<span style="font-size:15.0pt;mso-fareast-font-family:"Times New Roman";mso-ansi-language: EN-GB;mso-fareast-language:EN-US;mso-bidi-language:HI" lang="EN-GB">Purification, characterization and synthetic application of a thermally stable laccase from<i> Hexagonia tenuis</i> MTCC-1119</span>

Abstract

A thermally stable laccase was purified from the culture filtrate of <i style="mso-bidi-font-style: normal">Hexagonia tenuis</i> MTCC-1119. 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 100 kDa. The purification fold and percentage recovery of the enzyme activity were 12.75 and 30.12%, respectively. The <i style="mso-bidi-font-style:normal">p</i>H and the temperature optima were 3.5 and 45^○C, respectively. The enzyme was most stable at <i style="mso-bidi-font-style:normal">p</i>H 4.0 when exposed for 1 h. Using 2,6-dimethoxyphenol (DMP), 2,2^’ [azino-bis-(3-ethylbonzthiazoline-6-sulphonic acid) diammonium salt] (ABTS) and

3,5-dimethoxy-4-hydroxybenzaldehyde azine (syringaldazine) as the substrates, the <i style="mso-bidi-font-style:normal">K_m</i>, <i style="mso-bidi-font-style:normal">k_cat</i>and <i style="mso-bidi-font-style:normal">k_cat</i>/<i style="mso-bidi-font-style: normal">K_m </i>values of the laccase were 80 μM, 2.54 s^-1, 3.17 × 10⁴M^-1s^-1, 36 μM, 2.54 s^-1, 7.05 × 10⁴M^-1s^-1and 87 μM, 2.54 s^-1, 2.92 × 10⁴M^-1s^-1, respectively. The purified laccase was finally used for the selective biotransformation of aromatic methyl group to aldehyde group in presence of diammonium salt of ABTS as the mediator and products were characterized by HPLC, IR and ¹H NMR. The percentage yields of these transformed products were >91%.