| 1. |
- Engelmark Cassimjee, Karim, et al.
(författare)
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A general protein purification and immobilization method on controlled porosity glass : biocatalytic applications
- 2014
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Ingår i: Chemical Communications. - : Royal Society of Chemistry. - 1359-7345 .- 1364-548X. ; 50:65, s. 9134-9137
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Tidskriftsartikel (refereegranskat)abstract
- A general combined purification and immobilization method to facilitate biocatalytic process development is presented. The support material, EziG (TM), is based on controlled porosity glass (CPG) or polymer-coated versions thereof (HybCPG) and binds protein affinity tags. Biocatalytic reactions in aqueous and organic media with seven enzymes of biocatalytic interest are shown.
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| 2. |
- Svedendahl Humble, Maria, et al.
(författare)
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Crystal structures of the Chromobacterium violaceumω-transaminase reveal major structural rearrangements upon binding of coenzyme PLP.
- 2012
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 279:5, s. 779-792
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Tidskriftsartikel (refereegranskat)abstract
- The bacterial ω-transaminase from Chromobacterium violaceum (Cv-ωTA, EC2.6.1.18) catalyses industrially important transamination reactions by use of the coenzyme pyridoxal 5'-phosphate (PLP). Here, we present four crystal structures of Cv-ωTA: two in the apo form, one in the holo form and one in an intermediate state, at resolutions between 1.35 and 2.4 Å. The enzyme is a homodimer with a molecular mass of ∼ 100 kDa. Each monomer has an active site at the dimeric interface that involves amino acid residues from both subunits. The apo-Cv-ωTA structure reveals unique 'relaxed' conformations of three critical loops involved in structuring the active site that have not previously been seen in a transaminase. Analysis of the four crystal structures reveals major structural rearrangements involving elements of the large and small domains of both monomers that reorganize the active site in the presence of PLP. The conformational change appears to be triggered by binding of the phosphate group of PLP. Furthermore, one of the apo structures shows a disordered 'roof ' over the PLP-binding site, whereas in the other apo form and the holo form the 'roof' is ordered. Comparison with other known transaminase crystal structures suggests that ordering of the 'roof' structure may be associated with substrate binding in Cv-ωTA and some other transaminases. DATABASE: The atomic coordinates and structure factors for the Chromobacterium violaceumω-transaminase crystal structures can be found in the RCSB Protein Data Bank (http://www.rcsb.org) under the accession codes 4A6U for the holoenzyme, 4A6R for the apo1 form, 4A6T for the apo2 form and 4A72 for the mixed form STRUCTURED DIGITAL ABSTRACT: • -transaminases and -transaminases bind by dynamic light scattering (View interaction) • -transaminase and -transaminase bind by x-ray crystallography (View interaction) • -transaminase and -transaminase bind by x-ray crystallography (View interaction).
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| 3. |
- Svedendahl Humble, Maria, et al.
(författare)
-
Crystal structures of the Chromobacterium violaceumω-transaminase reveal major structural rearrangements upon binding of coenzyme PLP.
- 2012
-
Ingår i: The FEBS Journal. - 1742-464X. ; 279:5, s. 779-792
-
Tidskriftsartikel (refereegranskat)abstract
- SUMMARY: The bacterial ω-transaminase from Chromobacterium violaceum (Cv-ωTA, EC 2.6.1.18) catalyzes industrially important transamination reactions by use of the coenzyme pyridoxal 5'-phosphate (PLP). Here, we present four crystal structures of Cv-ωTA: two in the apo form, one in the holo form and one in an intermediate state, at resolutions between 1.35 and 2.4 Å. The enzyme is a homodimer with a molecular weight of approximately 100 kDa. Each monomer has an active site at the dimeric interface that involves amino acid residues from both subunits. The apo-Cv-ωTA structure reveals unique "relaxed" conformations of three critical loops involved in structuring the active site, that have not previously been seen in a transaminase. Analysis of the four crystal structures reveals major structural rearrangements involving elements of the large and small domains of both monomers that reorganize the active site in the presence of PLP. The conformational change appears to be triggered by binding of the phosphate group of PLP. Furthermore, one of the apo structures shows a disordered "roof" over the PLP binding site, while in the other apo form and the holo form the "roof" is ordered. Comparison with other known transaminase crystal structures suggests that ordering of the "roof" structure may be associated with substrate binding in Cv-ωTA and some other transaminases. STRUCTURED DIGITAL ABSTRACT: -transaminases and -transaminases bind by dynamic light scattering (View interaction) -transaminase and -transaminase bind by x-ray crystallography (View interaction) -transaminase and -transaminase bind by x-ray crystallography (View interaction).
