Characterization of 4-alpha-glucanotransferase from Synechocystis sp. PCC 6803 and its application to various corn starches.
A putative 4-alpha-glucanotransferase (alphaGTase) gene from Synechocystis sp. PCC 6803 was identified being composed of 1505 nucleotides, and the overexpressed protein was purified with an affinity chromatography. The recombinant alphaGTase had about 57kDa of molecular mass when judged by SDS-PAGE analysis. The optimum reaction condition of the alphaGTase was shown to be pH 7 at 45 degrees C in 50mm phosphate buffer. This enzyme displayed transglycosylating activity on various maltooligosaccharides, of which the smallest donor and acceptor molecules were determined to be maltose and glucose, respectively. Various corn starches consisting of different proportions of amylopectin and amylose were incubated with the recombinant alphaGTase. The change in molecular weight distribution of alphaGTase-modified starch was analyzed by HPSEC. The reaction pattern of alphaGTase showed substantial decrease in amylopectin and increase in the peak corresponding to cycloamylose (CA). The production yield of CA tended to increase from 5 to 30% along with the increase in the apparent amylose content in corn starch, which suggested that linear amylose chain would be preferred to produce CA in the alphaGTase treatment. The detectable minimum degree of polymerization (DP) of CA was shown to be 22 by MALDI-TOF-MS analysis. As another action mode of alphaGTase, the rearrangement of amylopectin branch-chain distribution occurred without hydrolysis to small oligosaccharides. After isoamylolysis, alphaGTase-treated starch displayed the increase in DP 4-9 and longer than DP 21 when the relative proportion of branch chains in amylopectin was determined by HPAEC.
Lee
Byung-Hoo
BH
Department of Food Science & Technology, BK21 Project Team, and Carbohydrate Bioproduct Research Center, Sejong University, 98 Gunja-Dong, Gwangjin-Gu, Seoul 143-747, Republic of Korea.
Oh
Deok-Kun
DK
Yoo
Sang-Ho
SH
D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D003505:Cyclodextrins / Q000096:biosynthesis / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006004:Glycogen Debranching Enzyme System / Q000302:isolation & purification / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D009842:Oligopeptides / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013213:Starch / Q000378:metabolism*; D013379:Substrate Specificity; D046939:Synechocystis / Q000201:enzymology*; D013696:Temperature
D003505:Cyclodextrins; D006004:Glycogen Debranching Enzyme System; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011994:Recombinant Proteins; D006639:Histidine; D013213:Starch; C020569:4 alpha-glucanotransferase