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Abstract Xylanases are in important class of industrial enzymes that are essential for the complete hydrolysis of lignocellulosic biomass into fermentable sugars. In the present study, we report the cloning of novel xylanases with interesting properties from compost metagenomics libraries. Controlled composting of lignocellulosic materials was used to enrich the microbial population in lignocellulolytic organisms. DNA extracted from the compost samples was used to construct metagenomics libraries, which were screened for xylanase activity. In total, 40 clones exhibiting xylanase activity were identified and the thermostability of the discovered xylanases was assayed directly from the library clones. Five genes, including one belonging to the more rare family GH8, were selected for subcloning and the enzymes were expressed in recombinant form in E. coli. Preliminary characterization of the metagenome-derived xylanases revealed interesting properties of the novel enzymes, such as high thermostability and specific activity, and differences in hydrolysis profiles. One enzyme was found to perform better than a standard Trichoderma reesei xylanase in the hydrolysis of lignocellulose at elevated temperatures.

Abstract The oxidative d-xylose pathway, i.e. Dahms pathway, can be utilised to produce from cheap biomass raw material useful chemical intermediates. In vitro metabolic pathways offer a fast way to study the rate-limiting steps and find the most suitable enzymes for each reaction. We have constructed here in vitro multi-enzyme cascades leading from d-xylose or d-xylonolactone to ethylene glycol, glycolic acid and lactic acid, and use simple spectrophotometric assays for the read-out of the efficiency of these pathways. Based on our earlier results, we focussed particularly on the less studied xylonolactone ring opening (hydrolysis) reaction. The bacterial Caulobacter crescentus lactonase (Cc XylC), was shown to be a metal-dependent enzyme clearly improving the formation of d-xylonic acid at pH range from 6 to 8. The following dehydration reaction by the ILVD/EDD family d-xylonate dehydratase is a rate-limiting step in the pathway, and an effort was made to screen for novel enolase family d-xylonate dehydratases, however, no suitable replacing enzymes were found for this reaction. Concerning the oxidation of glycolaldehyde to glycolic acid, several enzyme candidates were also tested. Both Escherichia coli aldehyde dehydrogenase (Ec AldA) and Azospirillum brasilense α-ketoglutarate semialdehyde dehydrogenase (Ab AraE) proved to be suitable enzymes for this reaction.