Overproduction of laccase by a monokaryotic strain of Pycnoporus cinnabarinus using ethanol as inducer

Abstract

Aims: Laccase production by the monokaryotic strain Pycnoporus cinnabarinus ss3 was studied using ethanol as inducer in the culture medium. Methods and Results:The effect of ethanol was tested at 10, 20, 30, 35 and 45 g l )1 and compared with that of ferulic acid, known until now as the most efficient inducer for laccase expression by P. cinnabarinus ss3. In the presence of 35 g l )1 ethanol, laccase activity (266 600 U l )1 ) and productivity (19 000 U l )1 day )1 ) were nine and fivefold higher compared with ferulic acid-induced cultures, and 155-and 65-fold higher compared with noninduced cultures, respectively. In vivo, ethanol added to the culture medium of P. cinnabarinus ss3 favoured a continuous and high expression of laccase gene. Under these conditions, P. cinnabarinus ss3 produced preferentially the isoenzyme LAC I. Ethanol added in vitro to the purified P. cinnabarinus ss3 laccase typically inhibited the enzymatic activity. Conclusions: In spite of an initial inhibitory effect on mycelial growth, ethanol was shown to be a very strong inducer for laccase expression by P. cinnabarinus ss3 allowing an average yield of 1-1AE5 g l )1 laccase.Significance and Impact of the Study: This study identified P. cinnabarinus ss3 as an outstanding producer of laccase in the presence of ethanol as inducer. Ethanol is an inexpensive agricultural by-product and the process is simple to scale-up for industrial production.

Expression of Melanocarpus albomyces laccase in Trichoderma reesei and characterization of the purified enzyme

“…The production levels reported in this work for M. albomyces laccase in T. reesei in shake-flask cultures (230 mg l 21 ), batch fermentations (290 mg l 21 ) and in the fed-batch fermentation (920 mg l 21 ) are thus the highest heterologous laccase expression levels reported so far. Comparable laccase yields have previously been achieved with homologous laccase production systems in a shake-flask cultivation of P. cinnabarinus which yielded 1000-1500 mg laccase l 21 (Lomascolo et al, 2003) and a fermenter cultivation of Trametes pubescens which yielded 700 mg laccase l 21 (Galhaup et al, 2002). The wild-type M. albomyces is not an efficient laccase producer and therefore heterologous expression of the lac1 gene was required in order to obtain high laccase amounts.…”

Highly Efficient Production of Laccase by the Basidiomycete Pycnoporus cinnabarinus

“…The consumption of maltose was somewhat delayed when ethanol was added to the medium. This can be explained by the fact that this organic solvent suppresses growth of the mycelium and can thus be considered a stress factor (17). In all cases, laccase activity appeared in the medium on days 4 to 6 (Fig.…”

“…The transformants with the highest activities are shown in Table 2. Ethanol was recently reported to induce the lac1 gene in the monokaryotic SS3 strain of P. cinnabarinus, resulting in a 155-fold increase in laccase activity in the medium (17). In the liquid shaken cultures of the recombinant strains L12-7 and L12-8, which express lac1 behind its own promoter, laccase activity was increased 7 and 33 times, respectively, upon the addition of the inducer to the medium.…”

“…The monokaryotic laccase-deficient P. cinnabarinus strain BRFM 44 (Banque de Resources Fongiques de Marseille, Marseille, France) was routinely grown at 30°C in liquid or solid (1.5% agar) yeast malt medium (YM) containing the following ingredients per liter: 10 g of glucose, 5 g of peptone, 3 g of yeast extract, and 3 g of malt extract. For laccase production, conditions were used that are optimal for lac1 production in wildtype P. cinnabarinus (17). Strains were grown in 250 ml of minimal medium (MM) with or without filter-sterilized ethanol in 1- 4 , and 1 ml of a vitamin solution (33).…”

Wood production, wood technology, and biotechnological impacts

“…Higher yields however require induction of expression. Several inducers have been applied to increase yields of laccase: copper, phenols, 2,5-xylidine and related compounds in T. versicolor (Collins & Dobson 1997, Tavares et al 2005, Kollmann et al 2005, Domínguez et al 2007, Ryan et al 2007), ethanol in P. cinnabarinus (Lomascolo et al 2003, Meza et al 2006, aromatic compounds including veratryl alcohol in Botryosphoraeria sp. (Vasconcelos et al 2000, Dekker & Barbosa 2001, copper and phenolic compounds in Panus tigrinus (Chernykh et al 2005), copper, cotton stalk extract, dimethyl sulphoxide, and the synthetic substrate ABTS [2,2'-azino-di-(3-ethylbenzothialozin-6-sulphonic acid)] in P. ostreatus (Ardon et al 1996, Hou et al 2004, Shah et al 2006, and copper and lignin-related compounds in Pleurotus pulmonarius .…”