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| 4. |
- Berglund, Per, et al.
(författare)
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7.18 C-X Bond Formation : Transaminases as Chiral Catalysts: Mechanism, Engineering, and Applications
- 2012
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Ingår i: Comprehensive Chirality. - : Elsevier. - 9780080951683 ; , s. 390-401
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Bokkapitel (refereegranskat)abstract
- Enantiomerically pure amines and amino acids are important building blocks in academic research as well as in industrial-scale chemical production. Transaminases are versatile enzymes providing access to such compounds of high enantiomeric excess. This chapter illustrates the available strategies with transaminases such as kinetic resolution or stereoselective synthesis and highlights many successful examples for amino acid and chiral amines synthesis. There are some known challenges linked to the use of transaminases, for example in terms of unfavorable equilibria and inhibition. Several successful examples to overcome these limitations are presented. Also, the classification of transaminases, mechanistic details, and various strategies for optimization are discussed.
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| 5. |
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| 6. |
- Cassimjee, Karim Engelmark, et al.
(författare)
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Active Site Quantification of an omega-Transaminase by Performing a Half Transamination Reaction
- 2011
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Ingår i: ACS Catalysis. - : American Chemical Society (ACS). - 2155-5435. ; 1:9, s. 1051-1055
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Tidskriftsartikel (refereegranskat)abstract
- Measurement of the active enzyme fraction in a given enzyme preparation is a requirement for accurate kinetic measurements and activity comparisons of, for example, engineered mutants. omega-Transaminases, enzymes capable of interconverting ketones and amines by use of pyridoxal-5'-phosphate (PIP), can be used for the production of pharmaceutically important chiral amines but are subject to engineering to meet the practical requirements in synthesis reactions. Therefore, an active site quantification method is needed. Such a method was developed by quantifying the amount of consumed substrate in a virtually irreversible half transamination reaction. (S)-1-phenylethylamine was converted to acetophenone, while the holo enzyme (E-PLP) was converted to apo enzyme with bound pyridoxamine-5'-phosphate (E:PMP). Further, the mass of active enzyme was correlated to the absorbance of the holo enzyme to achieve a direct measurement method. The active Chromobacterium violaceum omega-transaminase with bound PLP can be quantified at 395 nm with an apparent extinction coefficient of 8.1 mM(-1) cm(-1).
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| 7. |
- Cassimjee, Karim Engelmark, et al.
(författare)
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Chromobacterium violaceum omega-transaminase variant Trp60Cys shows increased specificity for (S)-1-phenylethylamine and 4 '-substituted acetophenones, and follows Swain-Lupton parameterisation
- 2012
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Ingår i: Organic and biomolecular chemistry. - : Royal Society of Chemistry (RSC). - 1477-0520 .- 1477-0539. ; 10:28, s. 5466-5470
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Tidskriftsartikel (refereegranskat)abstract
- For biocatalytic production of pharmaceutically important chiral amines the.-transaminase enzymes have proven useful. Engineering of these enzymes has to some extent been accomplished by rational design, but mostly by directed evolution. By use of a homology model a key point mutation in Chromobacterium violaceum omega-transaminase was found upon comparison with engineered variants from homologous enzymes. The variant Trp60Cys gave increased specificity for (S)-1-phenylethylamine (29-fold) and 4'-substituted acetophenones (similar to 5-fold). To further study the effect of the mutation the reaction rates were Swain-Lupton parameterised. On comparison with the wild type, reactions of the variant showed increased resonance dependence; this observation together with changed pH optimum and cofactor dependence suggests an altered reaction mechanism.
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| 8. |
- 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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| 9. |
- 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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| 10. |
- 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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