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.
•The paper reports on two novel extracellular peroxidases from Bjerkandera adusta CX-9.•These enzymes were purified (MnP BA30 and LiP BA45) and biochemically characterized.•The molecular weights and ...the NH2-terminal sequences of the enzymes were determined.•Their catalytic efficiency, solvent-stability, and dye-decolorization were studied.•These enzymes may be considered as potential candidates in distaining synthetic dyes.
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.
Proteases are hydrolytic enzymes that catalyze peptide linkage cleavage reactions at the level of proteins and peptides with different degrees of specificity. This group draws the attention of ...industry. More than one protease in three is a serine protease. Classically, they are active at neutral to alkaline pH. The serine proteases are researched for industrial uses, especially detergents. They are the most commercially available enzyme group in the world market. Overall, fungi produced extracellular proteases, easily separated from mycelium by filtration.
A new basidiomycete fungus CTM10057, a hyperproducer of a novel protease (10,500 U/mL), was identified as Pleurotus sajor-caju (oyster mushroom). The enzyme, called SPPS, was purified to homogeneity by heat-treatment (80 °C for 20 min) followed by ammonium sulfate precipitation (35-55%)-dialysis, then UNO Q-6 FPLC ion-exchange chromatography and finally HPLC-ZORBAX PSM 300 HPSEC gel filtration chromatography, and submitted to biochemical characterization assays. The molecular mass was estimated to be 65 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Native-PAGE, casein-zymography, and size exclusion by HPLC. A high homology with mushroom proteases was displayed by the first 26 amino-acid residues of the NH
-terminal aminoacid sequence. Phenylmethanesulfonyl fluoride (PMSF) and diiodopropyl fluorophosphates (DFP) strongly inhibit SPPS, revealing that it is a member of the serine-proteases family. The pH and temperature optima were 9.5 and 70 °C, respectively. Interestingly, SPPS possesses the most elevated hydrolysis level and catalytic efficiency in comparison with SPTC, Flavourzyme® 500 L, and Thermolysin type X proteases. More remarkably, a high tolerance towards organic solvent tolerance was exhibited by SPPS, together with considerable detergent stability compared to the commercial proteases Thermolysin type X and Flavourzyme® 500 L, respectively.
This proves the excellent proprieties characterizing SPPS, making it a potential candidate for industrial applications especially detergent formulations.
The keratin-degrading bacterium
Actinomadura viridilutea
DZ50 secretes a keratinase (KERDZ) with potential industrial interest. Here, the
ker
DZ gene was extracellularly expressed in
Escherichia coli
...BL21(DE3)pLysS using pTrc99A vector. The recombinant enzyme (rKERDZ) was purified and biochemically characterized. Results showed that the native and recombinant keratinases have similar biochemical characteristics. The conventional dehairing with lime and sodium sulfide degrades the hair to the extent that it cannot be recovered. Thus, these chemical processes become a major contributor to wastewater problem and create a lot of environmental concern. The complete dehairing was achieved with 2000 U/mL rKERDZ for 10 h at 40 °C. In fact, keratinase assisted dehairing entirely degraded chicken feather (45 mg) and removed wool/hair from rabbit, sheep, goat, or bovine’ hides (1.6 kg) while preserving the collagen structure. The enzymatic process is the eco-friendly option that reduces biological (BOD) (50%) and chemical (COD) oxygen demands (60%) in leather processing. Consequently, the enzymatic hair removal process could solve the problem of post-treatments encountering the traditional leather processing. The enzymatic (rKERDZ) dehaired leather was analyzed by scanning electron microscopic (SEM) studies, which revealed similar fiber orientation and compactness compared with control sample. Those properties support that the rKERDZ enzyme–mediated process is greener to some extent than the traditional one.
