A Laccase of Fomes durissimus MTCC-1173 and Its Role in the Conversion of Methylbenzene to Benzaldehyde

Abstract

A laccase has been purified from the liquid culture growth medium containing bagasse particles of Fomes durissimus. The method involved concentration of the culture filtrate by ultrafiltration and anion exchange chromatography on diethyl aminoethyl cellulose. The sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and native polyacrylamide gel electrophoresis both gave single protein band indicating that the enzyme preparation was pure. The molecular mass of the purified laccase determined from SDS-PAGE analysis was 75 kDa. Using 2,6-dimethoxyphenol as the substrate, the determined K (m) and k (cat) values of the laccase are 182 μM and 0.35 s(-1), respectively, giving a k (cat)/K (m) value of 1.92 × 10(3) M(-1) s(-1). The pH and temperature optimum were 4.0 and 35 °C, respectively. The purified laccase has yellow colour and does not show absorption band around 610 nm found in blue laccases. Moreover, it transformed methylbenzene to benzaldehyde in the absence of mediator molecules, property exhibited by yellow laccases.

Syntheses of aromatic aldehydes by laccase without the help of mediators

“…The conversion done with laccase occurs under milder conditions, yield is >90%, and the process is ecofriendly. The use of pure laccase for this purpose has been studied (17,18) in the presence of mediator molecules like 2,2'-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (17), but the role of laccase for such conversions in the absence of mediator molecule was done only for conversion of toluene to benzaldehyde (19), and its role was not reported for other substituted toluenes.…”

“…Since, laccase from this source has already been purified and characterized by Sahay et al (19), authors have checked the purity of laccase by using SDS-PAGE analysis for the purpose of their synthetic work. The results of SDS-PAGE analysis are shown in Figure 1.…”

“…The biotechnological importance of laccases has increased after the discovery of small readily oxidizable molecules called mediators (11,12) that extend the substrate range for laccases. During the last two decades, laccases have turned out to be the most promising enzymes for industrial uses (9, 10) having applications in food, pulps, paper, textile, and cosmetics industries and in synthetic organic chemistry (13)(14)(15)(16)(17)(18)(19).…”

“…It was previously reported that blue laccases transform aromatic methyl group to aromatic aldehyde group in the presence of mediator molecules (17,18), but yellow laccases can do it without the help of any mediator molecules (19). Only conversion of toluene to benzaldehyde was done previously by using yellow laccase without the help of any mediator (19).…”

“…Only conversion of toluene to benzaldehyde was done previously by using yellow laccase without the help of any mediator (19). Thus, the main objective of this communication was to demonstrate the conversions of different substituted toluenes to corresponding benzaldehydes by considering 3-nitrotoluene, 2-fluorotoluene, 4-fluorotoluene, 2-chlorotoluene, and 4-chlorotoluene as reacting substrates in the absence of mediator molecules using pure laccase of Fomes durissimus Microbial type culture collection-1173.…”