Cloning of a plastidic rye (Secale cereale) β‐glucosidase cDNA and its expression in Escherichia coli

The cDNA for a β‐glucosidase (EC3.2.1.21) was isolated from rye (Secale cereale, cv Motto) and the sequence corresponding to the mature protein cloned into pET21a expression vector and used for transformation of Escherichia coli. The recombinant β‐glucosidase expressed in E. coli was recognized by antibodies to maize β‐glucosidase and exhibited the same kinetic properties on the endogenous substrates hydroxamic acid glucosides and artificial substrates as the native enzyme purified from rye. The enzyme monomer had an apparent molecular weight of about 67 kDa. The isolated cDNA was analysed with web‐based chloroplast targeting prediction programs. The programs predicted a chloroplast targeting peptide with a cleavage site between amino acid 49 and 50. Sequence alignment of the plastidic rye β‐glucosidase showed that the putative sites for substrate specificity of maize Glu1, W378 and F198 (F197) are conserved in the rye enzyme, whereas F205, F466 and A467 of maize Glu1 are exchanged for histidine, glycine and serine, respectively, in rye. The plastidic β‐glucosidase is expressed in all plant parts and the highest levels were found in the coleoptile and mesocotyl.