Bakry M. Haroun

Purification and properties of an endoglucanase of Aspergillus terreus DSM 826.

Endoglucanase (EG) from A. terreus DSM 826 grown on sugar cane bagasse as a carbon source was purified using acetone fractionation, then a Sepharose‐4B chromatographic column, with purification of about 27‐fold and 10.5% recovery. The optimum temperature and pH for activity of the purified EG were found to be 50 °C and pH 4.8, respectively. The purified enzyme can stand heating up to 50 °C for 1 h without apparent loss of activity. However, the enzyme, incubated at 80 °C for 5 min, showed about 56% loss of activity. Optimum EG activity was recorded with a citrate buffer system (pH 4.8; 0.05 M). Co2+ (2.5 × 10–2 M) and Zn2+ (5 × 10–2 M) were found to activate the purified EG of A. terreus DSM 826 by about 83 and 25%, respectively. On the other hand, Hg2+ inhibited the activity of the purified EG by about 50 and 71% at a concentration of 2.5 × 10–2 and 5 × 10–2 M, respectively. Carboxymethyl cellulose was found to be the best substrate for the purified EG, with Vmax values of 4.35 μmol min–1 mg–1 protein. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Optimization of Cultural and Nutritional Parameters for the Production of Laccase by Pleurotus ostreatus ARC280

Aims: To optimize laccase production by submerged fermentation using an edible mushroom Pleurotus ostreatus ARC280. Study Design: Laccase activity was assayed by monitoring the product formation rate of enzymatic oxidation of syringaldazine spectrophotometrically at 525 nm. Place and Duration of Study: Department of Microbial Chemistry, Genetic Engineering and Biotechnology Division, National Research Centre, Dokki, Cairo, Egypt, between May 2009 and October 2010. Methodology: Pleurotus ostreatus ARC280 was maintained on potato dextrose agar medium. The liquid medium used for the laccase production by the fungal culture during its growth in submerged fermentation was selected from eight liquid media for inducing laccase production. Parameters such as incubation period, temperature, pH of the production medium, carbon and nitrogen sources and other nutritional parameters were studied using syringaldazine as a model substrate for laccase activity determination. Results: In the present work, Eight media with different components were screened. The enzyme formed by Pl. ostreatus ARC280 was localized mainly in the extra-cellular fraction. Laccase formation reaches its maximum value with specific activity of about 140 U/mg protein at the twenty-sixth day of incubation, pH 5.0 and 28oC. Among the various wastes used, corn stover induces the highest laccase production with specific activity of Research Article British Biotechnology Journal, 2(3): 115-132, 2012 116 75.48 U/mg protein. Soluble starch at 1.5% (w/v) and ammonium sulfate was found to be the best carbon and nitrogen sources for laccase formation, respectively. The optimal concentrations of Tween-80 and CuSO4. 5H2O, were found to be 0.1% (v/v) and 100μM and cause enzyme induction by about 44% and 19% than control, respectively. Conclusion: Laccase production by Pl. ostreatus ARC280 has been shown to depend markedly on the composition of the culture medium, carbon, nitrogen content and inducer compounds and governed by parameters such as pH of the production medium and other nutrition parameters.

Production and Characterization of Keratinolytic Protease from New Wool-Degrading Bacillus Species Isolated from Egyptian Ecosystem

Novel keratin-degrading bacteria were isolated from sand soil samples collected from Minia Governorate, Egypt. In this study, the isolates were identified as Bacillus amyloliquefaciens MA20 and Bacillus subtilis MA21 based on morphological and biochemical characteristics as well as 16S rRNA gene sequencing. B. amyloliquefaciens MA20 and B. subtilis MA21 produced alkaline keratinolytic serine protease when cultivated in mineral medium containing 1% of wool straight off sheep as sole carbon and nitrogen source. The two strains were observed to degrade wool completely to powder at pH 7 and 37°C within 5 days. Under these conditions the maximum activity of proteases produced by B. amyloliquefaciens MA20 and B. subtilis MA21 was 922 and 814 U/ml, respectively. The proteases exhibited optimum temperature and pH at 60°C and 9, respectively. However, the keratinolytic proteases were stable in broad range of temperature and pH values towards casein Hammerstein. Furthermore the protease inhibitor studies indicated that the produced proteases belong to serine protease because of their sensitivity to PMSF while they were inhibited partially in presence of EDTA. The two proteases are stable in most of the used organic solvents and enhanced by metals suggesting their potential use in biotechnological applications such as wool industry.

