| 1. |
- Cassimjee, Karim Engelmark, et al.
(författare)
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Streamlined Preparation of Immobilized Candida antarctica Lipase B
- 2017
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Ingår i: ACS Omega. - : American Chemical Society (ACS). - 2470-1343. ; 2:12, s. 8674-8677
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Tidskriftsartikel (refereegranskat)abstract
- Candida antarctica lipase B (CalB) was efficiently expressed (6.2 g L-1) in Escherichia coli by utilizing an N-terminal tag cassette and the XylS/Pm expression system in a fed-batch bioreactor; subsequent direct binding to EziG from crude extracts resulted in an immobilized catalyst with superior activity to Novozym 435.
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| 2. |
- Chen, Shan, et al.
(författare)
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Characterization of the stability of Vibrio fluvialis JS17 amine transaminase
- 2018
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Ingår i: Journal of Biotechnology. - : Elsevier. - 0168-1656 .- 1873-4863. ; 282, s. 10-17
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Tidskriftsartikel (refereegranskat)abstract
- The amine transaminase from Vibrio fluvialis (Vf-ATA) is an attractive enzyme with applications within Biocatalysis for the preparation of chiral amines. Various catalytic properties of Vf-ATA have been investigated, but a biophysical characterization of its stability has been lacking. Today, the industrial application of Vf-ATA is limited by its low operational stability. In order to enhance the knowledge regarding the structural stability of ATAs, general characterizations of different ATAs are required. In this work, the stability of Vf-ATA was explored. First, the affinity between enzyme and pyridoxal-5’-phosphate (PLP) (KD value of 7.9 ΌM) was determined. Addition of PLP to enzyme preparations significantly improved the enzyme thermal stability by preventing enzyme unfolding. With the aim to understand if this was due to the PLP phosphate group coordination into the phosphate group binding cup, the effect of phosphate buffer on the enzyme stability was compared to HEPES buffer. Low concentrations of phosphate buffer showed a positive effect on the enzyme initial activity, while higher phosphate buffer concentrations prevented cofactor dissociation. Additionally, the effects of various amine or ketone substrates on the enzyme stability were explored. All tested amines caused a concentration dependent enzyme inactivation, while the corresponding ketones showed no or stabilizing effects. The enzyme inactivation due to the presence of amine can be connected to the formation of PMP, which forms in the presence of amines in the absence of ketone. Since PMP is not covalently bound to the enzyme, it could readily leave the enzyme upon formation. Exploring the different stability effects of cofactor, substrates, additives and buffer system on ATAs seems to be important in order to understand and improve the general performance of ATAs.
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| 3. |
- Chen, Shan, et al.
(författare)
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Stabilization of an amine transaminase for biocatalysis
- 2016
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Ingår i: Journal of Molecular Catalysis B. - : Elsevier. - 1381-1177 .- 1873-3158. ; 124, s. 20-28
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Tidskriftsartikel (refereegranskat)abstract
- The amine transaminase from Chromobacterium violaceum (Cv-ATA) is a well-known enzyme to achievechiral amines of high enantiomeric excess in laboratory scales. However, the low operational stabilityof Cv-ATA limits the enzyme applicability on larger scales. In order to improve the operational stabilityof Cv-ATA, and thereby extending its applicability, factors (additives, co-solvents, organic solvents anddifferent temperatures) targeting enzyme stability and activity were explored in order to find out how tostore and apply the enzyme. The present investigation shows that the melting point of Cv-ATA is improvedby adding sucrose or glycerol, separately. Further, by storing the enzyme at higher concentrations and inco-solvents, such as; 50% glycerol, 20% methanol or 10% DMSO, the active dimeric structure of Cv-ATAis retained. Enzyme stored in 50% glycerol at −20◦C was e.g., still fully active after 6 months. Finally,the enzyme performance was improved 5-fold by a co-lyophilization with surfactants prior to usage inisooctane.
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| 4. |
- Chen, Shan, et al.
(författare)
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The effect of phosphate group binding cup coordination on the stability of the amine transaminase from Chromobacterium violaceum
- 2018
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Ingår i: Molecular Catalysis. - : Elsevier. - 2468-8231. ; 446, s. 115-123
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Tidskriftsartikel (refereegranskat)abstract
- The amine transaminase from Chromobacterium violaceum (Cv-ATA) is a pyridoxal-5’-phosphate (PLP)dependent enzyme. The biological activity of this enzyme requires the formation of a holo homo dimer.The operational stability of Cv-ATA is, however, low due to dimer dissociation. At the enzyme dimeric interface, two phosphate group binding cups (PGBC) are located. Each cup coordinates the phosphate group of PLP by hydrogen bonds originating from both subunits. Hypothetically, molecular coordination of phosphate groups (PLP or free inorganic phosphate) into the PGBC can affect both dimer stabilization and enzyme activity. To test this assumption, the influence of phosphate (as a functional group in PLP or as free inorganic anions) on the stability and activity of Cv-ATA was explored by various biophysical techniques. The results show that Cv-ATA has a relatively low affinity towards PLP, which results in an excess of apo dimeric enzyme after enzyme purification. Incubation of the apo dimer in buffer solution supplemented with PLP restored the active holo dimer. The addition of PLP or inorganic phosphate into the enzyme storage solutions protected Cv-ATA from both chemical and long term storage unfolding. The use of phosphate buffer leads to faster inactivation of the holo enzyme, compared to the use of HEPES buffer. These results open up for new perspectives on how to improve the stability of PLP-dependent enzymes.
