Identification of Novel Laccase from Ganoderma lucidum and Application in Biotransformation to Bio-based Fragrances Using Alkaline Lignin as Raw Material.

Abstract

A novel laccase, Lac3, was purified from Ganoderma lucidum fermentation broth by salting out, gel filtration chromatography, and Native-PAGE protein recovery. The molecular mass of Lac3 was 58.4 kDa as estimated by SDS-PAGE and exhibited catalytic properties with 2,2'-Biazobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) as substrate. The specific enzyme activity of Lac3 was determined to be 313.69 U/mg. The laccase was stable at temperatures < 65 °C and at pH of 2.5-4.5. The pH, temperature optima, K_m and V_max of the enzyme for ABTS oxidation were 3.0, 55 °C, 0.077 mM, and 2.98 mM/min, respectively. The metal ions and anions showed inhibitory effects on Lac3 activity except Cu²⁺ (1 mM). GC-MS analysis showed that various aroma products were generated by Lac3 treatment of alkaline lignin. The Lac3 and lignin model compounds had negative binding energy and hydrogen bonding. The analysis of docking suggested that Asp207, Asn256, and His459 play a key role in substrate binding and catalysis.