Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Increased activity of enzymatic transacylation of acrylates through rational design of lipases
KTH, School of Biotechnology (BIO), Biochemistry. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.ORCID iD: 0000-0002-4066-2776
KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
Show others and affiliations
2010 (English)In: Journal of Molecular Catalysis B: Enzymatic, ISSN 1381-1177, E-ISSN 1873-3158, Vol. 65, no 1-4, 3-10 p.Article in journal (Refereed) Published
Abstract [en]

A rational design approach was used to create the mutant Candida antarctica lipase B (CALB, also known as Pseudozyma antarctica lipase B) V190A having a k(cat) three times higher compared to that of the wild type (wt) enzyme for the transacylation of the industrially important compound methyl methacrylate. The enzymatic contribution to the transacylation of various acrylates and corresponding saturated esters was evaluated by comparing the reaction catalysed by CALB wt with the acid (H2SO4) catalysed reaction. The performances of CALB wt and mutants were compared to two other hydrolases, Humicola insolens cutinase and Rhizomucor mihei lipase. The low reaction rates of enzyme catalysed transacylation of acrylates were found to be caused mainly by electronic effects due to the double bond present in this class of molecules. The reduction in rate of enzyme catalysed transacylation of acrylates compared to that of the saturated ester methyl propionate was however less than what could be predicted from the energetic cost of breaking the pi-system of acrylates solely. The nature and concentration of the acyl acceptor was found to have a profound effect on the reaction rate. (C) 2009 Elsevier B.V. All rights reserved.

Place, publisher, year, edition, pages
2010. Vol. 65, no 1-4, 3-10 p.
Keyword [en]
Lipase, CALB, Point mutations, Kinetics, Proficiency
National Category
Biochemistry and Molecular Biology Biochemistry and Molecular Biology Physical Chemistry
Identifiers
URN: urn:nbn:se:kth:diva-29688DOI: 10.1016/j.molcatb.2009.11.016ISI: 000278926300002Scopus ID: 2-s2.0-77952673518OAI: oai:DiVA.org:kth-29688DiVA: diva2:398558
Note
QC 20110218Available from: 2011-02-18 Created: 2011-02-11 Last updated: 2017-12-11Bibliographically approved
In thesis
1. On electrostatic effects, minimal motion and other catalytic strategies used by enzymes
Open this publication in new window or tab >>On electrostatic effects, minimal motion and other catalytic strategies used by enzymes
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Enzymes are powerful biocatalysts that provide rate accelerations of up to 1019 fold compared to the corresponding uncatalyzed reaction in solution. The origin of the remarkable performance displayed by enzymes has fascinated and puzzled researchers for over a hundred years. It is clear that the catalytic effect is a consequence of the higher degree of transition state stabilization for the enzyme catalyzed reaction compared to the corresponding uncatalyzed reaction. It is still not well understood exactly how this transition state stabilization occurs and the relative importance of various catalytic effects are discussed. Catalytic effects involving electrostatics, near attack conformers, dynamic effects and an economy in atomic motion are discussed in this thesis.

The importance of electrostatic effects is corroborated in this thesis. A single hydrogen bond in transition state constitutes an important difference between amidases and esterases. A hydrogen bond in transition state is found in all sixteen analyzed amidases representing ten different reaction mechanisms and eleven different folding families. The hydrogen bond is shown to be either substrate assisted or enzyme assisted. The role of this hydrogen bond is to assist nitrogen inversion in amidases. Esterases lack this interaction in transition state and therefore they are very poor catalysts in the hydrolysis of amides. Electrostatic interactions are found to facilitate proton transfer that enhances the rate of lipase catalyzed N-acylation of amino alcohols.

In this thesis electrostatic effects in the substrate are shown to be important for the lipase catalyzed transacylation of acrylates The α,β-double bond present in acrylates introduce electronic effects that has the consequence of restricting the conformational freedom of the substrate in its ground state to two flat conformations, s-cis and s-trans. It is shown that acrylates form near attack conformers (NACs) from their ground state s-cis/s-trans planar conformations. The ability of the enzyme to accommodate such apparent s-cis/s-trans substrate conformations dictates the probability to form productive transition states and thus the reaction rate.

Dynamic effects are important in enzymes. In this thesis it is found that a point mutation increases the flexibility of a neighbouring residue in Candida antarctica lipase B. This allows the mutated enzyme to explore conformations not accessible for the wild-type enzyme. The dynamics has the effect to decrease steric interactions in transition state with concomitant rate increase for the transacylation of methyl methacrylate.

In this thesis an economy of atomic motion during enzyme catalysis is observed. Nitrogen inversion in amidases constitutes an interesting example. A rotation as part of the reaction mechanism for amide bond hydrolysis would involve much more motion.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2011. 42 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 14
National Category
Biochemistry and Molecular Biology
Research subject
SRA - Molecular Bioscience
Identifiers
urn:nbn:se:kth:diva-33334 (URN)978-91-7415-976-9 (ISBN)
Public defence
2011-05-27, D2, Lindstedtsvägen 5, Stockholm, 13:00 (English)
Opponent
Supervisors
Note
QC 20110512Available from: 2011-05-12 Created: 2011-05-03 Last updated: 2011-05-12Bibliographically approved

Open Access in DiVA

No full text

Other links

Publisher's full textScopus

Authority records BETA

Syren, Per-Olof

Search in DiVA

By author/editor
Syren, Per-OlofLindgren, EbbaHult, Karl
By organisation
BiochemistryAlbanova VinnExcellence Center for Protein Technology, ProNova
In the same journal
Journal of Molecular Catalysis B: Enzymatic
Biochemistry and Molecular BiologyBiochemistry and Molecular BiologyPhysical Chemistry

Search outside of DiVA

GoogleGoogle Scholar

doi
urn-nbn

Altmetric score

doi
urn-nbn
Total: 112 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf