长双歧杆菌α-唾液酸苷酶在大肠杆菌中的表达及酶学性质

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

摘要:

【背景】唾液酸苷酶是一类水解唾液酸糖复合物末端唾液酸残基的糖苷水解酶，广泛存在于动物和微生物中，具有重要的生物学功能。【目的】克隆一个长双歧杆菌(Bifidobacterium longum)唾液酸苷酶基因(blsia42)并在大肠杆菌(Escherichia coli)中表达，探讨该重组酶的酶学性质。【方法】从长双歧杆菌中克隆唾液酸苷酶基因(blsia42)，构建重组表达质粒pET-28a-blsia42并在大肠杆菌BL21(DE3)中异源表达。BlSia42粗酶液经Ni-NTA亲和层析纯化后研究其酶学性质。【结果】 BlSia42纯酶的比酶活为164 935.2 U/mg。SDS-PAGE法和凝胶过滤法测定BlSia42的分子量分别为42.8 kDa和41.5 kDa。该酶的最适pH和温度分别为6.0和50 ℃，在pH 3.5-9.0和45 ℃以下稳定。BlSia42底物特异性广泛，对α2,3、α2,6和α2,8糖苷键均显示出水解活性，对3'-唾液酸乳糖和多聚唾液酸的水解活性分别为6'-唾液酸乳糖水解活性的87.50%和67.19%。BlSia42水解多聚唾液酸12 h后，唾液酸浓度为2.4 g/L，水解率为23.6%。【结论】唾液酸苷酶BlSia42优良的酶学特性使其在唾液酸及其衍生物的制备中具有潜在的应用价值。

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.