Biochemical Characterization of a Novel Cysteine Protease Purified from the Medicinal Plant Kaempferia galanga L

Abstract

Plant-derived cysteine proteases have emerged as a compelling subject of investigation, capturing scientific interest owing to their potential applications in diverse industries, including food and biotechnology. This study focused on isolating Kaempferia galanga cysteine protease (KgCP) from rhizomes of Kaempferia galanga, followed by a comprehensive characterization of the protease. It was purified and characterized using various biochemical and biophysical techniques, including anion-exchange chromatography, gel filtration, SDS-PAGE electrophoresis, and enzyme assays. With a yield of 23.2%, the purification process generated a 6.03-fold increase in specific activity. KgCP's molecular weight was determined to be around 33 kDa and exhibited optimal catalytic performance at 55 °C and pH 5.5. Values of its catalytic parameters, V_max and K_m, were found to be 103.7 Units min^-1 and 0.025 μmol, respectively. Inhibition of KgCP by various cysteine protease inhibitors - E-64, iodoacetamide, and mercury chloride confirmed it to be a cysteine protease. The inclusion of detergents and organic solvents did not affect the stability of KgCP. Although proteolytic activity was compromised by metal ions such as Cd²⁺, Co²⁺, and Fe³⁺, other metal ions (Ca²⁺, Mg²⁺, Mn²⁺, Sn²⁺, Sr²⁺, etc.) showed negligible impact on its proteolytic activity. These findings expand our understanding of the biological characteristics of this cysteine protease, highlighting its potential for applications in the dairy industry, bioactive peptide synthesis, detergents industry, etc. The entire work can be graphically presented as follows.

Keywords: Kaempferia galanga; Chromatographic purification; Cysteine protease; Kinetic parameters; Milk-clotting activity.