Mohamed M. Hassan

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.

Purification, Characterization and Antitumor Activity of L-asparaginase from Penicillium brevicompactum NRC 829

Aim: The aims of this study were to attempt to extract, purify and characterize of Lasparaginase, an antitumor agent, from Penicillium brevicompactum NRC 829. Study Design: Testing of antitumor activity of L-asparaginase against four different tumor human cell lines.

Purification and some kinetic properties of b- glucosidase from Aspergillus terreus NRRL 265

An intracellular b-glucosidase (EC 3.2.1.21) from Aspergillus terreus NRRL 265 grown on whey permeate was purified to homogeneity as indicated by disc acrylamide gel electrophoresis with an apparent molecular mass of about 116 kDa. Optimal activity was observed at pH 5.0 and 60°C. The b- glucosidase had K m values of 2.5, 3.7 and 5.5 mM for p-nitrophenyl-β-D-glucopyranoside (p-NPG), cellobiose and salicin, respectively. Glucose and glucono-δ-lactone were found to be competitive inhibitors with apparent K i of 13.6 and 1.9 mM, respectively, when p-NPG was used as the substrate. Different metal cations had little or no effect on the enzyme activity, while Ag + and Hg 2+ had an inhibitory effect when used at high concentrations. In addition, b-glucosidase was found to be a glycoprotein containing 69% carbohydrate by weight and free from any myco-toxins. SH groups do not seem to play a role in the catalytic action of β-glucosidase as addition of iodoacetate, reduced glutathione or mercaptoethanol did not affect the activity. Key words : Aspergillus terreus , b-glucosidase, metal cations, iodoacetate.

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.

Purification, kinetic properties and antitumor activity of L-glutaminase from Penicillium brevicompactum NRC 829.

Aim: The aims of the present study were to purify and characterize L-glutaminase from Penicillium brevicompactum NRC 829; and to evaluate the antitumor activity of the purified enzyme against different tumor human cell lines. Study Design: Testing of antitumor activity of L-glutaminase, purified from a filamentous fungal strain, against four different tumor human cell lines. Place and Duration of Study: Department of Microbial Chemistry, Genetic Engineering and Biotechnology Division, National Research Centre (NRC), Cairo, Egypt, between January 2011 and February 2012. Methodology: P. brevicompactum NRC 829 was grown and maintained on modified Czapek Dox agar (MCD) medium. Cell-free extract was directly used as the source of crude enzyme. L-glutaminase was purified by heat treatment for 20 min at 50oC, followed by gel filtration on Sephadex G-100 and G-200 columns. Results: An intracellular L-glutaminase from Penicillium brevicompactum NRC 829 was purified to homogeneity (162.75 fold) with an apparent molecular mass (Mr) of 71 kDa. The purified enzyme showed its maximal activity against L-glutamine when incubated at pH 8.5 Research Article British Microbiology Research Journal, 4(1): 97-115, 2014 98 at 50oC for 30 min. The purified enzyme retained about 92 % of its initial activity after incubation at 70oC for 30 min indicating the thermo-stability nature of this enzyme. The highest activity was reported towards its natural substrate, L-glutamine, with an apparent Km value of 1.66 mM. The purified enzyme inhibited the growth of human cell line hepatocellular carcinoma (Hep-G2), with IC50 value of 63.3μg/ml. Conclusion: L-glutaminase purified from Penicillium brevicompactum NRC 829 is a potential candidate in food and pharmaceutical industries.

Application of response surface methodology to optimize the extracellular fungal mediated nanosilver green synthesis

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Purification and biochemical characterization of two isolated laccase isoforms from Agaricus bisporus CU13 and their potency in dye decolorization

Agaricus bisporus CU13 laccase was purified using ammonium sulfate precipitation (40-80%), Sephadex G100, and DEAE Sephadex A50 anion exchange column chromatography, respectively. Two laccase isoenzymes (Lacc1 & Lacc2) with purification folds of 1.40 and 5.81 respectively, were obtained from DEAE Sephadex A50 column. Optimal temperature and pH were recorded at 55 °C and pH 5.0 for both laccase isoenzymes using ABTS as substrate. Lacc1 was more thermostable than Lacc2 with residual activity of 95, 80 and 6%, while Lacc2 only retained 72, 25 and 0.4% of its activity after incubation for 90 min. at 50, 60 and 70 °C, respectively. Lacc2 retained about 93 and 86% of the initial activity at pH 9.0 and 7.0, whereas Lacc1 was stable at pH 7.0 and 5.0 followed by pH 9.0 and retained about 87, 76, and 36% of its activity respectively, after 4 h of incubation. Lacc1 was activated by 40% in the presence of Cu2+ (10 mM). Km and Vmax values found to be 0.394 and 0.158 μM, and 0.1351 and 0.4755 μmol min-1 for Lacc1 and Lacc2, respectively. The efficiency of both isoenzymes to decolorize Acid blue dye, make the enzyme seems to be a prospective for further biotechnological applications.

Degradation of purine ribonucleosides by extracts of Penicillium viridicatum

could catalyze the hydrolytic cleavage of N-glycosidic bond of adenosine, guanosine and inosine to the corresponding base and ribose by a ribonucleoside hydrolase; however, there is no evidence for the degradation of these compounds through phosphorylation. The rate of hydrolysis of the three ribonucleosides was in the order inosine> guanosine> adenosine. It was proven that adenosine hydrolyzing enzyme is not associated with the cell membrane. Maximum enzyme activity was observed at pH 4 and 50°C. Heat inactivation kinetics and the effect of the nature of the buffer on enzyme activity revealed that, the cleavage of the three purine ribonucleosides is affected by one intracellular nucleoside hydrolase. It was proven experimentally that, all the metal ions tested had a remarkable inhibitory effect on the activity of the ribonucleoside hydrolase. Results obtained indicate that extracts of