A novel organic solvent- and detergent-stable serine alkaline protease from Trametes cingulata strain CTM10101

• Published in: International journal of biological macromolecules 2016-10, Vol.91, p.961-972
• Main Authors: Omrane Benmrad, Maroua, Moujehed, Emna, Ben Elhoul, Mouna, Zaraî Jaouadi, Nadia, Mechri, Sondes, Rekik, Hatem, Kourdali, Sidali, El Hattab, Mohamed, Badis, Abdelmalek, Sayadi, Sami, Bejar, Samir, Jaouadi, Bassem
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Bacterial Proteins - chemistry; Bacterial Proteins - isolation & purification; Blood Stains; Detergent; Detergents - pharmacology; Endopeptidases - chemistry; Endopeptidases - isolation & purification; Enzyme Stability - drug effects; Hydrogen-Ion Concentration; Hydrolysis; Ions; Kinetics; Metals - pharmacology; Molecular Weight; Organic Chemicals - pharmacology; Organic solvent; Phylogeny; Protease Inhibitors - pharmacology; Serine Proteases - chemistry; Serine Proteases - isolation & purification; Solvents - pharmacology; Substrate Specificity - drug effects; Temperature; Textiles; Trametes - enzymology; Trametes cingulata
• ISSN: 0141-8130; 1879-0003
• DOI: 10.1016/j.ijbiomac.2016.06.025

•A novel protease (SPTC) from Trametes cingulata strain CTM10101 was studied.•SPTC with a molecular mass of 31405.16-Da was purified and characterized.•The optimum pH and temperature values for activity were pH 9 and 60°C, respectively.•SPTC displayed higher enzymatic performance than Flavourzyme® 500L and Thermolysin.•SPTC is a potential candidate for peptide synthesis and detergent formulations. A protease-producing fungus was isolated from an alkaline wastewater of chemical industries and identified as Trametes cingulata strain CTM10101 on the basis of the ITS rDNA gene-sequencing. It was observed that the fungus strongly produce extracellular protease grown at 30°C in potato-dextrose-broth (PDB) optimized media (13500U/ml). The pure serine protease isolated by Trametes cingulata (designated SPTC) was purified by ammonium sulfate precipitation-dialysis followed by heat-treatment and UNO S-1 FPLC cation-exchange chromatography. The chemical characterization carried on include phisico-chemical determination and spectroscopie analysis. The MALDI-TOF/MS analysis revealed that the purified enzyme was a monomer with a molecular mass of 31405.16-Da. The enzyme had an NH2-terminal sequence of ALTTQTEAPWALGTVSHKGQAST, thus sharing high homology with those of fungal-proteases. The optimum pH and temperature values of its proteolytic activity were pH 9 and 60°C, respectively, and its half-life times at 60 and 70°C were 9 and 5-h, respectively. It was completely inhibited by PMSF and DFP, which strongly suggested its belonging to the serine protease family. Compared to Flavourzyme®500L from Aspergillus oryzae and Thermolysin typeX from Geobacillus stearothermophilus, SPTC displayed higher levels of hydrolysis, substrate specificity, and catalytic efficiency as well as elevated organic solvent tolerance and considerable detergent stability. Finally, SPTC could potentially be used in peptide synthesis and detergent formulations.