Microbial β-glucosidases from cow rumen metagenome enhance the saccharification of lignocellulose in combination with commercial cellulase cocktail

Abstract

BackgroundA complete saccharification of plant polymers is the critical step in the efficient production of bio-alcohols. Beta-glucosidases acting in the degradation of intermediate gluco-oligosaccharides produced by cellulases limit the yield of the final product.ResultsIn the present work, we have identified and then successfully cloned, expressed, purified and characterised 4 highly active beta-glucosidases from fibre-adherent microbial community from the cow rumen. The enzymes were most active at temperatures 45–55°C and pH 4.0-7.0 and exhibited high affinity and activity towards synthetic substrates such as p-nitrophenyl-beta-D-glucopyranoside (pNPbetaG) and pNP-beta-cellobiose, as well as to natural cello-oligosaccharides ranging from cellobiose to cellopentaose. The apparent capability of the most active beta-glucosidase, herein named LAB25g2, was tested for its ability to improve, at low dosage (31.25 units g-1 dry biomass, using pNPbetaG as substrate), the hydrolysis of pre-treated corn stover (dry matter content of 20%; 350 g glucan kg-1 dry biomass) in combination with a beta-glucosidase-deficient commercial Trichoderma reseei cellulase cocktail (5 units g-1 dry biomass in the basis of pNPbetaG). LAB25g2 increased the final hydrolysis yield by a factor of 20% (44.5 ± 1.7% vs. 34.5 ± 1.5% in control conditions) after 96–120 h as compared to control reactions in its absence or in the presence of other commercial beta-glucosidase preparations. The high stability (half-life higher than 5 days at 50°C and pH 5.2) and 2–38000 fold higher (as compared with reported beta-glucosidases) activity towards cello-oligosaccharides may account for its performance in supplementation assays.ConclusionsThe results suggest that beta-glucosidases from yet uncultured bacteria from animal digestomes may be of a potential interest for biotechnological processes related to the effective bio-ethanol production in combination with low dosage of commercial cellulases.

Structural and Functional Characterization of a Ruminal β-Glycosidase Defines a Novel Subfamily of Glycoside Hydrolase Family 3 with Permuted Domain Topology

“…A subset of 14,000 clones from resident microbial communities of strained ruminal fluid (SRF) collected from rumen-fistulated, non-lactating Holstein cows (28) was screened for its ability to hydrolyze p -nitrophenyl-β- d -glucoside ( p NPβGlc) and p -nitrophenyl-β- d -cellobioside ( p NPβCel). We identified a positive clone (designated SRF4) that is highly active against both substrates.…”

“…The construction and characteristics of the library were described previously (28). A subset of 14,000 clones were plated onto large (22.5 × 22.5 cm) Petri plates with Luria Bertani (LB) agar containing chloramphenicol (12.5 μg/ml) and an arabinose-containing induction solution (Epicenter Biotechnologies) at a concentration (0.01% w/v) recommended by the supplier to induce a high fosmid copy number.…”

“…One positive clone, herein designated as SRF4, was selected, and its DNA insert was fully sequenced with a Roche 454 GS FLX Ti sequencer (454 Life Sciences, Branford, CT) at Life Sequencing S.L. (Valencia, Spain), and the predicted genes were identified as described previously (28). …”

Expression of novel glucose tolerant β-glucosidase on cell surface by Rhodotorula glutinis isolate

“…However, for production of ethanol from lignocellulosic biomass, various steps like pretreatment, hydrolysis, fermentation, separation and purification are involved (Banerjee et al, 2010). Of all these steps, enzymatic hydrolysis is most crucial as efficient conversion of polymers to sugars determines efficient ethanol production (Del Pozo et al, 2012).…”

Advances in consolidated bioprocessing systems for bioethanol and butanol production from biomass: a comprehensive review

“…Newly, many metagenomics studies have been focused on discovering novel cellulases from different sources, such as cow rumen (Hess et al, 2011;Del Pozo et al, 2012;, camel rumen (Gharachaei et al, 2014), and termite digestive system (Warnecke et al, 2007). Surface-display technology, as one of the most successful strategies has been used for designing CBP organisms.…”