Coupled bioconversion for preparation of N-acetyl-D: -neuraminic acid using immobilized N-acetyl-D: -glucosamine-2-epimerase and N-acetyl-D: -neuraminic acid lyase.
N-Acetyl-D: -neuraminic acid (Neu5Ac) can be produced from N-acetyl-D: -glucosamine (GlcNAc) and pyruvate by a chemoenzymatic process in which an alkaline-catalyzed epimerization transforms GlcNAc to N-acetyl-D: -manosamine (ManNAc). ManNAc is then condensed biocatalytically with pyruvate in the presence of N-acetyl-D: -neuraminic acid lyase (NAL) or by a two-step, fully enzymatic process involving bioconversions of GlcNAc to ManNAc and ManNAc to Neu5Ac using N-acetyl-D: -glucosamine 2-epimerase (AGE) and NAL. There are some drawbacks to this technique, such as lengthy reaction time, and the low conversion rate when the soluble forms of the enzymes are used in the two-step enzymatic process. In this study, the Escherichia coli-expressed AGE and NAL in the supernatant were purified by FP-based affinity chromatography and then immobilized on Amberzyme oxirane resin. These two immobilized enzymes, with a specific activity of 78.18 U/g for AGE and 69.30 U/g for NAL, were coupled to convert GlcNAc to Neu5Ac directly in one reactor. The conversion rate of the two-step reactions from GlcNAc to Neu5Ac was approximately 73% within 24 h. Furthermore, the immobilized AGE and NAL could both be used up to five reaction cycles without loss of activity or significant decrease of the conversion rate.
Hu
Shiyuan
S
Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, People's Republic of China.
Chen
Jun
J
Yang
Zhongyi
Z
Shao
Lijun
L
Bai
Hua
H
Luo
Jiali
J
Jiang
Weihong
W
Yang
Yunliu
Y
D000117:Acetylglucosamine / Q000378:metabolism*; D019149:Bioreactors; D002238:Carbohydrate Epimerases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002384:Catalysis; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004800:Enzymes, Immobilized / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006595:Hexosamines / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D019158:N-Acetylneuraminic Acid / Q000378:metabolism*; D007652:Oxo-Acid-Lyases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019289:Pyruvic Acid / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D002352:Carrier Proteins; D004269:DNA, Bacterial; D004800:Enzymes, Immobilized; D006595:Hexosamines; D011994:Recombinant Proteins; D019289:Pyruvic Acid; D007652:Oxo-Acid-Lyases; C018728:N-acetylneuraminate lyase; D002238:Carbohydrate Epimerases; C024519:N-acyl-D-glucosamine 2-epimerase; D019158:N-Acetylneuraminic Acid; D000117:Acetylglucosamine; C002022:N-acetylmannosamine