Samy A. El-Assar

Production of k-carrageenase by Cellulosimicrobium cellulans isolated from Egyptian Mediterranean coast

The screening for isolates was done from different sources: sea water, sediment and algal tissue. Only two strains showed the capability of degrading k-carrageenan. Both strains were identified genetically using 16S rDNA. The isolates were classified to be Cellulosimicrobium cellulans and Isoptericola sp. Morphological and biochemical properties for C. cellulans were selected for the study. The results showed that C. cellulans exhibited an optimal growth at 10% NaCl and pH 7.5. It showed capability of growing on different carbon sources and to ferment different sugars except mannitol and cellulose. Different media for growing the bacterial cells of selected strain was tested in shaken condition. The highest specific activity (19 U/mg) for k-carrageenase was obtained in mineral medium supplemented with k-carrageenan. The optimum temperature for growing the cells of C. cellulans was found to be 37°C. It gave the highest k-carrageenase (2.14 U/ml) after 18 h incubation. Sensitivity of C. cellulans towards different antibiotics was conducted with different concentrations. It showed a high resistance toward all tested antibiotics except chloramphenicol at a concentration of 40 µg/ml. Experimental design was studied for production of k-carrageenase from free cells of C. cellulans. The statistical design raise the k-carrageenase activity to about 2.3 times higher than that obtained from the basal medium. The production of k-carrageenase by immobilized cells of C. cellulans using adsorption technique was conducted as the cells adsorbed on different supporting materials. The results showed that the cultures adsorbed on luffa pieces gave a relative high k-carrageenase activity (2.22 U/ml) comparing to the other supporting materials and even to free cells. This activity was found to be 1.5 times more than that obtained from cell-free medium.

Purification of kappa (k)-carrageenase from locally isolated Cellulosimicrobium cellulans

Partial purification of the crude kappa (k)-carrageenase present in the culture filtrates of Cellulosimicrobium cellulans was carried out by fractional precipitation, using ammonium sulphate, acetone and ethanol individually. The highest recovered protein (37.08%) combined with enzyme activity was obtained with ammonium sulphate. The fraction precipitated by 90% ammonium sulphate was re-purified by anion exchange chromatography diethylaminoethyl (DEAE) cellulose, A-52 and 79 fractions were obtained. The loaded protein was separated into 4 peaks. The third protein peak was the major one which contained the most recovered enzyme activity (84.95%) from the eluted fractions. The collected fractions of this peak were subjected to further purification by re-chromatography on Sephadex G-100. The k-carrageenase activity was fractionated into 2 peaks. The first peak was the major one containing 95.622% of the total recovered activity. The pooled fractions of the major protein component showed a specific k-carrageenase activity of 46.22 U/mg protein, yielding about 4.6 fold purification of the crude enzyme preparation. Some properties of purified k-carrageenase obtained from cellusimicrobium cellulans cultures were studied. The optimum reaction temperature of the purified k-carrageenase was 30°C and the maximum activity occurred at a reaction pH of 6. Key words : Cellulosimicrobium cellulans, k-carrageenase, purification, sephadex G-100, diethylaminoethyl (DEAE) sephadex A-52.

Ligninolytic Oxidative System of Fungal Egyptian Isolates and their Applications in the Decolorization of Industrial Dyes

ABSTRACT The aim of this study was to investigate the ligninolytic system of fungal strains isolated from Egyptian agricultural soil which are efficient in biodegradation and mineralization of lignin. They were identified morphologically, microscopically and confirmed by RAPD profiles. Based on PCR amplification and sequencing of the 18S rDNA gene an analytical phylogenetic tree was drawn for species confirmation. The screening experiment revealed that Emericella nidulans, Aspergillus fumigatus, Phoma betae, Penicillium oxalicum and Humicola grisea exhibited maximum potential for high lignin degradation. They showed higher lignin peroxidase and laccase activities. By process optimization, enhanced lignin degradation (94%) was achieved within 7 days in lignin-glucose medium when compared with lignin degradation (70%) obtained in glucose-free medium. The ligninolytic enzymatic activities had a great potential for decolorization of chemically different synthetic dyes (Azure B, Safranin, Crystal Violet and Malachite Green). The highest enzymatic activities and the highest decolorization rates were detected at a dye concentration of 0.2 g/L. The dye decolorization rate significantly increased with tryptophan addition as (1 mmol/L). Humicola grisea showed the highest decolorization rate (99%) with azure B. Phoma beta also showed a high decolorization rate (99% with crystal violet and 90% with safranin). The observed activity enhancement resulted from the protective effect of tryptophan against H2O2 inactivation.

