Cloning and Expression of Isoprimeverose-producing Oligoxyloglucan Hydrolase from Actinomycetes Species, Oerskovia sp. Y1

Abstract

Isoprimeverose-producing oligoxyloglucan hydrolase (IPase; EC 3.2.1.120) is a unique β-glycosidase that cleaves xyloglucan oligosaccharides at the nonreducing end, producing isoprimeverose. Here, we report the first gene cloning and expression of IPase. Previously, we reported that IPase from an actinomycetes, Oerskovia sp. Y1, has a molecular mass of 105 kDa. In this study, the full-length DNA encoding IPase was cloned and sequenced, and shown to have a 3,054-bp open reading frame encoding a protein of 1,018 amino acid residues. Based on the amino acid sequence, the catalytic domain was classified as glycoside hydrolase family 3 enzyme, and it has a family 6 carbohydrate-binding module at its C-terminus. IPase was expressed in Escherichia coli and the recombinant protein was purified. Although the recombinant protein was expressed as an inclusion body, renaturation was successful, and enzymatically active recombinant IPase was obtained. An analysis of the substrate specificity revealed that IPase strictly recognizes the isoprimeverose unit at the nonreducing end of a xyloglucan oligosaccharide. In addition, the binding assay revealed that IPase had binding ability to xyloglucan.