Improvement in Saccharification Yield of Mixed Rumen Enzymes by Identification of Recalcitrant Cell Wall Constituents Using Enzyme Fingerprinting

Abstract

Identification of recalcitrant factors that limit digestion of forages and the development of enzymatic approaches that improve hydrolysis could play a key role in improving the efficiency of meat and milk production in ruminants. Enzyme fingerprinting of barley silage fed to heifers and total tract indigestible fibre residue (TIFR) collected from feces was used to identify cell wall components resistant to total tract digestion. Enzyme fingerprinting results identified acetyl xylan esterases as key to the enhanced ruminal digestion. FTIR analysis also suggested cross-link cell wall polymers as principal components of indigested fiber residues in feces. Based on structural information from enzymatic fingerprinting and FTIR, enzyme pretreatment to enhance glucose yield from barley straw and alfalfa hay upon exposure to mixed rumen-enzymes was developed. Prehydrolysis effects of recombinant fungal fibrolytic hydrolases were analyzed using microassay in combination with statistical experimental design. Recombinant hemicellulases and auxiliary enzymes initiated degradation of plant structural polysaccharides upon application and improved the in vitro saccharification of alfalfa and barley straw by mixed rumen enzymes. The validation results showed that microassay in combination with statistical experimental design can be successfully used to predict effective enzyme pretreatments that can enhance plant cell wall digestion by mixed rumen enzymes.

Identification of novel enzymes to enhance the ruminal digestion of barley straw

“…Crude commercial enzymes and fungal enzymes were screened for their ability to enhance the saccharification yield from barley straw incubated with RME using a microassay as described previously (Badhan et al, 2014(Badhan et al, , 2015. Ground barley straw was mixed in buffered suspension (composition as shown above) at a concentration of 0.5% w/v at pH 6; and 200 µl of this suspension was incubated with additive enzymes (5 mg/g substrate) and RME (5 mg/g substrate) at 39°C for 48 h. The reactions were then centrifuged at 1,500×g for 3 min; the supernatants transferred into a clean microplate and heated at 90°C for 10 min to inactivate enzymes.…”

“…Limited penetration of cellulolytic microbes into the interior of the plant cell, insufficient retention time of feed within rumen and rate-limiting enzyme activities, have all been reported to act as constraints to ruminal cellulose digestion (Weimer, 1996;Ribeiro et al, 2016). Characterization of total tract indigestible fiber residues (TTIR) could help identify those undigested plant cell wall moieties that escape ruminal digestion and provide insight into factors that limit plant cell wall digestion (Badhan et al, 2015). Metagenomics and metatranscriptomic studies have indicated an absence or scarcity of GH7 (endoglucanase and cellobiohydrolase), GH44 (endoglucanase and xyloglucanase), GH12 (xyloglucanase and endoglucanase), GH52 (β-xylosidase) and GH62 (arabinofuranosidase) activity within the rumen (Dai et al, 2015).…”