Control of enzyme activity and stability in nonaqueous media
(1996)- Abstract
- The use of enzymes in nonaqueous media has considerably expanded their potential areas of application. Limitations however arise that are inherent to the low conformational flexibility of enzymes in dry organic media. Enzyme activity is often unfavorably low due to kinetic barriers. Enzyme behavior, with respect to heat and long-term inactivation, may deviate from their behavior in aqueous media. This study investigates the significance of controlling the water activity, distribution of available water, and dielectric environment of the enzyme. Furthermore, the importance of the accessibility of the substrate to the enzyme is examined.
Increasing hydration of the enzymes, i.e. alpha-chymotrypsin and lipases from... (More) - The use of enzymes in nonaqueous media has considerably expanded their potential areas of application. Limitations however arise that are inherent to the low conformational flexibility of enzymes in dry organic media. Enzyme activity is often unfavorably low due to kinetic barriers. Enzyme behavior, with respect to heat and long-term inactivation, may deviate from their behavior in aqueous media. This study investigates the significance of controlling the water activity, distribution of available water, and dielectric environment of the enzyme. Furthermore, the importance of the accessibility of the substrate to the enzyme is examined.
Increasing hydration of the enzymes, i.e. alpha-chymotrypsin and lipases from Chromobacterium viscosum and Candida rugosa, resulted generally in the enhancement of reaction rates. This enhancement presumably stems from an increase in the local polarity of the enzyme. Polarity could also be tuned by the addition of polar cosolvents to the dry hydrophobic medium. A twofold increase in the rate of the Candida rugosa lipase-catalyzed transesterification reaction was achieved by appropriate additions of N,N-dimethyl acetamide. The rate of the hydrolytic side reaction was maintained low by securing favorable equilibria. Solid additives to the enzyme, such as different salts and sorbitol, improved its performance. Increases in activity of up to eightfold were obtained for alpha-chymotrypsin and Candida antarctica lipase. Buffer salts, i.e. EPPS and sodium phosphate, were particularly successful. These salts are thought not only to preserve the correct ionization state of the macromolecule, but also participate in the dielectric screening of the protein and to serve as immobilization matrices. Spreading the enzyme on a large surface facilitates contact with the substrate. The larger the surface area the more powerful the catalyst, when a series of polyacrylamides wew used for immobilization. Diffusional limitations were less pronounced for the polymers with large area. Immobilization, either on celite or on a sorbitol matrix, afforded an improved operational stability of the catalyst.
The stability of alpha-chymotrypsin preparations against thermal inactivation was found to depend on the water content and its distribution among the components of the enzyme preparation. Control of the water activity was therefore essential. Hydrophilic agents, e.g. salts and sorbitol, increased the water content of the enzyme preparation and suppressed the transition temperature considerably, as determined by means of differential scanning calorimetry. Additions that lead to suppressed hydration of the macromolecule resulted in stabilization of the enzyme preparation at high water activities. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/28769
- author
- Öste Triantafyllou, Angeliki
- supervisor
- opponent
-
- Dr. Carrea, Giacomo, Italy
- publishing date
- 1996
- type
- Thesis
- publication status
- published
- subject
- keywords
- polyacrylamide, polar solvent, lyophilization, immobilization, enzymes in nonaqueous media, differential scanning calorimetry, celite, additives, buffers, salts, sorbitol, Biotechnology, Bioteknik
- pages
- 120 pages
- publisher
- Angeliki Öste Triantafyllou, Dept. of Biotechnology, Lund University, Box 124, 221 00 Lund, Sweden
- defense location
- Center for Chemistry and Chemical Engineering, hall C, Sölvegatan 39, Lund, 13.15 h.,
- defense date
- 1996-10-28 10:15:00
- external identifiers
-
- other:ISRN: LUTKDH/TKBT--96/1031--SE
- language
- English
- LU publication?
- no
- id
- a0e1f68b-7be6-435c-9d08-8c26a9eea2d7 (old id 28769)
- date added to LUP
- 2016-04-04 12:24:04
- date last changed
- 2018-11-21 21:10:44
@phdthesis{a0e1f68b-7be6-435c-9d08-8c26a9eea2d7,
abstract = {{The use of enzymes in nonaqueous media has considerably expanded their potential areas of application. Limitations however arise that are inherent to the low conformational flexibility of enzymes in dry organic media. Enzyme activity is often unfavorably low due to kinetic barriers. Enzyme behavior, with respect to heat and long-term inactivation, may deviate from their behavior in aqueous media. This study investigates the significance of controlling the water activity, distribution of available water, and dielectric environment of the enzyme. Furthermore, the importance of the accessibility of the substrate to the enzyme is examined.<br/><br>
<br/><br>
Increasing hydration of the enzymes, i.e. alpha-chymotrypsin and lipases from Chromobacterium viscosum and Candida rugosa, resulted generally in the enhancement of reaction rates. This enhancement presumably stems from an increase in the local polarity of the enzyme. Polarity could also be tuned by the addition of polar cosolvents to the dry hydrophobic medium. A twofold increase in the rate of the Candida rugosa lipase-catalyzed transesterification reaction was achieved by appropriate additions of N,N-dimethyl acetamide. The rate of the hydrolytic side reaction was maintained low by securing favorable equilibria. Solid additives to the enzyme, such as different salts and sorbitol, improved its performance. Increases in activity of up to eightfold were obtained for alpha-chymotrypsin and Candida antarctica lipase. Buffer salts, i.e. EPPS and sodium phosphate, were particularly successful. These salts are thought not only to preserve the correct ionization state of the macromolecule, but also participate in the dielectric screening of the protein and to serve as immobilization matrices. Spreading the enzyme on a large surface facilitates contact with the substrate. The larger the surface area the more powerful the catalyst, when a series of polyacrylamides wew used for immobilization. Diffusional limitations were less pronounced for the polymers with large area. Immobilization, either on celite or on a sorbitol matrix, afforded an improved operational stability of the catalyst.<br/><br>
<br/><br>
The stability of alpha-chymotrypsin preparations against thermal inactivation was found to depend on the water content and its distribution among the components of the enzyme preparation. Control of the water activity was therefore essential. Hydrophilic agents, e.g. salts and sorbitol, increased the water content of the enzyme preparation and suppressed the transition temperature considerably, as determined by means of differential scanning calorimetry. Additions that lead to suppressed hydration of the macromolecule resulted in stabilization of the enzyme preparation at high water activities.}},
author = {{Öste Triantafyllou, Angeliki}},
keywords = {{polyacrylamide; polar solvent; lyophilization; immobilization; enzymes in nonaqueous media; differential scanning calorimetry; celite; additives; buffers; salts; sorbitol; Biotechnology; Bioteknik}},
language = {{eng}},
publisher = {{Angeliki Öste Triantafyllou, Dept. of Biotechnology, Lund University, Box 124, 221 00 Lund, Sweden}},
title = {{Control of enzyme activity and stability in nonaqueous media}},
year = {{1996}},
}