Determination of optimal conditions for laccase production by Pleurotus ostreatus using sawdust as solid medium and its use in phenol degradation

The ability of four local fungal isolates for extracellular laccase production has been tested with five grams 1:1(w/v) humidified sawdust as substrate in mineral salt medium. After 21 day of incubation at 25±1 ? C and using one mycelial plug (5mm), higher level of laccase activity (0.15U/ml) and specific activity (15U/mg) were observed by Pleurotus ostreatus in comparison with other fungal isolates. The results of optimum conditions for laccase production from selected isolate showed that, the maximum laccase activity (0.55U/ml) and specific activity (55U/mg) were obtained at moisture ratio 1:3 (w/v), using 3 mycelial plugs (5 mm), after 15 days incubation period at 25±1 ? C. The results of phenol degradation by crud laccase revealed that, 100% degradation of phenol occurred after 24 hr of incubation at 25 ? C using shaking water bath except at 200mg/l, the remaining phenol was 10.13%

PCR Detection of Aspergillus flavus Isolates for Aflatoxin B1 producer

The ability of five Aspergillus flavus that produce Aflatoxin B1 have been detected using coconut medium as substrate. Chromatographical analysis by TLC and HPLC revealed that, three out of five isolates were a good producer for the Aflatoxin B1. In this study, rapid assessment of five isolates of A. flavus was accomplished using an indigenously designed primer pair for the Aflatoxin regulatory gene aflR in polymerase chain reaction (PCR). Specificity was assayed in pure culture systems using DNA extracted from five different A. flavus isolates as PCR template. Positive amplification was achieved only with DNA from A. flavus that produce Aflatoxin B1

Extruction of keratinase from the local isolate Bacillus licheniformis and partially purified and characterized

The keratinase produced from local isolate Bacillus licheniformis was purified by two steps included precipitation by ammonium sulphate with 40% saturation; followed by ion exchange using CM-Cellulose column. The enzyme was purified to 12.6 times in the last step with an enzyme yield of 17%. Enzyme characterization results indicated that: The optimal pH for enzyme activity was 7.5 and it was stable at 7-9.5. The optimal temperature for enzyme activity was 50°C and it was stable for 30 min at 25-45 °C. Substrate specifity was tested using casein, Bovine serum albumin, gelatin, hooves, human hair, chicken feathers and wool; higher specifity was recorded using casein gave 0.6 unit /ml. The enzyme was inhibited by PMSF and metal ions like Hg+2, Fe+2, Cu+2 and Mn+2, and activated by Ca+2, Mg+2, Zn+2and Al+3

Cytotoxic Effect of Gliotoxin, Hemolysin, Protease and Melanin purified from Aspergillus fumigates on REF Cell Line, in vitro Study

Aspergillus fumigates especially clinical isolate produces a series of toxic substances and proteinaceous hemolysin, protease and pigment like melanin which appear to act in an additive and synergic way on cells. In this study, gliotoxin, hemolysin, Protease, and Melanin were used in an experimental model to study their Cytotoxic activity by evaluating their effect on REF cell line (Rat embryonic fibroblast ), for exposure time of 24 hrs at three different concentrations of each compounds triplicate of each concentration were used, cytotoxicity of the purified compounds were active against REF cell line under study and a toxic effect was clear with a significant difference at the level of probability (p≤ 0.05) and this effect was contrasted among different concentrations for each purified compound growth inhibition of REF cell line was increase gradually with the increase of compound concentration

Improving Conditions for Gliotoxin Production by Local Isolates of Aspergillus fumigatus

Thirty two isolates of Aspergillus fumigatus were obtained from a total of 44 samples of sputum, nose swab and tracheal aspirate from suspected patient with aspergillosis were also collected from February 2014 to June 2014.  A morphological examination of A. fumigatus was first made with naked eye and at low magnification power of microscope after that detailed examination was done by measuring the dimensions of the microscopic structures, photographing the microscopic structures and using relevant literature. Results appeared conical-shaped terminal vesicles, uniseriate row of phialides on the upper two thirds of the vesicle, conidiophore stipes were short, phialides arrange uniseriate upper vesicle conidia and parallel to axis of conidiophore, produced in chains of  spore basipetally from phialides, the chains of spore were borne directly in the absence of metulae and represented by septet and branching hyphae. The ability of A. fumigatus for GT production was investigated using thin layer Chromatography (TLC) and High Performance Liquid Chromatography (HPLC) techniques and results showed that GT was produced by 81.25% of A. fumigatus isolates. Optimum conditions for GT production by A. fumigatus 16 (AF-16) isolate were determined by submerged fermentation using Yeast extract sucrose medium. Results indicated that AF-16 isolate was the highest GT producer on Yeast Exract Sucrose medium with inoculum size 2×107 conidium and incubation at 32ºC for 15 days and the concentration of gliotoxin was (4511µg mL-1).                                                                                                                               

