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.
Yoshigi
N
N
Brewing Research Laboratories, Sapporo Breweries Ltd., Shizuoka.
Sahara
H
H
Koshino
S
S
D001483:Base Sequence; D004586:Electrophoresis; D004795:Enzyme Stability; D005822:Genetic Vectors; D001467:Hordeum / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007525:Isoelectric Focusing; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013379:Substrate Specificity; D013696:Temperature; D001614:beta-Amylase / Q000737:chemistry*
D007202:Indicators and Reagents; D008670:Metals; D001614:beta-Amylase