Recombinant sucrose phosphorylase from Leuconostoc mesenteroides: characterization, kinetic studies of transglucosylation, and application of immobilised enzyme for production of alpha-D-glucose 1-phosphate.
Sucrose phosphorylase catalyzes the reversible conversion of sucrose (alpha-D-glucopyranosyl-1,2-beta-D-fructofuranoside) and phosphate into D-fructose and alpha-D-glucose 1-phosphate. We report on the molecular cloning and expression of the structural gene encoding sucrose phosphorylase from Leuconostoc mesenteroides (LmSPase) in Escherichia coli DH10B. The recombinant enzyme, containing an 11 amino acid-long N-terminal metal affinity fusion peptide, was overproduced 60-fold in comparison with the natural enzyme. It was purified to apparent homogeneity using copper-loaded Chelating Sepharose and obtained in 20% yield with a specific activity of 190 Umg(-1). LmSPase was covalently attached onto Eupergit C with a binding efficiency of 50% and used for the continuous production of alpha-D-glucose 1-phosphate from sucrose and phosphate (600 mM each) in a packed-bed immobilised enzyme reactor (30 degrees C, pH 7.0). The reactor was operated at a stable conversion of 91% (550 mM product) and productivity of approximately 11 gl(-1)h(-1) for up to 600 h. A kinetic study of transglucosylation by soluble LmSPase was performed using alpha-d-glucose 1-phosphate as the donor substrate and various alcohols as acceptors. D- and L-arabitol were found to be good glucosyl acceptors.
Goedl
Christiane
C
Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12, A-8010 Graz, Austria.
Schwarz
Alexandra
A
Minani
Alphonse
A
Nidetzky
Bernd
B
D019149:Bioreactors; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000378:metabolism*; D004926:Escherichia coli / Q000254:growth & development; D015870:Gene Expression; D005958:Glucosephosphates / Q000096:biosynthesis*; D005964:Glucosyltransferases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006031:Glycosylation; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007934:Leuconostoc / Q000201:enzymology*; D009842:Oligopeptides; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013395:Sucrose / Q000378:metabolism; D013696:Temperature
D004800:Enzymes, Immobilized; D005958:Glucosephosphates; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011994:Recombinant Proteins; D006639:Histidine; D013395:Sucrose; C031590:glucose-1-phosphate; D005964:Glucosyltransferases; C025901:sucrose phosphorylase