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Lipase and ω-Transaminase: Biocatalytic Investigations
KTH, Skolan för bioteknologi (BIO), Biokemi.ORCID-id: 0000-0003-2371-8755
2010 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

In a lipase investigation, Candida antarctica lipase B (CALB) are explored for enzyme catalytic promiscuity. Enzyme catalytic promiscuity is shown by enzymes catalyzing alternative catalytic transformations proceeding via different transition state structures than normal. CALB normally performs hydrolysis reactions by activating and coordinating carboxylic acid/ester substrates in an oxyanion hole prior to nucleophilic attack from an active-site serine resulting in acyl enzyme formation. The idea of utilizing the carbonyl activation oxyanion hole in the active-site of CALB to catalyze promiscuous reactions arose by combining catalytic and structural knowledge about the enzyme with chemical imagination. We choose to explore conjugate addition and direct epoxidation activities in CALB by combining molecular modeling and kinetic experiments. By quantum-chemical calculations, the investigated promiscuous reactions were shown to proceed via ordered reaction mechanisms that differ from the native ping pong bi bi reaction mechanism. The investigated promiscuous activities were shown to take place in the enzyme active-site by various kinetic experiments, but despite this, no enantioselectivity was displayed. The reason for this is unknown, but can be a result of a too voluminous active-site or the lack of covalent coordination of the substrates during enzyme-catalysis (Paper I-IV). Combining enzyme structural knowledge with chemical imagination may provide numerous novel enzyme activities to be discovered. In an ω-transaminase investigation, two (S)-selective ω-transaminases from Arthrobacter citreus (Ac-ωTA) and Chromobacterium violaceum (Cv-ωTA) are explored aiming to improve their catalytic properties. Structural knowledge of these enzymes was provided by homology modeling. A homology structure of Ac-ωTA was successfully applied for rational design resulting in enzyme variants with improved enantioselectivity. Additionally, a single-point mutation reversed the enantiopreference of the enzyme from (S) to (R), which was further shown to be substrate dependent (Paper V). A homology structure of Cv-ωTA guided the creation of an enzyme variant showing reduced isopropyl amine inhibition.

Ort, förlag, år, upplaga, sidor
Stockholm: KTH , 2010. , s. 67
Serie
Trita-BIO-Report, ISSN 1654-2312 ; 2010:10
Nyckelord [en]
Candida antarctica lipase B, enzyme catalysis, enzyme catalytic promiscuity, molecular modeling, ω-transaminase
Nationell ämneskategori
Biokemi och molekylärbiologi
Identifikatorer
URN: urn:nbn:se:kth:diva-13279ISBN: 978-91-7415-648-5 (tryckt)OAI: oai:DiVA.org:kth-13279DiVA, id: diva2:323077
Disputation
2010-06-11, Svedbergssalen FD5, Roslagstullsbacken 21, AlbaNova, Stockholm, 10:00 (Engelska)
Opponent
Handledare
Anmärkning
QC20100609Tillgänglig från: 2010-06-09 Skapad: 2010-06-09 Senast uppdaterad: 2010-07-02Bibliografiskt granskad
Delarbeten
1. Exploring the Active-Site of a Rationally Redesigned Lipase for Catalysis of Michael-Type Additions
Öppna denna publikation i ny flik eller fönster >>Exploring the Active-Site of a Rationally Redesigned Lipase for Catalysis of Michael-Type Additions
Visa övriga...
2005 (Engelska)Ingår i: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 6, s. 331-336Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Michael-type additions of various thiols and alpha,beta-unsaturated carbonyl compounds were performed in organic solvent catalyzed by wild-type and a rationally redesigned mutant of Candida antarctica lipase B. The mutant locks the nucleophilic serine 105 in the active-site; this results in a changed catalytic mechanism of the enzyme. The possibility of utilizing this mutant for Michael-type additions was initially explored by quantum-chemical calculations on the reaction between acrolein and methanethiol in a model system. The model system was constructed on the basis of docking and molecular-dynamics simulations and was designed to simulate the catalytic properties of the active site. The catalytic system was explored experimentally with a range of different substrates. The k(cat) values were found to be in the range of 10(-3) to 4 min(-1), similar to the values obtained with aldolase antibodies. The enzyme proficiency was 10(7). Furthermore, the Michael-type reactions followed saturation kinetics and were confirmed to take place in the enzyme active site.

