Role of calcium in enhancing the activity and thermal stability of a new cationic peroxidase purified from euphorbia tirucalli latex

A new cationic peroxidase from Euphorbi and firucalli [pencil cactus] latex was purified to homogeneity using benzene fractionation, gel filtration and cation-exchange chromatography. The purified enzyme was found to be monomeric with a molecular weight of 44 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]. The purified enzyme had a broad specificity towards some phenolic substrates in the order of 2,2'-azino-bis [3-ethylbenzothiazoline-6-sulfonicacid] [ABTS] > guaiacol > 0-phenylenediamine > 4-aminoantipyrene, whereas no affinity towards ascorbic acid and o-dianisidine was recorded. The enzyme had pH and temperature optima at 7.0 and 40°C, respectively. Study of kinetic parameters demonstrated that ABTS had the highest affinity towards ELP, where K[m], F[max] and V[ms]/K[m] values were 0.503 mM, 500 U/assay and 994.04 U/mM, respectively. ELP was stable from 10°C up to 60°C and lost about 70% of its activity at 70°C. The thermal inactivation profile of ELP in absence of Ca[2+] is biphasic and characterized by a rapid decline in activity on exposure to heat, followed by a more gradual decrease in activity on continued exposure. However, the purified enzyme exhibited increased thermal stability in the presence of calcium ions. Furthermore, the activity of purified enzyme was enhanced by 550% in the presence of 15 mM CaCl[2], suggesting a pivotal role for Ca[2+] in conferring structural stability to the heme environment and in retaining the active site of ELP. Most of the examined metal ions [except for Ca and Mg] and compounds had differential inhibitory effects on ELP activity. In conclusion, a locally available plant [Euphorbia tirucalli] could be a potential candidate source for peroxidase, the most widely used enzyme in industrial and biomedical applications. In addition, calcium was found to be essential for enhancing enzymatic activity and thermal stability of the purified Euphorbia tirucalli latex peroxidase

Purification and Properties of a-Amylase from Trichoderma reesei

Two alpha-amylases AIV and AV from Trichoderma reesei, cultured on citrus peel, were purified with specific activity 2082 and 611 units/mg protein, by chromatography on DEAE-Sepharose and Sephacryl S-200 columns, respectively. The purified enzymes gave an apparent single protein band on SDS-polyacrylamide gel electrophoresis [SDS-PAGE]. The molecular mass of purified enzymes AIV and AV as estimated by SDS-PAGE and by gel filtration on Sephacryl S-200 to be 24 and 30 kDa, respectively, indicated that two enzymes are monomers. The same pH and temperature optima were detected at 5.5 and 40°C and are stable up to 40°C for AIV and AV. The K[m] for hydrolysis of soluble starch were 1.3 and 2.38 mg starch/ml for AIV and AV, respectively. AIV and AV display highest specificity towards starch followed by amylose, amylopectin, glycogen and beta-cyclodextrin. While Ba[+2] and Ca[+2] showed an activation effect for AIV and AV, Fe[+3] and Hg[+2] had a complete inhibition effect. At 20 Mm concentration, oxalic acid inhibited the two enzymes, and EDTA and citric acid had moderate inhibition effects for AIV and AV