Abstract
BackgroundProteases are hydrolytic enzymes that catalyze peptide linkage cleavage reactions at the level of proteins and peptides with different degrees of specificity. This group draws the attention of industry. More than one protease in three is a serine protease. Classically, they are active at neutral to alkaline pH. The serine proteases are researched for industrial uses, especially detergents. They are the most commercially available enzyme group in the world market. Overall, fungi produced extracellular proteases, easily separated from mycelium by filtration.ResultsA new basidiomycete fungus CTM10057, a hyperproducer of a novel protease (10,500 U/mL), was identified as Pleurotus sajor-caju (oyster mushroom). The enzyme, called SPPS, was purified to homogeneity by heat-treatment (80 °C for 20 min) followed by ammonium sulfate precipitation (35–55%)-dialysis, then UNO Q-6 FPLC ion-exchange chromatography and finally HPLC-ZORBAX PSM 300 HPSEC gel filtration chromatography, and submitted to biochemical characterization assays. The molecular mass was estimated to be 65 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Native-PAGE, casein-zymography, and size exclusion by HPLC. A high homology with mushroom proteases was displayed by the first 26 amino-acid residues of the NH2-terminal aminoacid sequence. Phenylmethanesulfonyl fluoride (PMSF) and diiodopropyl fluorophosphates (DFP) strongly inhibit SPPS, revealing that it is a member of the serine-proteases family. The pH and temperature optima were 9.5 and 70 °C, respectively. Interestingly, SPPS possesses the most elevated hydrolysis level and catalytic efficiency in comparison with SPTC, Flavourzyme® 500 L, and Thermolysin type X proteases. More remarkably, a high tolerance towards organic solvent tolerance was exhibited by SPPS, together with considerable detergent stability compared to the commercial proteases Thermolysin type X and Flavourzyme® 500 L, respectively.ConclusionsThis proves the excellent proprieties characterizing SPPS, making it a potential candidate for industrial applications especially detergent formulations.
Highlights
Proteases are hydrolytic enzymes that catalyze peptide linkage cleavage reactions at the level of proteins and peptides with different degrees of specificity
Screening of alkaline protease-producing strains The new basidiomycete fungus, Pleurotus P10, was newly isolated from the symptomatic wood of the camphor trees Quercus faginea (L.) at the El Feïdja National Park, Aïn Draham (Jendouba, Tunisia). It was registered in the Collection Tunisienne de Microorganismes (CTM)-Centre of Biotechnology of Sfax (CBS) under the authentic culture number: “CTM10057” and was retained, for further and advanced study, as the highest protease producer fungal strain on skimmed milk agar after 24 h and on potato dextrose broth (PDB) liquid medium after 72 h (Fig. 1) at 28 ± 2 °C
Effects of pH on protease activity and stability According to the results presented in Fig. 3a, SPPS possesses an optimum activity at pH 9.5
Summary
Proteases are hydrolytic enzymes that catalyze peptide linkage cleavage reactions at the level of proteins and peptides with different degrees of specificity. The serine proteases are researched for industrial uses, especially detergents They are the most commercially available enzyme group in the world market. The microbial proteases production is beneficial thanks to their distinctive features such as short generation time, easy manipulation of microorganisms genes, and large availability in nature [1] Their preferred properties in biotechnology are widely approved. The 3D structural and functional proprieties of basidiomycetes proteases occurred more than 30 years ago in scientific research, the variety and complexity of xylotrophic basidiomycetes enzymes action has resulted in interesting studies. In this respect, our researches are focused on fungi protease thanks to their easy cell separation. In addition to laccase production, this genus is known for the hydrolytic enzymes production by Pleurotus spp. that have been designated, resulting in these properties of this genus for future research [1]
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