Nyckelord
DENSITY-FUNCTIONAL THEORY; ENZYMATIC-REACTIONS; HYDROLYTIC ENZYMES; ALKALINE PROTEASE; BACILLUS-SUBTILIS; ORGANIC MEDIA; GROUND-STATE; MECHANISM
Identifikatorer
urn:nbn:se:kth:diva-5113 (URN)10.1002/cbic.200400213 (DOI)000226957100014 ()2-s2.0-20544452621 (Scopus ID)
Anmärkning
QC20100609Tillgänglig från: 2005-05-15 Skapad: 2005-05-15 Senast uppdaterad: 2017-12-04Bibliografiskt granskad
2. Fast carbon-carbon bond formation by a promiscuous lipase
Öppna denna publikation i ny flik eller fönster >>Fast carbon-carbon bond formation by a promiscuous lipase
2005 (Engelska)Ingår i: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 127, nr 51, s. 17988-17989Artikel i tidskrift (Refereegranskat) Published
Nyckelord
ACTIVE TRIFLUOROMETHYLATED COMPOUNDS, CATALYZED MICHAEL ADDITION, ORGANIC MEDIA, HYDROLYTIC ENZYMES, 1, 3-DICARBONYL COMPOUNDS, CANDIDA-ANTARCTICA, ALKALINE PROTEASE, BACILLUS-SUBTILIS, BIOCATALYSIS, SITE
Identifikatorer
urn:nbn:se:kth:diva-13271 (URN)10.1021/ja056660r (DOI)000234258700012 ()2-s2.0-29844436474 (Scopus ID)
Anmärkning
QC20100609Tillgänglig från: 2010-06-09 Skapad: 2010-06-09 Senast uppdaterad: 2017-12-12Bibliografiskt granskad
3. Direct Epoxidation in Candida antarctica Lipase B Studied by Experiment and Theory
Öppna denna publikation i ny flik eller fönster >>Direct Epoxidation in Candida antarctica Lipase B Studied by Experiment and Theory
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2008 (Engelska)Ingår i: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 9, nr 15, s. 2443-2451Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Candida antarctica lipase B (CALB) is a promiscuous serine hydrolase that, besides its native function, catalyzes different side reactions, such as direct epoxidation. A single-point mutant of CALB demonstrated a direct epoxidation reaction mechanism for the epoxidation of alpha,beta-unsaturated aldehydes by hydrogen peroxide in aqueous and organic solution. Mutation of the catalytically active Ser105 to alanine made the previously assumed indirect epoxidation reaction mechanism impossible. Gibbs free energies, activation parameters, and substrate selectivities were determined both computationally and experimentally. The energetics and mechanism for the direct epoxidation in CALB Ser105Ala were investigated that the reaction proceeds through a two step-mechanism with formation of an oxyanionic intermediate. The active-site residue His224 functions as a general acid-base catalyst with support from Asp187. Oxyanion stabilization is facilitated by two hydrogen bonds from Thr40.

Nyckelord
catalytic promiscuity, enzyme catalysis, epoxidation, hydrolases, molecular dynamics, HYDROGEN-PEROXIDE, ACTIVE-SITE, PEROXYCARBOXYLIC ACIDS, EFFICIENT GENERATION, AM1-BCC MODEL, CATALYSIS, ALKENES, ENZYMES, OXIDATION, CHLOROPEROXIDASE
Identifikatorer
urn:nbn:se:kth:diva-13270 (URN)10.1002/cbic.200800318 (DOI)000260591100015 ()2-s2.0-54349127157 (Scopus ID)
Anmärkning
QC20100608Tillgänglig från: 2010-06-09 Skapad: 2010-06-09 Senast uppdaterad: 2017-12-12Bibliografiskt granskad
4. Suppressed Native Hydrolytic Activity of a Lipase to Reveal Promiscuous Michael Addition Activity in Water
Öppna denna publikation i ny flik eller fönster >>Suppressed Native Hydrolytic Activity of a Lipase to Reveal Promiscuous Michael Addition Activity in Water
2009 (Engelska)Ingår i: CHEMCATCHEM, ISSN 1867-3880, Vol. 1, nr 2, s. 252-258Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Suppression of,the,native hydrolytic activity of Pseudozyma antarctica lipase B (PalB) (formerly Candida antarctica lipase B) in water is demonstrated. By replacing the catalytic Ser 105 residue with an alanine unit, promiscuous Michael addition activity is favored. A Michael addition reaction between methyl acrylate and acetylacetone was explored as a model system. For the PalB Ser 105 Ala mutant, the hydrolytic activity was suppressed more than 1000 times and at the same time, the Michael addition activity was increased by a factor of 100. Docking studies and molecular dynamics simulations revealed an increased ability of the PalB Ser 105 Ala mutant to harbor the substrates close to a catalytically competent conformation.

Nyckelord
enzyme catalysis, enzyme promiscuity, lipases, Michael addition, molecular modeling, CANDIDA-ANTARCTICA, PYRIMIDINE-DERIVATIVES, CATALYTIC PROMISCUITY, ALKALINE PROTEASE, BACILLUS-SUBTILIS, ORGANIC MEDIA, OLD ENZYMES, FORCE-FIELD, BIOCATALYSIS
Identifikatorer
urn:nbn:se:kth:diva-13269 (URN)10.1002/cctc.200900041 (DOI)000274153900009 ()2-s2.0-77958074145 (Scopus ID)
Anmärkning
QC20100609Tillgänglig från: 2010-06-09 Skapad: 2010-06-09 Senast uppdaterad: 2010-10-29Bibliografiskt granskad
5. Reversed enantiopreference of an ω-transaminase by a single-point mutation
Öppna denna publikation i ny flik eller fönster >>Reversed enantiopreference of an ω-transaminase by a single-point mutation
(Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
Nationell ämneskategori
Medicinsk bioteknologi (med inriktning mot cellbiologi (inklusive stamcellsbiologi), molekylärbiologi, mikrobiologi, biokemi eller biofarmaci)
Identifikatorer
urn:nbn:se:kth:diva-13921 (URN)
Anmärkning
QC20100702Tillgänglig från: 2010-07-02 Skapad: 2010-07-02 Senast uppdaterad: 2010-07-02Bibliografiskt granskad

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