A Novel Glucosylation Enzyme: Molecular Cloning, Expression, and Characterization of Trichoderma viride JCM22452 α-Amylase and Enzymatic Synthesis of Some Flavonoid Monoglucosides and Oligoglucosides

It was found that commercial cellulase preparations from Trichoderma viride showed transglucosylation activity toward (+)-catechin and (-)-epigallocatechin gallate (EGCG) using dextrin as a glucosyl donor. To isolate the enzyme exhibiting transglucosylation activity toward (+)-catechin and EGCG, the present study isolated the cDNA encoding the T. viride JCM22452 a-amylase homologue (TRa2), which showed high amino acid sequence identity to functionally uncharacterized (x-amylase homologues from other ascomycetes, which also produced some (+)-catechin and EGCG glucosides. TRa2 was able to glucosylate a wide range of natural flavonoids, particularly (+)-catechin and EGCG, and to hydrolyze maltooligosaccharides (k(cat)/K-m for maltotriose, maltotetraose, maltopentaose, maltohexaose, and maltoheptaose were 1.98, 45.2, 58.3, 97.4, and 92.6 s(-1) mM(-1), respectively) except maltose but could not transfer the monoglucosyl residue to maltooligosaccharides. By H-1 NMR and C-13 NMR, the structures of several novel glucosides obtained by commercial cellulase preparations from T. viride and TRa2 were determined as (+)-catechin 5-O-alpha-D-glucopyranoside, (+)-catechin 5-O-alpha-D-maltoside, (+)-catechin 4'-O-alpha-D-maltoside, EGCG 5-O-alpha-D-glucopyranoside, and EGCG 7-O-alpha-D-maltoside. One of these glucosides, EGCG 5-O-alpha-D-glucopyranoside, showed higher heat stability and solubility and lower astringency and astringent stimulation than its aglycon, suggesting that EGCG glucosides are functionally superior to EGCG as food additives.