In vitro assessment of two novel Cellulases from Trabulsiella odontotermitis for agricultural waste utilization.

BACKGROUND: The production of agricultural wastes still growing as a consequence of the population growing. However, the majority of these residues are under-utilized due their chemical composition, which is mainly composed by cellulose. Actually, the search of cellulases with high efficiency to degrade this carbohydrate remains as the challenge. In the present experiment, two genes encoding an endoglucanase (EC 3.2.1.4) and ß-glucosidase (EC 3.2.1.21) were overexpressed in Escherichia coli and their recombinant enzymes (egl-FZYE and cel-FZYE, respectively) characterized. Those genes were found in Trabulsiella odontermitis which was isolated from the gut of termite Heterotermes sp. Additionally, the capability to release sugars from agricultural wastes was evaluated in both enzymes, alone and in combination. RESULTS: The results have shown that optimal pH was 6.0 and 6.5, reaching an activity of 1051.65 ± 47.78 and 607.80 ± 10.19 U/mg at 39 °C, for egl-FZYE and cel-FZYE, respectively. The Km and Vmax for egl-FZYE using CMC as substrate were 11.25 mg/mL and 3921.57 U/mg, respectively, whereas using Avicel were 15.39 mg/mL and 2314.81 U/mg, respectively. The Km and Vmax for cel-FZYE using Avicel as substrate were 11.49 mg/mL and 2105.26 U/mg, respectively, whereas using CMC the enzyme did not had activity. Both enzymes had effect on agricultural wastes, and their effect was improved when they were combined reaching an activity of 955.1 ± 116.1, 4016.8 ± 332 and 1124.2 ± 241 U/mg on corn stover, sorghum stover and pine sawdust, respectively. CONCLUSIONS: Both enzymes were capable of degrading agricultural wastes, and their effectiveness was improved up to 60% of glucose released when combined. In summary, the results of the study demonstrate that the recombinant enzymes exhibit characteristics that indicate their value as potential feed additives and that the enzymes could be used to enhance the degradation of cellulose in the poor-quality forage generally used in ruminant feedstuffs.

In vitro reduction of methane production by 3-nitro-1-propionic acid is dose-dependent1.

Methanogenesis is a metabolic process that allows the rumen ecosystem the ability to maintain the low hydrogen partial pressures needed for proper digestive function. However, rumen methanogenesis is considered to be an inefficient process because it can result in the loss of 4% to 12% of the total energy consumed by the host. Recent studies have shown that some short-chain nitrocompounds such as nitroethane, 2-nitroethanol, 2-nitro-1-propanol, and 3-nitro-1-propionic acid (3NPA) are capable of inhibiting the production of methane during in vitro culture; nevertheless, optimal supplementation doses have yet to be determined. In the present study, in vitro cultures of freshly collected mixed populations of ruminal microbes were supplemented with the naturally occurring nitrocompound, 3NPA, to achieve 0, 3, 6, 9, or 12 mM. Analysis of fermentation products after 24 h of incubation revealed that methane (CH4) production was reduced in a dose-dependent manner by 29% to 96% (P < 0.05) compared with the amount produced by untreated controls (15.03 ± 0.88 µmol mL-1 incubated liquid). Main effects of the supplement were also observed, which resulted in a reduction (P < 0.05) on amounts of total gas and volatile fatty acids (VFA) produced, as well as in an increase of 0.07 to 0.30 µmol mL-1 on rates of 3NPA degradation. Changes in production of metabolites as CH4, hydrogen (H2), VFA, and NH3 indicated that the fermentation efficiency was not compromised dramatically by 3NPA treatment in moderate doses of 6 and 9 mM. Results further revealed that the metabolism of the 3NPA by microbial populations is also dose-dependent. The microbes were able to metabolize more than 75% of the added nitrocompound, with the greatest degradation rates in cultures treated with 9-mM 3NPA. Finally, from a practical standpoint, and considering the magnitude of CH4 reduction, effect on VFA, and percentage of metabolized supplement, the most efficacious dose for 3NPA administration may be between 3 and 9 mM.