| 1. |
- Larsen, Marianne Wittrup, et al.
(författare)
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Suppression of Water as a Nucleophile in Candida antarctica Lipase B Catalysis
- 2010
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Ingår i: ChemBioChem. - : Wiley. - 1439-4227 .- 1439-7633. ; 11:6, s. 796-801
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Tidskriftsartikel (refereegranskat)abstract
- A water tunnel in Candida antarctica lipase B that provides the active site with substrate water is hypothesized. A small, focused library created in order to prevent water from entering the active site through the tunnel was screened for increased transacylation over hydrolysis activity. A single mutant, S47L, in which the inner part of the tunnel was blocked, catalysed the transacylation of vinyl butyrate to 20 mm butanol 14 times faster than hydrolysis. The single mutant Q46A, which has a more open outer end of the tunnel, showed an increased hydrolysis rate and a decreased hydrolysis to transacylation ratio compared to the wild-type lipase. Mutants with a blocked, tunnel could be very useful in applications in which hydrolysis is unwanted, such as the acylation of highly hydrophilic compounds in the presence of water.
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| 2. |
- Marton, Z., et al.
(författare)
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Mutations in the stereospecificity pocket and at the entrance of the active site of Candida antarctica lipase B enhancing enzyme enantioselectivity
- 2010
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Ingår i: Journal of Molecular Catalysis B. - : Elsevier BV. - 1381-1177 .- 1873-3158. ; 65:1-4, s. 11-17
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Tidskriftsartikel (refereegranskat)abstract
- Two different parts of Candida antarctica lipase B (stereospecificity pocket at the bottom of the active site and hydrophobic tunnel leading to the active site) were redesigned by single- or double-point mutations, in order to better control and improve enzyme enantioselectivity toward secondary alcohols. Single-point isosteric mutations of Ser47 and Thr42 situated in the stereospecificity pocket gave rise to variants with doubled enantioselectivity toward pentan-2-ol, in solid/gas reactor. Besides, the width and shape of the hydrophobic tunnel leading to the active site was modified by producing the following single-point mutants: Ile189Ala, Leu278Val and Ala282Leu. For each of these variants a significant modification of enantioselectivity was observed compared to wild-type enzyme, indicating that discrimination of the enantiomers by the enzyme could also arise from their different accessibilities from the enzyme surface to the catalytic site. (C) 2010 Elsevier B.V. All rights reserved.
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| 3. |
- Syren, Per-Olof, et al.
(författare)
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Increased activity of enzymatic transacylation of acrylates through rational design of lipases
- 2010
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Ingår i: Journal of Molecular Catalysis B. - : Elsevier BV. - 1381-1177 .- 1873-3158. ; 65:1-4, s. 3-10
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Tidskriftsartikel (refereegranskat)abstract
- A rational design approach was used to create the mutant Candida antarctica lipase B (CALB, also known as Pseudozyma antarctica lipase B) V190A having a k(cat) three times higher compared to that of the wild type (wt) enzyme for the transacylation of the industrially important compound methyl methacrylate. The enzymatic contribution to the transacylation of various acrylates and corresponding saturated esters was evaluated by comparing the reaction catalysed by CALB wt with the acid (H2SO4) catalysed reaction. The performances of CALB wt and mutants were compared to two other hydrolases, Humicola insolens cutinase and Rhizomucor mihei lipase. The low reaction rates of enzyme catalysed transacylation of acrylates were found to be caused mainly by electronic effects due to the double bond present in this class of molecules. The reduction in rate of enzyme catalysed transacylation of acrylates compared to that of the saturated ester methyl propionate was however less than what could be predicted from the energetic cost of breaking the pi-system of acrylates solely. The nature and concentration of the acyl acceptor was found to have a profound effect on the reaction rate. (C) 2009 Elsevier B.V. All rights reserved.
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| 4. |
- Berglund, Per, et al.
(författare)
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Switched enantiopreference of Humicola lipase for 2-phenoxyalkanoic acid ester homologs can be rationalized by different substrate binding modes
- 1999
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Ingår i: Tetrahedron. - 0957-4166 .- 1362-511X. ; 10:21, s. 4191-4202
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Tidskriftsartikel (refereegranskat)abstract
- Humicola lanuginosa lipase was used for enantioselective hydrolyses of a series of homologous 2-phenoxyalkanoic acid ethyl esters. The enantioselectivity (E-value) of the enzyme changed from an (R)-enantiomer preference for the smallest substrate, 2-phenoxypropanoic acid ester, to an (S)-enantiomer preference for the homologous esters with longer acyl moieties. The E-values span the range from E=13 (R) to E=56 (S). A molecular modeling study identified two different substrate-binding modes for each enantiomer. We found that the enantiomers favored different modes. This discovery provided a model that offered a rational explanation for the observed switch in enantioselectivity. (C) 1999 Elsevier Science Ltd. All rights reserved.
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| 5. |
- Bernhardt, Peter, et al.
(författare)
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Molecular basis of perhydrolase activity in serine hydrolases
- 2005
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Ingår i: Angewandte Chemie International Edition. - : Wiley. - 1433-7851 .- 1521-3773. ; 44:18, s. 2742-2746
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Tidskriftsartikel (refereegranskat)abstract
- (Chemical Equation Presented) Changing substrates: A mutation that forms a cis-proline-peptide bond in a loop structure close to the active site of an aryl esterase from Pseudomonas fluorescens converts the enzyme into a perhydrolase (see picture). The switch in activity is explained by a new hydrogen bond formed between a backbone carbonyl oxygen atom and the peroxy deacylation intermediate.
