Characterization of chimeric enzymes constructed between two distinct alpha-amylase cDNAs from cultured rice cells.
Cultured cells of rice (Oryza sativa cv Sasanishiki) produce two alpha-amylase isozymes, AMY-I and AMY-III. Using a bacterial expression system, eight chimeric genes constructed with various combination of AMY-I and AMY-III cDNA fragments were expressed, and each recombinant chimeric protein was characterized. Four of the eight recombinant enzymes having region c (one of the four regions having unconserved base sequences between AMY-I and AMY-III cDNAs) of AMY-I showed the same enzyme characteristics as that of native AMY-I, which had high temperature optimum at 50 degrees C. The other four chimeric proteins carrying region c of AMY-III showed the AMY-III type characteristics, which were a low temperature optimum at 25 degrees C and susceptibility to a higher maltooligosaccharide (G17) substrate. The unconserved region c is involved in the decision of the characteristic of AMY-I or AMY-III.
Abe
R
R
Laboratory of Enzymology, Division of Life Science, Graduate School of Agricultural Science, Tohoku University, Sendai-shi, Japan.
Yoshida
K
K
Aoyagi
M
M
Kasahara
S
S
Ichishima
E
E
Nakajima
T
T
D000595:Amino Acid Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis*; D018744:DNA, Plant / Q000032:analysis*; D004926:Escherichia coli; D007527:Isoenzymes / Q000235:genetics*; D008969:Molecular Sequence Data; D012275:Oryza / Q000166:cytology / Q000201:enzymology*; D011993:Recombinant Fusion Proteins / Q000032:analysis*; D017386:Sequence Homology, Amino Acid; D000516:alpha-Amylases / Q000235:genetics*
D018076:DNA, Complementary; D018744:DNA, Plant; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D000516:alpha-Amylases