Characterization of the highly efficient sucrose isomerase from Pantoea dispersa UQ68J and cloning of the sucrose isomerase gene.
Sucrose isomerase (SI) genes from Pantoea dispersa UQ68J, Klebsiella planticola UQ14S, and Erwinia rhapontici WAC2928 were cloned and expressed in Escherichia coli. The predicted products of the UQ14S and WAC2928 genes were similar to known SIs. The UQ68J SI differed substantially, and it showed the highest isomaltulose-producing efficiency in E. coli cells. The purified recombinant WAC2928 SI was unstable, whereas purified UQ68J and UQ14S SIs were very stable. UQ68J SI activity was optimal at pH 5 and 30 to 35 degrees C, and it produced a high ratio of isomaltulose to trehalulose (>22:1) across its pH and temperature ranges for activity (pH 4 to 7 and 20 to 50 degrees C). In contrast, UQ14S SI showed optimal activity at pH 6 and 35 degrees C and produced a lower ratio of isomaltulose to trehalulose (<8:1) across its pH and temperature ranges for activity. UQ68J SI had much higher catalytic efficiency; the Km was 39.9 mM, the Vmax was 638 U mg(-1), and the Kcat/Km was 1.79 x 10(4) M(-1) s(-1), compared to a Km of 76.0 mM, a Vmax of 423 U mg(-1), and a Kcat/Km of 0.62 x 10(4) M(-1) s(-1) for UQ14S SI. UQ68J SI also showed no apparent reverse reaction producing glucose, fructose, or trehalulose from isomaltulose. These properties of the P. dispersa UQ68J enzyme are exceptional among purified SIs, and they indicate likely differences in the mechanism at the enzyme active site. They may favor the production of isomaltulose as an inhibitor of competing microbes in high-sucrose environments, and they are likely to be highly beneficial for industrial production of isomaltulose.
Wu
Luguang
L
Botany Department, The University of Queensland, Brisbane 4072, Australia.
Birch
Robert G
RG
D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004269:DNA, Bacterial / Q000235:genetics; D004187:Disaccharides / Q000096:biosynthesis; D004795:Enzyme Stability; D004885:Erwinia / Q000201:enzymology / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007534:Isomaltose / Q000031:analogs & derivatives* / Q000096:biosynthesis; D007700:Kinetics; D007709:Klebsiella / Q000201:enzymology / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020636:Pantoea / Q000201:enzymology* / Q000235:genetics*; D010802:Phylogeny; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature
D004269:DNA, Bacterial; D004187:Disaccharides; D011994:Recombinant Proteins; D007534:Isomaltose; D005964:Glucosyltransferases; C116853:sucrose isomerase; C042003:trehalulose; C008189:isomaltulose