Identification and characterization of two xyloglucan-specific endo-1,4-glucanases in Aspergillus oryzae

Aspergillus oryzae produces glycoside hydrolases to degrade xyloglucan. We identified and characterized two xyloglucan-specific endo-1,4-glucanases (xyloglucanases) named Xeg12A and Xeg5A. Based on their amino acid sequences, Xeg12A and Xeg5A were classified into glycoside hydrolase families GH12 and GH5, respectively. Xeg12A degrades tamarind seed xyloglucan polysaccharide into xyloglucan oligosaccharides containing four glucopyranosyl residues as main chains, including heptasaccharides (XXXG: Glc₄Xyl₃), octasaccharides (XXLG and XLXG: Glc₄Xyl₃Gal₁), and nonasaccharides (XLLG: Glc₄Xyl₃Gal₂). By contrast, Xeg5A produces various xyloglucan oligosaccharides from xyloglucan. Xeg5A hydrolyzes xyloglucan into not only XXXG, XXLG/XLXG, and XLLG but also disaccharides (isoprimeverose: Glc₁Xyl₁), tetrasaccharides (XX: Glc₂Xyl₂ and LG: Glc₂Xyl₁Gal₁), and so on. Xeg12A is a typical endo-dissociative-type xyloglucanase that repeats hydrolysis and desorption from xyloglucan. Conversely, Xeg5A acts as an endo-processive-type xyloglucanase that hydrolyzes xyloglucan progressively without desorption. These results indicate that although both Xeg12A and Xeg5A contribute to the degradation of xyloglucan, they have different modes of activity toward xyloglucan, and the hydrolysis machinery of Xeg5A is unique compared with that of other known GH5 enzymes. KEY POINTS: • We identified two xyloglucanases, Xeg12A and Xeg5A, in A. oryzae. • Modes of activity and regiospecificities of Xeg12A and Xeg5A were clearly different. • Xeg5A is a unique xyloglucanase that produces low-molecular-weight oligosaccharides.