Catalyzed Mediator-Based Decolorization of Five Synthetic Dyes by Pleurotus ostreatus ARC280 Laccase.

Aim: The aim of the present study was to evaluate qualitatively the decolorization of five dyes by Pleurotus ostreatus (P. ostreatus) ARC280 using solid medium. The laccase produced by the fungus was used in terms of its concentration and thermal stability for enzymatic decolorization and also in combination with Hydroxybenzotriazole (HBT) as a redox mediator. Study Design: Qualitative evaluation of decolorization of dyes and determining the best conditions required for decolorization in the presence and absence of HBT. Place and Duration of Study: Department of Microbial Chemistry, Genetic Engineering and Biotechnology Division, National Research Centre (NRC), Cairo, Egypt, between January 2013 and February 2014. Methodology: P. ostreatus ARC280 fungal ability for dyes decolorization was qualitatively evaluated on solid medium containing (g/L): dye, 0.1; glucose, 10; agar, 30; 100 mL mineral solution and 100 mL wheat bran washing water obtained by boiling 50 g of wheat bran in 1000 mL of distilled water. The efficiency of decolorization was expressed in terms of decolorization percentage as follows: Decolorization (%) = 100 × Absorbance t0 - Absorbance tf Absorbance t0 Where Absorbance t0 is the absorbance at the optimumwavelength of the reaction mixture before incubation with the enzyme and Absorbance tf is the absorbance at the optimum wavelength after incubation time. Results: The enzyme was efficient in decolorizing Acid Blue C.I. 220 (100%), Dichlorophenol indophenol sodium salt D 5110 (92.6%) and Brilliant Green C.I. 42040 (78.6%) after 6 h of incubation at 30ºC. In the presence of HBT (1 mM), Lanasol Red 6G was greatly affected by HBT as a laccase mediator system with decolorization percentage of 53.85% instead of 10.90 in case of laccase alone, however the enzyme could not efficiently decolorize Foron Yellow Brown S 2RFLI dye even in presence of HBT. The decolorization efficiency of all dyes was decreased by increasing reaction temperature from 30 to 50ºC. The absorbance reduction at the maximum wavelength was recorded with all the tested dyes. Conclusion: The results obtained clearly confirmed the role of P. ostreatus ARC280 laccase and its mediated system in the decolorization of structurally different dyes.

Purification, Biochemical Characterization and Applications of Pleurotus ostreatus ARC280 Laccase.

Aims: To purify, characterize, and apply the laccase produced by submerged fermentation using an edible mushroom Pleurotus ostreatus ARC280. Study Design: Laccase purification and characterization were designed using the most recent approaches and statistical studies of triplicate results values. Place and Duration of Study: Department of Microbial Chemistry, Genetic Engineering and Biotechnology Division, National Research Centre, Dokki, Cairo, Egypt, between May 2011 and January 2013. Methodology: P. ostreatus ARC280 laccase was purified using ammonium sulfate precipitation (40-80%), followed by gel filtration using Sephadex G100 column chromatography. The resulted pure laccase was analyzed on SDS-PAGE (12%). Laccase activity parameters such as temperature, pH, stability, metal ions and kinetic constants were studied. Laccase was applied to reduce four tumor cell lines growth and as antibacterial and antifungal agent. Results: P. ostreatus ARC280 laccase was purified using ammonium sulphate followed by Sephadex G-100 chromatographic column by about 148 purification fold with Mr of 85kDa. Optimum P. ostreatus ARC280 purified laccase activity was recorded at 50ºC and at pH 6.0, 3.0, 4.5 for Syringaldazine (SGZ), 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic) acid (ABTS) and 2, 6-dimethoxyphenol (DMP) as substrates, respectively. The purified enzyme was more stable in alkaline pH range and retained about 37.42, 73.51, 85.65, 87.7, 88.49, 93.65, 92.86 and 100.0 % of the initial activity after 5hrs of incubation at pH 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0, respectively. Hg2+ caused complete inhibition at all tested concentrations; however Mn2+ (2.5x10-3M) caused laccase activation by about 190 and 330% after 1 and 24 hrs, respectively. Km and Vmax were calculated and found to be 0.074, 2.857 and 0.476 μM and 1.563, 2.500 and 2.632 μmol min-1 for SGZ, DMP and ABTS, respectively. The purified enzyme has the ability to reduce four tested cell lines growth in vitro with percentage reduction of 16.8, 23.4, 15.2 and 23.4% for HePG2, HCT116, A549 and MCF7, respectively. On the other hand, the enzyme was found to have antibacterial and antifungal activities against Escherichia coli and Candida albicans respectively. Conclusion: This enzyme seems to be a prospective enzyme for further biotechnological exploitation such as anticancer and antimicrobial activity applications.

Synthesis of Silver Nanoparticles and Incorporation with Certain Antibiotic Using Gamma Irradiation.

Aims: The study was explaining that silver nanoparticles (AgNPs) were synthesized biologically by Bacillus megaterium culture supernatant (as reducing and stabilizing agents) by the optimization of medium components for nitrate reductase production to enhance the synthesis of AgNPs. And use of gamma irradiation for the synthesis and incorporation of AgNPs with selected antibiotics at distinct dose. Place and Duration of Study: The study was carried out in 2012 in the Department of Drug Radiation Research, Egyptian Atomic Energy Authority. Methodology: The optimized conditions for AgNPs formation by B. megaterium culture supernatant were as follows; media containing: (%) yeast extract: 0.15, peptone: 0.25, KNO3:0.1 temp: 30ºC and incubation period 24 h with maximum nitrate reductase activity of (680.89U/ml). Physical synthesis of AgNPs and incorporation with antibiotics such as (Sodium Cefotaxime, Gentamycin sulphate and Amoxicillin) by γ-rays doses such as (0.5, 1, 1.5, 2, 2.5 and 3 kGy) were studded. AgNPs were characterized by (UV-Vis.), (DLS), (FT-IR) and (TEM) analysis. Combined and individual antibacterial activities of Amoxicillin and AgNPs were investigated against different pathogenic bacterial species by measuring the (ZOI) and by determining the (MIC). Results: This method shows that Aqueous Ag+ ions were reduced to AgNPs when added to the cell-free supernatant of B. megaterium this is indicated by the color change from whitish yellow to brown and the control showed no color change. In physical method Amoxicillin was incorporated with AgNPs perfectly at 2.5kGy. The decreasing order of the average antibacterial activity against bacterial group was observed to be AgNPs > Amoxicillin > Amoxicillin + AgNPs. Conclusion: The radiation-induced AgNPs synthesis is a simple, clean which involves radiolysis of aqueous solution that provides an efficient method to reduce metal ions. B. megaterium was found to be an effective biological tool for the extracellular biosynthesis of AgNPs. The bactericidal activity have proved that AgNPs in combination with amoxicillin kill bacteria at such low concentrations (units of ppm), which do not reveal acute toxic effects on human cell, in addition to overcoming resistance, and lowering cost when compared to conventional antibiotics.

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 Mg2+, Ca2+ whereas inhibited strongly by F-, Mo04 -, Cu2+ and Fe2+. 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 10-4 M.

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 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.

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 30ºC 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.