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.

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.

Characterization of a novel biosurfactant produced by Staphylococcus sp. strain 1E with potential application on hydrocarbon bioremediation.

A biosurfactant-producing bacterium (Staphylococcus sp. strain 1E) was isolated from an Algerian crude oil contaminated soil. Biosurfactant production was tested with different carbon sources using the surface tension measurement and the oil displacement test. Olive oil produced the highest reduction in surface tension (25.9 dynes cm(-1)). Crude oil presented the best substrate for 1E biosurfactant emulsification activity. The biosurfactant produced by strain 1E reduced the growth medium surface tension below 30 dynes cm(-1). This reduction was also obtained in cell-free filtrates. Biosurfactant produced by strain 1E showed stability in a wide range of pH (from 2 to 12), temperature (from 4 to 55 °C) and salinity (from 0 to 300 g l(-1)) variations. The biosurfactant produced by strain 1E belonged to lipopeptide group and also constituted an antibacterial activity againt the pathogenic bacteria such as Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis. Phenanthrene solubility in water was enhanced by biosurfactant addition. Our results suggest that the 1E biosurfactant has interesting properties for its application in bioremediation of hydrocarbons contaminated sites.