Enzymatic properties and expression of an α-sialidase from Bifidobacterium longum in Escherichia coli

Abstract:

[Background] Sialidases are a type of glycoside hydrolases that hydrolyze the terminal sialic acid residue from sialic acid-containing complex. Sialidases are ubiquitous in animals and microorganisms and have important biological functions.[Objective] To clone a novel sialidase gene blsia42 from Bifidobacterium longum express it in Escherichia coli BL21(DE3), and characterize the enzymatic properties of the expressed protein. [Methods] A novel sialidase gene blsia42 was cloned from B. longum, and the recombinant expression plasmid pET-28a-blsia42 was constructed and expressed heterologously in E. coli BL21(DE3). After the crude enzyme was purified by Ni-NTA affinity chromatography, the enzymatic properties were studied. [Results] The specific activity of purified BlSia42 was determined to be 164 935.2 U/mg. The molecular weight of BlSia42 was determined as 42.8 kDa and 41.5 kDa by SDS-PAGE and gel filtration, respectively. The optimum conditions for BlSia42 were pH 6.0 and 50℃, and this enzyme was stable within pH 3.5-9.0 and below 45℃. BlSia42 showed a broad range of substrate specificity and had hydrolysis activity towards α2,3, α2,6, and α2,8 glycosidic bonds. The activity of BlSia42 with 3'-SL and colominic acid as substrates was 87.50% and 67.19% of that with 6'-SL as the substrate. After colominic acid was hydrolyzed by BlSia42 for 12 h, the sialic acid concentration and hydrolysis rate was 2.4 g/L and 23.6%, respectively. [Conclusion] The excellent enzymatic properties make BlSia42 potentially suitable for the preparation of sialic acid and its derivatives.