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| 3. |
- Fiorati, Andrea, et al.
(författare)
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Application of Transaminases in a Disperse System for the Bioamination of Hydrophobic Substrates
- 2020
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Ingår i: Advanced Synthesis and Catalysis. - : Wiley-VCH Verlagsgesellschaft. - 1615-4150 .- 1615-4169. ; 362:5, s. 1156-1166
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Tidskriftsartikel (refereegranskat)abstract
- Abstract The challenging bioamination of hydrophobic substrates has been attained through the employment of a disperse system consisting of a combination of a low polarity solvent (e. g. isooctane or methyl-tert-butylether), a non-ionic surfactant and a minimal amount of water. In these conditions, amine transaminases (ATA) were shown to efficiently carry out the reductive amination of variously substituted cyclohexanones, providing good conversions often coupled with a superior stereoselectivity if compared with the corresponding chemical reductive amination. An array of synthetically useful 4-substituted aminocyclohexanes was consequentially synthesized through biocatalysis, analyzed and stereochemically characterized.
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| 4. |
- Löfgren, J., et al.
(författare)
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Transesterification of a Tertiary Alcohol by Engineered Candida antarctica Lipase A
- 2019
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Ingår i: ChemBioChem. - : Wiley. - 1439-4227 .- 1439-7633. ; 20:11, s. 1438-1443
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Tidskriftsartikel (refereegranskat)abstract
- Tertiary alcohols are known to be challenging substrates for applications in asymmetric synthesis due to their complexity and steric hinderance. The occurrence of tertiary alcohols and their esters in nature indicates the presence of natural biocatalytic synthetic routes for their preparation. Lipase A from Candida antarctica (CalA) is a hydrolase that has previously been shown to catalyze the transesterification of racemic 2-phenylbut-3-yn-2-ol at a low rate. In this work, the activity of that enzyme was improved by protein engineering through a semi-rational design strategy. An enzyme library was created and screened for transesterification activity towards racemic 2-phenylbut-3-yn-2-ol in an organic solvent. One successful enzyme variant (L367G) showed a tenfold increased reaction rate compared to the wild-type enzyme, while maintaining a high enantioselectivity.
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| 5. |
- Svedendahl, Maria, 1975-, et al.
(författare)
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Direct Epoxidation in Candida antarctica Lipase B Studied by Experiment and Theory
- 2008
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Ingår i: ChemBioChem. - : Wiley. - 1439-4227 .- 1439-7633. ; 9:15, s. 2443-2451
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 7. |
- Svedendahl, Maria, 1975-
(författare)
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Lipase and ω-Transaminase : Biocatalytic Investigations
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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