A Novel Monomodular and Multifunctional Processive β‐1,4‐Endoglucanase Has Been Identified and Characterized from Porcine Gut Microbiome

Abstract

Cellulose‐degrading enzymes play an important role in dietary fibre digestion of humans and animals, as well as in multiple industrial applications. Herein, a plasmid metagenomic expression library was constructed for mining cellulose‐degrading enzymes from the pig hindgut microbiota that was modulated by feeding a purified crystalline cellulose supplemented diet (Journal of Animal Science, 2012, 90 (Suppl. 4):400–402). One of the cellulase genes screened from this library was localized to a 2949‐base pair insert fragment p4818. Within the sequence preceding this enzyme‐coding region, ‐10 and ‐35 promoter elements were identified along with three transcription‐factor binding sites. The enzyme possesses a catalytic domain consistent with those of the glycoside hydrolase family 5 subfamily 2 (GH5_2), thus named as p4818Cel5_2A. The protein was overexpressed, purified, and characterized; and it displayed optimal enzymatic activity at pH 6.0 and 50 °C. The enzyme also exhibited resilience to trypsin digestion, where >70 % activity was retained after 6.0 h incubation with trypsin at 37 °C. p4818Cel5_2A exhibited a broad substrate profile with the highest specific activity towards β‐glucan, carboxymethyl cellulose (CMC), followed by hydroxyethyl‐cellulose and regenerated amorphous cellulose (RAC). The enzyme was also active on other substrates including crystalline cellulose substrates such as Avicel and Solkafloc, as well as the hemicelluloses of glucomannan and xyloglucan. Furthermore, based on viscosity and hydrolysis product analyses, the enzyme behaved like a typical endo‐β‐1,4‐glucanase, and displayed processivity towards RAC. In summary, p4818Cel5_2A exhibited novel enzyme properties for potential industrial applications.Support or Funding InformationRelated research activities have been supported by grants (to M.Z.F.) from the Ontario Ministry of Agriculture, Food and Rural Affairs (OMAFRA) New Directions & Alternative Renewable Fuels Research Program; and the University of Guelph Gryphon's LAAIR (Leading to Accelerated Adoption of Innovative Research) program.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.