Heterologous Expression and Activity Study of a Novel Sialic Acid Aldolase Gene Derived from Pedobacter heparinus

In order to develop a new type of sialic acid aldolase and express it effectively in Escherichia coli, the gene PhNeuLy3300 putatively encoding sialic acid aldolase from Pedobacter heparinus was cloned. A constitutive pRSF-EM5 vector containing self-initiating promotors was constructed. The cloned sialic acid aldolase gene was transferred into the constitutive vector pRSF-ME5 and another commonly used inducible vector pET-30a to construct the recombinant plasmids pRSF-EM5-PhNeuLy3300 and pET30a-PhNeuLy3300, respectively. The constructs were heterologously expressed in E. coli, further electrophoretic analysis and activity studies were performed on the two recombinant proteases. The results showed that the gene fragment encoding sialic acid aldolase was 945 bp and encodes a total of 314 amino acid residues. Polyacrylamide gel electrophoresis showed that the apparent molecular weight of the two recombinant proteins was about 36.4 kDa. Bradford method measured that the concentration of purified pET30a-PhNeuLy3300 recombinant protein was 3.29 mg/mL, which proved that two recombinant proteases were obtained by heterologous expression. The activity studies showed that both recombinant sialic acid aldolases can catalyze N-Acetylneuraminic acid to produce the N-Acetylmannosamine within 1 h, which means that both recombinant proteases have high activity.