Expression, Purification and Biocatalytic Studies of a Plant (Arabidopsis thaliana) Hydroxynitrile Lyase

Abstract: Hydroxynitrile lyases are the versatile group of enzymes, which play a significant defensive role in plant system against microbial attack by producing cyanide via cyanogenesis of Cyanogenic Glycoside. In chemical industries, HNL is employed as an important industrial biocatalyst for the synthesis of chiral cyanohydrins. Cyanohydrins are biologically active compounds used in production of ?-amino alcohols, ?-hydroxy ketones and ?-hydroxy acids that have importance as fine chemicals, agrochemicals and pharmaceuticals. Enzymes are present in cellular environment. Cell consists of biomolecules like carbohydrates, lipids, nucleic acids along with proteins. To analyse the functions and applications of enzymes, protein purification is necessary. Important part of biotechnology research is to use protein engineering techniques to design proteins with optimized properties for specific industrial applications. Sonication and high-speed centrifugation separate out protein from the non-protein component in the cell. Isolation of protein is achieved by precipitation with ammonium sulphate (NH4) 2SO4. Different proteins precipitate in different concentrations of (NH4) 2SO4. Proteins of higher molecular weight precipitate in lower concentrations of (NH4) 2SO4. Affinity chromatography is a vital technique for protein purification. Beads in chromatography column in cross-linkage to ligands, bind specifically to the target proteins. Protein is then removed from the column by rinsing with a solution, free of ligands. This method gives the purest result and highest specific activity. Aim of protein purification is to study biocatalytic activity, enzyme kinetics, stereo selectivity, enzyme biophysical and bio chemical properties etc.

Keywords: Hydroxynitrile lyase, Biocatalyst, Cyanohydrin, Affinity chromatography, Enzyme kinetics, Stereo-selectivity

Edition: Volume 12 Issue 4, April 2023,

Pages: 355 - 362