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An S-selective lipase was created by rational redesign and the enantioselectivity increased with temperature
KTH, School of Biotechnology (BIO), Biochemistry.
KTH, School of Biotechnology (BIO), Biochemistry.
KTH, School of Biotechnology (BIO), Biochemistry.
KTH, School of Biotechnology (BIO), Biochemistry.
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.

Place, publisher, year, edition, pages
2005. Vol. 44, no 29, 4582-4585 p.
Keyword [en]
enantioselectivity, enzyme catalysis, hydrolases, protein engineering, thermodynamics, DYNAMIC KINETIC RESOLUTION, SECONDARY ALCOHOLS, STEREOSELECTIVITY, STEREOCHEMISTRY, RECOGNITION, ENANTIOMERS, CATALYSTS, ENTROPY, CEPACIA
Identifiers
URN: urn:nbn:se:kth:diva-13009DOI: 10.1002/anie.200500971ISI: 000230737500019Scopus ID: 2-s2.0-22744445622OAI: oai:DiVA.org:kth-13009DiVA: diva2:320246
Note
QC20100524Available from: 2010-05-24 Created: 2010-05-24 Last updated: 2010-10-06Bibliographically approved
In thesis
1. Serine Hydrolase Selectivity: Kinetics and applications in organic and analytical chemistry
Open this publication in new window or tab >>Serine Hydrolase Selectivity: Kinetics and applications in organic and analytical chemistry
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The substrate selectivities for different serine hydrolases were utilized in various applications, presented in papers I-VI. The articles are discussed in the thesis in view of the kinetics of the enzyme catalysis involved.

In paper I the enantioselectivities towards a range of secondary alcohols were reversed for Candida antarctica lipase B by site directed mutagenesis. The thermodynamic components of the enantioselectivity were determined for the mutated variant of the lipase.

In papers II-III Candida antarctica lipase B was engineered for selective monoacylation using two different approaches. A variant of the lipase created for substrate assisted catalysis (paper II) and three different variants with mutations which decreased the volume of the active site (paper III) were evaluated. Enzyme kinetics for the different variants were measured and translated into activation energies for comparison of the approaches.

In papers IV and V three different enzymes were used for rapid analysis of enantiomeric excess and conversion of O-acylated cyanohydrins synthesized by a defined protocol. Horse liver alcohol dehydrogenase, Candida antarctica lipase B and pig liver esterase were sequentially added to a solution containing the O-acylated cyanohydrin. Each enzyme caused a drop in absorbance from oxidation of NADH to NAD+. The product yield and enantiomeric excess was calculated from the relative differences in absorbance.

In paper VI a method for C-terminal peptide sequencing was developed based on conventional Carboxypeptidase Y digestion combined with matrix assisted laser desorption/ionization mass spectrometry. An alternative nucleophile was used to obtain a stable peptide ladder and improve sequence coverage.

Place, publisher, year, edition, pages
Stockholm: KTH, 2010. viii, 62 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2010:12
Keyword
Candida antarctica lipase B, monoacylation of diols, kinetic resolution, thermodynamics in enzyme catalysis, enzyme engineering
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-12831 (URN)978-91-7415-663-8 (ISBN)
Public defence
2010-06-04, FD5, AlbaNova University Center, Roslagstullsbacken 21, Stockholm, 15:42 (English)
Opponent
Supervisors
Note
QC20100629Available from: 2010-05-24 Created: 2010-05-12 Last updated: 2010-06-29Bibliographically approved
2. Rational redesign of Candida antarctica lipase B
Open this publication in new window or tab >>Rational redesign of Candida antarctica lipase B
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.

Keyword
Biochemistry, Candida antarctica lipase B, rational redesign, secondary alcohols, substrate-assisted catalysis, S-selective, entropy, aldolase, stereospecificity pocket, oxyanion hole., Biokemi
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-186 (URN)91-7178-012-2 (ISBN)
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
3. Lipase Specificity and Selectivity: Engineering, Kinetics and Applied Catalysis
Open this publication in new window or tab >>Lipase Specificity and Selectivity: Engineering, Kinetics and Applied Catalysis
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The specificity and selectivity of the enzyme Candida antarctica lipase B (CALB) were studiedfor several substrates and applications.With help of molecular modeling, the active site of CALB was redesigned for the ring openingpolymerization of D,D‐lactide. Two mutants, with about 90‐fold increase in activity ascompared to the wild‐type enzyme, were created. Changing a glutamine into alanineaccounted for this increase in both mutants by creating a larger space in the acyl donorpocket. The new space made it possible to accommodate the bulky substrate and improvethe transition state‐active site complementarity during polymer chain propagation.The enantioselectivity of CALB towards secondary alcohols was engineered by rationalredesign of the stereoselectivity pocket in the enzyme active site. A larger space created by asingle point mutation resulted in an 8’300’000 times change in enantioselectivity towards 1‐phenylethanol and the enantiopreference was inverted into S‐preference. The activitytowards the S‐enantiomer increased 64’000 times in the mutant as related to the wild‐type.The solvent and temperature effects on the enantioselectivity were studied for severalsubstrates and revealed the importance of entropy in the change in enantioselectivity.Substrate selectivity is of great importance for the outcome of enzyme catalyzed polymersynthesis. Ring opening polymerization (ROP) of γ‐acyloxy‐ε‐caprolactones will result in apolyester chain with pendant functional groups. CALB was found to have activity not onlytowards the lactone but also towards the γ‐ester leading to rearrangement of the monomersyielding γ‐acetyloxyethyl‐γ‐butyrolactone. This selectivity between the lactone and the γ‐ester was dependent on the type of group in the γ position and determined the ratio ofpolymerization and rearrangement of the monomers. Molecular dynamics simulations wereused to gain molecular understanding of the selectivity between the lactone and γ‐ester.In order to obtain (meth)acrylate functional polyesters we investigated the use of 2‐hydroxyethyl (meth)acrylate (HEA and HEMA) as initiators for ring opening polymerization.We found that, in addition to the ring opening polymerization activity, CALB catalyzed thetransacylation of the acid moiety of the initiators. The selectivity of CALB towards thedifferent acyl donors in the reaction resulted in a mixture of polymers with different endgroups. A kinetic investigation of the reaction showed the product distribution with timewhen using HEA or HEMA with ε‐caprolactone or ω‐pentadecalactone.The high selectivity of CALB towards lactones over (meth)acrylate esters such as ethyleneglycol di(meth)acrylate was used to design a single‐step route for the synthesis ofdi(meth)acrylated polymers. By mixing ω‐pentadecalactone with the ethylene glycoldi(meth)acrylate and the enzyme in solvent free conditions, we obtained >95 % ofdi(meth)acrylated polypentadecalactone.Taking advantage of the high chemoselectivity of CALB, it was possible to synthesizepolyesters with thiol and/or acrylate functional ends. When using a thioalcohol as initiatorCALB showed high selectivity towards the alcohol group over the thiol group as acyl acceptorfor the ROP reaction. The enzymatic ability of catalyzing simultaneous reactions (ROP andtransacylation) it was possible to develop a single‐step route for the synthesis ofdifunctionalized polyesters with two thiol ends or one thiol and one acrylate end by mixingthe initiator, lactone and a terminator.

Place, publisher, year, edition, pages
Stockholm: KTH, 2010. viii, 48 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2010:17
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-25039 (URN)978-91-7415-729-1 (ISBN)
Public defence
2010-10-22, F3, Lindstedtsvägen 26, KTH, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
QC 20101006Available from: 2010-10-06 Created: 2010-10-06 Last updated: 2010-10-06Bibliographically approved

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