Role of the C-terminal region of �-amylase from barley

To investigate the role of the C-terminal region of barley #-amylase, plasmid J54 was constructed with an expression vector (pBETA92) of barley #-amylase by site-directed mutagenesis. Escherichia coli JM109 harboring plasmid //54 was expected to express z/54 #-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. J54 /S-amylase gave a single activity band on isoelectric focusing (pi 6.85). J54 #-amylase was purified from the cells by consecutive a-cyclodextrin/Sepharose 6B column chromatography. A comparison of the properties of the mutant enzyme with those of the original recombinant ^-amylase [Biosci. Biotech. Biochem. (1994) 58,1080-1086] revealed two major differences. First, the original recombinant /?-amylase showed heterogeneity on isoelectric focusing, but J54 #-amylase gave a single main band of protein (pi 6.85). Therefore, the isoelectrophoretic heterogeneity of the original recombinant 0- amylase was apparently due to its C-terminal region. Secondly, z/54 #-amylase lacked thermostability. Therefore, it was concluded that the C-terminal region was significantly involved in the thermostability of ^-amylase