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Enzyme substrate solvent interactions: a case study on serine hydrolases
KTH, School of Biotechnology (BIO), Biochemistry. (Biocatalysis)
2008 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

Reaction rates and selectivities were measured for transacylation of fatty acid esters in solvents catalysed by Candida antarctica lipase B and by cutinase from Humicola insolens. With these enzymes classical water-based enzymology can be expanded to many different solvents allowing large variations in interaction energies between the enzymes, the substrates and the surrounding. Further ,hydrolysis reactions catalysed by Bacillus subtilis esterase 2 were investigated.

Thermodynamics analyses revealed that the enzyme contribution to reaction rate acceleration compared to acid catalysis was purely entropic. On the other hand, studies of differences in activation entropy and enthalpy between enantiomers and between homologous esters showed that high substrate specificity was favoured by enthalpic stabilisation.

Solvent was found to have a profound effect on enzyme catalysis, affecting both reaction rate and selectivity. Differences in substrate solubility will impact enzyme specificity since substrate binding is an equilibrium between enzyme-bound substrate and substrate in free solution. In addition, solven tmolecules were found to act as enzyme inhibitors, showing both competitive and non-competitive behaviour.

In several homologous data series enthalpy-entropy compensation relationships were encountered. A possible extrathermodynamic relationship between enthalpy and entropy can easily be lost under co-varying errors propagated from the experiments. From the data in this thesis, one instance was found of a real enthalpy-entropy compensation that could be distinguished from statistical errors, while other examples could not be verified.

Place, publisher, year, edition, pages
Stockholm: KTH , 2008. , 43 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2008:15
Keyword [en]
lipase, esterase, specificity
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-4867ISBN: 978-91-7415-094-0 (print)OAI: oai:DiVA.org:kth-4867DiVA: diva2:148
Public defence
2008-09-05, Stockholm, 13:00 (English)
Opponent
Supervisors
Note
QC 20100722Available from: 2008-09-10 Created: 2008-09-05 Last updated: 2011-07-07Bibliographically approved
List of papers
1. 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
2. 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
3. Solvent as a competitive inhibitor for Candida antarctica lipase B
Open this publication in new window or tab >>Solvent as a competitive inhibitor for Candida antarctica lipase B
Show others...
2007 (English)In: Biochimica et Biophysica Acta - Proteins and Proteomics, ISSN 1570-9639, E-ISSN 1878-1454, Vol. 1774, no 8, 1052-1057 p.Article in journal (Refereed) Published
Abstract [en]

In enzyme-catalyzed reactions, the choice of solvent often has a marked effect on the reaction outcome. In this paper, it is shown that solvent effects could be explained by the ability of the solvent to act as a competitive inhibitor to the substrate. Experimentally, the effect of six solvents, 2-pentanone, 3-pentanone, 2-methyl-2-pentanol, 3-methyl-3-pentanot, 2-methylpentane and 3-methylpentane, was studied in a solid/gas reactor. As a model reaction, the CALB-catalyzed transacylation between methyl propanoate and I -propanol, was studied. It was shown that both ketones inhibited the enzyme activity whereas the tertiary alcohols and the hydrocarbons did not. Alcohol inhibition constants, K-il were changed to "K-i", determined in presence of 2-pentanone, 3-pentanone, and 3-methyl-3-pentanol, confirmed the marked inhibitory character of the ketones and an absence of inhibition of 3-methyl-3-pentanol. The molecular modeling study was performed on three solvents, 2-pentanone, 2-methyl-2-pentanol and 2-methyl pentane. It showed a clear inhibitory effect for the ketone and the tertiary alcohol, but no effect for the hydrocarbon. No change in enzyme conformation was seen during the simulations. The study led to the conclusion that the effect of added organic component on lipase catalyzed transacylation could be explained by the competitive inhibitory character of solvents towards the first binding substrate methyl propanoate.

Keyword
kinetics, organic solvent, molecular modeling, solid/gas biocatalysis, conformational change, solubility
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-14179 (URN)10.1016/j.bbapap.2007.05.013 (DOI)000249149600012 ()2-s2.0-34547544566 (Scopus ID)
Note
QC 20100722Available from: 2010-07-22 Created: 2010-07-22 Last updated: 2017-12-12Bibliographically approved
4. A water molecule in the stereospecificity pocket of Candida antarctica lipase B enhances enantioselectivity towards pentan-2-ol
Open this publication in new window or tab >>A water molecule in the stereospecificity pocket of Candida antarctica lipase B enhances enantioselectivity towards pentan-2-ol
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2007 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 8, no 6, 662-667 p.Article in journal (Refereed) Published
Abstract [en]

The effect of water activity on enzyme-catalyzed enantioselective transesterification was studied by using a solid/gas reactor. The experimental results were compared with predictions from molecular modelling. The system studied was the esterification of pentan-2-ol with methylpropanoate as acyl donor and lipase B from Candida antarctica as catalyst. The data showed a pronounced water-activity effect on both reaction rote and enantioselectivity. The enantioselectivity increased from 100, at water activity close to zero, to a maximum of 320, at a water activity of 0.2. Molecular modelling revealed how a water molecule could bind in the active site and obstruct the binding of the slowly reacting enantiomer. Measurements of enantioselectivity at different water-activity values and temperatures showed that the water molecule had a high affinity for the stereospecificity pocket of the active site with a binding energy of 9 kJ mol(-1), and that it lost all its degrees of rotation, corresponding to an entropic energy of 37 Jmol(-1)K(-1).

Keyword
enantioselectivity, enzyme catalysis, hydrolases, thermodynamics, water
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-14180 (URN)10.1002/cbic.200600479 (DOI)000245907100015 ()2-s2.0-34250309643 (Scopus ID)
Note
QC 20100722Available from: 2010-07-22 Created: 2010-07-22 Last updated: 2017-12-12Bibliographically approved
5. Understanding promiscuous amidase activity of an esterase from Bacillus subtilis
Open this publication in new window or tab >>Understanding promiscuous amidase activity of an esterase from Bacillus subtilis
Show others...
2008 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 9, no 1, 67-69 p.Article in journal (Refereed) Published
Abstract [en]

Water works. Bacillus subtilis esterase BS2 is a promiscuous esterase that shows amidase activity. This amidase activity was shown to depend on a hydrogen-bond network with the substrate amide hydrogen (indicated by arrow). When this stabilising hydrogen bond network was removed by a point mutation, the amide activity was significantly lowered in comparison with the esterase activity. (Figure Presented)

Keyword
amidases, Bacillus subtilis, catalysis, esterases, molecular modeling
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-14181 (URN)10.1002/cbic.200700521 (DOI)000252292200013 ()
Note
QC 20100722Available from: 2010-07-22 Created: 2010-07-22 Last updated: 2017-12-12Bibliographically approved
6. On the benefit of an active site
Open this publication in new window or tab >>On the benefit of an active site
(English)Manuscript (preprint) (Other academic)
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-14182 (URN)
Note
QC 20100722Available from: 2010-07-22 Created: 2010-07-22 Last updated: 2010-07-22Bibliographically approved

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