Purification and kinetics of two novel thermophilic extracellular proteases from Lactobacillus helveticus, from kefir with possible biotechnological interest

Two thermophilic extracellular proteases, designated Lmm-protease-Lh (29 kDa) and Hmm-protease-Lh (62 kDa), were purified from the Lactobacillus helveticus from kefir, and found active in media containing dithiothreitol; the activity of Lmm-protease-Lh was increased significantly in media containing also EDTAK₂. Both novel proteases maintained full activity at 60 °C after 1-h incubation at 10 °C as well as at 80 °C, showing optimum k_cat/K_m values at pH 7.00 and 60 °C. Only irreversible inhibitors specific for cysteine proteinases strongly inhibited the activity of both novel enzymes, while they remained unaffected by irreversible inhibitors specific for serine proteinases. Both enzymes hydrolyzed the substrate Suc-FR-pNA via Michaelis–Menten kinetics; conversely, the substrate Cbz-FR-pNA was hydrolyzed by Lmm-protease-Lh via Michaelis–Menten kinetics and by Hmm-protease-Lh via substrate inhibition kinetics. Valuable rate constants and activation energies were estimated from the temperature-(k_cat/K_m) profiles of both enzymes, and useful results were obtained from the effect of different metallic ions on their Michaelis–Menten parameters.