Role of the C-terminal region of beta-amylase from barley.

To investigate the role of the C-terminal region of barley beta-amylase, plasmid delta 54 was constructed with an expression vector (pBETA92) of barley beta-amylase by site-directed mutagenesis. Escherichia coli JM109 harboring plasmid delta 54 was expected to express delta 54 beta-amylase in which 54 amino acid residues were deleted from the C-terminus. The enzyme production started in the logarithmic phase, increased linearly, and reached a maximum after 12 h. delta 54 beta-amylase gave a single activity band on isoelectric focusing (pI 6.85). delta 54 beta-amylase was purified from the cells by consecutive alpha-cyclodextrin/Sepharose 6B column chromatography. A comparison of the properties of the mutant enzyme with those of the original recombinant beta-amylase [Biosci. Biotech. Biochem. (1994) 58, 1080-1086] revealed two major differences. First, the original recombinant beta-amylase showed heterogeneity on isoelectric focusing, but delta 54 beta-amylase gave a single main band of protein (pI 6.85). Therefore, the isoelectrophoretic heterogeneity of the original recombinant beta-amylase was apparently due to its C-terminal region. Secondly, delta 54 beta-amylase lacked thermostability. Therefore, it was concluded that the C-terminal region was significantly involved in the thermostability of beta-amylase.