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Aim: the present study aims to optimize Cibacron Blue 3G-A decolorization as a model dye through laccase enzymatic biocatalysis presenting the role of HBT as a redox mediator via RSM approach. Study Design: RSM using Central Composite Design (CCD) was used in order to determine the most effective variables levels in Cibacron Blue 3G-A decolorization and to investigate their interactions. Place and Duration of Study: Department of Microbial Chemistry, Genetic Engineering and Biotechnology Research Division, National Research Centre (NRC), Cairo, Egypt, between August 2017 and January 2018. Methodology: The evaluation of Cibacron Blue 3G-A decolorization by A. bisporus CU13 crude laccase was conducted through different trials using a 1.5 mL reaction mixture containing different concentration of crude laccase, Cibacron Blue 3G-A, and HBT in 0.1 M sodium citrate buffer (pH 4.5) at room temperature for different incubation periods. Results: Hydroxybenzotriazole (HBT) as a mediator enhanced Cibacron Blue 3G-A decolorization levels significantly, where decolorization percentage caused by laccase enzyme alone were 11.92 and 23.78%, whereas that caused by laccase HBT mediator system under the same conditions were 43.43 and 76.34% after 1 and 22 h of incubation, respectively. HBT concentration, dye concentration, enzyme activity, and incubation time were chosen as study variables to optimize Cibacron Blue 3G-A dye decolorization through RSM approach via central composite design (CCD). The optimum conditions for Cibacron Blue 3G-A decolorization were found to be under using 0.50 U/mL of Agaricus bisporus CU13 laccase, 92.19 ppm of Cibacron Blue 3G-A, and 1 mM of HBT in order to get decolorization percentage of 29.29% in 35 min. Conclusion: Agaricus bisporus CU13 crude laccase was used as a biocatalyst to decolorize Cibacron Blue 3G-A in presence of HBT as a mediator through utilizing the response surface methodology approach. HBT concentration, dye concentration, enzyme activity, and incubation time affects the decolorization levels considerably.
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Background: Urinary tract infections (UTIs) represent one of the most common diseases that are encountered in clinical practice and are caused mainly by Escherichia coli (E. coli). Aims: The objectives of this study were to identify and compare the blaTEM, blaSHV and blaCTX-M as marker of beta-lactamase genes in E. coli strains isolated from patients with UTIs collecting from King Abdul-Aziz hospital in Taif region, Saudi Arabia. Study Design: In vitro experimental and molecular study. Place and Duration of Study: Genetic engineering and biotechnology unit, Taif University, from September, 2016 to November, 2017. Methodology: Beta-lactame antibiotics are prescribed in most infectious disease including UTIs. Twenty one isolates identified as E. coli using microbial identification and confirmed by 16S rDNA. Results: These isolates were susceptible to Imipenem (100%), Ampicillin (90%) and Cefoxitin, but resistant to Cefepime (38%). Existance of selected bla-genes (blaTEM, blaSHV and blaCTX-M) were detected in the 21 isolates by PCR. Moreover, phylogeny tree was drawn based on 16S rDNA sequence. The results of this study show significant differences in susceptibility to different beta-lactam antibiotics among the bla-genes in E. coli isolates. Conclusion: Therefore, our findings instead of our data provide some new epidemiological information about the clonal nature of E. coli isolated from patients with UTIs in Taif region, KSA.
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Aims: We designed this work to confirm if the PCR technique is more rapid and specific than traditional diagnostic method by culture. Study Design: In vitro experimental and molecular study. Place and Duration of Study: Genetic engineering and biotechnology unit, Taif University, Saudi Arabia from October, 2016 to September, 2017. Methodology: Ninety three nasal and tracheal swabs and lung samples were collected from camel in Taif slaughterhouse, Saudi Arabia. All samples were tested by culture and PCR method using universal primer of 16S rRNA gene. Results: There was no positive result obtained by culture method, but 30 (32.2%) of nasal swabs were positive using PCR method. Moreover, we used species-specific primers for Mycoplasma arginine, M. bovis and M. mycoides subspecies mycoides to identify the isolates at species level, but no positive results obtained with specific primers. These positive samples could be other Mycoplasma species. Conclusion: These results indicate that PCR technique is a specific molecular detection technique for Mycoplasma identification, and more sensitive test. These techniques are simple and fast methods to detect and isolate infected animals.
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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.
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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.
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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.
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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.