Removal of the four C-terminal glycine-rich repeats enhances the thermostability and substrate binding affinity of barley beta-amylase.
Barley beta-amylase undergoes proteolytic cleavage in the C-terminal region after germination. The implication of the cleavage in the enzyme's characteristics is unclear. With purified native beta-amylases from both mature barley grain and germinated barley, we found that the beta-amylase from germinated barley had significantly higher thermostability and substrate binding affinity for starch than that from mature barley grain. To better understand the effect of the proteolytic cleavage on the enzyme's thermostability and substrate binding affinity for starch, recombinant barley beta-amylases with specific deletions at the C-terminal tail were generated. The complete deletion of the four C-terminal glycine-rich repeats significantly increased the enzyme's thermostability, but an incomplete deletion with one repeat remaining did not change the thermostability. Although different C-terminal deletions affect the thermostability differently, they all increased the enzyme's affinity for starch. The possible reasons for the increased thermostability and substrate binding affinity, due to the removal of the four C-terminal glycine-rich repeats, are discussed in terms of the three-dimensional structure of beta-amylase.
Ma
Y F
YF
Department of Plant Science, Waite Campus, The University of Adelaide, Glen Osmond, SA 5064, Australia.
Eglinton
J K
JK
Evans
D E
DE
Logue
S J
SJ
Langridge
P
P
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D005998:Glycine / Q000378:metabolism*; D001467:Hordeum / Q000201:enzymology* / Q000235:genetics; D006358:Hot Temperature; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D020449:Repetitive Sequences, Amino Acid / Q000235:genetics; D017384:Sequence Deletion; D013213:Starch / Q000737:chemistry; D013379:Substrate Specificity / Q000235:genetics; D001614:beta-Amylase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D010446:Peptide Fragments; D011994:Recombinant Proteins; D013213:Starch; D001614:beta-Amylase; D005998:Glycine