Using Random Amplified Polymorphic DNA (RAPD) analysis to investigation of genetic diversity, and relationships among a set of clinical Aspergillus fumigatus isolates

This study is an attempt to determine the genetic diversity and relationships among fourteen local isolate isolated from patients with Aspergillosis (Aspergillus fumigatus) by using the Random Amplified Polymorphic DNA (RAPD) technique. Twelve universal primers used in this study produced 94 bands across fourteen isolates. Of these bands, 67 bands or 71.2% were polymorphic. The size of the amplified bands ranged between 100-2000 bp. The genetic polymorphism value of each primer was determined and ranged between 33-100%. In terms of unique banding patterns, determine the finger print for six isolates the most characteristic banding pattern was for the (AFU1, AFU2, AFU3, AFU4, AFU8 and AFU14) with primer (OP F-16 , OP I-06, OP F-16, OP X-01, OP X-01and OP A-06). Genetic distances ranged from 0.12419 to 0.64404 among A. fumigatus isolates. Cluster analyses were performed to construct a dendrogram among studied A. fumigatus isolates. The cluster analysis places most of the A.fumigatus isolates isolated from patient come from yhe same area into a close relation (subcluster) showing a high level of genetic relatedness and were distinct from isolates from another area (the other subcluster). Interestingly, a number of isolates originating from the same sources did form well defined groups, indicating association between the RAPD patterns and the geographic origin of the isolates. The information generated from this study can be used in the future for controlling of Aspergillosis programs

Decolorization of textile dyes by partially purified Pleurotus ostreatus laccase

Pleurotus ostreatus produced 2.93 U/mg of laccase in solid state fermentation (SSF) using barley bran as substrate under optimum conditions. The optimum SSF conditions were: pH 6.5; temperature, 25Cº; inoculums size 3.5 mm and moisture content, 1:1.5 w/v. Laccase was partially purified 8.29 fold with specific activity 17.5 U/mg by ion exchange chromatography after curd enzyme concentrated by dialysis against the solid sucrose. Partially purified laccase had an optimum pH of 6.5 and was stable in the pH range from 6.5 to 7.5. The optimum temperature was 45 Cº and it displayed considerable stability within the range 15 to 45 Cº with 1h incubation as well as The ability of partial purified laccase to decolorize of textile dyes showed that the blue H3R dye was completely decolorized in all concentrations within first min while yellow FG and red 3B dyes were decolorized in different percentage

Optimal conditions for gliotoxin production from Aspergillus fumigatus using solid state fermentation

The ability of ten Aspergillus fumigatus isolates to produce gliotoxin was assessed by solid state fermentation using rice medium as a substrate. The results indicated that the AF-5 Aspergillus fumigatus isolate produced the most gliotoxin. The optimum conditions for gliotoxin production were a moisturising ratio of 5:1 (w: v) with distilled water, an inoculum size of 1.5 ml, containing

PCR Detection of Aspergillus flavus Isolates for Aflatoxin B1 producer

The ability of five Aspergillus flavus that produce Aflatoxin B1 have been detected using coconut medium as substrate. Chromatographical analysis by TLC and HPLC revealed that, three out of five isolates were a good producer for the Aflatoxin B1. In this study, rapid assessment of five isolates of A. flavus was accomplished using an indigenously designed primer pair for the Aflatoxin regulatory gene aflR in polymerase chain reaction (PCR). Specificity was assayed in pure culture systems using DNA extracted from five different A. flavus isolates as PCR template. Positive amplification was achieved only with DNA from A. flavus that produce Aflatoxin B1.</jats:p

Improving Conditions for Gliotoxin Production by Local Isolates of Aspergillus fumigatus

Thirty two isolates of Aspergillus fumigatus were obtained from a total of 44 samples of sputum, nose swab and tracheal aspirate from suspected patient with aspergillosis were also collected from February 2014 to June 2014.  A morphological examination of A. fumigatus was first made with naked eye and at low magnification power of microscope after that detailed examination was done by measuring the dimensions of the microscopic structures, photographing the microscopic structures and using relevant literature. Results appeared conical-shaped terminal vesicles, uniseriate row of phialides on the upper two thirds of the vesicle, conidiophore stipes were short, phialides arrange uniseriate upper vesicle conidia and parallel to axis of conidiophore, produced in chains of  spore basipetally from phialides, the chains of spore were borne directly in the absence of metulae and represented by septet and branching hyphae. The ability of A. fumigatus for GT production was investigated using thin layer Chromatography (TLC) and High Performance Liquid Chromatography (HPLC) techniques and results showed that GT was produced by 81.25% of A. fumigatus isolates. Optimum conditions for GT production by A. fumigatus 16 (AF-16) isolate were determined by submerged fermentation using Yeast extract sucrose medium. Results indicated that AF-16 isolate was the highest GT producer on Yeast Exract Sucrose medium with inoculum size 2×107 conidium and incubation at 32ºC for 15 days and the concentration of gliotoxin was (4511µg mL-1).                                                                                                                                </jats:p