Molecular Cloning, Expression And Activity Analysis Of N-Acetylneuraminate Aldolase

N-acetyl-D-neuraminic acid (Neu5Ac), a major species of the sialic acids, is widely distributed in the animal kingdom at the terminal positions of glycoproteins and glycolipids, and plays important roles in a variety of physiological and pathological processes. Neu5Ac has an important medical value and was expected to be utilized extensively as pharmaceuticals in the treatment of influenza, neurological disorders and diagnostic reagents in the detection of malignant and inflammatory diseases. To enzymatically produce Neu5Ac from N-acetyl-D-glucosamine (GlcNAc), a two-enzyme, sequential system of GlcNAc 2-epimerase (EC 5.1.3.8, Age) and Neu5Ac aldolase (EC 4.1.3.3, NanA) is commonly used. Age catalyzes the interconversion of GlcNAc to ManNAc, which then reacts with pyruvate to form Neu5Ac through the enzymatic action of NanA.These two reactions are reversible reactions. It is important to regulate the expression of NanA and Age proteins on the same level that can make the reversible reaction toward positive to get the maximum production of Neu5Ac. In this study, On the one hand we construct a component by the introduction of rare codons to regulate the expression of NanA which achieves the purpose that regulate the expression level of protein with not change the promoter, and also improves the expression of the protein in soluble forms. This construction strategy for the regulation of protein provide a very important theoretical guidance and has a very large role in analyses of protein expression; on the other hand, Neu5Ac aldolase is a bottleneck in the synthesis of Neu5Ac. We cloned and purified five NanA proteins from different microbial sources and analyzed the enzymatic property, the result showed we have found an enzyme which have a good specific activity and enzymatic property and can increase the production of Neu5Ac.It provides some theoretical guidance on the industrial production of Neu5Ac and has broad application prospects and higher market values.