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| 2. |
- Kasrayan, Alex, et al.
(författare)
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Prediction of the Candida antarctica lipase A protein structure by comparative modeling and site-directed mutagenesis
- 2007
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Ingår i: ChemBioChem. - : Wiley. - 1439-4227 .- 1439-7633. ; 8:12, s. 1409-1415
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Tidskriftsartikel (refereegranskat)abstract
- A number of model structures of the CalA suggested by comparative modeling were tested by site-directed mutagenesis. Enzyme variants were created where amino acids predicted to play key roles for the lipase activity in the different models were replaced by an inert amino acid (alanine). The results from activity measurements of the overproduced and purified mutant enzymes indicate a structure where the active site consists of amino acid residues Ser184, His366, and Asp334 and in which there is no lid. This model can be used for future targeted modifications of the enzyme to obtain new substrate acceptance, better thermostability, and higher enantioselectivity.
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| 3. |
- Nyhlén, Jonas, et al.
(författare)
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Influence of delta-functional groups on the enantiorecognition of secondary alcohols by Candida antarctica lipase B
- 2008
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Ingår i: ChemBioChem. - : Wiley. - 1439-4227 .- 1439-7633. ; 9:12, s. 1968-1974
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Tidskriftsartikel (refereegranskat)abstract
- The selectivity of acetylation of delta-functionalized secondary alcohols catalyzed by Candida antarctica lipase B has been examined by molecular dynamics. The results from the simulation show that a delta-alcohol functionality forms a hydrogen bond with the carbonyl group of Thr 40. This interaction stabilizes the tetrahedral intermediate and thus leads to selective acetylation of the R enantiomer. A stabilizing interaction of the delta-(R)-acetoxy group with the peptide NH of alanine 282 was also observed. No stabilizing interaction could be found for the delta-keto functionality, and it is proposed that this is the reason for the experimentally observed decrease in enantioselectivity. From these results, it was hypothesized that the enantioselectivity could be restored by mutating the alanine in position 281 for serine. The mutation was made experimentally, and the results show that the E value increased from 9 to 120.
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| 4. |
- Reetz, Manfred T., et al.
(författare)
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Directed Evolution of an Enantioselective Epoxide Hydrolase : Uncovering the Source of Enantioselectivity at Each Evolutionary Stage
- 2009
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Ingår i: Journal of the American Chemical Society. - : ACS. - 0002-7863 .- 1520-5126. ; 131:21, s. 7334-7343
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Forskningsöversikt (refereegranskat)abstract
- Directed evolution of enzymes as enantioselective catalysts in organic chemistry is an alternative to traditional asymmetric catalysis using chiral transition-metal complexes or organocatalysts, the different approaches often being complementary. Moreover, directed evolution studies allow us to learn more about how enzymes perform mechanistically. The present study concerns a previously evolved highly enantioselective mutant of the epoxide hydrolase from Aspergillus niger in the hydrolytic kinetic resolution of racemic glycidyl phenyl ether. Kinetic data, molecular dynamics calculations, molecular modeling, inhibition experiments, and X-ray structural work for the wild-type (WT) enzyme and the best mutant reveal the basis of the large increase in enantioselectivity (E = 4.6 versus E = 115). The overall structures of the WT and the mutant are essentially identical, but dramatic differences are observed in the active site as revealed by the X-ray structures. All of the experimental and computational results support a model in which productive positioning of the preferred (S)-glycidyl phenyl ether, but not the (R)-enantiomer, forms the basis of enhanced enantioselectivity. Predictions regarding substrate scope and enantioselectivity of the best mutant are shown to be possible.
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