TY - JOUR
T1 - Combined biosynthetic pathway for de novo production of UDP-galactose
T2 - Catalysis with multiple enzymes immobilized on agarose beads
AU - Liu, Ziye
AU - Zhang, Jianbo
AU - Chen, Xi
AU - Wang, Peng G.
PY - 2002/4/2
Y1 - 2002/4/2
N2 - Regeneration of sugar nucleotides is a critical step in the biosynthetic pathway for the formation of oligosaccharides. To alleviate the difficulties in the production of sugar nucleotides, we have developed a method to produce uridine diphosphate galactose (UDP-galactose). The combined biosynthetic pathway, which involves seven enzymes, is composed of three parts: i) the main pathway to form UDP-galactose from galactose, with the enzymes galactokinase, galactose-1-phosphate uridyltransferase, UDP-glucose pyrophosphorylase, and inorganic pyrophosphatase, ii) the uridine triphosphate supply pathway catalyzed by uridine monophosphate (UMP) kinase and nucleotide diphosphate kinase, and iii) the adenosine triphosphate (ATP) regeneration pathway catalyzed by polyphosphate kinase with polyphosphate added as an energy resource. All of the enzymes were expressed individually and immobilized through their hexahistidine tags onto nickel agarose beads ("super beads"). The reaction requires a stoichiometric amount of UMP and galactose, and catalytic amounts of ATP and glucose 1-phosphate, all inexpensive starting materials. After continuous circulation of the reaction mixture through the super-bead column for 48 h, 50% of the UMP was converted into UDP-galactose. The results show that de novo production of UDP-galactose on the super-bead column is more efficient than in solution because of the stability of the immobilized enzymes.
AB - Regeneration of sugar nucleotides is a critical step in the biosynthetic pathway for the formation of oligosaccharides. To alleviate the difficulties in the production of sugar nucleotides, we have developed a method to produce uridine diphosphate galactose (UDP-galactose). The combined biosynthetic pathway, which involves seven enzymes, is composed of three parts: i) the main pathway to form UDP-galactose from galactose, with the enzymes galactokinase, galactose-1-phosphate uridyltransferase, UDP-glucose pyrophosphorylase, and inorganic pyrophosphatase, ii) the uridine triphosphate supply pathway catalyzed by uridine monophosphate (UMP) kinase and nucleotide diphosphate kinase, and iii) the adenosine triphosphate (ATP) regeneration pathway catalyzed by polyphosphate kinase with polyphosphate added as an energy resource. All of the enzymes were expressed individually and immobilized through their hexahistidine tags onto nickel agarose beads ("super beads"). The reaction requires a stoichiometric amount of UMP and galactose, and catalytic amounts of ATP and glucose 1-phosphate, all inexpensive starting materials. After continuous circulation of the reaction mixture through the super-bead column for 48 h, 50% of the UMP was converted into UDP-galactose. The results show that de novo production of UDP-galactose on the super-bead column is more efficient than in solution because of the stability of the immobilized enzymes.
KW - Biosynthesis
KW - Enzymes
KW - Immobilization
KW - Polyphosphate
KW - UDP-galactose
UR - http://www.scopus.com/inward/record.url?scp=0037007176&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0037007176&partnerID=8YFLogxK
U2 - 10.1002/1439-7633(20020402)3:4<348::AID-CBIC348>3.0.CO;2-K
DO - 10.1002/1439-7633(20020402)3:4<348::AID-CBIC348>3.0.CO;2-K
M3 - Article
C2 - 11933236
AN - SCOPUS:0037007176
VL - 3
SP - 348
EP - 355
JO - ChemBioChem
JF - ChemBioChem
SN - 1439-4227
IS - 4
ER -