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Rational redesign of Candida antarctica lipase B
KTH, School of Biotechnology (BIO).
2005 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

This thesis describes the use of rational redesign to modify the properties of the enzyme Candida antarctica lipase B. Through carefully selected single-point mutations, we were able to introduce substrate-assisted catalysis and to alter the reaction specificity. Other single-point mutations afforded variants with greatly changed substrate selectivity and enantioselectivity.

Mutation of the catalytic serine changed the hydrolase activity into an aldolase activity. The mutation decreased the activation energy for aldol addition by 4 kJ×mol-1, while the activation energy increased so much for hydrolysis that no hydrolysis activity could be detected. This mutant can catalyze aldol additions that no natural aldolases can catalyze.

Mutation of the threonine in the oxyanion hole proved the great importance of its hydroxyl group in the transition-state stabilization. The lost transition-state stabilization was partly replaced through substrate-assisted catalysis with substrates carrying a hydroxyl group. The poor selectivity of the wild-type lipase for ethyl 2-hydroxypropanoate (E=1.6) was greatly improved in the mutant (E=22), since only one enantiomer could perform substrate-assisted catalysis.

The redesign of the size of the stereospecificity pocket was very successful. Mutation of the tryptophan at the bottom of this pocket removed steric interactions with secondary alcohols that have to position a substituent larger than an ethyl in this pocket. This mutation increased the activity 5 500 times towards 5-nonanol and 130 000 times towards (S)-1-phenylethanol. The acceptance of such large substituents (butyl and phenyl) in the redesigned stereospecificity pocket increases the utility of lipases in biocatalysis. The improved activity with (S)-1-phenylethanol strongly contributed to the 8 300 000 times change in enantioselectivity towards 1-phenylethanol; example of such a large change was not found in the literature. The S-selectivity of the mutant is unique for lipases. Its enantioselectivity increases strongly with temperature reaching a useful S-selectivity (E=44) at 69 °C.

Thermodynamics analysis of the enantioselectivity showed that the mutation in the stereospecificity pocket mainly changed the entropic term, while the enthalpic term was only slightly affected. This pinpoints the importance of entropy in enzyme catalysis and entropy should not be neglected in rational redesign.

Place, publisher, year, edition, pages
2005.
Keyword [en]
Biochemistry, Candida antarctica lipase B, rational redesign, secondary alcohols, substrate-assisted catalysis, S-selective, entropy, aldolase, stereospecificity pocket, oxyanion hole.
Keyword [sv]
Biokemi
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
URN: urn:nbn:se:kth:diva-186ISBN: 91-7178-012-2 (print)OAI: oai:DiVA.org:kth-186DiVA: diva2:7850
Public defence
2005-05-13, FR4, AlbaNova, Roslagstullsbacken 21, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2005-05-10 Created: 2005-05-10 Last updated: 2012-03-21Bibliographically approved
List of papers
1. Creation of an Enantioselective Hydrolase by Engineered Substrate-Assisted Catalysis
Open this publication in new window or tab >>Creation of an Enantioselective Hydrolase by Engineered Substrate-Assisted Catalysis
2001 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 123, no 18, 4354-4355 p.Article in journal (Refereed) Published
Keyword
CANDIDA-ANTARCTICA, OXYANION HOLE, CHEMICAL MODIFICATION, TRANSITION-STATE, MOLECULAR-BASIS, SIDE-CHAIN, LIPASE-B, SERINE, STABILIZATION, SPECIFICITY
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-5089 (URN)10.1021/ja015604x (DOI)000168495800038 ()
Note
QC 20100907Available from: 2005-05-10 Created: 2005-05-10 Last updated: 2017-12-05Bibliographically approved
2. Carbon-Carbon Bonds by Hydrolytic Enzymes
Open this publication in new window or tab >>Carbon-Carbon Bonds by Hydrolytic Enzymes
Show others...
2003 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 125, no 4, 874-875 p.Article in journal (Refereed) Published
Abstract [en]

