Purification and Biochemical Characterization of a New Thermostable and Detergent-Stable Serine Protease From a Novel Thermo-Halotolerant Bacterial Strain, Laceyella sacchari Strain BK-TM.

Abstract

Laceyella sacchari strain BK-TM, isolated from the Salt Lake of Chott Ech Chergui (El-Bayadh, Algeria), was found to produce an extracellular thermostable serine protease (SPLS). The highest level of protease activity detected after 6 days of incubation at 50°C was 44,600 U/mL. SPLS was purified after heat treatment for 5 min at 80°C, subjected to ammonium sulfate fractionation (80%), and Sephacryl S-200 High Resolution (HR) column purification. The purified enzyme consists of a single protein with an approximate molecular weight of 29 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The sequence of the 24 N-terminal residues of SPLS was highly similar to those of Thermoactinomycetaceae proteases. Furthermore, the complete inhibition by phenylmethanesulfonyl fluoride (PMSF) and diiodopropyl fluorophosphates (DIFP) indicates that SPLS is a member of the serine protease family. The enzyme exhibits optimal activity at pH 9 and 70°C. The thermostability of SPLS increased when 1 mM of Ca²⁺ was added, with a half-life of 8 h at 80°C and 3 h at 90°C. Its catalytic activity was superior to that of SPSM from Streptomyces mutabilis strain TN-X30, PREFERENZ P300, and PROTEASE Type XIV. Interestingly, SPLS demonstrated high compatibility with ISIS and Maison Det, serving as solid and liquid laundry detergents, respectively. Furthermore, performance evaluations revealed that SPLS effectively removes blood stains. Overall, SPLS exhibited interesting biochemical features, suggesting its potential use as a cleaning bio-additive in detergent formulations.