Abstract
BackgroundThe bioconversion of lignocellulosic biomass in various industrial processes, such as the production of biofuels, requires the degradation of hemicellulose. Clostridium stercorarium is a thermophilic bacterium, well known for its outstanding hemicellulose-degrading capability. Its genome comprises about 50 genes for partially still uncharacterised thermostable hemicellulolytic enzymes. These are promising candidates for industrial applications.ResultsTo reveal the hemicellulose-degrading potential of 50 glycoside hydrolases, they were recombinantly produced and characterised. 46 of them were identified in the secretome of C. stercorarium cultivated on cellobiose. Xylanases Xyn11A, Xyn10B, Xyn10C, and cellulase Cel9Z were among the most abundant proteins. The secretome of C. stercorarium was active on xylan, β-glucan, xyloglucan, galactan, and glucomannan. In addition, the recombinant enzymes hydrolysed arabinan, mannan, and galactomannan. 20 enzymes are newly described, degrading xylan, galactan, arabinan, mannan, and aryl-glycosides of β-d-xylose, β-d-glucose, β-d-galactose, α-l-arabinofuranose, α-l-rhamnose, β-d-glucuronic acid, and N-acetyl-β-d-glucosamine. The activities of three enzymes with non-classified glycoside hydrolase (GH) family modules were determined. Xylanase Xyn105F and β-d-xylosidase Bxl31D showed activities not described so far for their GH families. 11 of the 13 polysaccharide-degrading enzymes were most active at pH 5.0 to pH 6.5 and at temperatures of 57–76 °C. Investigation of the substrate and product specificity of arabinoxylan-degrading enzymes revealed that only the GH10 xylanases were able to degrade arabinoxylooligosaccharides. While Xyn10C was inhibited by α-(1,2)-arabinosylations, Xyn10D showed a degradation pattern different to Xyn10B and Xyn10C. Xyn11A released longer degradation products than Xyn10B. Both tested arabinose-releasing enzymes, Arf51B and Axh43A, were able to hydrolyse single- as well as double-arabinosylated xylooligosaccharides.ConclusionsThe obtained results lead to a better understanding of the hemicellulose-degrading capacity of C. stercorarium and its involved enzyme systems. Despite similar average activities measured by depolymerisation tests, a closer look revealed distinctive differences in the activities and specificities within an enzyme class. This may lead to synergistic effects and influence the enzyme choice for biotechnological applications. The newly characterised glycoside hydrolases can now serve as components of an enzyme platform for industrial applications in order to reconstitute synthetic enzyme systems for complete and optimised degradation of defined polysaccharides and hemicellulose.
Highlights
The bioconversion of lignocellulosic biomass in various industrial processes, such as the production of biofuels, requires the degradation of hemicellulose
Hemicellulose‐degrading potential of the extracellular enzymes of C. stercorarium The hemicellulose-degrading capabilities of C. stercorarium derive from the glycoside hydrolases secreted by the bacterium into the extracellular environment
It was shown that a synthetic culture of C. thermocellum and C. stercorarium degraded 76% of maize silage and performed only slightly less effectively than the best thermophilic bacterial consortium isolated from a biogas plant [14]
Summary
The bioconversion of lignocellulosic biomass in various industrial processes, such as the production of biofuels, requires the degradation of hemicellulose. Hemicelluloses are heterogeneous polysaccharides in plant cell walls that are characterised by β-(1,4)-linked backbones with an equatorial configuration [1] After cellulose, they are the second most common polysaccharides representing about 20–35% of lignocellulosic biomass [2, 3]. According to the predominant monosaccharide in the main polymeric chain, hemicelluloses are classified into four types of polysaccharides: xylans, mannans, β-(1,3;1,4)-glucans, and xyloglucans. Further polysaccharides, such as galactans, arabinans, and arabinogalactans, are rather parts of pectin substances and do not share the equatorial β-(1,4)-linked backbone structure [1, 5]. Xylans are the most abundant polysaccharides and the major hemicelluloses in hardwood and grasses [3, 6]
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