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Exploring the Active-Site of a Rationally Redesigned Lipase for Catalysis of Michael-Type Additions
KTH, Skolan för kemivetenskap (CHE), Kemi, Fysikalisk kemi.
KTH, Skolan för bioteknologi (BIO), Biokemi.ORCID-id: 0000-0003-2371-8755
KTH, Skolan för bioteknologi (BIO), Biokemi.
KTH, Skolan för bioteknologi (BIO), Biokemi.
Vise andre og tillknytning
2005 (engelsk)Inngår i: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 6, s. 331-336Artikkel i tidsskrift (Fagfellevurdert) 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.

sted, utgiver, år, opplag, sider
2005. Vol. 6, s. 331-336
Emneord [en]
DENSITY-FUNCTIONAL THEORY; ENZYMATIC-REACTIONS; HYDROLYTIC ENZYMES; ALKALINE PROTEASE; BACILLUS-SUBTILIS; ORGANIC MEDIA; GROUND-STATE; MECHANISM
Identifikatorer
URN: urn:nbn:se:kth:diva-5113DOI: 10.1002/cbic.200400213ISI: 000226957100014Scopus ID: 2-s2.0-20544452621OAI: oai:DiVA.org:kth-5113DiVA, id: diva2:7880
Merknad
QC20100609Tilgjengelig fra: 2005-05-15 Laget: 2005-05-15 Sist oppdatert: 2017-12-04bibliografisk kontrollert
Inngår i avhandling
1. Exploiting enzyme promiscuity for rational design
Åpne denne publikasjonen i ny fane eller vindu >>Exploiting enzyme promiscuity for rational design
2005 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

Enzymes are today well recognized in various industrial applications, being an important component in detergents, and catalysts in the production of agrochemicals, foods, pharmaceuticals, and fine chemicals. Their large use is mainly due to their high selectivity and environmental advantage, compared to traditional catalysts. Tools and techniques in molecular biology offer the possibility to screen the natural sources and engineer new enzyme activities which further increases their usefulness as catalysts, in a broader area.

Although enzymes show high substrate and reaction selectivity many enzymes are today known to catalyze other reactions than their natural ones. This is called enzyme promiscuity. It has been suggested that enzyme promiscuity is Nature’s way to create diversity. Small changes in the protein sequence can give the enzyme new reaction specificity.

In this thesis I will present how rational design, based on molecular modeling, can be used to explore enzyme promiscuity and to change the enzyme reaction specificity. The first part of this work describes how Candida antarctica lipase B (CALB), by a single point mutation, was mutated to give increased activity for aldol additions, Michael additions and epoxidations. The activities of these reactions were predicted by quantum chemical calculations, which suggested that a single-point mutant of CALB would catalyze these reactions. Hence, the active site of CALB, which consists of a catalytic triad (Ser, His, Asp) and an oxyanion hole, was targeted by site-directed mutagenesis and the nucleophilic serine was mutated for either glycine or alanine. Enzymes were expressed in Pichia pastoris and analyzed for activity of the different reactions. In the case of the aldol additions the best mutant showed a four-fold initial rate over the wild type enzyme, for hexanal. Also Michael additions and epoxidations were successfully catalyzed by this mutant.

In the last part of this thesis, rational design of alanine racemase from Geobacillus stearothermophilus was performed in order to alter the enzyme specificity. Active protein was expressed in Escherichia coli and analyzed. The explored reaction was the conversion of alanine to pyruvate and 2-butanone to 2-butylamine. One of the mutants showed increased activity for transamination, compared to the wild type.

