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 beta -D-glucan exohydrolase [EC 3.2.1.73], and the purified 68 kDa protein had beta -glucosidase activity. The purified protein released glucose from a mixture of laminaridextrins (beta -1,3-oligoglucosides). In addition to beta -1,3-linkages, the enzyme also hydrolyzed beta -1,4- and beta -1,6-linkages of glucose. A cDNA encoding tobacco beta -D-glucan exohydrolase was isolated using polymerase chain reaction (PCR) based on the internal amino acid sequence. The deduced amino acid sequence of tobacco beta -glucan exohydrolase showed 73 %, 74% and 44% amino acid identity with barley beta -D-glucan exohydrolase, nasturtium beta -D-glucosidase and 1,4-beta -D-glucan glucohydrolase D (CELD) of Pseudomonas fluorescens, respectively.