A new uricase from Bacillus cereus SKIII: Characterization, gene identification and genetic improvement.

Twenty-five microbial isolates were investigated for uricase production on uric acid medium. All isolates were obtained from Jeddah, Saudi Arabia. The highest uricase producer was identified as Bacillus cereus SKIII. Using glucose peptone broth at pH 7.5, incubation temperature 30 °C for 3 days with shaking of 150 rpm were the best conditions for maximum enzyme production. Glucose and peptone were the best carbon and nitrogen sources. The molecular weight of the purified enzyme was34.5 KDa, and isoelectric point was 7.9. The optimum pH and temperature were pH 8.0 and 35 °C, respectively. It was stable at 35 °C for 60 min, but thermally inactivated at 60 °C after 60 min Its enzymatic activity was enhanced by Mg2+, Ca2+,Fe2+, Mn2+, Zn2+ions and inhibited by Co2+, Na+, Hg2+, Ag+ ions and EDTA at 1 mM. Uricase production was enhanced using UV mutation and the obtained mutant produced six times higher than the original isolate. An amplicon 900 bp of uricase gene (Pucl) was sequenced (accession number MF417635). No remarkable difference was noticed in B. cereus SKIII and SKm mutant nucleotide sequences. In conclusion, SKIII and SKm are promising strains in uricase production for biotechnological applications.

Isolation and Molecular Identification of Two Chitinase Producing Bacteria from Marine Shrimp Shell Wastes.

BACKGROUND AND OBJECTIVE: Chitinase enzymes have a various application in the field of environmental, biotechnology and medical aspects. This study aimed to the production of the chitinolytic enzymes from different species of bacteria. MATERIALS AND METHODS: Bacterial isolation from different habitats was carried out on agar medium containing chitin as carbon and nitrogen sources. The obtained bacteria (20) were characterized and screened again in chitin broth medium. RESULTS: Out of 20 bacterial isolate, 2 new isolates, belonged to Streptomyces laurentii SN5 and Cellulosimicrobium funkei SN20, were the most active in chitin degradation compared to the other isolates. They have been characterized for the first time for their chitinase activity. They were identified using 16S rRNA gene analysis and in the liquid medium, the 2 isolates have enzyme activities of 0.533 and 0.537 U mL-1, respectively. The maximum chitinase production was obtained when those bacterial strains were grown in Luria-Bertani (LB) broth amended with 1% colloidal chitin, for 1 day and at temperature of 30°C. The optimum pH value for chitinase production was pH 7 for both S. laurentii and C. funkei. The enzyme has been purified using Sephadex G-100 and DEAE-Cellulose chromatography column and found to have a similar molecular size of ~50 kDa. CONCLUSION: Those two bacterial species could be used in chitinase production and in the environmental recycling of disposable chitin wastes such as chitin from shrimp shell waste.

A new l-glutaminase from Streptomyces pratensis NRC 10: Gene identification, enzyme purification, and characterization.

In the current study, the purified l-glutaminase from Streptomyces pratensis NRC10 (GenBank number KC857622) was characterized. Its molecular weight was estimated to be 46kDa and isoelectric point 7.4. Its Vmax was calculated to be 2.19U/mg/min, while Km was 0.175mM. The optimum pH and temperature were 9 and 45°C, respectively. It was thermostable at 45°C but thermally inactivated at 60°C after 50min. Moreover, its enzymatic activity was enhanced by K+ ions and inhibited by Mg2+, Cu2+, Ag+, Hg2+, Ni2+, Fe2+, Cr2, Na+, Ca2+, and EDTA. A PCR fragment of 1550bp of S. pratensis NRC10 l-glutaminase gene (glsA) was purified and its sequence was determined (GenBank number KJ567136). l-glutaminase from NRC10 was induced mainly by l-glutamic acid. Model 3-D structure was composed of two domains, the serine - dependent beta-lactamase dominant the small STAS domain (Sulphate Transporter and anti-sigma factor antagonist) which had probably functioned as a general NTP binding domain. The two domains are linked by a linker peptide (GLHLMRNPALPGST), but sequence alignment between salt-tolerant glutaminase and the obtained glutaminase showed 44.75% of identity and 57% of similarity. This enzyme appears to have a distinctive structure compared to the rest of glutaminase family, and seems to construct a new subgroup of glutaminase.

Isolation, antimicrobial and antitumor activities of a new polyhydroxysteroid and a new diterpenoid from the soft coral Xenia umbellata.

A new C-30 steroid, 3ß-,5α-,6ß-,11α-,20ß-pentahydroxygorgosterol (1), and a new diterpenoid, xeniumbellal (2), along with three known aromadendrane-type sesquiterpenes, aromadendrene (3), palustrol (4) and viridiflorol (5), were isolated from the soft coral Xenia umbellata. Chemical structures were determined by analyzing their NMR and MS data. The antimicrobial and antitumor activities of the isolated compounds were examined. Both 1 and 2 showed moderate antibacterial activities, especially against the multidrug-resistant Acinetobacter baumannii (MIC 0.22 and 0.28 mM, respectively); while 2 showed antitumor activity against a lymphoma cell line with LD50 0.57 mM and was nontoxic to Artemia salina at all tested concentrations up to about 4 mM.

