Feruloyl and acetyl esterase production of an anaerobic rumen fungus Neocallimastix sp. YQ2 effected by glucose and soluble nitrogen supplementations and its potential in the hydrolysis of fibrous feedstuffs

摘要

To obtain an optimal feruloyl and acetyl esterase production of an anaerobic Neocallimastix sp. YQ2 grown on either Chinese wildrye grass hay (CW) or alfalfa hay (AH) or corn stalk (CS), three simultaneous 2x4 factorial experiments of 10-day pure cultures were applied with two levels of glucose (G: glucose added at a level of 1.0g/L and G: without glucose) and four levels of N source (N1: 1.0g/L yeast extract, 1.0g/L tryptone and 0.5g/L (NH)SO; N2: 2.8g/L yeast extract and 0.5g/L (NH)SO; N3: 1.6g/L tryptone and 0.5g/L (NH)SO; N4: 1.4g/L yeast extract, 1.7g/L tryptone). Optimal combinations of glucose and N supplements for YQ2 to produce ferulic acid esterase, acetyl esterase, xylanase, carboxymethyl cellulase and avicelase were GN2 for CS, GN3 for CW, GN3 for CW, GN2 for CS, and GN2 for CW, respectively. These five kinds of fibrolytic enzyme had different variation tendencies and maximum activity at different times of incubation. The fungus produced more total volatile fatty acids, and less propionic acids and iso-valeric acids, when CS was used as growth substrate, as well as more acetic acids when CW was used than when AH was used (P<0.05). As for the enzymological characteristics of feruloyl and acetyl esterase, Michaelis constants (K m) and maximum velocities (V max) of ferulic acid esterase against methyl ferulate at pH 6.0 and 39pC were 90oM and 1.15mU, and the Michaelis constants and maximum velocities of acetyl esterase at pH 7.0 and 39pC were 2.97mM and 294mU with specific substrate of p-nitrophenyl acetate. The crude enzyme preparation of the fungi grown on CS with the GN1 combination had higher potential to release ferulic acid, and p-coumaric acid in CS up to 129.8g/kg the alkali-extractable ferulic acid and 88.1g/kg the alkali-extractable p-coumaric acid, respectively (P<0.05). The strong capability to hydrolyse plant materials by the fungus can be used to understand the degradation of fibre-rich feedstuffs in the rumen and also open prospects for development of direct fed feed enzyme products for ruminants.