Sam Mathew

Biochemical characterization of thermostable ω-transaminase from Sphaerobacter thermophilus and its application for producing aromatic β- and γ-amino acids.

An (S)-ω-transaminase from the thermophilic eubacterium Sphaerobacter thermophilus was expressed and functionally characterized. The enzyme showed good stability at high temperature and in the presence of various substrates. Substrate specificity analysis showed that the enzyme had activity towards a broad range of substrates including amines, β- and γ-amino acids. The purified enzyme showed a specific activity of 3.3U/mg towards rac-β-phenylalanine at 37°C. The applicability of this enzyme as an attractive biocatalyst was demonstrated by synthesizing optically pure β- and γ-amino acids. Among the various beta and gamma amino acids produced via asymmetric synthesis, (S)-4-amino-4-(4-methoxyphenyl)-butanoic acid showed highest analytical yield (82%) with excellent enantiomeric excess (>99%).

Engineering Transaminase for Stability Enhancement and Site‐Specific Immobilization through Multiple Noncanonical Amino Acids Incorporation

In general, conventional enzyme engineering utilizes 20 canonical amino acids to alter and improve the functional properties of proteins such as stability, and activity. In this study, we utilized the noncanonical amino acid incorporation technique to enhance the functional properties of ω‐transaminase (ω‐TA). Herein, we enhanced the stability of ω‐TA by residue‐specific incorporation of (4R)‐fluoroproline [(4R)‐FP] and successfully immobilized onto chitosan or polystyrene (PS) beads with site‐specifically incorporated L‐3,4‐dihydroxyphenylalanine (DOPA) moiety. The immobilization of ω‐TAdopa and ω‐TAdp[(4R)‐FP] onto PS beads showed excellent reusability for 10 cycles in the kinetic resolution of chiral amines. Compared to the ω‐TAdopa, the ω‐TAdp[(4R)‐FP] immobilized onto PS beads exerted more stability that can serve as suitable biocatalyst for the asymmetric synthesis of chiral amines.

High throughput screening methods for ω-transaminases

Recently, ω-transaminases have been increasingly used to synthesize amine compounds by reductive amination of prochiral ketones which are of high pharmacological significance. However, the conventional methods for evaluating these enzymes are time consuming and have often been regarded as a bottle neck in developing these enzymes as industrial biocatalysts. In the past few years, several high throughput screening methods have been developed for fast evaluation and identification of ω-transaminase. This review summarizes the various methodologies developed for rapidly screening ω-transaminases.

Asymmetric synthesis of aromatic β-amino acids using ω-transaminase: Optimizing the lipase concentration to obtain thermodynamically unstable β-keto acids

Synthesized aromatic β‐amino acids have recently attracted considerable attention for their application as precursors in many pharmacologically relevant compounds. Previous studies on asymmetric synthesis of aromatic β‐amino acids using ω‐transaminases could not be done efficiently due to the instability of β‐keto acids. In this study, a strategy to circumvent the instability problem of β‐keto acids was utilized to generate β‐amino acids efficiently via asymmetric synthesis. In this work, thermodynamically stable β‐ketoesters were initially converted to β‐keto acids using lipase, and the β‐keto acids were subsequently aminated using ω‐transaminase. By optimizing the lipase concentration, we successfully overcame the instability problem of β‐keto acids and enhanced the production of β‐amino acids. This strategy can be used as a general approach to efficiently generate β‐amino acids from β‐ketoesters.

Production of chiral β-amino acids using ω-transaminase from Burkholderia graminis

Optically pure β-amino acids are of high pharmacological significance since they are used as key ingredients in many physiologically active compounds. Despite a number of enzymatic routes to these compounds, an efficient synthesis of β-amino acids continues to pose a major challenge for researchers. ω-Transaminase has emerged as an important class of enzymes for generating amine compounds. However, only a few ω-transaminases have been reported so far which show activity towards aromatic β-amino acids. In this study, (S)-ω-transaminase from Burkholderia graminis C4D1M has been functionally characterized and used for the production of chiral aromatic β-amino acids via kinetic resolution. The enzyme showed a specific activity of 3.1 U/mg towards rac-β-phenylalanine at 37°C. The Km and Kcat values of this enzyme towards rac-β-phenylalanine with pyruvate as the amino acceptor were 2.88 mM and 91.57 min(-1) respectively. Using this enzyme, racemic β-amino acids were kinetically resolved to produce (R)-β-amino acids with an excellent enantiomeric excess (> 99%) and ∼ 50% conversion. Additionally, kinetic resolution of aromatic β-amino acids was performed using benzaldehyde as a cheap amino acceptor.

An in silico approach to evaluate the polyspecificity of methionyl-tRNA synthetases

Residue-specific incorporation is a technique used to replace natural amino acids with their close structural analogs, unnatural amino acids (UAAs), during protein synthesis. This is achieved by exploiting the substrate promiscuity of the wild type amino acyl tRNA synthetase (AARS) towards the close structural analogs of their cognate amino acids. In the past few decades, seleno-methionine was incorporated into proteins, using the substrate promiscuity of wild type AARSs, to resolve their crystal structures. Later, the incorporation of many UAAs showed that the AARSs are polyspecific to the close structural analogs of their cognate amino acids and that they maintain fidelity for the 19 natural amino acids. This polyspecificity helps to expand the use of this powerful tool to incorporate various UAA residues specifically through in vivo and in vitro approaches. Incorporation of UAAs is expensive, tedious and time-consuming. For the efficient incorporation of UAAs, it is important to screen substrate selectivity prior to their incorporation. As an initial study, using a docking tool, we analyzed the polyspecificity of the methionyl-tRNA synthetases (MetRSs) towards multiple reported and virtually generated methionine analogs. Based on the interaction result of these docking simulations, we predicted the substrate selectivity of the MetRS and the key residues responsible for the recognition of methionine analogs. Similarly, we compared the active site residues of the MetRSs of different species and identified the conserved amino acids in their active sites. Given the close similarity in the active site residues of these systems, we evaluated the polyspecificity of MetRSs.