Antimicrobial Activity of Biocompatible Microemulsions Against Aspergillus niger and Herpes Simplex Virus Type 2.

BACKGROUND: Microemulsions (MEs), which consist of oil, water, surfactants, and cosurfactants, have recently generated considerable interest as antimicrobial agents. OBJECTIVES: To determine the antifungal and antiviral activities of three ME formulations (MEa, MEb, and MEc) that differ in their hydrophilicity. METHODS: The ME formulas were produced by mixing different fractions of Tween 80, Span 20, ethanol, oil, isopropyl myristate, and distilled water. The antifungal activity of the ME formulas against Aspergillus niger, A. flavus, Bacillus, Candida albicans, and C. glabrata were determined by the solid medium diffusion cytotoxicity test against the mitochondria, measuring the minimum inhibitory concentration, dry biomass, and leakage of potassium, and characterizing the cell morphology. The antiviral activities of the ME formulas against the herpes simplex virus type 2 (HSV-2) were determined using the cytopathic effect assay. RESULTS: Significant antimicrobial activities were recorded against A. niger and herpes simplex virus type 2 (HSV-2) when treated with MEb that had hydrophobic nanodroplets with an average diameter of 4.7 ± 1.22 nm. A volume of 0.1 mL of MEb (10 mL of potato dextrose broth) inhibited the germination of A. niger cells, reduced their dry biomass, enhanced the leakage of potassium from the cell membranes, affected their mitochondria, and altered the shape of their conidia, in addition to enlarging them. MEb was able to destroy the HSV-2 virus at a 200-fold dilution in Dulbecco's modified eagle medium. CONCLUSIONS: The water-in-oil ME with equivalent surfactant-to-oil ratio (MEb) has great potential as an antifungal and antiviral agent.

Purification and partial characterization of serine-metallokeratinase from a newly isolated Bacillus pumilus NRC21.

A serine metallokeratinase enzyme (30 kDa) produced by a newly isolated Bacillus strain (Bacillus pumilus NRC21) cultivated under optimized conditions in medium containing chicken feather meal was purified and characterized in a set of biochemical assays. The purification was carried out using two successive chromatographic steps; cation exchange chromatography on CM-cellulose and gel filtration on sephadex G-100 columns. The purified enzyme showed a specific activity of 2000 units/mg protein against 170 units/mg protein for crude extract with 12 fold purification. The enzymatic activity of the keratinase stimulated by (Na(+), K(+), Mg(2+)), Hg(+2) had no effect, and inhibited by entire tested cations, serine and metalloproteinase inhibitors, therefore it can be considered as a serine metalloenzyme. The optimum pH and temperature for the purified enzyme were (7.5, 8.5) and (50, 45 °C) when using keratin azure and azocasein as substrates, respectively. The purified enzyme was highly stable at broad pH and temperature ranged (5-10) and (20-60 °C), respectively and its thermoactivity and thermostability were enhanced in the presence of 5 mM Mg(+2). These results suggest that the purified keratinase may be used in several industrial applications.

Two new polyacetylene derivatives from the Red Sea sponge Xestospongia sp.

Two new polyacetylenes (1 and 2), along with two known C-30 steroids (3 and 4) were identified from the Red Sea sponge, Xestospongia sp. The chemical structures were determined based on extensive spectroscopic measurements 1D (1H, 13C and DEPT) and 2D (COSY, HSQC and HMBC) NMR, UV, IR and MS. The new compounds 1 and 2 were evaluated for their antimicrobial and antitumor activities. 1 and 2 were active against multidrug- resistant bacteria with MICs ranged from 2.2 to 4.5 µM. No toxicity was recorded for the two tested compounds up to 5 µM using Artemia salina as a test organism. Compound 2 showed excellent antifungal activity against some pathogenic fungi such as Aspergillus niger and Candida albicans (MIC 2.2-2.5 µM) and antitumor activity against both Ehrlich ascites carcinoma and lymphocytic leukemia (LD50 5.0 µM).

Purification and characterization of gamma poly glutamic acid from newly Bacillus licheniformis NRC20.

γ-poly glutamic acid (γ-PGA) has received considerable attention for pharmaceutical and biomedical applications. γ-PGA from the newly isolate Bacillus licheniformis NRC20 was purified and characterized using diffusion distance agar plate, mass spectrometry and thin layer chromatography. All analysis indicated that γ-PGA is a homopolymer composed of glutamic acid. Its molecular weight was determined to be 1266 kDa. It was composed of L- and D-glutamic acid residues. An amplicon of 3050 represents the γ-PGA-coding genes was obtained, sequenced and submitted in genbank database. Its amino acid sequence showed high similarity with that obtained from B. licheniformis strains. The bacterium NRC 20 was independent of L-glutamic acid but the polymer production enhanced when cultivated in medium containing L-glutamic acid as the sole nitrogen source. Finally we can conclude that γ-PGA production from B. licheniformis NRC20 has many promised applications in medicine, industry and nanotechnology.