•Using statistical methodology, protease production by strain C250R was optimized.•RSM was used to determine optimum levels of screened factors and their interaction.•New extracellular protease from ...strain C250R was purified (SAPLF) and characterized.•SAPLF displayed a higher catalytic efficiency than SPVP and Alcalase Ultra 2.5L.•SAPLF offers an interesting potential for its use in the laundry detergent industry.
In this study, we aimed to optimize the cultural and nutritional conditions for protease production by Lysinibacillus fusiformis strain C250R in submerged fermentation process using statistical methodology. The most significant factors (gruel, wheat bran, yeast extract, and FeSO4) were identified by Plackett-Burman design. Response surface methodology (RSM) was used to determine the optimum levels of the screened factors and their interaction. Under the optimized conditions, protease yield 3100U/mL was 4.5 folds higher than those obtained by the use of the initial conditions (680U/mL). Additionally, a new extracellular 51kDa-protease, designated SAPLF, was purified and biochemically characterized from strain C250R. It shows optimum activity at 70°C and pH 10. Its half-life times at 70 and 80°C were 10 and 6-h, respectively. Irreversible inhibition of enzyme activity of SAPLF with serine protease inhibitors demonstrated that it belongs to the serine protease family. Interestingly, its catalytic efficiency was higher than that of SPVP from Aeribacillus pallidus strain VP3 and Alcalase Ultra 2.5L from Bacillus licheniformis. This study demonstrated that SAPLF has a high detergent compatibility and an excellent stain removal compared to Alcalase Ultra 2.5L; which offers an interesting potential for its application in the laundry detergent industry.
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 32019.10 Da. 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.
•A novel extracellular Bacillus circulans keratinase was purified (SAPDZ) and characterized.•Optimum pH and temperature values for activity were pH 12.5 and 85 °C, respectively.•The sapDZ gene encoding SAPDZ was cloned, sequenced, and expressed in Escherichia coli.•SAPDZ is a strong candidate for use as a biocatalyst for detergent and leather.•It seems to be an efficient eco-friendly enzyme in feather keratin-biodegradation.
The sapB gene, encoding Bacillus pumilus CBS protease, and seven mutated genes (sapB-L31I, sapB-T33S, sapB-N99Y, sapB-L31I/T33S, sapB-L31I/N99Y, sapB-T33S/N99Y, and sapB-L31I/T33S/N99Y) were ...overexpressed in protease-deficient Bacillus subtilis DB430 and purified to homogeneity. SAPB-N99Y and rSAPB displayed the highest levels of keratinolytic activity, hydrolysis efficiency, and enzymatic depilation. Interestingly, and at the semi-industrial scale, rSAPB efficiently removed the hair of goat hides within a short time interval of 8 h, thus offering a promising opportunity for the attainment of a lime and sulphide-free depilation process. The efficacy of the process was supported by submitting depilated pelts and dyed crusts to scanning electron microscopic analysis, and the results showed well opened fibre bundles and no apparent damage to the collagen layer. The findings also revealed better physico-chemical properties and less effluent loads, which further confirmed the potential candidacy of the rSAPB enzyme for application in the leather industry to attain an ecofriendly process of animal hide depilation. More interestingly, the findings on the substrate specificity and kinetic properties of the enzyme using the synthetic peptide para-nitroanilide revealed strong preferences for an aliphatic amino-acid (valine) at position P1 for keratinases and an aromatic amino-acid (phenylalanine) at positions P1/P4 for subtilisins. Molecular modeling suggested the potential involvement of a Leu31 residue in a network of hydrophobic interactions, which could have shaped the S4 substrate binding site. The latter could be enlarged by mutating L31I, fitting more easily in position P4 than a phenylalanine residue. The molecular modeling of SAPB-T33S showed a potential S2 subside widening by a T33S mutation, thus suggesting its importance in substrate specificity.
