菊糖蔗糖酶的性质鉴定及菊糖的酶法合成

Characterization of inulosucrase and the enzymatic synthesis of inulin

摘要:

菊糖作为益生元和膳食纤维，具有许多重要的生理功能，广泛应用于食品、医药等领域。微生物菊糖蔗糖酶可以以蔗糖为底物合成较植物菊糖具有更高分子量的菊糖。文中通过基因数据库筛选获得一段拟表达菊糖蔗糖酶的基因。通过N-端和C-端截断的方式，保留中间催化域，构建重组质粒。将重组质粒在大肠杆菌表达系统中表达，粗酶液经Ni2+亲和层析纯化，获得分子量约为65 kDa的重组酶。以蔗糖为唯一底物时，重组酶的最适pH和温度分别为5.5和45 ℃。金属离子在不同程度上抑制酶的活性。产物多糖分离纯化后，使用核磁共振鉴定产物多糖为β-(2,1)糖苷键连接的菊糖。最后对菊糖合成的条件进行优化，结果表明：以700 g/L的蔗糖为底物，加酶量4 U/mL时，7 h后菊糖产量达到最大，约为287 g/L，蔗糖到菊糖的转化率约为41%。

Abstract:

As a type of prebiotics and dietary fiber, inulin performs plenty of significant physiological functions and is applied in food and pharmaceutical fields. Inulosucrase from microorganisms can use sucrose as the substrate to synthesize inulin possessing higher molecular weight than that from plants. In this work, a hypothetical gene coding inulosucrase was selected from the GenBank database. The catalytic domain was remained by N- and C- truncation strategies, constructing the recombinant plasmid. The recombinant plasmid was expressed in E. coli expression system, and after purifying the crude enzyme by Ni2+ affinity chromatography, a recombinant enzyme with a molecular weight of approximately 65 kDa was obtained. The optimal pH and temperature of the recombinant enzyme were 5.5 and 45 °C, respectively, when sucrose was used as the sole substrate. The activity of this enzyme was inhibited by various metal ions at different degrees. After purifying the produced polysaccharide, nuclear magnetic resonance analysis was used to determine that the polysaccharide was inulin connected by β-(2,1) linkages. Finally, the conditions for the production of inulin were optimized. The results showed that the inulin production reached the maximum, approximately 287 g/L after 7 h, when sucrose concentration and enzyme dosage were 700 g/L and 4 U/mL, respectively. The conversion rate from sucrose to inulin was approximately 41%.