Expression, purification and characterization of recombinant sucrose synthase 1 from Solanum tuberosum L. for carbohydrate engineering.
The gene sus1 from Solanum tuberosum L. encoding for sucrose synthase 1 was cloned into the plasmid pDR195 under the control of the PMA1 promotor. After transformation of Saccharomyces cerevisiae strain 22574d sus1 was constitutively expressed giving a specific activity of 0.3Umg(-1) protein in the crude extract. A one-step purification by Q-Sepharose resulted in an 14-fold purified enzyme preparation in 74% yield. SuSy1 was subsequently purified by immobilized metal ion affinity chromatography and characterized for its utilization in synthesizing different nucleotide sugars and sucrose analogues. The kinetic constants for the cleavage and synthesis reaction were determined: K(m) (UDP) 4microM; K(iS) (UDP) 0.11mM; K(m) (sucrose) 91.6mM; K(m) (UDP-Glc) 0.5mM; K(iS) (UDP-Glc) 2.3mM; K(m) (D-fructose) 2.1mM; K(iS) (D-fructose) 35.9mM. Different nucleoside diphosphates as well as different donor substrate were accepted as follows: UDP>dTDP>ADP>CDP>GDP in the cleavage reaction and UDP-Glc>dTDP-Glc>ADP-Glc>CDP-Glc in the synthesis reaction. SuSy1 shows also a broad acceptance of D- and L-ketoses and D- and L-aldoses. The acceptance of aldoses was deduced from the binding of the inhibitor 5-deoxy-D-fructose (K(i) 0.3mM), an analogue of the natural substrate D-fructopyranoside. The broad substrate spectrum renders SuSy1 from potato a versatile biocatalyst for carbohydrate engineering.
Römer
Ulrike
U
Department of Biotechnology/Biomaterial Sciences, Helmholtz-Institute for Biomedical Engineering, RWTH Aachen, Germany.
Schrader
Henning
H
Günther
Nicole
N
Nettelstroth
Nadja
N
Frommer
Wolf B
WB
Elling
Lothar
L
D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005818:Genetic Engineering; D005964:Glucosyltransferases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D007525:Isoelectric Focusing; D007700:Kinetics; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D011198:Solanum tuberosum / Q000201:enzymology*; D013379:Substrate Specificity
D011994:Recombinant Proteins; D005964:Glucosyltransferases; C022699:sucrose synthase