Production of N-acetyl-D-neuraminic acid using two sequential enzymes overexpressed as double-tagged fusion proteins.
BACKGROUND
Two sequential enzymes in the production of sialic acids, N-acetyl-D-glucosamine 2-epimerase (GlcNAc 2-epimerase) and N-acetyl-D-neuraminic acid aldolase (Neu5Ac aldolase), were overexpressed as double-tagged gene fusions. Both were tagged with glutathione S-transferase (GST) at the N-terminus, but at the C-terminus, one was tagged with five contiguous aspartate residues (5D), and the other with five contiguous arginine residues (5R).


RESULTS
Both fusion proteins were overexpressed in Escherichia coli and retained enzymatic activity. The fusions were designed so their surfaces were charged under enzyme reaction conditions, which allowed isolation and immobilization in a single step, through a simple capture with either an anionic or a cationic exchanger (Sepharose Q or Sepharose SP) that electrostatically bound the 5D or 5R tag. The introduction of double tags only marginally altered the affinity of the enzymes for their substrates, and the double-tagged proteins were enzymatically active in both soluble and immobilized forms. Combined use of the fusion proteins led to the production of N-acetyl-D-neuraminic acid (Neu5Ac) from N-acetyl-D-glucosamine (GlcNAc).


CONCLUSIONS
Double-tagged gene fusions were overexpressed to yield two enzymes that perform sequential steps in sialic acid synthesis. The proteins were easily immobilized via ionic tags onto ionic exchange resins and could thus be purified by direct capture from crude protein extracts. The immobilized, double-tagged proteins were effective for one-pot enzymatic production of sialic acid.
Wang
Tzu-Hsien
TH
Department of Chemical Engineering, National Chung Cheng University, Chiayi 621, Taiwan. d8944009@yahoo.com.tw
Chen
Ying-Yin
YY
Pan
Hsin-Hung
HH
Wang
Feng-Pao
FP
Cheng
Chung-Hsien
CH
Lee
Wen-Chien
WC
D002238:Carbohydrate Epimerases / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D004800:Enzymes, Immobilized / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D019158:N-Acetylneuraminic Acid / Q000096:biosynthesis*; D007652:Oxo-Acid-Lyases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D046939:Synechocystis / Q000201:enzymology
D002352:Carrier Proteins; D004800:Enzymes, Immobilized; D011993:Recombinant Fusion Proteins; D007652:Oxo-Acid-Lyases; C018728:N-acetylneuraminate lyase; D002238:Carbohydrate Epimerases; C024519:N-acyl-D-glucosamine 2-epimerase; D019158:N-Acetylneuraminic Acid