黄杆菌褐藻胶裂解酶的高效表达及其在褐藻寡糖制备中的应用

研究黄杆菌褐藻胶裂解酶基因(rFlAlyA)在枯草芽孢杆菌中的高效表达,高密度发酵48 h最高酶活力为2550 U/mL,蛋白质量浓度达4.5 mg/mL.经(NH4)2SO4沉淀和SP强阳离子交换柱纯化后得到电泳级纯酶,分子质量约为30 kDa.褐藻胶裂解酶rFlAlyA最适pH值为7.5,在pH 4.0～11.0范...

High Level Expression of Alginate Lyase from Flavobacterium sp.and Its Application in Preparation of Alginate Oligosaccharides

High-efficiency expression of the alginate lyase gene (rFlAlyA) from Flavobacterium sp. in Bacillus subtilis was studied. After high cell-density fermentation for 48 h, the enzymatic activity reached the highest level of 2 550 U/mL with a protein content of 4.5 mg/mL. The rFlAlyA, with a molecular mass of about 30 kDa, was purified to electrophoretic homogeneity after fractionation with (NH4)2SO4 and ion exchange chromatography on SP strong cation exchange column. Maximal enzymatic activity was observed at pH 7.5 and 50 ℃. The enzyme showed good stability at pH 4.0?11.0 and was thermostable up to 50 ℃, retaining over 80% of its activity. The substrate specificity indicated that rFlAlyA was a polymannuronate lyase with trisaccharide being the shortest-chain substrate. For alginate oligosaccharides production, sodium alginate was reacted with rFlAlyA under the optimal conditions for 4 h, giving a final concentration of reducing sugar of 33 mg/mL in the reaction mixture and a conversion rate was 70.1%. Electrospray ionization tandem mass spectrometry showed that the degree of polymerization (DP) of the hydrolysates was 1 to 8 and trisaccharide was the most abundant, which accounted for 34.0% of the total components when the DP was 3. The findings of this study indicated that the recombinant alginate lyase could be applied for the production of alginate oligosaccharides.