Purification of β-Glucosidase, One of the Flavor-enhancing Food Enzymes, from Peppermint (Mentha piperita L.) and Its Biochemical Characterization

Abstract

Aims: Determination of the biochemical properties of β-glucosidase in peppermint, which is rich in aromatic compounds. Study Design: β-glucosidase was purified from mint, and biochemical characterization of the purified enzyme was performed. Place and Duration of Study: This study was carried out in the Faculty of Arts and Sciences Biochemistry laboratory. Methodology: Enzyme purification was performed by hydrophobic interaction chromatography using a Sepharose 4B-L-tyrosine-1-naphthylamine gel. Optimum pH, temperature, and substrate specificity of the purified enzyme were determined. The effects of glucose, δ-gluconolactone and some heavy metals on the enzyme activity were investigated. Results: The enzyme was purified with 8-fold and 28% yield. The purified protein from mint was visualized at 65 kDa on SDS-PAGE. The substrate specificity of the purified β-glucosidase from mint was determined against para- and ortho-nitrophenyl β-D-glucopyranoside (p/o-NPG) substrates. The Km values were 0.4 and 0.9 mM, and the Vmax values were 102.2 EU and 96.6 EU, respectively. While the optimum pH for the purified enzyme was 6, the optimum temperature was 35°C. Effects of heavy metals Ag+2, Fe+3, Zn+2, Cu+2, and Pb+2 on the purified enzyme activity were investigated. Relative activities of heavy metals were introduced into the reaction medium as 0.75 mM samples without any known inhibitors in the environment. Fe+3 increased the enzyme activity, and Ag+2, Pb+2, Cu+2, and Zn+2 inhibited the enzyme, and their relative activities were 78, 76, 22, and 31%, respectively. Glucose and δ-gluconolactone competitively inhibited the enzyme activity when p-NPG was the substrate. Ki values of glucose and δ-gluconolactone were determined as 0.034±0.001 and 0.038±0.002 mM, respectively. Conclusion: Determination of the biochemical properties of β-glucosidase from mint, which has commercial and pharmacological importance due to the phenolic substances it contains, will contribute to studies on improving food quality.