Py/GC-MS to evaluate the activity of a thermostable laccase produced by Streptomyces ipomoeae in the degradation of lignin from wheat straw

Abstract

Streptomycetes are mycelial microorganisms with a high growth rate on solid substrate that have been studied for their potential to degrade lignocellulosic grass residues in solid-state fermentation and therefore with interest for biopulping purposes. In these studies, it has been demonstrated the ability of selected Streptomyces strains to solubilise lignin from wheat straw and produce a high depolymerization of this polymer. Among the enzymes involved in lignin degradation, oxidoreductases such as peroxidases and laccases have been involved. In recent studies a thermostable laccase (SilA) produced by Streptomyces ipomoeae CECT 3341 presenting both physico-chemical and structural chracteristics usually uncommon among microbial laccases was isolated. Thus, its high resistance to alkaline conditions and to high concentrations of sodium chloride opened up the biotechnological interest of this enzyme [1]. Moreover, the presence of two cupredoxine domains in its structure in contrast with most of fungal laccases drive us to elucidate the possible biological role of this laccase in lignin degradation of wheat straw. For this study, the wild strain (SilA) and a deficient laccase mutant (SilA¿) obtained through gene disruption were grown on wheat straw as substrate under solid-state fermentation (SSF) conditions. In addition, a non-inoculated substrate was used as control. After 7 days of incubation the alkali-lignin was extracted from fermented substrates, quantified and analyzed through analytical Py-GC/MS at 350 ºC. The alkali-lignin extracted was 12 times and 6 times higher for the wild-type strain than for the control and SilA¿ mutant strain, respectively. The analytical pyrolysis of the alkali-lignin extracts revealed conspicuous differences in their composition which is evident at a first sight of the pyrograms normalized to the polysaccharides- derived pyrolysis compound furfural (Fig. 1). The chromatogram obtained can be divided in three parts where main biogenic compounds elute; a first part from min 2-5 is dominated by polysaccharides-derived compounds, from min 5-14 are lignin-derived compounds including those from G, S and H units and from min 14 to the end of the chromatogram lipids-derived compounds including high fatty acid peaks (FA14, 16 & 18), plant derived long chain alkanes with odd homologous dominance (C27-C33) and sterols could be identified. From the results obtained, both from the qualitative alkali-lignin extracted from the fermented substrateds and from the qualitative information provided by the analytical pyrolysis, it could be inferred that SilA laccase seems to be the main enzyme responsible for the lignin degradation activity of Streptomyces ipomoeae in wheat straw.