sted, utgiver, år, opplag, sider
Stockholm: KTH, 2005. s. 58
Emneord
Biochemistry, Candida antarctica lipase B, alanine racemase, substrate specificity, reaction specificity, enzyme catalytic promiscuity, Biokemi
HSV kategori
Identifikatorer
urn:nbn:se:kth:diva-199 (URN)91-7178-008-4 (ISBN)
Disputas
2005-05-20, Oskar Kleins auditorium, AlvaNova, 13:00 (engelsk)
Opponent
Veileder
Merknad
QC 20100929Tilgjengelig fra: 2005-05-15 Laget: 2005-05-15 Sist oppdatert: 2010-09-29bibliografisk kontrollert
2. Lipase and ω-Transaminase: Biocatalytic Investigations
Åpne denne publikasjonen i ny fane eller vindu >>Lipase and ω-Transaminase: Biocatalytic Investigations
2010 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
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.

sted, utgiver, år, opplag, sider
Stockholm: KTH, 2010. s. 67
Serie
Trita-BIO-Report, ISSN 1654-2312 ; 2010:10
Emneord
Candida antarctica lipase B, enzyme catalysis, enzyme catalytic promiscuity, molecular modeling, ω-transaminase
HSV kategori
Identifikatorer
urn:nbn:se:kth:diva-13279 (URN)978-91-7415-648-5 (ISBN)
Disputas
2010-06-11, Svedbergssalen FD5, Roslagstullsbacken 21, AlbaNova, Stockholm, 10:00 (engelsk)
Opponent
Veileder
Merknad
QC20100609Tilgjengelig fra: 2010-06-09 Laget: 2010-06-09 Sist oppdatert: 2010-07-02bibliografisk kontrollert
3. Exploring Conjugate Addition Activity in Pseudozyma antarctica Lipase B
Åpne denne publikasjonen i ny fane eller vindu >>Exploring Conjugate Addition Activity in Pseudozyma antarctica Lipase B
2009 (engelsk)Licentiatavhandling, med artikler (Annet vitenskapelig)
Abstract [en]

Multifunctional enzymes have alternative functions or activities, known as “moonlighting” or “promiscuous”, which are often hidden behind a native enzyme activity and therefore only visible under special environmental conditions. In this thesis, the active-site of Pseudozyma (formerly Candida) antarctica lipase B was explored for a promiscuous conjugate addition activity. Pseudozyma antarctica lipase B is a lipase industrially used for hydrolysis or transacylation reactions. This enzyme contains a catalytic triad, Ser105-His224-Asp187, where a nucleophilic attack from Ser105 on carboxylic acid/ester substrates cause the formation of an acyl enzyme. For conjugate addition activity in Pseudozyma antarctica lipase B, replacement of Ser105 was assumed necessary to prevent competing hemiacetal formation. However, experiments revealed conjugate addition activity in both wild-type enzyme and the Ser105Ala variant. Enzyme-catalyzed conjugate additions were performed by adding sec-amine, thiols or 1,3-dicarbonyl compounds to various α,β-unsaturated carbonyl compounds in both water or organic solvent. The reactions followed Michaelis-Menten kinetics and the native ping pong bi bi reaction mechanism of Pseudozyma antarctica lipase B for hydrolysis/transacylation was rerouted to a novel ordered bi uni reaction mechanism for conjugate addition (Paper I, II, III). The lipase hydrolysis activity was suppressed more than 1000 times by the replacement of the nucleophilic Ser105 to Ala (Paper III).

sted, utgiver, år, opplag, sider
Stockholm: KTH, 2009. s. 42
Serie
Trita-BIO-Report, ISSN 1654-2312 ; 2009:20
Serie
TRITA-BIO-Report 2009:20, ISSN 1654-2312
Emneord
Candida antarctica lipase B · conjugate addition · enzyme catalysis · enzyme catalytic promiscuity · molecular modeling
HSV kategori
Identifikatorer
urn:nbn:se:kth:diva-11598 (URN)978-91-7415-435-1 (ISBN)
Presentation
2009-10-16, M22, Brinellvägen 64, KTH, 10:00 (engelsk)
Opponent
Veileder
Tilgjengelig fra: 2009-11-30 Laget: 2009-11-23 Sist oppdatert: 2010-10-29bibliografisk kontrollert

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