Purification, molecular characterization and reactivity with aromatic compounds of a laccase from basidiomycete Trametes sp. strain AH28-2

Abstract

A recently isolated basidiomycete, Trametes sp. strain AH28-2, can be induced to produce a high level of laccases when grown on a cellobiose-asparagine liquid medium. After induction by kraft lignin, two major isozymes were detected in the fermentation supernatant of the fungus. The principal component laccase A, which accounts for about 85% of the total activity, can be purified to electrophoretic homogeneity by three chromatographic steps: DEAE-Sepharose FF, Superdex-200 and Mono-Q. The solution containing purified laccase is blue in color, and the ratio of absorbance at 280 nm to that at 600 nm is 22. The molecular mass of laccase A is estimated to be 62 kDa by SDS-PAGE, 57 kDa by FPLC, and measured as 58522 Da by MALDI mass spectrum. Laccase A is a monomeric glycoprotein with a carbohydrate content of 11-12% and an isoelectric point of 4.2. The optimum pH and temperature for oxidizing guaiacol are 4.5 and 50 degrees C, respectively. The half-life of the enzyme at 75 degrees C is 27 min. The enzyme shows a good stability from pH 4.2 to pH 8.0. The K(m) values of the enzyme toward substrates 2,2'-azino-bis (3-ethylbenzothazoline-6-sulfonate) (ABTS), guaiacol and 2,6-dimethoxyphenol are 25, 420 and 25.5 microM, respectively, and the corresponding V(max) values are 670, 66.8, and 79 microM min(-1) x mg(-1), respectively. Laccase A activity is strongly inhibited by 0.1 mM NaN(3) or 0.1 mM cyanide. Two units of laccase A alone is able to completely oxidize 100 micromol 2,6-chlorophenol in 6 h. In the presence of 1 mM ABTS and 1-hydroxybenzotriazole, 15.0 U laccase A is able to oxidize 45% and 70% of 50 micromol fluorene in 12 and 18 h, respectively. The laccase A gene was cloned by a PCR method, and preliminary analysis of its sequence indicates 87.0% similarity to the corresponding segment in the phenoloxidase gene from Coriolus hirsutus.

Thermotolerant and thermostable laccases

“…Two novel Trametes sp. isolates, AH282 and 420, have been reported to produce laccases with noticeable stabilities at 75°C (Xiao et al 2003) and at 60°C (Tong et al 2007) ( Table 1).…”

Additive effects of CuSO4 and aromatic compounds on laccase production by Pleurotus sajor-caju PS-2001 using sucrose as a carbon source

“…Laccases can be produced in submerged processes using glucose as the main carbon source and the enzyme titer can be increased by the addition of ethanol (Lee et al, 1999;Rodakiewicz-Nowak 1999), CuSO 4 (Giardina et al, 1999;Palmieri et al, 2000;Baldrian and Gabriel, 2002;Hess et al, 2002;Levin et al, 2002;Chen et al, 2003), gallic acid Peralta et al, 2004;Gnanamani et al, 2006;Revankar and Lele, 2006), tannic acid (Galhaup and Haltrich, 2001), syringaldazine (Koroljova-Skorobogat'ko et al, 1998), vanillin (Peralta et al, 2004;Xiao et al, 2003;Elisashvili et al, 2010), and xylidine (Lee et al, 1999;Elisashvili et al, 2006;Jang et al 2002;Mougin et al, 2002;Kollmann et al, 2005;Pazarlioglu et al, 2005;Jang et al, 2006;Murugesan et al, 2006).…”

Gongronella sp induces overproduction of laccase in Panus rudis

“…This laccase yield is much higher than those found in literature regarding microbial interactions, and even comparable with 140,000 U l -1 produced by recombinant yeast under high-density fermentation [19]. One of the advantages of microbial interactions, in comparison with the traditional strategy of chemical induction, is that there is no need to use a large number of toxic inducers including phenolic compounds and heavy metals which are usually efficient inducers for laccase production [13]. On the other hand, microbial interactions usually enhance co-expression of several cooperative enzymes such as lignin-modifying enzymes [9,18].…”

“…The suspension was spread on PDA agar plates [13] and then cultured at 28 °C untill colonies were observed. The primary screening was performed on 9 cm PDA plates containing 0.5 mM guaiacol.…”

“…Tests were performed in triplicates and mean values were obtained from those data with standard errors less than 15%. Isozymes of laccase in the supernatants of G. W5 cultures were analyzed by native polyacrylamide gel electrophoresis (PAGE) [13].…”