ROLE OF THE C-TERMINAL REGION OF BETA-AMYLASE FROM BARLEY

To investigate the role of the C-terminal region of barley beta-amylas e, plasmid Delta 54 was constructed with an expression vector (pBETA92 ) of barley beta-amylase by site-directed mutagenesis. Escherichia col i JM109 harboring plasmid Delta 54 was expected to express Delta 54 be ta-amylase in which 54 amino acid residues were deleted from the C-ter minus. The enzyme production started in the logarithmic phase, increas ed linearly, and reached a maximum after 12 h, Delta 54 beta-amylase g ave a single activity band on isoelectric focusing (pI 6.85). Delta 54 beta-amylase was purified from the cells by consecutive alpha-cyclode xtrin/Sepharose 6B column chromatography. A comparison of the properti es of the mutant enzyme with those of the original recombinant beta-am ylase [Biosci, Biotech. Biochem. (1994) 58, 1080-1086] revealed two ma jor differences. First, the original recombinant beta-amylase showed h eterogeneity on isoelectric focusing, but Delta 54 beta-amylase gave a single main band of protein (pI 6.85). Therefore, the isoelectrophore tic heterogeneity of the original recombinant beta-amylase was apparen tly due to its C-terminal region. Secondly, Delta 54 beta-amylase lack ed thermostability. Therefore, it was concluded that the C-terminal re gion was significantly involved in the thermostability of beta-amylase .