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Enthalpy and Entropy in Enzyme Catalysis: A Study of Lipase Enantioselectivity
KTH, Superseded Departments, Biotechnology.
2001 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

Biocatalysis has become a popular technique in organic synthesis due to high activity and selectivity of enzyme catalyzed reactions. Enantioselectivity is a particularly attractive enzyme property, which is utilized for the production of enantiopure substances. Determination of the temperature dependence of enzyme enantioselectivity allows for thermodynamic analyses that reveal the contribution of differential activation enthalpy, ΔR-SΔH, and entropy, ΔR-SΔS. In the present investigation the influence of substrate structure, variations on enzyme structure and of reaction media on the enantioselectivity of Candida Antarctica lipase B has been studied.

The contribution of enthalpy, ΔR-SΔH, and entropy, TΔR-SΔS, to the differential free energy, ΔR-SΔG, of kinetic resolutions of sec-alcohols were of similar magnitude. Generally the two terms were counteracting, meaning that the enantiomer favored by enthalpy was disfavored by entropy. 3-Hexanol was an exception where the preferred enantiomer was favored both by enthalpy and by entropy. Resolution of 1-bromo-2-butanol revealed non-steric interactions to influence both ΔR-SΔH and ΔR-SΔS. Molecular modeling of the spatial freedom of the enzyme-substrate transition state indicated correlation tothe transition state entropy. The acyl chain length was shown to affect enantioselectivity in transesterifications of a sec-alcohol.

Point mutations in the active site were found to decrease or increase enantioselectivity. The changes were caused by partly compensatory changes in both ΔR-SΔH and ΔR-SΔS. Studies on single and double mutation variants showed that the observed changes were not additive.

Enantioselectivity was strongly affected by the reaction media. Transesterifications of a sec-alcohol catalyzed by Candida Antarctica lipase B was studied in eight liquidorganic solvents and supercritical carbon dioxide. A correlation of enantioselectivity and the molecular volume of the solvent was found.

Differential activation enthalpy, ΔR-SΔH, and entropy, ΔR-SΔS, display a compensatory nature. However this compensation is not perfect, which allows for modifications of enantioselectivity. The components of the thermodynamic parameters are highly complex and interdependent but if their roles are elucidated rational design of enantioselective enzymatic processes may be possible.

 

 

Place, publisher, year, edition, pages
Stockholm: KTH , 2001. , [10], 49 p.
Keyword [en]
Biocatalysis, enzyme catalysis, Candida antarctica lipase B, enantioselectivity, enthalpy, entropy, CALB, enantiomeric ratio
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-3216ISBN: 91-7283-157-X (print)OAI: oai:DiVA.org:kth-3216DiVA: diva2:8994
Public defence
2001-09-28, 00:00
Note
QC 20100616Available from: 2001-09-12 Created: 2001-09-12 Last updated: 2010-07-22Bibliographically approved
List of papers
1. The temperature dependence of enzymatic kinetic resolutions reveals the relative contribution of enthalpy and entropy to enzymatic enantioselectivity
Open this publication in new window or tab >>The temperature dependence of enzymatic kinetic resolutions reveals the relative contribution of enthalpy and entropy to enzymatic enantioselectivity
Show others...
1999 (English)In: Biocatalysis and Biotransformation, ISSN 1024-2422, E-ISSN 1029-2446, Vol. 17, no 1, 61-79 p.Article in journal (Refereed) Published
Abstract [en]

The temperature dependence of the enantioselectivity of several lipase-calalyzed hydrolysis and acylation reactions of racemic esters and alcohols has been determined. From the results we estimated the difference in activation enthalpy (Delta Delta H-# and activation entropy (Delta Delta S-#) for the two enantiomers in the enantioselective reaction step. Contrary to earlier suggestions, we found that the enthalpic and entropic contributions to the enantioselectivity are of similar magnitude. A plot of Delta Delta H-# versus Delta Delta S-#-values of data available in the literature for various enzyme-substrate combinations revealed a tempting correlation between the enthalpic and entropic contributions. This observation would imply enthalpy-entropy compensation to be a general feature of enantioselective enzymatic catalysis. On closer inspection of the data set it was realized that this trend must be considered fortuitous. It originates from the non-random collection of those enzyme-substrate combinations for which the numerical value of the enantiomeric ratio can be measured with a suitable degree of accuracy at ambient temperatures. Indications for the occurrence of genuine enthalpy-entropy compensation, however, have been observed for series of homologous substrates and changes of solvent composition.

