Enzymatic Process For Preparing N-acetyl-D-neuraminic Acid

This work includes three parts: (1) Cloning and expression of Neu5Ac Aldolase. (2) Enzymatic synthesis of N-Acetyl-D-neuraminic acid. (3) Cloning and expression of N-acetyl-D-glucosamine2-epimerase.1 Cloning and expression of N-acetylneuraminate aldolaseThe gene ( nanA ) coding for Neu5Ac aldolase was cloned , sequenced, and expressed in Escherichia coli in this work. Sequencing data revealed that the open reading frame of this gene was 894 bp in size and predicted to encode a protein consisting of 298 amino acids. The patterns of SDS-PAGE showed that the purified enzyme protein migrated as a single protein band with a molecular weight of 33000 daltons being good agreement with those reported in the reference.In the recombinant plasmid pNA1, the expression of nanA gene is controlled by the lac promoter with the induction of IPTG or lactose.2 Enzymatic synthesis of N-Acetyl-D-neuraminic acidAfter fermentation of the recombinated bacteria E.coli BL21(DE3)/pNAl, collected cells were freeze-thawed in order to enhance permeability. The process for the enzymatic formation of N-Acetyl-D-neuraminic acid was carried out in a reaction system containing 0.8M of ManNAc, 1.2M of Pyr and 1.25g of wet cells containing N-acetylneuraminate aldolase in a total volumn of 5ml ion free water (pH7.5) at 28℃. It was shown that after 10 hours incubation,the reaction reached constant state and gave the conversion rate of 90% for Neu5Ac synthesis. The reaction mixture was mixed with 7 volumns of acetic acid and keep at 4℃ for 3 days. The crystal was collected by filtering,dried at 40℃.1.01g Neu5Ac was finially obtained with the rate of 90.7% in crystallization. The totol efficiency rate was 81.6%. The HPLC patterns of the Neu5Ac crystal prepared in this experiment was same with Simga product and its purity was slightly higher.3 Cloning and expression of N-acetyl-D-glucosamineZ-epimerase N-acetyl-D-glucosamine2-epimerase (EC 5.1.3.8) can be used to epimerised N-acetyl-D-glucosamine (GlcNAc) to ManNAc, one of the start material for the synthesis of Neu5Ac.The gene for N-acetyl-D-glucosamine2-epimerase has been cloned and sequenced and the recombinant strain for expressing the enzyme has been constructed. Sequencing data revealed that the ORF of this gene was 1206 bp in size and predicted to encode a protein consisting of 402 amino acids. The purified protein was a single band with a molecular weight of 45000 doltons on the SDS-PAGE, which is consistent to the analysis based on gene sequence. After optimizing of it's expression conditions, the recombinant strain gave the yield of enzyme activity of l.lU/ml in the fermentation.