[b] Purification, Characterization and Steady State Enzyme Kinetics of Laccase from Xylaria polymorpha MTCC-1100[/b]
Journal Title: International Journal of Research in Chemistry and Environment (IJRCE) - Year 2013, Vol 3, Issue 2
Abstract
[i] A laccase from the culture filtrate of Xylaria polymorpha MTCC-1100 has been purified to homogeneity. The method involved concentration of the culture filtrate by ammonium sulphate precipitation and an anion exchange chromatography on diethylaminoethyl (DEAE) cellulose. The sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and native polyacrylamide gel electrophoresis (native-PAGE) both gave single protein bands indicating that the enzyme preparation was pure. The molecular mass of the enzyme determined from SDS-PAGE analysis was 63 kDa. Using 2,6- dimethoxyphenol, 2,2’ [Azino-bis-(3-ethylbonzthiazoline-6-sulphonic acid) diammonium salt] (ABTS) and 4,4’ [Azino-bis-(methenylylidone) bis (2,6-dimethoxyphenol)] (syringaldazine) as the substrates, the Km, kcat and kcat/Km values of the laccase were found to be 120 μM, 9.30 s-1, 7.75×104 M-1s-1, 48 μM, 9.30 s-1, 19.38×104 M-1s-1 and 80 μM, 9.30 s-1, 11.65×104 M-1s-1, respectively. The pH and the temperature optima were 4.0 and 50○C, respectively. The enzyme was most stable also at pH 4.0 and temperature 50 ○C when exposed for one hour. Thermal denaturation energy of the purified laccase is 15.26 × 103[/i] J/mol/K.
Authors and Affiliations
Chaurasia Pankaj Kumar, , Yadav R. S. S , Yadava Sudha
Keywords
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