Baseline levels of integrated biomarker response index (IBR) in Mytilus galloprovincialis (Lamarck, 1819) of BouIsmail bay for monitoring Algerian Bays (Mediterranean Sea).

The Multiple Linear Regression (MLR) technique was used to determine the relationship between the integrated biomarker response index (IBR) with the most important confounding factors (Temperature, pH, Salinity and Dissolved oxygen), in order to define: (1) the baseline assessment criteria (BAC), (2) the environmental assessment criteria (EAC) and (3) the BouIsmail Bay (BIB) -Taxonomy by using Mytilus galloprovincialis sampled from two references sites in BIB (W. Tipaza, Algeria) during one season. Therefore, our results show that, the temperature appeared as a major factor for the explanation of the model. The coefficients of determination and F values for IBR validated the efficiency of model. In addition, the use MLR, BAC and EAC values in BIB-Taxonomy can deference between unpolluted and polluted areas in BouIsmail Bay in particular and Algerian Bays in general. This approach is therefore a valuable tool that could be used for monitoring the coastal sites.

Modeling integrated biomarker response (IBR) index for the mussel Mytilus galloprovincialis (Lamark 1819) exposed to heavy metal mixture using the CCF design.

Marine pollutants such as heavy metals (HMs) are considered among the most copious oxidative stress (OS) inducers in marine organisms which leads to reactive oxygen species (ROS) formation. Complementary to our previous bioassays studies, the present research focuses on Catalase (CAT), Glutathione S-transferase (GST) and Malondialdehyde (MDA) as oxidative stress biomarkers and the integrated biomarker response (IBR) indexes (IBR1 and IBR2) as an ecotoxicological assessment tool in Mytilus galloprovincialis using central composite face centered (CCF) design. The oxidative stress biomarkers were measured in adult mussels (45-55 mm) on 3 days-exposed under different sub-lethal concentrations of cadmium (Cd), zinc (Zn), and copper (Cu). Using multiple regressions, ANOVA analysis revealed that experimental data fitted to second-order (quadratic) polynomial equations. The results showed that types, concentrations and metals combinations has a direct effect in CAT and GST activities, MDA level and IBR indexes. Additionally, metal-metal interactions were found synergistic (supra-additive), antagonistic (infra-additive) or zero interaction in the toxicological effect. As necessary, the optimization of the experimental results was done in order to determine the optimal conditions for the oxidative stress responses and IBR indexes. It was demonstrated that the CCF design combined with the multi-biomarker approach and IBR index can be used as an appropriate tool in ecotoxicological modulation and prediction of oxidative stress and antioxidant status by heavy metals in the mussels Mytilus galloprovincialis.

Extraction and characterization of chitin, chitosan, and protein hydrolysate from the invasive Pacific blue crab, Portunus segnis (Forskål, 1775) having potential biological activities.

The diversity of marine biomasses is a set of exploitable and renewable resources with application in several sectors. In this context, a co-culture based on three protease-producing bacterial isolates, namely Aeribacillus pallidus VP3, Lysinibacillus fusiformis C250R, and Anoxybacillus kamchatkensis M1V strains, was carried out in a medium based on the blue swimming crab Portunus segnis bio-waste. Proteases production was optimized using a central composite design (CCD). The highest level of proteases production obtained was 8,809 U/mL in a medium comprising 75 g/L of Portunus segnis by-product powder (Pspp). The biological value of Pspp and its obtained derivatives were evidenced via accredited protocols. The recovered protein hydrolysate (PHyd) was found to be active towards radical scavenging power and against angiotensin I-converting enzyme (ACE). The blue crab chitin (BC) extraction efficiency was achieved with a yield of 32%. Afterwards, chitosan was prepared through chitin N-deacetylation with a yield of 52%, leading to an acetylation degree (AD) of 19% and solubility of 90%. In addition, chitosan is found to be active against the growth of all pathogenic bacteria tested.

Purification and biochemical characterization of two keratinases from Bacillus amyloliquefaciens S13 isolated from marine brown alga Zonaria tournefortii with potential keratin-biodegradation and hide-unhairing activities.

