title: Production of N-acetyl-D-neuraminic acid using two sequential enzymes overexpressed as double-tagged fusion proteins.
abstract: 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.
authors: Department of Chemical Engineering, National Chung Cheng University, Chiayi 621, Taiwan. d8944009@yahoo.com.tw
authors: Tzu-Hsien
authors: TH
authors: Wang
authors: Ying-Yin
authors: YY
authors: Chen
authors: Hsin-Hung
authors: HH
authors: Pan
authors: Feng-Pao
authors: FP
authors: Wang
authors: Chung-Hsien
authors: CH
authors: Cheng
authors: Wen-Chien
authors: WC
authors: Lee
mesh_terms: 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
chemical_list: 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