Hydrogel film loaded with new formula from manuka honey for treatment of chronic wound infections

OBJECTIVES Chronic wound infections can be effectively treated using hydrogel loaded with an extract from manuka honey. METHODS This study was performed on bacterial isolates from patients with infected wounds at Alexandria Main University Hospital (Alexandria, Egypt). Isolates were exposed to hydrogel sheets composed of chitosan and gelatin and loaded with a new formula from manuka honey. RESULTS The results illustrate the antibacterial activity of the formula extracted from manuka honey against Staphylococcus aureus, Streptococcus pyogenes, Acinetobacter baumannii, Pseudomonas aeruginosa and Proteus mirabilis. Screening of the hydrogel by electron microscopy showed the ultrastructure of the gel. CONCLUSIONS A hydrogel sheet composed of chitosan and gelatin loaded with a new formula extracted from manuka honey can be used as a dressing for chronic infected wounds.

Isolation, characterization, optimization, immobilization and batch fermentation of bioflocculant produced by Bacillus aryabhattai strain PSK1

Among others, isolate PSK1 was selected and identified by 16 S rDNA sequencing as Bacillus aryabhattai. Growth optimization of PSK1 and physicochemical parameters affected bioflocculant production was carried out by Plackett-Burman design and resulted in increasing in the activity by 4.5%. Bioflocculant production by entrapped and adsorbed immobilized microbial cells was performed using different techniques and revealed enhancement in the activity in particular with pumice adsorption. HPLC analysis of sugars and amino acids composition, FTIR and the effect of different factors on the purified PSK1 biopolymer such as presence of cations, thermal stability, pH range and clay concentration was carried out. Scanning electron microscopy (SEM) of free, immobilized cells, PSK1 bioflocculant and formed flocs were performed. The results revealed that bioflocculant PSK1 is mainly glycoprotein consists of glucose and rhamnose with a large number of amino acids in which arginine and phenylalanine were the major. SEM analysis demonstrated that PSK1 have a clear crystalline rod shaped structure. FTIR spectrum reported the presence of hydroxyl and amino groups which are preferred in flocculation process. PSK1 was soluble in water and insoluble in all other tested organic solvents, while it was thermally stable from 40 to 80 °C. Among examined cations, CaCl2 was the best coagulant. The maximum flocculation activity of the PSK1 recorded at 50 °C (92.8%), pH 2.0 (94.56%) with clay concentration range 5–9 g/l. To obtain a large amount of PSK1 bioflocculant with high flocculating activity, batch fermentation was employed. The results recorded ∼6 g/l yield after 24 h of fermentation.

Purification and characterization of alginate lyase from locally isolated marine Pseudomonas stutzeri MSEA04.

An alginate lyase with high specific enzyme activity was purified from Pseudomonas stutzeri MSEA04, isolated from marine brown algae. The alginate lyase was purified by precipitation with ammonium sulphate, acetone and ethanol individually. 70% ethanol fraction showed maximum specific activity (133.3 U/mg). This fraction was re-purified by anion exchange chromatography DEAE- Cellulose A-52. The loaded protein was separated into 3 peaks. The second protein peak was the major one which contained 48.2% of the total protein recovered and 79.4% of the total recovered activity. The collected fractions of this peak were subjected to further purification by re-chromatography on Sephadex G-100. Alginate lyase activity was fractionated in the Sephadex column into one major peak, and the specific activity of this fraction reached 116 U/mg. The optimal substrate concentration, pH and temperature for alginate lyase activity were 8 mg/ml, pH 7.5 and 37 °C, respectively. While, Km and Vmax values were 1.07 mg alginate/ ml and 128.2 U/mg protein, respectively. The enzyme was partially stable below 50 °C, and the activity of the enzyme was strongly enhanced by K(+), and strongly inhibited by Ba(+2), Cd(+2), Fe(+2) and Zn(+2). The purified enzyme yielded a single band on SDS-PAGE with molecular weight (40.0 kDa).