The current paper reports the purification and biochemical characterization of two extracellular keratinolytic enzymes, with moderate elastolytic activity, from Bacillus amyloliquefaciens strain S13 newly isolated from the brown alga Zonaria tournefortii. The enzymes were purified to homogeneity by precipitation with (NH4)2SO4-dialysis, followed by size exclusion HPLC column, and submitted to biochemical characterization assays. The findings revealed that the pure enzymes designated KERZT-A and B were monomers with molecular masses of 28 and 47 kDa, respectively. Their identified NH2-terminal amino acid displayed high homologies with those of Bacillus keratinases. While KERZT-A was optimally active at pH 6.5 and 50 °C, KERZT-B showed optimum activity at pH 8 and 60 °C. Both enzymes were completely inhibited by phenylmethanesulfonyl fluoride (PMSF) and diiodopropyl fluorophosphates (DFP), which suggests their belonging to the serine keratinases family. Interestingly, KERZT-A displayed higher levels of hydrolysis, substrate specificity, and catalytic efficiency than KERUS from Brevibacillus brevis strain US575, NUE 12 MG (commercial enzyme), and KERZT-B unhairing keratinases. Above all, the findings indicated that KERZT-A and B enzymes seems to be an effective and an eco-friendly alternative to the conventional chemicals used for the feather keratin-biodegradation and for the unhairing of hides or skins in the leather processing industry.

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

Purification, biochemical, and molecular characterization of novel protease from Bacillus licheniformis strain K7A.

A novel extracellular alkaline protease, called SAPHM, from Bacillus licheniformis strain K7A was purified by four steps procedure involving heat treatment (30 min at 70 °C) followed by ammonium sulfate precipitation (40-70%)-dialysis, UNO Q-12 FPLC, and ZORBAX PSM 300 HPLC, and submitted to biochemical characterization assays. The purified enzyme is a monomer of molecular mass of 30,325.12 Da. It was completely inhibited by phenylmethanesulfonyl fluoride (PMSF)and diiodopropyl fluorophosphates (DFP), which strongly suggested its belonging to the serine protease family. Its sequence of the 26 NH2-terminal residues showed high homology with those of Bacillus proteases. The purified enzyme was optimally active at pH 10 and temperature 70 °C. Its catalytic efficiency was higher than those of Alcalase and Thermolysin. SAPHM exhibited excellent stability to detergents and wash performance analysis revealed that it could remove blood-stains effectively. Data suggest also that SAPHM may be considered as potential candidate for future applications in non-aqueous peptide biocatalysis because it possesses an elevated organic solvent resistance. The sapHM gene encoding SAPHM was cloned, sequenced, and expressed in Escherichia coli strain BL21(DE3)pLysS. The biochemical properties of the extracellular purified recombinant enzyme (rSAPHM) were similar to those of native one. The deduced amino acid sequence showed strong homology with other Bacillus proteases. The highest sequence identity value (97%) was obtained with APRMP1 protease from Bacillus licheniformis strain MP1, with only 9 aa of difference.

A non-toxic microbial surfactant from Marinobacter hydrocarbonoclasticus SdK644 for crude oil solubilization enhancement.

This study aims to investigate the ability of a biosurfactant produced by Marinobacter hydrocarbonoclasticus strain SdK644 isolated from hydrocarbon contaminated sediment to enhance the solubilization rate of crude oil contaminated seawater. Phylogenetic analysis shows that strain SdK644 was very closely related to M. hydrocarbonoclasticus with 16S rRNA gene sequence similarity of 97.44%. Using waste frying oil as inducer carbon source, the producing biosurfactant by strain SdK644 was applied to improve crude oil solubilization in seawater. The preliminary characterization of the produced biosurfactant by FT-IR analysis indicates its possible classification in a glycolipids group. Results from crude oil solubilization assay showed that SdK644 strain biosurfactant was 2-fold greater than Tween 80 surfactant in crude oil solubilization and 12-fold higher than seawater control, as shown by GC-MS analysis of aliphatic compounds. Furthermore, this bioactive compound was shown to be nontoxic against Artemia larvae in short-term acute toxicity bioassay. Generally, the results showed the possible use of M. hydrocarbonoclasticus strain SdK644 biosurfactant in bioremediation processes of the marine environments.

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.

Preliminary characterization of biosurfactant produced by a PAH-degrading Paenibacillus sp. under thermophilic conditions.

