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| 2. |
- 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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| 3. |
- 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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| 4. |
- Cassimjee, Karim Engelmark, et al.
(författare)
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One-step enzyme extraction and immobilization for biocatalysis applications
- 2011
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Ingår i: Biotechnology Journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 6:4, s. 463-469
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Tidskriftsartikel (refereegranskat)abstract
- An extraction/immobilization method for HIs(6) -tagged enzymes for use in synthesis applications is presented. By modifying silica oxide beads to be able to accommodate metal ions, the enzyme was tethered to the beads after adsorption of Co(II). The beads were successfully used for direct extraction of C. antarctica lipase B (CalB) from a periplasmic preparation with a minimum of 58% activity yield, creating a quick one-step extraction-immobilization protocol. This method, named HisSi Immobilization, was evaluated with five different enzymes [Candida antarctica lipase B (CalB), Bacillus subtilis lipase A (BslA), Bacillus subtilis esterase (BS2), Pseudomonas fluorescence esterase (PFE), and Solanum tuberosum epoxide hydrolase 1 (StEH1)]. Immobilized CalB was effectively employed in organic solvent (cyclohexane and acetonitrile) in a transacylation reaction and in aqueous buffer for ester hydrolysis. For the remaining enzymes some activity in organic solvent could be shown, whereas the non-immobilized enzymes were found inactive. The protocol presented in this work provides a facile immobilization method by utilization of the common His(6) -tag, offering specific and defined means of binding a protein in a specific location, which is applicable for a wide range of enzymes.
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| 5. |
- 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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| 6. |
- Engelmark Cassimjee, Karim, et al.
(författare)
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Rational Redesign of omega-Transaminase
- 2010
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Ingår i: Biocat2010. - Hamburg, Germany : TuTech Verlag. - 9783941492240
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Konferensbidrag (refereegranskat)
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| 7. |
- Engelmark Cassimjee, Karim, et al.
(författare)
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Synthesis of cyclic polyamines by enzymatic generation of an amino aldehyde in situ
- 2012
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Ingår i: Macromolecular rapid communications. - : Wiley. - 1022-1336 .- 1521-3927. ; 33:18, s. 1580-1583
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Tidskriftsartikel (refereegranskat)abstract
- Multifunctional polycationic polyamines, for example, used in drug and gene delivery, have product range limitations in their synthesis methods. Here, we synthesize a polyamine by forming a self-assembling amino aldehyde from the corresponding amino alcohol with horse liver alcohol dehydrogenase (HLADH), followed by reduction. Circular polyamines were synthesized from 3-amino-propan-1-ol as starting material, analogous to cyclic polyamines formed from azetidin. The product had an isolated yield of 89.7% or 15.3 g L -1. The predicted range of possible polyamine products by this method is broad since many amino alcohols are putative substrates for HLADH. The enzyme also had activity for 2-amino-propan-1-ol and 2-amino-2-phenyl-ethanol, for which the enantioselectivity was 330 (S) and 32 (R), respectively.
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| 8. |
- Engelmark Cassimjee, Karim
(författare)
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Tools in biocatalysis : enzyme immobilisation on silica and synthesis of enantiopure amines
- 2010
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis presents two techniques in the field of biocatalysis: An enzyme immobilisation method based on the His6-tag for attachment on modified silica oxide beads, and it’s employment in aqueous and organic medium for synthesis applications. The method functions as a one step extraction and immobilisation protocol. An equilibrium displacement system which enables complete conversion in reactions with ω-transaminases where isopropylamine is the donor, a route for synthesis of pharmaceutically interesting enantiopure amines. Biocatalysis is predicted to be a paramount technology for an environmentally sustainable chemical industry, to which every newly developed method represents a small but important step. The work done here is aimed to be a part of this development.
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| 9. |
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| 10. |
- Engelmark Cassimjee, Karim
(författare)
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ω-Transaminase in Biocatalysis : Methods, Reactions and Engineering
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Biocatalysis offers an alternative to classic chemistry by using enzymes, the protein catalysts of Nature, for production of fine chemicals. Evolution has created enzymes capable of catalysis at moderate temperature of a specific reaction in the presence of a plethora of compounds in the aqueous cell environment. The focal point of biocatalysis is to utilise these traits in vitro, for creation of valuable molecules. The ω-transaminase is an enzyme capable of producing chiral amines, compounds used to great extent in pharmaceuticals. Much effort has in recent years been invested in the research and engineering of this enzyme type since the catalysed reaction offers an advantageous alternative to classical techniques. Nevertheless, there is a need for method development, adaptation of the enzyme and increased understanding of the catalytic mechanism for feasibility as an effective biocatalyst for unnatural substrates. This thesis addresses a chosen set of obstacles as a contribution to meeting the demands at hand. ω-Transaminase from Chromobacterium violaceum and Arthrobacter citreus was used. Many homologous ω-transaminases are available, which are also subject to engineering where variants are produced. To accurately compare their kinetic constants an active site quantification method is required but has not been available. Here such a method is presented (Paper 1) which encompasses a virtually irreversible half transamination reaction. In stereoselective synthesis the ω-transaminase catalysed equilibrium reaction inherently results in incomplete conversion. An equilibrium displacement system is presented (Paper II) where isopropylamine is the amino donor for transamination of acetophenone and derivatives thereof, coupled to an enzymatic cascade reaction. For many unnatural substrates the specificity and enantiospecificity is insufficient. Rationally redesigned variants were produced with improved properties for chosen substrates (Paper III and IV). The catalytic contributions of field and resonance of a variant compared to the wild type were investigated (Paper IV) for increased knowledge of the mechanism. For rational redesign of an enzyme the three-dimensional structure is required, of which only a few are available for the ω-transaminases. X-ray crystallographic structures of the holo and apo form of Chromobacterium violaceum ω-transaminase were made (Paper V) which revealed significant structural rearrangements upon coenzyme binding which may be of consequence for future engineering.
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