EXPRESSION OF A HYBRID (1-3,1-4)-BETA-GLUCANASE IN BARLEY PROTOPLASTS

Barley aleurone protoplasts were used to study expression and secretio n of a heat-stable hybrid, Bacillus (1-3,1-4)-beta-glucanase (H(A107-M )). The strength and hormone responsiveness of a barley, aleurone spec ific, low pI alpha-amylase promoter was analysed in comparison with th e synthetic, constitutive pEmu promoter, by expressing the reporter ge ne encoding chloramphenicol acetyl transferase (CAT) in aleurone proto plasts. Both promoters directed high levels of CAT expression after GA 3 addition. Plasmids containing either promoter upstream of the coding region for H(A107-M) were also constructed. To effect secretion of H( A107-M) the coding region for a low pI alpha-amylase signal peptide wa s inserted between each promoter and the (1-3,1-4)-beta-glucanase codi ng region. Activity derived from heat-stable (1-3,1-4)-beta-glucanase could not be detected in protoplasts transfected with the alpha-amylas e promoter containing plasmids, whereas both plasmids with the pEmu pr omoter directed expression of H(A107-M). Measurements of (1-3,1-4)-bet a-glucanase activity from protoplasts transfected with plasmids encodi ng the mature enzyme indicated that the majority of H(A107-M) was intr acellular. When the plasmid encoding the pre-enzyme was used, 90% of t he activity from H(A107-M) was extracellular. The intracellular H(A107 -M) also reacted with specific antibodies, was active and had the expe cted molecular mass of 24 kDa. Extracellular H(A107-M) was post-transl ationally modified resulting in three active forms of 22, 24 and 28 kD a.