The capacities of a biosurfactant producing and polycyclic aromatic hydrocarbon (PAH) utilizing bacterium, namely, strain 1C, isolated from an Algerian contaminated soil, were investigated. Strain 1C belonged to the Paenibacillus genus and was closely related to the specie Paenibacillus popilliae, with 16S rRNA gene sequence similarity of 98.4 %. It was able to produce biosurfactant using olive oil as substrate. The biosurfactant production was shown by surface tension (32.6 mN/m) after 24 h of incubation at 45 °C and 150 rpm. The biosurfactant(s) retained its properties during exposure to elevated temperatures (70 °C), relatively high salinity (20 % NaCl), and a wide range of pH values (2-10). The infrared spectroscopy (FTIR) revealed that its chemical structure belonged to lipopeptide class. The critical micelle concentration (CMC) of this biosurfactant was about 0.5 g/l with 29.4 mN/m. In addition, the surface active compound(s) produced by strain 1C enhanced PAH solubility and showed a significant antimicrobial activity against pathogens. In addition to its biosurfactant production, strain 1C was shown to be able to utilize PAHs as the sole carbon and energy sources. Strain 1C as hydrocarbonoclastic bacteria and its interesting surface active agent may be used for cleaning the environments polluted with polyaromatic hydrocarbons.

A novel detergent-stable solvent-tolerant serine thiol alkaline protease from Streptomyces koyangensis TN650.

An alkaline proteinase (STAP) was produced from strain TN650 isolated from a Tunisian off-shore oil field and assigned as Streptomyces koyangensis strain TN650 based on physiological and biochemical properties and 16S rRNA gene sequencing. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis revealed that the purified enzyme was a monomer with a molecular mass of 45125.17-Da. The enzyme had an NH2-terminal sequence of TQSNPPSWGLDRIDQTTAFTKACSIKY, thus sharing high homology with those of Streptomyces proteases. The results showed that this protease was completely inhibited by phenylmethanesulfonyl fluoride (PMSF), diiodopropyl fluorophosphates (DFP), and partially inhibited by 5,5-dithio-bis-(2-nitro benzoic acid) (DTNB), which strongly suggested its belonging to the serine thiol protease family. Using casein as a substrate, the optimum pH and temperature values for protease activity were pH 10 and 70 °C, respectively. The protease was stable at pH 7-10 and 30-60 °C for 24 h. STAP exhibited high catalytic efficiency, significant detergent stability, and elevated organic solvent resistance compared to the SG-XIV proteases from S. griseus and KERAB from Streptomyces sp. AB1. The stap gene encoding STAP was isolated, and its DNA sequence was determined. These properties make STAP a potential candidate for future application in detergent formulations and non-aqueous peptide biocatalysis.

A novel keratinase from Bacillus tequilensis strain Q7 with promising potential for the leather bating process.

The present paper reports on the purification and characterization of an extracellular keratinase (KERQ7) newly purified from Bacillus tequilensis Q7. Pure protein was obtained after ammonium sulfate fractionation (30-60%), followed by Mono S Sepharose cation-exchange chromatography. MALDI-TOF/MS analysis revealed that the purified enzyme was a monomer with a molecular mass of 28,355.07-Da. The sequence of the 21 N-terminal residues of KERQ7 showed high homology with those of Bacillus keratinases. Optimal activity was achieved at pH 7 and 30°C. KERQ7 was completely inhibited by PMSF and DFP, which suggests that it belongs to the serine keratinase family. KERQ7 displayed higher levels of hydrolysis and catalytic efficiency than Basozym(®) CS 10, Koropon(®) SC 5K, and Pyrase(®) 250 MP. The kerQ7 gene encoding KERQ7 was cloned, sequenced, and expressed in Escherichia coli BL21(DE3)pLysS. The biochemical properties of the extracellular purified recombinant enzyme (rKERQ7) were similar to those of native KERQ7. The deduced amino acid sequence showed strong homology with other Bacillus keratinases. The highest sequence identity value (97%) was obtained with KERUS from Brevibacillus brevis US575, with only 7 aa of difference. These properties make KERQ7 a potential promising and eco-friendly enzymatically enhanced process for animal hide bating in the leather processing industry.

Purification and biochemical characterization of two detergent-stable serine alkaline proteases from Streptomyces sp. strain AH4.

