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Abstract

Natural products of Anthracophyllum discolor: Ligninolytic enzymes and antifungal volatile Compounds

Author(s): Heidi Schalchli, Briceno G and Diez M C

 White-rot fungi play important roles in ecosystems mainly because of their extracellular enzymatic system and their production of chlorinated aromatic compounds that act as decomposers of organic matter, antibiotics for protecting fungi, methyl donors and/or substrates for H2 O2 -generating oxidases. In this study, we evaluated the production of ligninolytic enzymes and antifungal volatile organic compounds (VOCs) by A. discolor Sp4 using Potato Peels (PP) and Discarded Potato (DP) as nutritional support. The manganese-dependent peroxidase (MnP) was evaluated by monitoring the oxidation of 2, 6-dimethoxyphenol. Beside the production of MnP, the discoloration of remazol brilliant blue R (RBBR) was also determined using a qualitative assay. The antifungal activity of VOCs against Mucor miehei and Fusarium oxysporum was evaluated using a bi-compartmented plate assay. Finally, VOCs released from mycelial cultures were analyzed by headspace solid phase microextraction and gas chromatography mass spectrometry. The highest MnP and MiP activities (163 U L-1 and 24 U L-1) were obtained at day 15 of incubation and a complete RBBR discoloration was observed. Although both potato wastes supported the ligninolytic activity, a higher MnP activity was obtained using PP than DP. The A. discolor volatiles inhibited approximately 62% and 76% the mycelial growth of M. miehei on PP agar and DP agar media, respectively. Nevertheless, the plant pathogen F. oxysporum was slightly inhibited (approximately 10%). The major VOCs detected were chlorinated aromatic compounds (over 50% relative area). The obtained natural products have multiple biotechnological applications among which are pollutant degradation and plant protection increase efficacy. The valorization of agro-industrial wastes as nutritional supports for producing bio-products offers farmers and agro-industry product diversification and improved environmental sustainability. The use of these wastes as substrates for microbial cultivation is currently an active field of research, allowing for energy to be saved and reducing environmental waste disposal (Dhillon et al. 2011; Obruca et al. 2015; Gopalan and Nampoothiri 2016). Some of the most interesting bio-products are biofuels and high value chemical compounds (e.g. antimicrobials, antioxidants, pigments and industrial enzymes) (Gassara et al. 2010; Hughes et al. 2014; Rodrigues et al. 2014; Schalchli et al. 2015). Different food wastes from the agro-industry and their potential recoveries have been studied, with fruits and vegetables being the most promising sources of valuable compounds (Mirabella et al. 2014). Among them, potato solid wastes (PSW) have been reported as an interesting source of carbohydrates, proteins and micronutrients that can be used as raw materials for the growth of fungi and synthesis of their bio-products (Rosales et al. 2002; Schieber and Saldaña 2009). Currently, PSW represents a disposal problem in numerous potato production and processing systems, since ~40 to 50% of the total production is unsuitable for human consumption constituting a source of plant pathogens (Charmley et al. 2006). Microbial growth is supported by media with a high content of starch (approx. 50% dry weight), which is one of the best carbon sources for some white-rot fungi (WRF) for growth and degradation of organic molecules, such as recalcitrant dyes (Revankar and Lele 2007).

The WRF are recognized by their characteristic nonspecific extracellular enzymes that are usually produced in secondary metabolic processes, and can interact with a wide range of substrates, including ligninolytic wastes, recalcitrant dyes and organic contaminants. One of the most studied ligninolytic enzymes is laccase (Lac) because of its enormous potential for the treatment of industrial effluents containing various dyes and other aromatic compounds, even at high temperatures and high ionic concentrations (Dhillon et al. 2012; Yan et al. 2014). The multiple biotechnological applications of Lac have been reported in numerous studies, which have revealed the necessity of enhancing their production, improving their efficiency and reducing their production costs (Dhillon et al. 2012; Akpinar and Urek 2014). Thus, a considerable reduction in production costs of Lac can potentially be obtained using a mixture of potato peels and barley bran to promote microbial processes mediated by Trametes hirsuta in solid-state conditions (Rosales et al. 2002).
In addition to the ligninolytic activity, the WRF are able to synthetize antimicrobial compounds using composted agro-industrial wastes as nutritional support. Although the synthesis of antifungal compounds by WRF using agro-industrial and lignocellulosic wastes has been less studied than their ligninolytic enzymes, interesting antimicrobial activities (Akyus and Kirbag 2009) and the synthesis of interesting chemical products have been reported (Sánchez 2009; Schalchli et al. 2015). In previous studies, we reported the release of volatile compounds with antifungal activity by WRF grown on PSW, where chlorinated aromatic compounds and sesquiterpenes extracted from the headspace were identified (Schalchli et al. 2015). However, there are very few studies that relate the synthesis of metabolites by WRF to the activity of ligninolytic enzymes. Teunissen and Field (1998) demonstrated that WRF are able to synthesize chlorinated anisyl alcohols that play important physiological roles in the fungal ligninolytic system, acting as a redox mediator and helping to protect the fungus from its extracellular ligninolytic enzymes.
In this article, we hypothesize that PSW may be used as a sole nutrient source to obtain enzymes and antifungal compounds produced by WRF that could be useful for solving important environmental and agricultural problems. Therefore, the aim of this study was to evaluate the synthesis of ligninolytic enzymes and some diffusible antifungal compounds by WRF using peels or discarded potato as sole nutrient sources.

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