A novel organic solvent- and detergent-stable serine alkaline protease from Trametes cingulata strain CTM10101.
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.
Laboratory of Microbial Biotechnology and Engineering Enzymes (LMBEE), Centre of Biotechnology of Sfax (CBS), University of Sfax, Road of Sidi Mansour Km 6, PO Box 1177, Sfax 3018, Tunisia.
Maroua
M
Omrane Benmrad
Laboratory of Microbial Biotechnology and Engineering Enzymes (LMBEE), Centre of Biotechnology of Sfax (CBS), University of Sfax, Road of Sidi Mansour Km 6, PO Box 1177, Sfax 3018, Tunisia.
Emna
E
Moujehed
Laboratory of Microbial Biotechnology and Engineering Enzymes (LMBEE), Centre of Biotechnology of Sfax (CBS), University of Sfax, Road of Sidi Mansour Km 6, PO Box 1177, Sfax 3018, Tunisia.
Mouna
M
Ben Elhoul
Laboratory of Microbial Biotechnology and Engineering Enzymes (LMBEE), Centre of Biotechnology of Sfax (CBS), University of Sfax, Road of Sidi Mansour Km 6, PO Box 1177, Sfax 3018, Tunisia.
Nadia
N
Zaraî Jaouadi
Laboratory of Microbial Biotechnology and Engineering Enzymes (LMBEE), Centre of Biotechnology of Sfax (CBS), University of Sfax, Road of Sidi Mansour Km 6, PO Box 1177, Sfax 3018, Tunisia.
Sondes
S
Mechri
Laboratory of Microbial Biotechnology and Engineering Enzymes (LMBEE), Centre of Biotechnology of Sfax (CBS), University of Sfax, Road of Sidi Mansour Km 6, PO Box 1177, Sfax 3018, Tunisia.
Hatem
H
Rekik
National Centre for Research and Development of Fisheries and Aquaculture (CNRDPA) 11, Bd Amirouche PO Box 67, BouIsmaïl, 42415 Tipaza, Algeria.
Sidali
S
Kourdali
Laboratory of Natural Products Chemistry and Biomolecules (LNPCB), University of Blida 1, Road of Soumaâ, PO Box 270, 09000 Blida, Algeria.
Mohamed
M
El Hattab
National Centre for Research and Development of Fisheries and Aquaculture (CNRDPA) 11, Bd Amirouche PO Box 67, BouIsmaïl, 42415 Tipaza, Algeria; Laboratory of Natural Products Chemistry and Biomolecules (LNPCB), University of Blida 1, Road of Soumaâ, PO Box 270, 09000 Blida, Algeria.
Abdelmalek
A
Badis
Laboratory of Environmental Bioprocesses, LMI COSYS-Med, Centre of Biotechnology of Sfax, University of Sfax, University of Sfax, Road of Sidi Mansour Km 6, PO Box 1177, Sfax 3018, Tunisia.
Sami
S
Sayadi
Laboratory of Microbial Biotechnology and Engineering Enzymes (LMBEE), Centre of Biotechnology of Sfax (CBS), University of Sfax, Road of Sidi Mansour Km 6, PO Box 1177, Sfax 3018, Tunisia.
Samir
S
Bejar
Laboratory of Microbial Biotechnology and Engineering Enzymes (LMBEE), Centre of Biotechnology of Sfax (CBS), University of Sfax, Road of Sidi Mansour Km 6, PO Box 1177, Sfax 3018, Tunisia. Electronic address: bassem.jaouadi@cbs.rnrt.tn.
Bassem
B
Jaouadi
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification*; D001801:Blood Stains; D003902:Detergents / Q000494:pharmacology*; D010450:Endopeptidases / Q000737:chemistry / Q000302:isolation & purification*; D004795:Enzyme Stability / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007477:Ions; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D009930:Organic Chemicals / Q000494:pharmacology*; D010802:Phylogeny; D011480:Protease Inhibitors / Q000494:pharmacology; D057057:Serine Proteases / Q000737:chemistry / Q000302:isolation & purification*; D012997:Solvents / Q000494:pharmacology*; D013379:Substrate Specificity / Q000187:drug effects; D013696:Temperature; D013784:Textiles; D055454:Trametes / Q000201:enzymology*
D001426:Bacterial Proteins; D003902:Detergents; D007477:Ions; D008670:Metals; D009930:Organic Chemicals; D011480:Protease Inhibitors; D012997:Solvents; D010450:Endopeptidases; D057057:Serine Proteases; C087822:alkaline protease
Detergent; Organic solvent; Trametes cingulata