Karim Engelmark Cassimjee

Transaminations with isopropyl amine: equilibrium displacement with yeast alcohol dehydrogenase coupled to in situ cofactor regeneration

Enantiopure chiral amines synthesis using omega-transaminases is hindered by an unfavourable equilibrium, but when using isopropylamine as the amine donor the equilibrium can be completely displaced by using a specific dehydrogenase in situ for removal of formed acetone.

Crystal structures of the Chromobacterium violaceumω-transaminase reveal major structural rearrangements upon binding of coenzyme PLP.

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.

Key Amino Acid Residues for Reversed or Improved Enantiospecificity of an ω‐Transaminase

Transaminases inherently possess high enantiospecificity and are valuable tools for stereoselective synthesis of chiral amines in high yield from a ketone and a simple amino donor such as 2‐propylamine. Most known ω‐transaminases are (S)‐selective and there is, therefore, a need of (R)‐selective enzymes. We report the successful rational design of an (S)‐selective ω‐transaminase for reversed and improved enantioselectivity. Previously, engineering performed on this enzyme group was mainly based on directed evolution, with few exceptions. One reason for this is the current lack of 3D structures. We have explored the ω‐transaminase from Chromobacterium violaceum and have used a homology modeling/rational design approach to create enzyme variants for which the activity was increased and the enantioselectivity reversed. This work led to the identification of key amino acid residues that control the activity and enantiomeric preference. To increase the enantiospecificity of the C. violaceum ω‐transaminase, a possible single point mutation (W60C) in the active site was identified by homology modeling. By site‐directed mutagenesis this enzyme variant was created and it displayed an E value improved up to 15‐fold. In addition, to reverse the enantiomeric preference of the enzyme, two other point mutations (F88A/A231F) were identified. This double mutation created an enzyme variant, which displayed substrate dependent reversed enantiomeric preference with an E value shifted from 3.9 (S) to 63 (R) for 2‐aminotetralin.

One-step enzyme extraction and immobilization for biocatalysis applications

An extraction/immobilization method for HIs6‐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 His6‐tag, offering specific and defined means of binding a protein in a specific location, which is applicable for a wide range of enzymes.

Removing the Active-Site Flap in Lipase A from Candida antarctica Produces a Functional Enzyme without Interfacial Activation.

A mobile region is proposed to be a flap that covers the active site of Candida antarctica lipase A. Removal of the mobile region retains the functional properties of the enzyme. Interestingly interfacial activation, required for the wild‐type enzyme, was not observed for the truncated variant, although stability, activity, and stereoselectivity were very similar for the wild‐type and variant enzymes. The variant followed classical Michaelis–Menten kinetics, unlike the wild type. Both gave the same relative specificity in the transacylation of a primary and a secondary alcohol in organic solvent. Furthermore, both showed the same enantioselectivity in transacylation of alcohols and the hydrolysis of alcohol esters, as well as in the hydrolysis of esters chiral at the acid part.

Quantum Chemical Study of Dual-Substrate Recognition in ω-Transaminase

ω-Transaminases are attractive biocatalysts for the production of chiral amines. These enzymes usually have a broad substrate range. Their substrates include hydrophobic amines as well as amino acids, a feature referred to as dual-substrate recognition. In the present study, the reaction mechanism for the half-transamination of l-alanine to pyruvate in (S)-selective Chromobacterium violaceum ω-transaminase is investigated using density functional theory calculations. The role of a flexible arginine residue, Arg416, in the dual-substrate recognition is investigated by employing two active-site models, one including this residue and one lacking it. The results of this study are compared to those of the mechanism of the conversion of (S)-1-phenylethylamine to acetophenone. The calculations suggest that the deaminations of amino acids and hydrophobic amines follow essentially the same mechanism, but the energetics of the reactions differ significantly. It is shown that the amine is kinetically favored in the half-transamination of l-alanine/pyruvate, whereas the ketone is kinetically favored in the half-transamination of (S)-1-phenylethylamine/acetophenone. The calculations further support the proposal that the arginine residue facilitates the dual-substrate recognition by functioning as an arginine switch, where the side chain is positioned inside or outside of the active site depending on the substrate. Arg416 participates in the binding of l-alanine by forming a salt bridge to the carboxylate moiety, whereas the conversion of (S)-1-phenylethylamine is feasible in the absence of Arg416, which here represents the case in which the side chain of Arg416 is positioned outside of the active site.

Synthesis of Cyclic Polyamines by Enzymatic Generation of an Amino Aldehyde In Situ

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.

CHAPTER 13:EziG: A Universal Platform for Enzyme Immobilisation

EnginZyme, a Swedish biocatalysis company founded in 2014, aims to reduce the environmental impact of the chemical industry by developing products that make it easier and more cost efficient to use enzymes. Currently, the company is addressing the issues of high enzyme cost and process implementation by marketing EziG™, a universal enzyme immobilisation material. EziG eliminates several common issues with enzyme immobilisation and is workable for all enzyme types. This designed enzyme carrier can be used for direct extraction of enzyme from crude preparations, and high loadings of active enzyme are achieved. As an alternative to costly immobilisation testing towards an uncertain end, assured biocatalyst reusability gives benefits beyond the direct economic gain of lower biocatalyst cost, including focus changes in enzyme engineering and simpler work-up procedures. EziG can be used as a standard heterogeneous catalyst, as well as in flow chemistry with packed bed reactors. The importance of this technology in growing the field remains to be evaluated. However, the issues that are now resolved have been identified as key barriers to increased industrial implementation. EziG may play an important role in the ongoing greening of the chemical production industry.

Streamlined Preparation of Immobilized Candida antarctica Lipase B

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.