PURIFICATION AND CHARACTERIZATION OF RECOMBINANT LACCASE FROM ASPERGILLUS NIGERD15#26 lcc1 1.8B
Abstract
Laccase is an interesting enzym thanks to its wide application in the industry. Since complexity of reducers for natural enzymes, the production of recombinant laccase using simple reducers attracts scientists worldwide. Laccase from Trametes versicolor 06 was expressed in A. niger D15#26 using glucose as an ideal reducer, achievedhighest activity of 4,250 U/L after 7 days of incubation at 200 rpm, pH 6. The studied enzyme was purified by fractionation with 40 % - 80 % saturated ammonium sulfate then eluted on the Hitrap Q Fast Flow with a gradient NaCl 0 – 1 M. The purification yield was of 26 % achieving a specific activity of 34.7 U/mg protein, increased 41.19 folds to the native one. The recombinant enzyme had a molecular weight of 70 kDa and optimal temperature and pH for the reaction (with ABTS) of 450C and pH 4, respectively. The enzyme was stable at a temperature from 30 – 35 0C and pH 4 – 6, having Km1,35 µM; Vmax 53,14 × 106µM/min-1; Kcat10,42 × 106s-1and Kcat/Km7,72 × 106µM-1s-1
with ABTS showing its highly catalytic ability.