Identification of enzymes from genus Trichoderma that can accelerate formation of ferulic acid and ethyl ferulate in collaboration with rice koji enzyme in sake mash.

ABSTRACT

The enzymes responsible for acceleration of ferulic acid and ethyl ferulate formation in sake mash were studied. Ferulic acid and ethyl ferulate are formed during the sake brewing process from feruloylated glucuronoarabinoxylan. Cellulase reagent from genus Trichoderma was used instead of rice koji, because rice koji for sake brewing produces extremely low levels of xylan-degrading enzymes. A combination of the reagent with rice koji enzymes accelerated the formation of ferulic acid from α-rice powder. Addition of the reagent to sake mash increased ferulic acid and ethyl ferulate formation. The enzyme responsible for the accelerated formation was purified using a newly developed assay method and α-rice powder as a substrate. During the assay procedure, feruloylated oligosaccharide was converted to ferulic acid by feruloylesterase for HPLC analysis. Analysis of the N-terminal amino acid sequence of the purified samples was successfully conducted after pyroglutamyl aminopeptidase de-blocking. Purified enzymes were identified as members of the glycoside hydrolase family 10 (GH10) and family 11 (GH11) xylanases by BLASTP database research. The GH10 xylanase showed higher specific activity for α-rice powder and insoluble wheat arabinoxylan compared with GH11 xylanase; the GH11 xylanase showed higher specific activity for the other xylan substrates, especially glucuronoarabinoxylan. The GH10 xylanase showed higher accelerating activity than the GH11 xylanase in the sake mash. The results of this study provides useful knowledge on ferulic acid and ethyl ferulate formation in sake mash, the relative levels of these compounds and their influence on the sensory quality of sake.