Abstract
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.
Highlights
; inoculums size 3.5 mm and moisture content, 1:1.5 w/v
Laccase production on solid substrate: Solid state fermentation (SSF) medium consist of 7.5 gm barley bran has been used for producing the fungal enzyme .The substrate was humidified with a 1:1.5 (w/v) of mineral salt solution containing (0.2 gm KH2PO4, 0.1gm MgSO4.7H2O, 0.3gm NH4CL2,1.0 GM CaCO3 in one liter distilled water, pH 5.6).The humidified medium was placed in 250 ml Erlenmeyer flasks and autoclaved at (121 ◦C, 20min ).The sterilized medium was incubated with three mycelial plugs 5mm from 7 days culture of P.ostreatus
Purification of laccase was carried out by ion exchange chromatography after curd enzyme concentrated by dialysis against the solid sucrose, and loaded onto a DEAE –cellulose anion exchange column 3.5× 16 cm, equilibrated with 0.01 M citrate phosphate buffers pH 5.6 at flow rate 0.5 ml /min, with linearly increasing NaCl concentration gradient 0.2- 1M in the same buffer.The five fraction containing laccase activity of fractions were determined
Summary
The isolate Pleurotus ostreatus was obtained from Department of Biotechnology, Collage of Science, Baghdad University. Fungal isolate was cultivated at 25◦C on malt extract agar (MEA) and stored at 4◦C. Laccase production on solid substrate: Solid state fermentation (SSF) medium consist of 7.5 gm barley bran has been used for producing the fungal enzyme .The substrate was humidified with a 1:1.5 (w/v) of mineral salt solution containing (0.2 gm KH2PO4, 0.1gm MgSO4.7H2O, 0.3gm NH4CL2 ,1.0 GM CaCO3 in one liter distilled water, pH 5.6).The humidified medium was placed in 250 ml Erlenmeyer flasks and autoclaved at (121 ◦C , 20min ).The sterilized medium was incubated with three mycelial plugs 5mm from 7 days culture of P.ostreatus (two flasks). Flasks were incubated for 10 days at 25◦C. Flask without inoculation was used as control [7]
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