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| 5. |
- Dorau, Robin, et al.
(författare)
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Improved Enantioselectivity of Subtilisin Carlsberg Towards Secondary Alcohols by Protein Engineering
- 2018
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Ingår i: ChemBioChem (Print). - : Wiley. - 1439-4227 .- 1439-7633. ; 19:4, s. 338-346
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Tidskriftsartikel (refereegranskat)abstract
- Generally, the catalytic activity of subtilisin Carlsberg (SC) for transacylation reactions with secondary alcohols in organic solvent is low. Enzyme immobilization and protein engineering was performed to improve the enantioselectivity of SC towards secondary alcohols. Possible amino-acid residues for mutagenesis were found by combining available literature data with molecular modeling. SC variants were created by site-directed mutagenesis and were evaluated for a model transacylation reaction containing 1-phenylethanol in THF. Variants showing high E values (>100) were found. However, the conversions were still low. A second mutation was made, and both the E values and conversions were increased. Relative to that shown by the wild type, the most successful variant, G165L/M221F, showed increased conversion (up to 36 %), enantioselectivity (E values up to 400), substrate scope, and stability in THF.
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| 6. |
- Land, Henrik, et al.
(författare)
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B-factor Guided Proline Substitutions in Chromobacterium violaceum Amine Transaminase – An Evaluation of the Proline Rule as a Method for Enzyme Stabilization
- 2019
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Ingår i: ChemBioChem (Print). - : Wiley-VCH Verlagsgesellschaft. - 1439-4227 .- 1439-7633. ; 20:10, s. 1297-1304
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Tidskriftsartikel (refereegranskat)abstract
- Biocatalysis is attracting interest in the chemical industry as a sustainable alternative in large-scale chemical transformations. However, low operational stability of naturally evolved enzymes is a challenge and major efforts are required to engineer protein stability, usually by directed evolution. The development of methods for protein stabilization based on rational design is of great interest, as it would minimize the efforts needed to generate stable enzymes. We hereby present a rational design strategy based on proline substitutions in flexible areas of the protein identified by analyzing B-factors. Several proline substitutions in the amine transaminase from Chromobacterium violaceum were shown to have a positive impact on stability with increased half-life at 60°C by a factor of 2.7 (variant K69P/D218P/K304P/R432P) as well as increased melting temperature by 8.3°C (variant K167P). Finally, the presented method utilizing B-factor analysis in combination with the Proline rule was deemed successful at increasing the stability of this enzyme.
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| 7. |
- Land, Henrik, et al.
(författare)
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YASARA : A tool to obtain structural guidance in biocatalytic investigations
- 2018
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Ingår i: Protein Engineering. - New York, NY : Humana Press. - 9781493973644 ; , s. 43-67
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Bokkapitel (refereegranskat)abstract
- In biocatalysis, structural knowledge regarding an enzyme and its substrate interactions complements and guides experimental investigations. Structural knowledge regarding an enzyme or a biocatalytic reaction system can be generated through computational techniques, such as homology- or molecular modeling. For this type of computational work, a computer program developed for molecular modeling of proteins is required. Here, we describe the use of the program YASARA Structure. Protocols for two specific biocatalytic applications, including both homology modeling and molecular modeling such as energy minimization, molecular docking simulations and molecular dynamics simulations, are shown. The applications are chosen to give realistic examples showing how structural knowledge through homology and molecular modeling is used to guide biocatalytic investigations and protein engineering studies.
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| 8. |
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| 9. |
- Wikmark, Ylva, et al.
(författare)
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Combinatorial Library Based Engineering of Candida antarctica Lipase A for Enantioselective Transacylation of sec-Alcohols in Organic Solvent
- 2015
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Ingår i: Angewandte Chemie International Edition. - : Wiley. - 1433-7851 .- 1521-3773. ; 54:14, s. 4284-4288
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Tidskriftsartikel (refereegranskat)abstract
- A method for determining lipase enantioselectivity in the transacylation of sec-alcohols in organic solvent was developed. The method was applied to a model library of Candida antarctica lipase A (CalA) variants for improved enantioselectivity (E values) in the kinetic resolution of 1-phenylethanol in isooctane. A focused combinatorial gene library simultaneously targeting seven positions in the enzyme active site was designed. Enzyme variants were immobilized on nickel-coated 96-well microtiter plates through a histidine tag (His6 -tag), screened for transacylation of 1-phenylethanol in isooctane, and analyzed by GC. The highest enantioselectivity was shown by the double mutant Y93L/L367I. This enzyme variant gave an E value of 100 (R), which is a dramatic improvement on the wild-type CalA (E=3). This variant also showed high to excellent enantioselectivity for other secondary alcohols tested.
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