A thermostable extracellular alkaline protease (called SAPA) was produced (4600 U/mL) by
Anoxybacillus kamchatkensis
M1V, purified to homogeneity, and biochemically characterized. SAPA is a monomer ...with a molecular mass of 28 kDa estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Native-PAGE, casein-zymography, and size exclusion using high performance liquid chromatography (HPLC). The sequence of its NH
2
-terminal amino-acid residues showed high homology with those of
Bacillus
proteases. The SAPA irreversible inhibition by diiodopropyl fluorophosphates (DFP) and phenylmethanesulfonyl fluoride (PMSF) confirmed its belonging to the serine proteases family. Optimal activity of SAPA was at pH 11 and 70 °C. The s
apA
gene was cloned and expressed in the extracellular fraction of
E. coli
. The highest sequence identity value (95%) of SAPA was obtained with peptidase S8 from
Bacillus subtilis
WT 168, but with 16 amino-acids of difference. The biochemical characteristics of the purified recombinant extracellular enzyme (called rSAPA) were analogous to those of native SAPA. Interestingly, rSAPA exhibit a degree of hydrolysis that were 1.24 and 2.6 than SAPB from
Bacillus pumilus
CBS and subtilisin A from
Bacillus licheniformis
, respectively. Furthermore, rSAPA showed a high detergent compatibility and an outstanding stain removal capacity compared to commercial enzymes: savinase™ 16L, type EX and alcalase™ Ultra 2.5 L.
Graphic Abstract
Highlights
A new extracellular
Anoxybacillus kamchatkensis
protease was purified (SAPA) and characterized.
SAPA was a serine protease and a monomer with a molecular mass of ∼28 kDa.
Optimum pH and temperature values for activity were pH 11 and 70 °C, respectively.
The
sapA
gene encoding SAPA was cloned, sequenced, and expressed in
E. coli
.
rSAPA is a potential candidate for future application as laundry detergent additive.
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.
► Overexpression of rSAPB and SAPB-L31I/T33S/N99Y proteases in
Bacillus subtilis DB430. ► Purification and biochemical characteristics of the heterologously expressed enzymes. ► Same N-terminal ...sequences and biochemical properties than those expressed in
Escherichia coli. ► SAPB-L31I/T33S/N99Y showed extremely stability to detergents and organic solvents. ► SAPB-L31I/T33S/N99Y constitutes an attractive candidate for industrial applications.
The
sapB gene encoding for
Bacillus pumilus CBS protease (SAPB) and the triple mutated
sapB-L31I/T33S/N99Y gene were cloned and overexpressed in the protease-deficient
Bacillus subtilis DB430 using an
Escherichia coli–
Bacillus shuttle vector pBSMuL2. The 34,625.13 and 34,675.11-Da enzymes were purified from the culture supernatant of
B. subtilis expressing the wild-type and mutated genes, respectively. The purified proteases showed the same N-terminal sequences and biochemical properties of those expressed in
E. coli. Further investigations demonstrated that, compared to wild-type and other proteases, SAPB-L31I/T33S/N99Y had the highest catalytic efficiency and the best degree of hydrolysis. The mutant enzyme was also noted to exhibit a number of newly explored properties that are highly valued in the marketplace, namely considerable stability to detergents, higher resistance towards organic solvents, and potent dehairing ability. Overall, the findings indicated that SAPB-L31I/T33S/N99Y is a promising candidate for future use in a wide range of industrial and commercial applications.
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.
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.
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.
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.
Omics approaches are widely applied in the field of biology for the discovery of potential CAZymes including whole genome sequencing. The aim of this study was to identify protein encoding genes ...including CAZymes in order to understand glycans-degrading machinery in the thermophilic Caldicoprobacter algeriensis TH7C1.sup.T strain. Caldicoprobacter algeriensis TH7C1.sup.T is a thermophilic anaerobic bacterium belonging to the Firmicutes phylum, which grows between the temperatures of 55 degreesC and 75 degreesC. Next generation sequencing using Illumina technology was performed on the C. algeriensis strain resulting in 45 contigs with an average GC content of 44.9% and a total length of 2,535,023 bp. Genome annotation reveals 2425 protein-coding genes with 97 ORFs coding CAZymes. Many glycoside hydrolases, carbohydrate esterases and glycosyltransferases genes were found linked to genes encoding oligosaccharide transporters and transcriptional regulators; suggesting that CAZyme encoding genes are organized in clusters involved in polysaccharides degradation and transport. In depth analysis of CAZomes content in C. algeriensis genome unveiled 33 CAZyme gene clusters uncovering new enzyme combinations targeting specific substrates. This study is the first targeting CAZymes repertoire of C. algeriensis, it provides insight to the high potential of identified enzymes for plant biomass degradation and their biotechnological 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.
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.
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