A novel extracellular protease (SAPRH) was hyper-produced (9000 U/mL) from Bacillus safensis RH12, a newly isolated enzyme from a Tunisian offshore oil field. The enzyme was purified to homogeneity, ...using salt-precipitation, heat-treatment and FPLC anion-exchange chromatography. The purified enzyme was a monomer of molecular mass of ~28 kDa. The NH2-terminal 23 amino-acid sequence of SAPRH showed high homology with those of Bacillus-proteases. SAPRH displayed optimal activity at pH 9 and 60 °C. It was strongly inhibited by phenylmethanesulfonyl fluoride (PMSF) and diiodopropyl fluorophosphates (DFP), indicating that it belongs to the serine-proteases family. Moreover, SAPRH was extremely stable at a broad range of temperature and pH retaining 85% of its activity at 50 °C and 75% at pH 11. The enzyme exhibited excellent stability and compatibility with surfactants and commercial detergents, revealing 90% stability with SDS and 100% stability with Class commercial laundry detergent. One of the most distinctive properties is its catalytic efficiency, which is higher than that of Alcalase 2.5 L, typeDX (commercial enzyme) and SAPB from B. pumilus CBS. Interestingly, the results of the wash performance analysis demonstrated considerably good de-staining at 40 °C for 30 min with low supplementation (500 U/mL). Accordingly, such a protease could be considered as a good detergent-additive in detergent industry.
•A novel extracellular protease (SAPRH) from Bacillus safensis strain RH12 was studied.•SAPRH with a molecular mass of ~28 kDa was purified and biochemically characterized.•The optimum pH and temperature values for activity were pH 9 and 60 °C, respectively.•SAPRH displayed higher enzymatic performance than Alcalase 2.5 L, type DX and SAPB.•SAPRH is a future potential candidate as bio-additive for detergent formulations.
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.
•New peroxidase isoform from T. pubescens i8 was purified (MnP TP55) and characterized.•Optimum pH and temperature values for activity were pH 5 and 40 °C, respectively.•The molecular weight and the NH2-terminal sequence of the enzyme were determined.•Its catalytic efficiency, solvent-stability, and dye-decolorization were studied.•This enzyme may be considered as potential candidate in environmental applications.
The presence of textile dyes in wastewater of textile factories represents a major environmental problem, threatening aquatic life. The decolorisation and degradation of harmful textile-dyes ...therefore remains a key field of research. In this study, a novel extracellular heme-manganese-peroxidase (30 kDa-MnP AN30) produced by Aspergillus niger strain CTM10002, a strain which was isolated from contaminated-wastewater of a nitrogen-phosphate-potash (NPK) chemical company in Sfax (Tunisia), was evaluated for its efficiency in the decolorization of different textile-dyes. Purification yield reached 60%, with a specific activity of 372 U/mg and Reinheitzahl (RZ) value of 2.8 under the optimal conditions of 40 °C and pH 5. The MnP AN30 NH2-terminal sequence was characterized by a high degree of similarity with those of other fungal-peroxidases. The mnp AN30 gene was cloned, sequenced, and expressed in Escherichia coli strain BL21(DE3)pLysS. Biochemical properties of the recombinant enzyme (rMnP AN30) were similar to those of the native one. Interestingly, molecular modeling and docking of MnP AN30 heme binding site revealed the involvement of 19 amino acid residues in substrate binding. More interestingly, MnP AN30 is capable of decolorizing textile dyes with higher decolorization efficiency than MnP TC55 from Trametes pubescens strain i8. Accordingly, these properties showed the potential candidacy of MnP AN30 to be used in biological treatment and decolorization of synthetic textile dyes.
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•New 30 kDa heme manganese peroxidase from A. niger CTM10002 was purified (MnP AN30).•Optimum pH and temperature values for activity were pH 5 and 40 °C, respectively.•The mnp AN30 gene was cloned, sequenced, and expressed in E. coli BL21(DE3)pLysS.•Docking data provided insight into the involvement of 19 amino acid residues in the substrate binding.•MnP AN30 is considered a promising candidate for textile dyes biological treatment and decolorization.
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