Biochemical and molecular characterization of a novel metalloprotease from Pseudomonas fluorescens strain TBS09.

A novel extracellular protease called MPDZ was purified and characterized from Pseudomonas fluorescens strain TBS09. The enzymatic properties of MPDZ were investigated using biochemical and biophysical methods. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis revealed that it was a monomer with a molecular mass of 50013.17Da. The NH2-terminal 27 amino acid sequence of MPDZ showed high homology with those of Pseudomonas-proteases of the serralysin family. MPDZ showed optimal activity at pH 7 and 60°C. It was totally inhibited by EGTA, EDTA, and 1,10-phenanthroline, suggesting its belonging to the metalloprotease family. Because of the interesting properties, the mpDZ gene encoding MPDZ was cloned, sequenced, and expressed in E. coli. The deduced amino acid sequence showed a strong homology with other Pseudomonas-metalloproteases. The highest sequence identity value (97%) was obtained with AprX from P. fluorescens strain CY091, with only 12 different amino acid residues. The physico-chemical properties of the extracellular purified recombinant enzyme (rMPDZ) were similar to those of MPDZ. Overall, MPDZ is bestowed with a number of promising biochemical properties that might give new opportunities for its biocatalytic applications. These data constitute an essential first step towards an understanding of the properties of MPDZ enzyme.

Purification and biochemical characterization of two detergent-stable serine alkaline proteases from Streptomyces sp. strain AH4.

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.

Chemical composition and antimicrobial activity of the essential oil from leaves of Algerian Melissa officinalis L.

The essential oil obtained from leaves of Melissa officinalis L. (Family of Lamiaceae) growing in Algeria, was investigated for its chemical composition and in vitro antimicrobial activity. The chemical composition was determined by hydrodistillation and analyzed by GC/MS and GC-FID. Sixty-three compounds were identified in the essential oil, representing 94.10 % of the total oil and the yields were 0.34 %. The major component was geranial (44.20 %). Other predominant components were neral (30.20 %) and citronellal (6.30 %). The in vitro antimicrobial activity was determined by paper disk agar diffusion testing and minimum inhibitory concentration (MIC) using 7 bacteria (3 Gram-positive and 4 Gram-negative), 2 yeasts and 3 fungi. The results showed that the essential oil presented high antimicrobial activity against all microorganisms targeted mainly against five human pathogenic bacteria, one yeast Candida albicans and two phytopathogenic fungi tested. The minimum inhibitory concentrations (MIC) ranged from 1.00 to 5.00 µL/mL.

Taxonomy and chemical characterization of antibiotics of Streptosporangium Sg 10 isolated from a Saharan soil.

A new actinomycete strain designated Sg 10, producing antimicrobial substances was isolated from an Algerian soil. Morphological and chemical studies indicated that strain Sg 10 belonged to the genus Streptosporangium. The comparison of its physiological characteristics with those of known species of Streptosporangium showed significant differences with the nearest species Streptosporangium carneum. Analysis of the 16S rDNA sequence of strain Sg 10 showed a similarity level ranging between 96.3% and 97.8% within Streptosporangium species, with S. carneum the most closely related. However, the phylogenetic analysis indicated that strain Sg 10 represent a distinct phyletic line suggesting a new genomic species. The antimicrobial activity of strain Sg 10 showed an antibacterial activity against Gram-positive bacteria as well as an antifungal one. Four active products were isolated from the culture broth using various separation procedures. On the basis of UV-VIS spectrometry, infrared spectroscopy and chemical revelations, the antibiotics were classified in the group of glycosylated aromatics.

Nocardiopsis and Saccharothrix genera in Saharan soils in Algeria: isolation, biological activities and partial characterization of antibiotics.

Twenty-five soil samples were collected in the Algerian Sahara and analyzed to isolate rare actinomycetes. Eighty-six isolates with the same Nocardiopsis or Saccharothrix morphology were isolated on humic-vitamin B agar medium using dilution techniques and several antibiotics as selective agents. Certain of these antibiotics seemed to be very selective for some phenotypes. Morphological and chemotaxonomic characteristics led to identifying 54 isolates belonging to the Nocardiopsis genus and 32 isolates belonging to the Saccharothrix genus. An assessment of the antimicrobial properties of the isolates showed activities against Gram-positive bacteria, fungi and yeasts. Saccharothrix isolates possessed better antifungal activity than Nocardiopsis. One of them, labeled SA 103, was therefore selected for identification of its antifungal antibiotic activities. Production of overall antifungal and antibacterial activities was checked on the complex medium ISP2 and a synthetic medium (SM) that contains glucose or starch as carbon source, and ammonium or nitrate as nitrogen source. The SM medium containing ammonium sulfate (0.2%), supplemented with starch (0.5%) and yeast extract (0.3%), was retained for production of antibiotics. Active substances were purified by a G25-80 Sephadex column and reverse phase HPLC. Two pure substances were obtained and named ZA01 and ZA02; they were characterized on the basis of combined data resulting from chemical tests, UV visibile and IR spectra and mass spectrometry. The two antibiotics were found to be related and were partially characterized as nucleotidic or nucleosidic antibiotics. Their structures consisted of a chain of three sugar units linked to an aromatic base containing a phosphate residue.

Mutactimycin PR, a new anthracycline antibiotic from Saccharothrix sp. SA 103. I. Taxonomy, fermentation, isolation and biological activities.

In the course of screening for new antibacterial agents, a new isolate collected from a soil sample of an arid area in south Algeria, produced a red pigment which was shown an antagonistic action against a gram-positive bacterium Bacillus subtilis. The isolate was identified as Saccharothrix sp. and named SA 103. The red pigment, eluted by HPLC on reverse phase C18 column, contained two compounds of an anthracycline antibiotics group. The structure of the major product (2) was characterized as mutactimycin C, and PR (1) was a new member of this group, designated as mutactimycin PR. These compounds showed an antibiotic activity against certain gram-positive bacteria in vitro. This is the first report of mutactimycins production by the genus Saccharothrix.

New dithiolopyrrolone antibiotics from Saccharothrix sp. SA 233. I. Taxonomy, fermentation, isolation and biological activities.

Three new natural antibacterial and antifungal dithiolopyrrolone antibiotics were isolated along with the known iso-butyropyrrothine and thiolutine from the fermentation broth of an actinomycete strain which was isolated from a saharian palm grove soil collected at Adrar, south Algeria. The strain was identified as Saccharothrix sp. The three new antibiotics exhibited broad antimicrobial activity against gram-positive bacteria, yeasts and fungi in vitro.