National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-13386 (URN)000080165300006 ()
Note
QC 20100616Available from: 2010-06-16 Created: 2010-06-16 Last updated: 2017-12-12Bibliographically approved
2. Influence of acyl chain length on the enantioselectivity of Candida antarctica lipase B and its thermodynamic components in kinetic resolution of sec-alcohols
Open this publication in new window or tab >>Influence of acyl chain length on the enantioselectivity of Candida antarctica lipase B and its thermodynamic components in kinetic resolution of sec-alcohols
2001 (English)In: Journal of Molecular Catalysis B: Enzymatic, ISSN 1381-1177, E-ISSN 1873-3158, Vol. 11, no 4-6, 1025-1028 p.Article in journal (Refereed) Published
Abstract [en]

The enantioselectivity, E, of Candida antarctica lipase B (CALB) was found to be strongly influenced by the chain length of the achiral acyl donor employed in the transesterification of 3-methyl-2-butanol. Of the four studied acyl donors, the longest, vinyl octanoate, afforded the highest enantioselectivity. This was true over the temperature range studied, 6-70 degreesC. Measurements of the temperature dependence of E allows for separation of the enthalpic and entropic components of enantioselectivity. Changes in E with chain length were mainly caused by changes in the entropic component except for the reaction with vinyl propionate, which differed from the others also in the enthalpic component. Optimisation of acyl donor adds one more possibility to improve the yield of enantiopurity in the production of optically active compounds apart from optimisation of solvent, temperature, water activity, and choice of enzyme.

National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-13384 (URN)10.1016/S1381-1177(00)00088-6 (DOI)000167014100113 ()
Note
QC 20100616Available from: 2010-06-16 Created: 2010-06-16 Last updated: 2017-12-12Bibliographically approved
3. Rational design of enantio selective enzymes requires considerations of entropy
Open this publication in new window or tab >>Rational design of enantio selective enzymes requires considerations of entropy
2001 (English)In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 10, no 9, 1769-1774 p.Article in journal (Refereed) Published
Abstract [en]

Entropy was shown to play an equally important role as enthalpy for how enantioselectivity changes when redesigning an enzyme. By studying the temperature dependence of the enantiomeric ratio E of an enantioselective enzyme, its differential activation enthalpy (Delta (R-S)DeltaH(double dagger)) and entropy (Delta (R-S)DeltaS(double dagger)) components can be determined. This was done for the resolution of 3-methyl-2-butanol catalyzed by Candida antarctica lipase B and five variants with one or two point mutations. Delta (R-S)DeltaS(double dagger) was in all cases equally significant as Delta (R-S)DeltaH(double dagger) to E. One variant, T103G, displayed an increase in E, the others a decrease. The altered enantioselectivities of the variants were all related to simultaneous changes in Delta (R-S)DeltaH(double dagger) and Delta (R-S)DeltaS(double dagger). Although the changes in Delta (R-S)DeltaH(double dagger) and Delta (R-S)DeltaS(double dagger) were of a compensatory nature the compensation was not perfect, thereby allowing modifications of E. Both the W104H and the T103G variants displayed larger Delta (R-S)DeltaH(double dagger). than wild type but exhibited a decrease or increase, respectively, in E due to their different relative increase in Delta (R-S)DeltaS(double dagger).

