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Ben Elhoul M.,University of Sfax | Zarai Jaouadi N.,University of Sfax | Rekik H.,University of Sfax | Omrane Benmrad M.,University of Sfax | And 8 more authors.
International Journal of Biological Macromolecules | Year: 2016

A new extracellular thermostable keratinolytic protease, designated KERDZ, was purified and characterized from a thermophilic actinomycetes Actinomadura viridilutea DZ50 isolated from Algerian fishing port. The isolate exhibited high keratinase production when grown in chicken-feather meal media (18,000 U/ml) after 96-h of incubation at 45 °C. The enzyme was purified by ammonium sulfate precipitation (35–55%)-dialysis and heat treatment (30 min at 75 °C) followed by UNO S-1 FPLC cation exchange chromatography and size exclusion HPLC column. The biochemical characterizations carried on include physico-chemical determination and spectroscopic analysis. The MALDI-TOF/MS analysis revealed that the purified enzyme was a monomer with a molecular mass of 19536.10-Da. The sequence of the 25 N-terminal residues of KERDZ showed high homology with those of actinomycetes keratinases. Optimal activity was achieved at pH 11 and 80 °C. KERDZ was completely inhibited by PMSF and DFP suggested its belonging to the serine keratinase family. KERDZ displayed higher levels of hydrolysis and catalytic efficiency than bacterial keratinases (KERAK-29, Actinase E, and KERAB) and subtilisins (subtilisin Carlsberg and subtilisin Novo). The kerDZ gene encoding KERDZ was isolated and its DNA sequence was determined. These properties make KERDZ a potential, promising and eco-friendly alternative to the conventional chemicals used for industrial applications. © 2016 Elsevier B.V. Source


Omrane Benmrad M.,University of Sfax | Moujehed E.,University of Sfax | Ben Elhoul M.,University of Sfax | Zarai Jaouadi N.,University of Sfax | And 9 more authors.
International Journal of Biological Macromolecules | Year: 2016

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 (13500 U/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®500 L 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. © 2016 Elsevier B.V. Source


Jaouadi B.,University of Sfax | Rekik H.,University of Sfax | Badis A.,National Center for Research and Development of Fisheries and Aquaculture 11 | Badis A.,Blida University | And 6 more authors.
International Biodeterioration and Biodegradation | Year: 2014

This study reports on the production, purification, and characterization of a new extracellular humic acid peroxidase (termed HaP4, following the names given to HaP1-3) from decolorizing actinomycetes strain isolated from Tunisian off-shore oil field and assigned as Streptomyces albidoflavus strain TN644 based on physiological and biochemical characteristics and 16S rRNA gene sequencing. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis revealed that HaP4 is a monomer with a molecular mass of 50,011.17Da and a Reinheitzahl (RZ) value of 1.55. Its N-terminal 27 amino-acid sequence showed high homology with those of Streptomyces humic acid peroxidases. HaP4 showed optimal activity at pH 4 and 70°C using 2,4-dichlorophenol (2,4-DCP) as a substrate. The enzyme was inhibited by sodium azide, mercury, cadmium, cyanide, and l-cystine, suggesting that heme components were present in its tertiary structure. The findings revealed that it was a heme protein catalyzing the oxidation of various substrates in the presence of hydrogen peroxide (H2O2). HaP4 obeyed Michaelis-Menten kinetics, and its catalytic efficiency (kcat/Km) was higher than that of the commercial enzyme horseradish peroxidase (HRP). © 2014 Elsevier Ltd. Source


Kourdali S.,Blida University | Kourdali S.,National Center for Research and Development of Fisheries and Aquaculture 11 | Badis A.,Blida University | Badis A.,National Center for Research and Development of Fisheries and Aquaculture 11 | Boucherit A.,Blida University
Ecotoxicology and Environmental Safety | Year: 2014

The present study was undertaken to investigate the degradation and removal of direct yellow 9 (DY9) by the electro-Fenton (EF) process in batch reactor using iron and stainless steel electrodes. DY9 removal decreased with the increase in pH (3 to 8) and increased with the increase in current intensity (0.05 to 0.2A) and [H2O2] (0 to 0.5gL-1, but not with high doses which led to low rates of DY9 removal and OH{bullet operator} uptake). The regression quadratic models describing DY9 degradation yield "R (percent)" and electrical energy consumption "EEC (kWhkg-1)" were validated by the analysis of variance (ANOVA) and were both noted to fit well with the experimental data. The R2 correlation coefficients (0.995, 0.978), those adjusted coefficients (0.986, 0.939), and F values (110.7, 24.9) obtained for the responses validated the efficiency of model. The results revealed that among several other parameters, EEC depended essentially on the degradation yield. The eco-toxicity tests showed a positive correlation between catalase activity and DY9 concentration, and catalase could be qualitatively identified to assess the effect of dye and its by-products generated during the EF process. © 2014 Elsevier Inc. Source


Benkiar A.,Blida University | Benkiar A.,National Center for Research and Development of Fisheries and Aquaculture 11 | Nadia Z.J.,University of Sfax | Badis A.,Blida University | And 9 more authors.
International Biodeterioration and Biodegradation | Year: 2013

An extracellular keratinolytic protease (SAPDZ) was produced and purified from a newly isolated Bacillus circulans strain DZ100. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis revealed that the purified enzyme is a monomer with a molecular mass of 32019.10Da. The sequence of the 25 N-terminal residues of SAPDZ showed high homology with those of Bacillus proteases. Optimal activity was achieved at pH 12.5 and 85°C. This enzyme was completely inhibited by phenylmethanesulfonyl fluoride (PMSF) and diiodopropyl fluorophosphates (DFP), which suggests that it belongs to the serine protease family. Compared to the other tested proteases, SAPDZ exhibited broader substrate specificity, higher levels of catalytic efficiency, and greater keratinolytic activity, which made it able to accomplish the entire feather-biodegradation process on its own. The sapDZ gene encoding SAPDZ was cloned, sequenced, and expressed in Escherichia coli. The biochemical properties exhibited by the extracellular purified recombinant enzyme (rSAPDZ) were similar to those of the native one. Above all, SAPDZ exhibited marked stability to detergents, making it a potential candidate for future applications in detergent formulations and an efficient eco-friendly enzyme for the biodegradation of feather keratin. © 2013 Elsevier Ltd. Source

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