Streptomyces sp. strain AH4 exhibited a high ability to produce two extracellular proteases when cultured on a yeast malt-extract (ISP2)-casein-based medium. Pure proteins were obtained after heat treatment (30 min at 70 °C) and ammonium sulphate fractionation (30-60 %), followed by size exclusion HPLC column. Matrix assisted laser desorption ionization-time of flight mass spectrometry analysis revealed that the purified enzymes (named SAPS-P1 and SAPS-P2) were monomers with molecular masses of 36,417.13 and 21,099.10 Da, respectively. Their identified N-terminal amino acid displayed high homologies with those of Streptomyces proteases. While SAPS-P1 was optimally active at pH 12.0 and 70 °C, SAPS-P2 showed optimum activity at pH 10.0 and 60 °C. Both enzymes were completely stable within a wide range of temperature (45-75 °C) and pH (8.0-11.5). They were noted to be completely inhibited by phenylmethanesulfonyl fluoride and diisopropyl fluorophosphates, which confirmed their belonging to the serine proteases family. Compared to SAPS-P2, SAPS-P1 showed high thermostability and excellent stability towards bleaching, denaturing, and oxidizing agents. Both enzymes displayed marked stability and compatibility with a wide range of commercial laundry detergents and significant catalytic efficiencies compared to Subtilisin Carlsberg and Protease SG-XIV. Overall, the results indicated that SAPS-P1 and SAPS-P2 can be considered as potential promising candidates for future application as bioadditives in 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.

Purification and biochemical characterization of a detergent-stable keratinase from a newly thermophilic actinomycete Actinomadura keratinilytica strain Cpt29 isolated from poultry compost.

An extracellular thermostable keratinase (KERAK-29) was purified and biochemically characterized from a thermophilic actinomycete Actinomadura keratinilytica strain Cpt29 newly isolated from Algerian poultry compost. The isolate exhibited high keratinase production when grown in chicken feather meal media (24,000 U/ml). Based on matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis, the purified enzyme is a monomer with a molecular mass of 29,233.10-Da. The data revealed that the 25 N-terminal residue sequence displayed by KERAK-29 was TQADPPSWGLNNIDRQTAFTKATSI, which showed high homology with those of Streptomyces proteases. This keratinase was completely inhibited by phenylmethanesulfonyl fluoride (PMSF) and diiodopropyl fluorophosphates (DFP), which suggests that it belongs to the serine protease family. Using keratin azure as a substrate, the optimum pH and temperature values for keratinase activity were pH 10 and 70°C, respectively. KERAK-29 was stable between 20 and 60°C and pH 3 and 10 for 5 and 120 h, respectively, and its thermoactivity and thermostability were enhanced in the presence of 5 mM Mn(2+). Its catalytic efficiency was higher than that of the KERAB keratinase from Streptomyces sp. strain AB1. KERAK-29 was also noted to show high keratinolytic activity and significant stability in the presence of detergents, which made it able to accomplish the entire feather-biodegradation process on its own. The ability of the A. keratinilytica strain Cpt29 to grow and produce substantial levels of keratinase using feather as a substrate could open new promising opportunities for the valorization of keratin-containing wastes and reduction of its impacts on the environment.

Biochemical and molecular characterization of a serine keratinase from Brevibacillus brevis US575 with promising keratin-biodegradation and hide-dehairing activities.

Dehairing is one of the highly polluting operations in the leather industry. The conventional lime-sulfide process used for dehairing produces large amounts of sulfide, which poses serious toxicity and disposal problems. This operation also involves hair destruction, a process that leads to increased chemical oxygen demand (COD), biological oxygen demand (BOD), and total suspended solid (TSS) loads in the effluent. With these concerns in mind, enzyme-assisted dehairing has often been proposed as an alternative method. The main enzyme preparations so far used involved keratinases. The present paper reports on the purification of an extracellular keratinase (KERUS) newly isolated from Brevibacillus brevis strain US575. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis revealed that the purified enzyme was a monomer with a molecular mass of 29121.11 Da. The sequence of the 27 N-terminal residues of KERUS showed high homology with those of Bacillus keratinases. Optimal activity was achieved at pH 8 and 40°C. Its thermoactivity and thermostability were upgraded in the presence of 5 mM Ca(2+). The enzyme was completely inhibited by phenylmethanesulfonyl fluoride (PMSF) and diiodopropyl fluorophosphates (DFP), which suggests that it belongs to the serine protease family. KERUS displayed higher levels of hydrolysis, substrate specificity, and catalytic efficiency than NUE 12 MG and KOROPON® MK EG keratinases. The enzyme also exhibited powerful keratinolytic activity that made it able to accomplish the entire feather-biodegradation process on its own. The kerUS gene encoding KERUS was cloned, sequenced, and expressed in Escherichia coli. The biochemical properties of the extracellular purified recombinant enzyme (rKERUS) were similar to those of native KERUS. Overall, the findings provide strong support for the potential candidacy of this enzyme as an effective and eco-friendly alternative to the conventional chemicals used for the dehairing of rabbit, goat, sheep and bovine hides in the leather processing industry.