Physiological studies on carboxymethyl cellulase formation by Aspergillus terreus DSM 826

Physiological studies were conducted to determine the optimum cultural conditions for maximal carboxymethyl cellulase (CMCase) formation by Aspergillus terreus DSM 826. Shaking condition at 150 rpm is favorable for the production of CMCase from rice straw and sugar cane bagasse. The highest enzyme yield was obtained at the third day of incubation at 30 °C for both cases; however CMCase formation occurred at a broad range of pH values, with maximal formation of A. terreus DSM 826 CMCase at pH 4.5 and 5.0 when rice straw and sugar cane bagasse were used as sole carbon source, respectively. Carboxymethyl cellulose (CMC) was found to be a good inducer for CMCase formation in both agricultural wastes with CMC concentrations of 0.5 and 1.0 % (w/v) in case of rice straw and sugar cane bagasse, respectively. High level of enzyme formation was obtained with the addition of ammonium chloride as nitrogen source in both cases and at a concentration of 0.4 % (v/v Tween-80) as an addition to medium containing rice straw. However this addition did not influence the production of CMCase in case of using sugar cane bagasse as carbon source.

Studies on Biosurfcatants Produced by Microorganisms Isolated from Different Locations

Background: Biosurfactants are amphiphilic compounds which have the significant advantages over synthetic counterparts. The advantages of biosurfactants over their synthetic derivatives and a wide range of applications have attracted a strong interest of scientific community. These have wide range of potential applications in areas of environmental applications and management, crude oil recovery, antimicrobial agents in health care and food processing industries. Objectives: the present study describes the Collection of different samples and isolation of microorganisms utilizing different hydrocarbons , purification and identification of the microbial isolates , screening of the ability of the used isolates for the effeciency in producing biosurfactants and studies for achieving optimum production of the biosurfactants and their chemical identification. Methodology and Results: In this study, we collected 15 samples from different locations from crude oil reservoires, waste industrial water, waste water, agricultural soil and automobile workshop. totally 1834colonies were obtained from all 15 samples from which 149 morphologically different colonies were tested for biosurfactants production and 50 strains showed a promising biosurfactants production. The two strains W1-S55 and SA3-S107 that presented the highest values of oil spreading test (65 and67mm), emulsification index (80 and85%), parafilm M test(8and10mm) and dropcollapse test(5and6mm) were identified as Pseudomonas aeruginosa and Bacillus subtilis, respectively by 16s rRNA sequence analysis and by Biochemical tests with Biolog system. 3 % Glyceroland %0.15 Glycine as carbon and nitrogen source, respectively was the optimal for biosurfactants producing condition by the isolate W1-S55, 16% molass and 0.5%sodium nitrate as carbon and nitrogen source, respectively was the optimal for biosurfactants producing condition by the isolate SA3-S107. The effect of pH; salinity and temperature on the produced biosurfactant were also evaluated.

Purification and partial characterization of a non-specific acid phosphatase degrading NAD from Aspergillus oryzae NRRL447.

A non specific acid phosphatase from Aspergillus oryzae NRRL447 catalyzes the phosphate hydrolysis from nicotinamide adenine dinucleotide forming nicotinamide riboside, adenosine and Pi as the final products of the reaction. The enzyme was purified to homogeneity by a sequential treatment of acetone fractionation, DEAE-cellulose chromatography and gel filtration chromatography. The enzyme was purified 400-fold. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme showed a single protein band of MW 52 kDa. The enzyme displayed maximum activity at pH 5.0 and 40 °C with NAD as substrate. The enzyme activity appeared to be stable over pH 2.0–5.0 and up to 40 °C. The enzyme activity was enhanced slightly by Mg, Ca whereas inhibited strongly by F, Mo04, Cu and Fe. The enzyme hydrolyzes several phosphate esters, suggesting a probable non-specific nature. The substrate concentration-activity relationship is the hyperbolic type and the apparent Km for NAD was 6.25 x 10 M.

Production of Candida utilis on Slop By-Product of Fermentation Industries

The slop (vinas) liquor, a major by-product a alcohol fermentation industries, has been used as growth medium for production of the torula yeast, Candida utilis. Supplementation of the slop with 0.2% ammonium sulphate and 1% to 5% molasses improved the cell yield significantly. The crude slop gave better results than the diluted or centrifuged liquors. Under optimal conditions, more than 15 grams of yeast were obtained on dry weight basis. The application feasibility of these results is presented.