Cloning and expression of sucrose phosphorylase gene from Bifidobacterium longum in E. coli and characterization of the recombinant enzyme.
A DNA fragment, which complemented the growth of E. coli both on M9 medium containing raffinose and on LB medium containing ampicillin, IPTG and 5-bromo-4-chloro-3-indoxyl-alpha-D-galactoside, was isolated from the genomic library of Bifidobacterium longum SJ32, which had been digested with EcoRI. In the cloned DNA fragment, a gene encoding a sucrose phosphorylase (splP) and a partially cloned putative sucrose regulator gene (splR) were identified using the deletion analysis and sequence analysis. A 56 kDa protein was synthesized in E. coli and partially purified by DEAE-ion exchange chromatography. The partially purified enzyme did not react with melibiose, melezitoze and raffinose but did with sucrose. It had transglucosylation activity in addition to hydrolytic activity.
D000595:Amino Acid Sequence; D001644:Bifidobacterium / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics*; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D005964:Glucosyltransferases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry* / Q000145:classification; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D009844:Oligosaccharides; D011994:Recombinant Proteins; D005964:Glucosyltransferases; C025901:sucrose phosphorylase