Identification of a novel protease from the thermophilic Anoxybacillus kamchatkensis M1V and its application as laundry detergent additive.

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 NH2-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 sapA 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.

Purification and biochemical characterization of a novel thermostable protease from the oyster mushroom Pleurotus sajor-caju strain CTM10057 with industrial interest.

BACKGROUND: 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. RESULTS: 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 NH2-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. CONCLUSIONS: This proves the excellent proprieties characterizing SPPS, making it a potential candidate for industrial applications especially detergent formulations.

Production, purification, and biochemical characterization of serine alkaline protease from Penicillium chrysogenium strain X5 used as excellent bio-additive for textile processing.

A new ascomycete fungus X5, a hyperproducer (9000 U/mL) of a serine alkaline protease (SAPTEX) was identified as Penicillium chrysogenum. The experimental purification protocol comprises three steps: heat treatment (10 min at 80 °C) followed by an ammonium sulfate precipitation (30-50%)-dialysis, and a UNO Q-12 anion exchange chromatography using the FPLC system. The chemical characterizations performed 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 43,074.11 Da. The 25 residue NH2-terminal sequence of the enzyme showed high homology with Penicillium proteases. The optimum pH and temperature values for protease activity were pH 10 and 80 °C, respectively. Compared to other proteases (SPTC; Flavourzyme® 500 L; Proteinase, type XXIII; Proteinase K; and Alcalase® 2.4 L), SAPTEX has the highest catalytic efficacy, hydrolysis degree, and a powerful stability toward some commercial detergents. According to morphological, physico-chemical [scanning electron microscopy (SEM), energy dispersive X-Ray analysis (EDX), and FTIR-Fourier transform infrared spectroscopy], and mechanical evaluation, SAPTEX has no destructive impact on fibers after the enzyme treatment and a very slight effect on textile support. Obtained results suggested that SAPTEX may be considered as a potential candidate as a protein stain removal product for textile supports.

Optimized production and characterization of a detergent-stable protease from Lysinibacillus fusiformis C250R.

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.

Characterization of a novel protease from Aeribacillus pallidus strain VP3 with potential biotechnological interest.

The present study investigates the purification and physico-chemical characterization of an extracellular protease from the Aeribacillus pallidus strain VP3 previously isolated from a geothermal oil-field (Sfax, Tunisia). The maximum protease activity recorded after 22h of incubation at 45°C was 3000U/ml. Pure enzyme, designated as SPVP, was obtained after ammonium sulfate fractionation (40-60%)-dialysis followed by heat-treatment (70°C for 30min) and UNO Q-6 FPLC anion-exchange chromatography. The purified enzyme is a monomer of molecular mass about 29kDa. The sequence of the 25 NH2-terminal residues of SPVP showed a high homology with those of Bacillus proteases. The almost complete inhibition by PMSF and DIFP confirmed that SPVP is a member of serine protease family. Its optima of pH and temperature were pH 10 and 60°C, respectively. Its half-life times at 70 and 80°C were 8 and 4h, respectively. Its catalytic efficiency was higher than those of SAPCG, Alcalase Ultra 2.5L, and Thermolysin type X. SPVP exhibited excellent stability to detergents and wash performance analysis revealed that it could remove blood-stains effectively and high resistance against organic solvents. These properties make SPVP a potential candidate for applications in detergent formulations and non-aqueous peptide biocatalysis.

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.