Enzymes are efficient catalysts in synthetic chemistry, and their catalytic activity with unnatural substrates in organic reaction media is an area attracting much attention. Protein engineering has opened the possibility to change the reaction specificity of enzymes and allow for new reactions to take place in their active sites. We have used this strategy on the well-studied active-site scaffold offered by the serine hydrolase Candida antarctica lipase B (CALB, EC 3.1.1.3) to achieve catalytic activity for aldol reactions. The catalytic reaction was studied in detail by means of quantum chemical calculations in model systems. The predictions from the quantum chemical calculations were then challenged by experiments. Consequently, Ser105 in CALB was targeted by site-directed mutagenesis to create enzyme variants lacking the nucleophilic feature of the active site. The experiments clearly showed an increased reaction rate when the aldol reaction was catalyzed by the mutant enzymes as compared to the wild-type lipase. We expect that the new catalytic activity, harbored in the stable protein scaffold of the lipase, will allow aldol additions of substrates, which cannot be reached by traditional aldolases

National Category
Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-5090 (URN)10.1021/ja028056b (DOI)000180579600011 ()
Note
QC 20100901Available from: 2005-05-10 Created: 2005-05-10 Last updated: 2017-12-05Bibliographically approved
3. Creating Space for Large Secondary Alcohols by Rational Redesign of Candida antarctica Lipase B
Open this publication in new window or tab >>Creating Space for Large Secondary Alcohols by Rational Redesign of Candida antarctica Lipase B
2005 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 6, no 6, 1051-1056 p.Article in journal (Refereed) Published
Abstract [en]

The active site of Candida antarctica lipase B (CALB) hosts the catalytic triad (Ser-His-Asp), an oxyanion hole and a stereospecificity pocket. During catalysis, the fast-reacting enantiomer of secondary alcohols places its medium-sized substituent in the stereospecificity pocket and its large substituent towards the active-site entrance. The largest group to fit comfortably in the stereospecificity pocket is ethyl, and this restricts the number of secondary alcohols that are good substrates for CALB. In order to overcome this limitation, the size of the stereospecificity pocket was redesigned by changing Trp104. The substrate specificity of the Trp104Ala mutant compared to that of the wild-type lipase increased 270 times towards heptan-4-ol and 5500 times towards nonan-5-ol; this resulted in the high specificity constants 1100 and 830 s(-1)m(-1), respectively. The substrate selectivity changed over 400000 times for nonan-5-ol over propan-2-ol with both Trp104Ala and the Trp104Gln mutations.

Keyword
enzyme catalysis, protein engineering, rational design, specificity constant, substrate specificity
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:kth:diva-5091 (URN)10.1002/cbic.200400410 (DOI)000229730000016 ()2-s2.0-20444448921 (Scopus ID)
Note
QC 20100907. Uppdaterad från In press till Published (20100907)Available from: 2005-05-10 Created: 2005-05-10 Last updated: 2017-12-05Bibliographically approved
4. An S-selective lipase was created by rational redesign and the enantioselectivity increased with temperature
Open this publication in new window or tab >>An S-selective lipase was created by rational redesign and the enantioselectivity increased with temperature
2005 (English)In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 44, no 29, 4582-4585 p.Article in journal (Refereed) Published
Abstract [en]

Higher activity with larger pockets: The figure shows a superposition of intermediates that occur in acyl transfer to (S)-1-phenylethanol catalyzed by Candida antarctica lipase B (CALB). Wild-type CALB cannot accomodate the phenyl group (gray) in the stereospecificity pocket and form all of the catalytically essential H bonds. The Trp 104 Ala mutation liberates the volume in yellow, the S enantiomer is easily fitted, and the specificity constant increases by a factor of 130 000.

Keyword
enantioselectivity, enzyme catalysis, hydrolases, protein engineering, thermodynamics, DYNAMIC KINETIC RESOLUTION, SECONDARY ALCOHOLS, STEREOSELECTIVITY, STEREOCHEMISTRY, RECOGNITION, ENANTIOMERS, CATALYSTS, ENTROPY, CEPACIA
Identifiers
urn:nbn:se:kth:diva-13009 (URN)10.1002/anie.200500971 (DOI)000230737500019 ()2-s2.0-22744445622 (Scopus ID)
Note
QC20100524Available from: 2010-05-24 Created: 2010-05-24 Last updated: 2017-12-12Bibliographically approved

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