Abstract
Bacterial expansin-like proteins have synergistically increased cellulose hydrolysis by cellulolytic enzymes during the initial stages of biofuel production, but they have not been tested on livestock feeds. The objectives of this study were to: isolate and express an expansin-like protein (BsEXLX1), to verify its disruptive activity (expansion) on cotton fibers by immunodetection (Experiment 1), and to determine the effect of dose, pH and temperature for BsEXLX1 and cellulase to synergistically hydrolyze filter paper (FP) and carboxymethyl cellulose (CMC) under laboratory (Experiment 2) and simulated ruminal (Experiment 3) conditions. In addition, we determined the ability of BsEXLX1 to synergistically increase hydrolysis of corn and bermudagrass silages by an exogenous fibrolytic enzyme (EFE) (Experiment 4) and how different doses of BsEXLX1 and EFE affect the gas production (GP), in vitro digestibility and fermentation of a diet for dairy cows (Experiment 5). In Experiment 1, immunofluorescence-based examination of cotton microfiber treated without or with recombinant expansin-like protein expressed from Bacillus subtilis (BsEXLX1) increased the surface area by > 100% compared to the untreated control. In Experiment 2, adding BsEXLX1 (100 μg/g FP) to cellulase (0.0148 FPU) increased release of reducing sugars compared to cellulase alone by more than 40% (P < 0.01) at optimal pH (4.0) and temperature (50°C) after 24 h. In Experiment 3 and 4, adding BsEXLX1 to cellulase or EFE, synergistically increased release of reducing sugars from FP, corn and bermudagrass silages under simulated ruminal conditions (pH 6.0, 39°C). In Experiment 5, increasing the concentration of BsEXLX1 linearly increased (P < 0.01) GP from fermentation of a diet for dairy cows by up to 17.8%. Synergistic effects between BsEXLX1 and EFE increased in vitro NDF digestibility of the diet by 23.3% compared to the control. In vitro digestibility of hemicellulose and butyrate concentration were linearly increased by BsEXLX1 compared to the control. This study demonstrated that BsEXLX1 can improve the efficacy of cellulase and EFE at hydrolyzing pure substrates and dairy cow feeds, respectively.
Highlights
Forages are the main component of the diet of dairy cattle [1,2] and grazing beef cattle yet their high fiber concentrations limit their digestibility and ruminant productivity [3,4]
Previous studies have shown that expansin-like proteins from Bacillus subtilis share structural similarities with expansins secreted by plant cell walls like corn[15,25]; greater synergy between endoglucanases and cellulases has been reported with expansin-like proteins than with plant expansins [15]
In agreement with previous studies, no cellulose hydrolysis was detected when BsEXLX1 alone was incubated with carboxymethyl cellulose (CMC), filter paper, or cotton fibers (Table 2)
Summary
Forages are the main component of the diet of dairy cattle [1,2] and grazing beef cattle yet their high fiber concentrations limit their digestibility and ruminant productivity [3,4]. Tropical and subtropical grasses are usually lower in nutritional quality than temperate grasses because of their higher fiber concentrations and the presence of lignin [7]. To increase their digestibility, such forages are sometimes treated with exogenous fibrolytic enzymes (EFE) [8] that can degrade some of the recalcitrant components of plant cell walls [1,9]. While the efficacy of EFE at degrading cellulose and hemicellulose is consistent with pure substrates [15], inconsistent responses have been observed with forages due to various anatomical and biochemical constraints [1,3] that include poor substrate accessibility to enzymes, suboptimal cellulase: hemicellulase or enzyme: substrate ratios as well as cell-wall cross linkages with phenolic acids and lignin [3,9]
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