Characterization and purification of the crude laccase enzyme | ||||
| Egyptian Pharmaceutical Journal | ||||
| Volume 11, Issue 2, December 2012, Page 93-98 PDF (437.91 K) | ||||
| DOI: 10.7123/01.EPJ.0000419801.40087.2a | ||||
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| Authors | ||||
| Atalla M. Mabrouk; Zeinab H. Kheiralla; Eman R. Hamed; Amani A. Youssry; Abeer A. Abd El Aty | ||||
| Abstract | ||||
| Objectives The aim of this work was to study the purification and characterization of the crude extracellular laccase produced by the marine-derived fungus Methods The general properties of the crude laccase enzyme produced by were investigated. These include the effect of temperature, pH, thermal and pH stabilities, and enzyme and substrate concentrations on the laccase activity. Partial purification of the laccase enzyme was carried out by fractional precipitation with ammonium sulphate, ethanol and acetone. Further purification was carried out on a Sephadex G-100 column. Results and conclusion The results obtained showed that the crude enzyme reached its maximal activity at 35πC, pH 4.5, at an enzyme concentration of 5.429 mg protein/reaction mixture and at a substrate concentration of 40 mmol/l 2,2-azinobis-(3-ethylbenzthiazoline-6-sulphonic acid). The enzyme was stable for 60 min at 35πC and retained about 80–90% of its activity after treatment for 60 min from 40 to 50πC. The enzyme showed maximum stability (100%) at pH 4.5 and 91.6% at pH 4.0 after 60 min. Fractional precipitation of the fungal extracellular laccase enzyme with different methods showed that the enzyme fraction precipitated at 60% acetone was the most favourable enzyme fraction; it showed 4.84 purification fold. Laccase obtained from the 50–60% acetone fraction was purified by Sephadex G-100. The final preparation thus obtained reached 31.47-fold that of the culture filtrate (1466.49 U/mg protein) and showed a single band on native polyacrylamide gel electrophoresis. | ||||
| Keywords | ||||
| Characterization; Laccase; lignin-degrading enzymes; ligninolytic enzymes; Marine-derived Fungi; Purification | ||||
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