Physical and enzymatic properties of a new manganese peroxidase from the white-rot fungus Trametes pubescens strain i8 for lignin biodegradation and textile-dyes biodecolorization.

A new manganese peroxidase-producing white-rot basidiomycete fungus was isolated from symptomatic wood of the camphor trees Cinnamomum camphora (L.) at the Hamma Botanical Garden (Algeria) and identified as Trametes pubescens strain i8. The enzyme was purified (MnP TP55) to apparent electrophoretic homogeneity and biochemically characterized. The specific activity and Reinheitzahl value of the purified enzyme were 221 U/mg and 2.25, respectively. MALDI-TOF/MS analysis revealed that the purified enzyme was a monomer with a molecular mass of 55.2 kDa. The NH2-terminal sequence of the first 26 amino acid residues of MnP TP55 showed high similarity with those of white-rot fungal peroxidases. It revealed optimal activity at pH 5 and 40 °C. This peroxidase was completely inhibited by sodium azide and potassium cyanide, suggesting the presence of heme-components in its tertiary structure. Interestingly, MnP TP55 showed higher catalytic efficiency, organic solvent-tolerance, dye-decolorization ability, and detergent-compatibility than that of horseradish peroxidase (HRP) from roots of Armoracia rustanica, manganese peroxidase from Bjerkandera adusta strain CX-9 (MnP BA30), and manganese peroxidase from Phanerochaete chrysosporium (MnP PC). Overall, the findings provide strong support for the potential candidacy of MnP TP55 for environmental applications, mainly the development of enzyme-based technologies for lignin biodegradation, textile-dyes biodecolorization, and detergent formulations.

Electrochemical disinfection of bacterial contamination: Effectiveness and modeling study of E. coli inactivation by electro-Fenton, electro-peroxi-coagulation and electrocoagulation.

The present work undertakes an examination and comparison of electro-Fenton (EF), electro-peroxi-coagulation (EPC) and electrocoagulation (EC) applied to the E. coli inactivation in batch reactor. Indeed, platinum (Pt (anode), EF), stainless steel (SS (cathode), EF, EPC) and ordinary steel (Fe (anode), EPC) and aluminum (Al, EC) were used respectively. The current intensity, nature of electrolytic support, bacterial density and hydrogen peroxide (H2O2) concentration are the most influenced study parameters. The obtained results showed that the high current intensities were significant for better inactivation and destruction of E. coli cells and caused a maximum of energy consumption. Both disinfection and energy consumption were improved by adding NaCl (or Na2SO4) in the three processes. Higher cellular density limited the electrochemical process and has negative effect in E. coli inactivation and the energy consumption. Only in the EPC case, the disinfection was considerably increased in function with H2O2 concentration. The modeling parameters of the inactivation kinetics of E. coli showed a good fitting of the established model (0.9560 < R2 < 0.9979, 0.9267 < R2 adjusted <0.997 and 0.0189 < RMSE <0.4821), faster kinetics of E. coli inactivation (significant values of Kmax and Sl) in the case of high current intensity (0.2442

Biochemical and molecular characterization of a novel metalloprotease from Pseudomonas fluorescens strain TBS09.

A novel extracellular protease called MPDZ was purified and characterized from Pseudomonas fluorescens strain TBS09. The enzymatic properties of MPDZ were investigated using biochemical and biophysical methods. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis revealed that it was a monomer with a molecular mass of 50013.17Da. The NH2-terminal 27 amino acid sequence of MPDZ showed high homology with those of Pseudomonas-proteases of the serralysin family. MPDZ showed optimal activity at pH 7 and 60°C. It was totally inhibited by EGTA, EDTA, and 1,10-phenanthroline, suggesting its belonging to the metalloprotease family. Because of the interesting properties, the mpDZ gene encoding MPDZ was cloned, sequenced, and expressed in E. coli. The deduced amino acid sequence showed a strong homology with other Pseudomonas-metalloproteases. The highest sequence identity value (97%) was obtained with AprX from P. fluorescens strain CY091, with only 12 different amino acid residues. The physico-chemical properties of the extracellular purified recombinant enzyme (rMPDZ) were similar to those of MPDZ. Overall, MPDZ is bestowed with a number of promising biochemical properties that might give new opportunities for its biocatalytic applications. These data constitute an essential first step towards an understanding of the properties of MPDZ enzyme.

Purification and characterization of two novel peroxidases from the dye-decolorizing fungus Bjerkandera adusta strain CX-9.

Two extracellular peroxidases from Bjerkandera adusta strain CX-9, namely a lignin peroxidase (called LiP BA45) and manganese peroxidase (called MnP BA30), were purified simultaneously by applying successively, ammonium sulfate precipitation-dialysis, Mono-S Sepharose anion-exchange and Sephacryl S-200 gel filtration and biochemically characterized. The sequence of their NH2-terminal amino acid residues showed high homology with those of fungi peroxidases. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis revealed that the purified enzymes MnP BA30 and LiP BA45 were a monomers with a molecular masses 30125.16 and 45221.10Da, respectively. While MnP BA30 was optimally active at pH 3 and 70°C, LiP BA45 showed optimum activity at pH 4 and 50°C. The two enzymes were inhibited by sodium azide and potassium cyanide, suggesting the presence of heme-components in their tertiary structures. The Km and Vmax for LiP BA45 toward 2,4-Dichlorolphenol (2,4-DCP) were 0.099mM and 9.12U/mg, respectively and for MnP BA30 toward 2,6-Dimethylphenol (2,6-DMP), they were 0.151mM and 18.60U/mg, respectively. Interestingly, MnP BA30 and LiP BA45 demonstrated higher catalytic efficiency than that of other tested peroxidases (MnP, LiP, HaP4, and LiP-SN) and marked organic solvent-stability and dye-decolorization efficiency. Data suggest that these peroxidases may be considered as potential candidates for future applications in distaining synthetic-dyes.

Biochemical and molecular characterization of new keratinoytic protease from Actinomadura viridilutea DZ50.

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 (18,000U/ml) after 96-h of incubation at 45°C. The enzyme was purified by ammonium sulfate precipitation (35-55%)-dialysis and heat treatment (30min 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.

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.

Characterization of a purified decolorizing detergent-stable peroxidase from Streptomyces griseosporeus SN9.

A novel extracellular lignin peroxidase (called LiP-SN) was produced and purified from a newly isolated Streptomyces griseosporeus strain SN9. The findings revealed that the pure enzyme was a monomeric protein with an estimated molecular mass of 43 kDa and a Reinheitzahl value of 1.63. The 19 N-terminal residue sequence of LiP-SN showed high homology with those of Streptomyces peroxidases. Its optimum pH and temperature were pH 8.5 and 65 °C, respectively. The enzyme was inhibited by sodium azide and potassium cyanide, suggesting the presence of heme components in its tertiary structure. Its catalytic efficiency was higher than that of the peroxidase from Streptomyces albidoflavus strain TN644. Interestingly, LiP-SN showed marked dye-decolorization efficiency and stability toward denaturing, oxidizing, and bleaching agents, and compatibility with EcoVax and Dipex as laundry detergents for 48 h at 40 °C. These properties make LiP-SN a potential candidate for future applications in distaining synthetic dyes and detergent formulations.

Degradation of direct yellow 9 by electro-Fenton: process study and optimization and, monitoring of treated water toxicity using catalase.

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(∙) 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 R(2) 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.