Enzyme engineering improves catalytic efficiency and enantioselectivity of hydroxynitrile lyase for promiscuous retro-nitroaldolase activity.

Protein engineering to improve promiscuous catalytic activity is important for biocatalytic application of enzymes in green synthesis. We uncovered the significance of binding site residues in Arabidopsis thaliana hydroxynitrile lyase (AtHNL) for promiscuous retro-nitroaldolase activity. Engineering of AtHNL has improved enantioselective retro-nitroaldolase activity, a synthetically important biotransformation, for the production of enantiopure ß-nitroalcohols having absolute configuration opposite to that of the stereopreference of the HNL. The variant F179A has shown âˆ¼ 12 fold increased selectivity towards the retro-nitroaldol reaction over cyanogenesis, the natural activity of the parent enzyme. Screening of the two saturation libraries of Phe179 and Tyr14 revealed several variants with higher kcat, while F179N showed âˆ¼ 2.4-fold kcat/Km than the native enzyme towards retro-nitroaldol reaction. Variants F179N, F179M, F179W, F179V, F179I, Y14L, and Y14M have shown > 99% ee in the preparation of (S)-2-nitro-1-phenylethanol (NPE) from the racemic substrate, while F179N has shown the E value of 138 vs. 81 by the wild type. Our molecular docking and dynamics simulations (MDS) studies results provided insights into the molecular basis of higher enantioselectivity by the F179N toward the retro-nitroaldolase activity than the other mutants. Binding energy calculations also showed the higher negative binding free energy in the case of F179N-(R)-NPE compared to other complexes that support our experimental low Km by the F179N for NPE. A plausible retro-nitroaldol reaction mechanism was proposed based on the MDS study of enzyme-substrate interaction.

Preliminary evaluation of antimicrobial activity of cream formulated with essential oil of Trachyuspermum ammi.

In our present research we studied physiochemical properties and antimicrobial activity of T. ammi (Trachyuspermum ammi) seeds. The seeds yielded 2.86% essential oil. Refractive index, specific gravity, acid value, and easter values were 1.496, 0.9212, 1.12 and 2.80 respectively. GLC was used to determine the composition of oil. The major component of oil was Thymol (55.308%). Antimicrobial activity of the oil was carried out against four species of bacteria and fungs. The oil was used to formulate a cream and the pH, physiochemical stability, phase separation and dermal irritation of cream were investigated. The cream was examined for healing wound in rabbits in comparison with Iodine tincture.