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| 6. |
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| 7. |
- Eriksson, Magnus, et al.
(författare)
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Enzymatic One-Pot Route to Telechelic Polypentadecalactone Epoxide : Synthesis, UV Curing, and Characterization
- 2009
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Ingår i: Biomacromolecules. - : American Chemical Society (ACS). - 1525-7797 .- 1526-4602. ; 10:11, s. 3108-3113
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Tidskriftsartikel (refereegranskat)abstract
- In an enzymatic one-pot procedure immobilized lipase B from Candida antarctica was used to synthesize semicrystalline diepoxy functional macromonomers based on glycidol, pentadecalactone, and adipic acid. By changing the stoichiometry of the building blocks. macromonomers of controlled molecular weight front 1400 to 2700 g mol(-1) could be afforded. The enzyme-catalyzed reaction went to completion (conversion >= 95%) within 24 h at 60 degrees C. After removal of the enzyme, the produced macromonomers were used for photopolymerization without any purification. The macromonomers readily copolymerized cationically with a cycloaliphatic diepoxide (Cyracure UVR-6110; CA-dE) to high conversion. The cross-linked copolymers formed a durable film with a degree of crystallinity depending on the macromonomer size and amount of CA-dE used, without CA-dE the macromonomers homopolymerized only to a low degree. Combined with CA-dE conversions of 85-90% were determined by FT-Raman spectroscopy. The films became more durable once reinforced with CA-dE, increasing the cross-link density and reducing the crystallinity of the PDL segments in the films.
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| 8. |
- Fransson, Linda, 1971-
(författare)
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Molecular modelling - understanding and prediction of enzyme selectivity.
- 2009
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Molecular modelling strategies for evaluation of enzyme selectivity wereinvestigated with a focus on principles of how molecular interactionscould be evaluated to provide information about selectivity. Althoughmolecular modelling provides tools for evaluation of geometrical andenergy features of molecular systems, no general strategies for evaluationof enzyme selectivity exist. Geometrical analyses can be based uponinspection and reasoning about molecular interactions, which provide aneasily accessible way to gain information, but suffer from the risk of biasput in by the modeller. They can also be based on geometrical features ofmolecular interactions such as bond lengths and hydrogen-bond formation.Energy analyses are appealing for their modeller independenceand for the possibility to predict not only stereopreference, but also itsmagnitude.In this thesis, four examples of enantio- or regioselective serinehydrolase-catalysed reaction systems are presented together with developedmodelling protocols for explanation, prediction or enhancement ofselectivity. Geometrical as well as energy-based methodology were used,and provided an understanding of the structural basis of enzymeselectivity. In total, the protocols were successful in making qualitative explanationsand predictions of stereoselectivity, although quantitative determinationswere not achieved.
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| 9. |
- Gardossi, Lucia, et al.
(författare)
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Guidelines for reporting of biocatalytic reactions
- 2010
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Ingår i: Trends in Biotechnology. - : Elsevier BV. - 0167-7799 .- 1879-3096. ; 28:4, s. 171-180
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Forskningsöversikt (refereegranskat)abstract
- Enzymes and whole cells are being increasingly applied in research and industry, but the adoption of biocatalysis relies strongly on useful scientific literature. Unfortunately, too many published papers lack essential information needed to reproduce and understand the results. Here, members of the scientific committee of the European Federation of Biotechnology Section on Applied Biocatalysis (ESAB) provide practical guidelines for reporting experiments. The document embraces the recommendations of the STRENDA initiative (Standards for Reporting Enzymology Data) in the context of pure enzymology and provides further guidelines and explanations on topics of crucial relevance for biocatalysis. In particular, guidelines are given on issues such as the selectivity, specificity, productivity and stability of biocatalysts, as well as on methodological problems related to reactions in multiphase systems. We believe that adoption and use of these guidelines could greatly increase the value and impact of published work in biocatalysis, and hence promote the further growth of applications.
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| 10. |
- Hamberg, Anders, et al.
(författare)
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C-terminal ladder sequencing of peptides using an alternative nucleophile in carboxypeptidase Y digests
- 2006
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 357:2, s. 167-172
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Tidskriftsartikel (refereegranskat)abstract
- A method for improved sequence coverage in C-terminal sequencing of peptides, based on carboxypeptidase digestion, is described. In conventional carboxypeptidase digestions, the peptide substrate is usually extensively degraded and a full amino acid sequence cannot be obtained due to the lack of a complete peptide ladder. In the presented method, a protecting group is introduced at the C terminus of a fraction of the peptide fragments formed in the digest, and thereby further degradation of the C-terminally modified peptides are slowed down. The protecting group was attached to the C-terminal amino acid through a carboxypeptidase-catalyzed reaction with an alternative nucleophile, 2-pyridylmethylamine, added to the aqueous digestion buffer. Six peptides were digested by carboxypeptidase Y with and without 2-pyridylmethylamine present in the digest buffer, and the resulting fragments subsequently were analyzed with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Comparison of the two digestion methods showed that the probability of successful ladder sequencing increased, by more than 50% using 2-pyridylmethylamine as a competing nucleophile in carboxypeptidase Y digests.
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