Production and characterization of thermostable metallo-keratinase from newly isolated Bacillus subtilis NRC 3.

Novel keratinolytic enzyme (32kDa) secreted by a newly isolated Bacillus strain (Bacillus subtilis NRC3) cultivated in medium containing chicken feather meal was purified and partially characterized in a set of biochemical assays. The purification was carried out by applying a protocol of two successive chromatographic steps; cation exchange chromatography on CM-cellulose and gel filtration on sephadex G-75 columns. The purified enzyme showed a specific activity of 5233units/mg protein against 169units/mg protein for crude extract with 31 fold purification. The enzymatic activity of the purified keratinolytic enzyme stimulated by Na(+), K(+), Mg(2+), Ba(2+), Ca(2+), and inhibited by entire tested cations and metalloproteinase inhibitors, indicating that it belongs to metallo-keratinase enzymes. The optimum pH and temperature for the purified enzyme were (7.5, 8.0) and (50, 40°C) when using keratin azure and azocasein as substrates, respectively. The purified enzyme was highly stable at broad pH and temperature ranged (5-10) and (20-60°C), respectively. These results suggest that this keratinase may be a useful alternative and ecofriendly route for handling the abundant amount of waste feathers.

Mineral content and microbiological examination of some white cheese in Jeddah, Saudi Arabia during 2008.

Different local and exported white cheese samples were collected from different markets in Jeddah during September 2008. Trace and heavy metals including Pb, Zn, Mn, Cu, Fe and Cd were analyzed using atomic absorption spectrometry. The concentration of the tested metals was in the range, Fe(++)>Zn(+++)>Mn(++)>Pb(++)>Cu(++)>Cd(++). The mean concentration of 7.63, 7.19, 0.5, 0.47, 0.16 and 0.14 µg/g was recorded for Fe, Zn, Mn, Pb, Cu and Cd, respectively. The concentration of iron ranged from 3.5 to 11.9 µg/g, zinc from 3.4 to 10.5, manganese from 0.12 to 1.0, lead from 0.14 to 1.14, and copper from 0.09 to 0.22. Yeasts and fungi were counted on Sabouraud and Potato Dextrose media and incubation was carried out at 25°C for 7 and 5 days, respectively. Yeast count and fungi count of cheese were ranged from 0.1 to 0.44CFU/g and from 0.123 to 1.11 CFU/g, respectively. Three out of 20 samples of cheese were contaminated with toxigenic fungi with 5% contamination level. Aflatoxin G1 was recorded in three samples using immunoadsorbent column chromatography with a range from 7 to 13 ppm.

Anti-Candida and mode of action of two newly synthesized polymers: a modified poly (methylmethacrylate-co-vinylbenzoylchloride) and a modified linear poly (chloroethylvinylether-co-vinylbenzoylchloride) with special reference to Candida albicans and Candida tropicalis.

Polymeric antimicrobial agents represent a new and important direction that is developing in the field of antimicrobial agents. Antimicrobial activity of two newly synthesized polymers: a modified poly (methylmethacrylate-co-vinylbenzoylchloride) and a modified linear poly (chloroethylvinylether-co-vinylbenzoylchloride) have been investigated and found to be active. Both polymers have showed a broad antimicrobial activity against C. albicans and C. tropicalis. Minimal inhibitory concentrations (MIC's) for poly (methylmethacrylate-co-vinylbenzoyl chloride) were 100, 75 and 100 microg/ml in case of C. albicans (ATCC 2091), C. albicans (SC5314) and C. tropicalis, respectively. However, polycholoroethylvinylether-covinylbenzoylchloride inhibited C. albicans (ATCC 2091), C. albicans (SC5314) and C. tropicalis with minimum inhibitory concentration values (MIC's) of 150 microg/ml against the three tested Candida strains. Mode of action studies of both polymers on the medically important yeasts, C. albicans and C. tropicalis revealed that poly (methylmethacrylate-co-vinylbenzoylchloride) induced cytotoxicity, DNA damage, and altered cell permeability and morphology, which was manifested as aggregated and swollen yeast cells (C. albicans ATCC 2091) by fluorescent microscopy examination. Poly (chloroethylvinylether-co-vinylbenzoylchloride) increased cell permeability, and respiration for C. albicans and C. tropicalis. The tested polymers at 50 microg/ml had pronounced effects on C. albicans and C. tropicalis cell wall phosphopeptidomannane, proteins, sugars and phosphorus. Generally, the two polymers proved effective against the tested microorganisms, but growth inhibitory effect varied according to the composition of the polymer active group. Many investigators consider polymeric antimicrobial agents as a potential new approach for enhancing the efficiency of some existing antimicrobial agents, including prolonged activity, reduce their toxicity, as well as reduce the environmental issues associated with product use.