Abstract
Degradation of lignocellulosic materials to release fermentable mono- and disaccharides is a decisive step toward a sustainable bio-based economy, thereby increasing the demand of robust and highly active lignocellulolytic enzymes. Anaerobic fungi of the phylum Neocallimastigomycota are potent biomass degraders harboring a huge variety of such enzymes. Compared to cellulose, hemicellulose degradation has received much less attention; therefore, the focus of this study has been the enzymatic xylan degradation of anaerobic fungi as these organisms produce some of the most effective known hydrolytic enzymes. We report the heterologous expression of a GH43 xylosidase, Xyl43Nc, and a GH11 endoxylanase, X11Nc, from the anaerobic fungus Neocallimastix californiae in Escherichia coli. The enzymes were identified by screening of the putative proteome. Xyl43Nc was highly active against 4-Nitrophenol-xylopyranosides with a Km of 0.72 mM, a kcat of 29.28 s-1, a temperature optimum of 32°C and a pH optimum of 6. When combined, Xyl43Nc and X11Nc released xylose from beechwood xylan and arabinoxylan from wheat. Phylogenetic analysis revealed that Xyl43Nc shares common ancestry with enzymes from Spirochaetes and groups separately from Ascomycete sequences in our phylogeny, highlighting the importance of horizontal gene transfer in the evolution of the anaerobic fungi.
Highlights
The conversion of lignocellulose containing material into fermentable sugars is an important step toward a CO2 neutral economy and society (Bundeministerium für Bildung und Forschung, 2010)
GH43 enzymes have been detected in anaerobic fungi previously (Youssef et al, 2013; Couger et al, 2015; Henske et al, 2017; Hagen et al, 2020) but none has been kinetically and thermodynamically characterized to the best of our knowledge
Xyl43Nc showed a very low temperature optimum at 32◦C when compared to the growth optimum of 39◦C for anaerobic fungi (Theodorou et al, 2005)
Summary
The conversion of lignocellulose containing material into fermentable sugars is an important step toward a CO2 neutral economy and society (Bundeministerium für Bildung und Forschung, 2010). Substitutions of the xylose backbone can inhibit xylanase activity and thereby the whole process (Pollet et al, 2010) In this case, debranching enzymes like arabinofuranosidases, glucuronidases and acetyl xylan esterases are required (Biely et al, 2016), which remove the respective substitutions and thereby enable endoxylanase activity. In addition to their application in the degradation of lignocellulose, xylanolytic enzymes can be used in animal feed, food industry, paper production and preparation of textile fibers (Polizeli et al, 2005)
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