Abstract
Phenylalanine ammonia lyase (PAL) was first purified using affinity chromatography from the leaves of red-flowered clover, a highly antioxidant source. The characterization results of the PAL enzyme were determined, including the concentration of its activity buffer solution, pH, and temperature, which were 0.1 M, 7, and 25 °C, respectively. The Vmax and KM values of the enzyme were calculated to be 0.97 EU and 0.68 mM, respectively. L-phenylalanine was used as the substrate. All kinetic studies were performed spectrophotometrically with a wavelength of 283 nm. Sepharose-4B–L-tyrosine–4-aminocinnamic acid (S-4B-TACA) was also synthesized for the first time and used as an affinity gel. The activity of the PAL extract was measured as 267.9 (millienzyme unit) mU per mL. The yield % and purification fold in the purification step of affinity chromatography were determined to be 3.8% and 19.4, respectively. The experimental results indicate that the PAL enzyme was successfully purified using affinity chromatography. The purity of the enzyme was controlled via SDS-PAGE analysis, which indicated that PAL gave a clear, single band at the line of 45 kDa, while the PAL homogenate gave two bands at around 35 and 45 kDa. Enzyme stabilization was also investigated using PAL stored at 4 °C, which retained completely protected activity for the first 3 weeks. The synthesis of the S-4B-TACA affinity gel, the purification of PAL from red clover leaves using affinity chromatography, and its characterization and statistical analysis have not been previously investigated or reported in the literature.