Enzymatic Preparation Of N-acetyl-d-neuraminidase

This thesis includes three parts: (1) Cloning and expression of Neu5Ac aldolase in Escherichia coli. (2) Production of Neu5Ac aldolase in the Fermentation of E.coli DH5α/pRY3 and comparison of the characteristic of immobilized and free enzyme .(3)Enzymatic synthesis of N-Acetyl-D-neuraminic acid. I Cloning and expression of Neu5Ac aldolase in Escherichia coli The 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 a single protein band with a molecular weight of 33000 daltons being good agreement with those reported in the reference . In the recombinant plasmid pRY1, the expression of nanA gene is controlled by the lac promoter with the induction of IPTG or lactose. Furthermore,the plasmid pRY3 was constructed,in which the nanA gene is controlled by the tac promoter .The protein of Neu5Ac aldolase could be constitutively expressed using the recombinant strain, E.coli DH5α/pRY3, without induction of IPTG or lactose. The proteins expressed of those two engineering strains have a 6 His-tag at N-terminal for easier purificiation and immobilization. II Production of Neu5Ac aldolase in the Fermentation of E.coli DH5α/pRY3 and comparison of the characteristic of immobilized and free enzyme In order to study the properties of immobilized Neu5Ac aldolase , the combinant strain E.coli DH5α/pRY3 was fermented in the 5L fermentor containing 3L LB broth.Applied with desired amount of glucose, at 37℃for 35 hours.The yield of wet cell and enzyme activity were 13.3g/L and 167 u/L ,respectively. It was demonstrated that the optimal reaction temperatures were 67℃for immobilized aldolase and 72℃for free form.Both of them showed the same optimal pH7.5. The value of Km and...