Purification, characterization, and sequence analysis of two alpha-amylase isoforms from azuki bean, Vigna angularis, showing different affinity towards beta-cyclodextrin sepharose.
Two alpha-amylase isoforms designated VAAmy1 and VAAmy2 were purified from cotyledons of germinating seedlings of azuki bean (Vigna angularis). VAAmy1 apparently had lower affinity towards a beta-cyclodextrin Sepharose column than VAAmy2. Molecular weights of VAAmy1 and VAAmy2 were estimated to be 47,000 and 44,000, respectively. However, no considerable difference was found between them in effects of pH, temperature, CaCl2, and EDTA, as well as the kinetic parameters for amylose (average degree of polymerization 17): kcat, 71.8 and 55.5 s(-1), Km, 0.113 and 0.097 mg/ml; for blocked 4-nitrophenyl alpha-D-maltoheptaoside: kcat, 62.4 and 85.3 s(-1), Km, 0.22 and 0.37 mM, respectively. Primary structures of the two enzymes were analyzed by N-terminal sequencing, cDNA cloning, and MALDI-TOF mass spectrometry, implying that the two enzymes have the same peptide. The results indicated that the low affinity of VAAmy1 towards beta-cyclodextrin Sepharose was due to some modification on/near carbohydrate binding site in the limited sequence regions, resulting in higher molecular weight.
Mar
San San
SS
Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan.
Mori
Haruhide
H
Lee
Jin-Ha
JH
Fukuda
Kenji
K
Saburi
Wataru
W
Fukuhara
Arinobu
A
Okuyama
Masayuki
M
Chiba
Seiya
S
Kimura
Atsuo
A
D000595:Amino Acid Sequence; D002118:Calcium / Q000494:pharmacology; D002614:Chelating Agents / Q000494:pharmacology; D002846:Chromatography, Affinity; D003505:Cyclodextrins; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D007887:Fabaceae / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification; D007700:Kinetics; D008969:Molecular Sequence Data; D010936:Plant Extracts / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012685:Sepharose; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013213:Starch; D013379:Substrate Specificity; D013696:Temperature; D014357:Trypsin; D000516:alpha-Amylases / Q000737:chemistry* / Q000302:isolation & purification; D047392:beta-Cyclodextrins
D002614:Chelating Agents; D003505:Cyclodextrins; D007527:Isoenzymes; D010936:Plant Extracts; D047392:beta-Cyclodextrins; D013213:Starch; D012685:Sepharose; D004492:Edetic Acid; D000516:alpha-Amylases; D014357:Trypsin; C031215:betadex; D002118:Calcium