D O I
10.1021/ja9622923
中图分类号
O6 [化学];
学科分类号
0703 ;
摘要
We have investigated the effect of physical properties of 30 nonaqueous solvents on the specificity of Subtilisin Carlsberg for nucleophiles in the transesterification of N-acetyl-L-phenylalanine ethyl ester by methanol, 1-propanol, and 1-butanol at fixed thermodynamic water activity. in organic solvents, enzyme activity and nucleophile specificity are solvent-dependent, while in supercritical fluoroform, the activity and specificity are pressure-dependent. Losses in catalytic efficiency and substrate specificity are observed when subtilisin is exposed to hydrophilic organic solvents such as dioxane, tetrahydrofuran, and acetonitrile as compared to hydrophobic solvents (hexane and heptane). Log P is an important descriptor for correlating both the rate and the specificity of deacylation with solvent properties. A linear model of log initial rate against both log P and nonpolar unsaturated area provides the best two-variable fit to the data for solvents of high log P. A nonlinear model of specificity against log P provides the best fit for the complete data set. Correcting the activity for partitioning of nucleophilic substrates shows a similar trend for the intrinsic activity dependence of nucleophiles as a function of log P. In propane, under subcritical conditions, there is no significant effect of pressure on either the activity or the nucleophilic specificity of subtilisin In fluoroform, however, where the physical properties of the solvent are pressure-dependent, the specificity of the enzyme is solvent density-dependent.
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收藏
页码:12891 / 12901
页数:11
相关论文
共 55 条
[11]   A FAST EMPIRICAL-METHOD FOR THE CALCULATION OF MOLECULAR POLARIZABILITY [J].
GLEN, RC .
JOURNAL OF COMPUTER-AIDED MOLECULAR DESIGN, 1994, 8 (04) :457-466
[12]  
GREEN TW, 1981, PROTECTIVE GROUPS OR, P1153
[13]   LYOPHILIZATION-INDUCED REVERSIBLE CHANGES IN THE SECONDARY STRUCTURE OF PROTEINS [J].
GRIEBENOW, K ;
KLIBANOV, AM .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1995, 92 (24) :10969-10976
[14]   THERMODYNAMIC PREDICTIONS FOR BIOCATALYSIS IN NONCONVENTIONAL MEDIA - THEORY, TESTS, AND RECOMMENDATIONS FOR EXPERIMENTAL-DESIGN AND ANALYSIS [J].
HALLING, PJ .
ENZYME AND MICROBIAL TECHNOLOGY, 1994, 16 (03) :178-206
[15]   HIGH-AFFINITY BINDING OF WATER BY PROTEINS IS SIMILAR IN AIR AND IN ORGANIC-SOLVENTS [J].
HALLING, PJ .
BIOCHIMICA ET BIOPHYSICA ACTA, 1990, 1040 (02) :225-228
[16]  
Halling PJ., 1992, Biotechnol Tech, V6, P271, DOI DOI 10.1007/BF02439357
[17]   SPECIFICITIES OF ENZYMES CORRECTED FOR SOLVATION DEPEND ON THE CHOICE OF THE STANDARD STATE [J].
JANSSEN, AEM ;
HALLING, PJ .
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 1994, 116 (22) :9827-9830
[18]   BIOCATALYTIC SYNTHESIS OF ACRYLATES IN ORGANIC-SOLVENTS AND SUPERCRITICAL FLUIDS .1. OPTIMIZATION OF ENZYME ENVIRONMENT [J].
KAMAT, S ;
BARRERA, J ;
BECKMAN, EJ ;
RUSSELL, AJ .
BIOTECHNOLOGY AND BIOENGINEERING, 1992, 40 (01) :158-166
[19]   BIOCATALYTIC SYNTHESIS OF ACRYLATES IN SUPERCRITICAL FLUIDS - TUNING ENZYME-ACTIVITY BY CHANGING PRESSURE [J].
KAMAT, SV ;
IWASKEWYCZ, B ;
BECKMAN, EJ ;
RUSSELL, AJ .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1993, 90 (07) :2940-2944
[20]   CONTROL OF ENZYME ENANTIOSELECTIVITY WITH PRESSURE CHANGES IN SUPERCRITICAL FLUOROFORM [J].
KAMAT, SV ;
BECKMAN, EJ ;
RUSSELL, AJ .
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 1993, 115 (19) :8845-8846