Role of the C-Terminal Region of β-Amylase from Barley

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To investigate the role of the C-terminal region of barley β-amylase, plasmid Δ54 was constructed with an expression vector (pBETA92) of barley β-amylase by site-directed mutagenesis. Escherichia coli JM109 harboring plasmid Δ54 was expected to express Δ54 β-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. Δ54 β-amylase gave a single activity band on isoelectric focusing (pI 6.85). Δ54 β-amylase was purified from the cells by consecutive α-cyclodextrin/Sepharose 6B column chromatography. A comparison of the properties of the mutant enzyme with those of the original recombinant β-amylase [Biosci. Biotech. Biochem. (1994) 58, 1080-1086] revealed two major differences. First, the original recombinant β-amylase showed heterogeneity on isoelectric focusing, but Δ54 β-amylase gave a single main band of protein (pI 6.85). Therefore, the isoelectrophoretic heterogeneity of the original recombinant β-amylase was apparently due to its C-terminal region. Secondly, Δ54 β-amylase lacked thermostability. Therefore, it was concluded that the C-terminal region was significantly involved in the thermostability of β-amylase.

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