Keyword
enthalpy; entropy; enantiomeric ratio; lipase; Candida antarctica; site-directed mutagenesis
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-13385 (URN)10.1110/ps.13501 (DOI)000170821500007 ()
Note
QC 20100616Available from: 2010-06-16 Created: 2010-06-16 Last updated: 2017-12-12Bibliographically approved
4. Substrate entropy in enzyme enantioselectivity: An experimental and molecular modeling study of a lipase
Open this publication in new window or tab >>Substrate entropy in enzyme enantioselectivity: An experimental and molecular modeling study of a lipase
2002 (English)In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 11, no 6, 1462-1471 p.Article in journal (Refereed) Published
Abstract [en]

The temperature dependence of the enantioselectivity of Candida antarctica lipase B for 3-hexanol, 2-butanol, 3-methyl-2-butanol, 3,3-dimethyl-2-butanol, and 1-bromo-2-butanol revealed that the differential activation entropy, Delta(R-S)Delta(S)(divided bydivided by)., was as significant as the differential activation enthalpy, Delta(R-S)DeltaH(divided bydivided by), to the enantiomeric ratio, E. 1-Bromo-2-butanol, with isosteric substituents, displayed the largest Delta(R-S)DeltaS(divided bydivided by) 3-Hexanol displayed, contrary to other sec-alcohols, a positive Delta(R-S)DeltaS(divided bydivided by). In other words, for 3-hexanol the preferred R-enantiomer is not only favored by enthalpy but also by entropy. Molecular dynamics (MID) simulations and systematic search calculations of the substrate accessible volume within the active site revealed that the (R)-3-hexanol transition state (TS) accessed a larger volume within the active site than the (S)-3-hexanol TS. This correlates well with the hi-her TS entropy of (R)-3-hexanol. In addition, this enantiomer did also yield a higher number of allowed conformations, N, from the systematic search routines, than did the S-enantiomer. The substrate accessible volume was greater for the enantiomer preferred by entropy also for 2-butanol. For 3,3-dimethyl-2-butanol, however, neither MD-simulations nor systematic search calculations yielded substrate accessible volumes that correlate to TS entropy. Ambiguous results were achieved for 3-methyl-2-butanol.

Keyword
molecular dynamics; systematic search; enthalpy; enantiomeric ratio; Candida antarctica
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-13383 (URN)10.1110/ps.3480102 (DOI)000175757900018 ()
Note
QC 20100616Available from: 2010-06-16 Created: 2010-06-16 Last updated: 2017-12-12Bibliographically approved
5. Size as a parameter for solvent effects on Candida antarctica lipase B enantioselectivity
Open this publication in new window or tab >>Size as a parameter for solvent effects on Candida antarctica lipase B enantioselectivity
2002 (English)In: Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology, ISSN 0167-4838, E-ISSN 1879-2588, Vol. 1594, no 2, 325-334 p.Article in journal (Refereed) Published
Abstract [en]

Changes in solvent type were shown to yield significant improvement of enzyme enantioselectivity. The resolution of 3-methyl-2-butanol catalyzed by Candida antarctica lipase B, CALB, was studied in eight liquid organic solvents and supercritical carbon dioxide, SCCO2. Studies of the temperature dependence of the enantiomeric ratio allowed determination of the enthalpic (Delta(R-S)Delta H-double dagger) as well as the entropic (Delta(R-S)Delta S-double dagger) contribution to the overall enantioselectivity (Delta(R-S)Delta G(double dagger) = -RTlnE). A correlation of the enantiomeric ratio, E. to the van der Waals volume of the solvent molecules was observed and suggested as one of the parameters that govern solvent effects on enzyme catalysis. An enthalpy-entropy compensation relationship was indicated between the studied liquid solvents. The enzymatic mechanism must be of a somewhat different nature in SCCO2, as this reaction in this medium did not follow the enthalpy-entropy compensation relation.

Keyword
enantiomeric ratio; enthalpy; entropy; lipase; resolution
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-13382 (URN)10.1016/S0167-4838(01)00324-7 (DOI)000174690100012 ()
Note
QC 20100616Available from: 2010-06-16 Created: 2010-06-16 Last updated: 2017-12-12Bibliographically approved

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