Isolation of DNA encoding sucrase genes from Streptococcus salivarius and partial characterization of the enzymes expressed in Escherichia coli.
Restriction enzyme fragments containing two sucrase genes have been isolated from a cosmid library of Streptococcus salivarius DNA. The genes were expressed in Escherichia coli cells, and the properties of both enzymes were studied in partially purified protein extracts from E. coli. One gene encoding an invertase-type sucrase was subcloned on a 2.4-kilobase-pair fragment. The sucrase enzyme had a Km for sucrose of 48 mM and a pH optimum of 6.5. The S. salivarius sucrase clone showed no detectable hybridization to a yeast invertase clone. Two overlapping subclones which had 1 kilobase pair of DNA in common were used to localize a fructosyltransferase gene. The fructosyltransferase had a Km of 93 mM and a pH optimum of 7.0. The product of the fructosyltransferase was a levan. A fructosyltransferase clone from Bacillus subtilis did not hybridize to S. salivarius DNA. The properties of the enzymes were compared with those of previously characterized sucrases.
D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics* / Q000302:isolation & purification; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005798:Genes, Bacterial; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism; D006602:Hexosyltransferases / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009693:Nucleic Acid Hybridization; D013291:Streptococcus / Q000201:enzymology / Q000235:genetics*; D013393:Sucrase / Q000235:genetics* / Q000378:metabolism; D013395:Sucrose / Q000378:metabolism; D015003:Yeasts / Q000201:enzymology / Q000235:genetics; D043324:beta-Fructofuranosidase; D001426:Bacterial Proteins
D004269:DNA, Bacterial; D004274:DNA, Recombinant; D006026:Glycoside Hydrolases; D006602:Hexosyltransferases; D013393:Sucrase; D013395:Sucrose; D043324:beta-Fructofuranosidase; C021451:inulosucrase; D001426:Bacterial Proteins