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 new extracellular B. licheniformis protease was purified (SAPHM) and characterized.•SAPHM was a serine protease and a monomer with a molecular mass of 30,325.12 Da.•Optimum pH and temperature values for activity were pH 10 and 70 °C, respectively.•The sapHM gene encoding SAPHM was cloned, sequenced, and expressed in E. coli.•SAPHM is a potential candidate for peptide synthesis and detergent formulations.
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.
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 Mn2+. 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.
•Extracellular A. keratinilytica keratinase was purified (KERAK-29) and characterized.•KERAK-29 was a serine enzyme and a monomer with a molecular mass of 29,233.10 Da.•Optimum pH and temperature values for activity were pH 10 and 70°C, respectively.•Its thermoactivity and thermostability were enhanced in the presence of 5 mM Mn2+.•KERAK-29 can be used in the valorization processes of keratin-containing wastes.
•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.
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 (P
spp
). The biological value of P
spp
and its obtained derivatives were evidenced via accredited protocols. The recovered protein hydrolysate (P
Hyd
) 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.
Graphical abstract
The aim of this study consists of the production of a bio-surfactant from a new bacterial strain, Marinobacter hydrocarbono clasticus SF (96.76 % similarity) isolated from soil contaminated by ...hydrocarbons in Hassi-Messaoud (Southern Algeria) to treat liquid effluent from slaughterhouse water. The characteristics of organic matter biodegradation tests were discussed. Despite the high pollutant load and the unfavorable physicochemical composition of the effluent, the specific growth rate of the isolated strain after 10 days of incubation in the range of 0?30 g L-1 of NaCl was at neutral pH 7.4 and temperature of 45?C. The best bio-surfactant production yield was obtained after 72 h of incubation and under the optimal production conditions such as diesel as carbon source, ammonium chloride as nitrogen source, and a C/N ratio of 5. The bio-surfactant produced is of glycolipid type with a low critical micellar concentration (CMC), good emulsifying power, and chemical and functional stability. Significant pollutant removal efficiency was obtained using the bacterial strain (up to 82 %) and the bio-surfactant (up to 96 %). Several anions, such as nitrates, phosphates, ammonium, and suspended solids, were measured.
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.
This study aimed at isolating acylphloroglucinol derivatives with potential antioxidant effect from the brown alga
Zonaria tournefortii
and to ascertain their structure–activity relationship (SAR). ...Fractionation and HPLC purification led to the isolation of four major metabolites that were screened for their antioxidant activity via five complementary methods. Results indicated that all the tested compounds proved to have potential antioxidant activities. In DPPH and ABTS methods, the acylphloroglucinols were found to be the most active compounds with IC
50
values of 42.76 ± 0.26 and 50.46 ± 0.12 µg cm
−3
in DPPH assay and 12.04 ± 1.60 and 12.73 ± 1.47 µg cm
−3
in ABTS assay, respectively. In contrast, in β-carotene and CUPRAC methods, the chromone derivatives have revealed the high antioxidant activities with IC
50
values of 23.76 ± 0.63 and 20.99 ± 2.70 µg cm
−3
in β-carotene assay and 19.06 ± 0.11 and 17.72 ± 0.26 µg cm
−3
in CUPRAC assay, respectively. However, in metal chelating method all the investigated compounds demonstrated potent ferrous ion-chelating capability, with approximately the same level of activity.
Graphic abstract
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.
In this paper, biosurfactant production by free and immobilized cells of Ochrobactrum intermedium has been studied. This bacterium strain was isolated from an Algerian crude oil-contaminated soil; ...hexadecane was used for the production as the sole carbon and energy source. The process was monitored by measuring the surface tension and emulsification index E24 for one week at 37 °C and neutral pH. For the production by immobilized cells, the concentrations of sodium alginate, calcium chloride and biomass were optimized. Results showed that O. intermedium entrapped in calcium alginate beads is able to preserve its viability and produce biosurfactants but with an effect on the production kinetics due to diffusional limitations of the alginate beads with greater stability with up to 75%. The product biosurfactant reduced the surface tension below 33 and emulsification index were 68%-93% after 48 and 72 h with free and immobilized cells, respectively. Also, the product belongs to the family of glycolipids and showed stability in a wide range of pH (2-12), temperature (25-120 °C), and to high salinity. Both products by strain O. intermedium, based on spectral features, have a chemical structure identical to that of glycolipids. The production yield of biosurfactant versus concentration of the hexadecane is 1292 g/g. Second, the surfactants are able to promote the solubility of polycyclic aromatic hydrocarbons (PAHs). Results show that the use of biosurfactant, produced by the isolated bacterial strain O. intermedium, obtained a better solubility of naphthalene and phenanthrene.
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