Function-unknown glycoside hydrolase family 31 proteins, mRNAs of which were expressed in rice ripening and germinating stages, are α-glucosidase and α-xylosidase

In rice (Oryza sativa L., var Nipponbare) seeds, there were three mRNAs encoding for function-unknown hydrolase family 31 homologous proteins (ONGX-H1, ONGX-H3 and ONGX-H4): ONGX-H1 mRNA was expressed in ripening stage and mRNAs of ONGX-H3 and ONGX-H4 were found in both the ripening and germinating stages [Nakai et al., (2007) Biochimie 89, 49-62]. This article describes that the recombinant proteins of ONGX-H1 (rONGXG-H1), ONGX-H3 (rONGXG-H3) and ONG-H4 (rONGXG-H4) were overproduced in Pichia pastoris as fusion protein with the alpha-factor signal peptide of Saccharomyces cerevisiae. Purified rONGXG-H1 and rONGXG-H3 efficiently hydrolysed malto-oligosaccharides, kojibiose, nigerose and soluble starch, indicating that ONGX-H1 and ONGX-H3 are alpha-glucosidases. Their substrate specificities were similar to that of ONG2, a main alpha-glucosidase in the dry and germinating seeds. The rONGXG-H1 and rONGX-H3 demonstrated the lower ability to adsorb to and degradation of starch granules than ONG2 did, suggesting that three a-glucosidases, different in action to starch granules, were expressed in ripening stage. Additionally, purified rONGXG-H4 showed the high activity towards alpha-xylosides, in particular, xyloglucan oligosaccharides. The enzyme hardly hydrolysed alpha-glucosidic linkage, so that ONGX-H4 was an alpha-xylosidase. alpha-Xylosidase encoded in rice genome was found for the first time.