Isolation, characterization and molecular cloning of β-D-glucan exohydrolase from cultured tobacco cells

We purified an abundant cationic 68 kDa protein from the culture medium of tobacco BY2 (Nicotiana tabacum L. cv. Bright Yellow 2) cells and determined the amino acid sequence of its N-terminus and some internal peptides. Similar sequences were found in barley β-D-glucan exohydrolase [EC 3.2.1.73], and the purified 68 kDa protein had β-glucosidase activity. The purified protein released glucose from a mixture of laminaridextrins (β-1 ,3-oligoglucosides). In addition to β-1 ,3-linkages, the enzyme also hydrolyzed β-1,4- and β-1,6-linkages of glucose. A cDNA encoding tobacco β-D-glucan exohydrolase was isolated using polymerase chain reaction (PCR) based on the internal amino acid sequence. The deduced amino acid sequence of tobacco β-glucan exohydrolase showed 73%, 74% and 44% amino acid identity with barley β-D-glucan exohydrolase, nasturtium β-D-glucosidase and 1,4-β-D-glucan glucohydrolase D (CELD) of Pseudomonas fluorescens, respectively.