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Aldol Additions with Mutant Lipase: Analysis by Experiments and Theoretical Calculations
KTH, Tidigare Institutioner                               , Bioteknologi.
KTH, Tidigare Institutioner                               , Kemi.
KTH, Tidigare Institutioner                               , Bioteknologi.
KTH, Tidigare Institutioner                               , Kemi.ORCID-id: 0000-0003-2673-075X
Vise andre og tillknytning
2004 (engelsk)Inngår i: Journal of Molecular Catalysis B: Enzymatic, ISSN 1381-1177, E-ISSN 1873-3158, Vol. 31, nr 4-6, s. 123-128Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

A Ser105Ala mutant of Candida antarctica lipase B has previously been shown to catalyze aldol additions. Quantum chemical calculations predicted a reaction rate similar to that of natural enzymes, whereas experiments showed a much lower reaction rate. Molecular dynamics simulations, presented here, show that the low reaction rate is a consequence of the low frequencies of near attack complexes in the enzyme. Equilibrium was also considered as a reason for the slow product formation, but could be excluded by performing a sequential reaction to push the reaction towards product formation. In this paper, further experimental results are also presented, highlighting the importance of the entire active site for catalysis.

sted, utgiver, år, opplag, sider
2004. Vol. 31, nr 4-6, s. 123-128
Emneord [en]
Ab initio calculations; Aldolase; Lipase; Oxyanion hole; Reaction specificity
HSV kategori
Identifikatorer
URN: urn:nbn:se:kth:diva-5112DOI: 10.1016/j.molcatb.2004.08.005ISI: 000225517400008Scopus ID: 2-s2.0-8644236642OAI: oai:DiVA.org:kth-5112DiVA, id: diva2:7879
Merknad
QC 20100827Tilgjengelig 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

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