具有极高热稳定性的β-（2，6）果聚糖蔗糖酶及其应用

An Extreme Thermostable Levansucrase and Its Application

摘要:

挖掘具有高热稳定性的β-（2，6）果聚糖蔗糖酶（levansucrase，LS），并应用果聚糖蔗糖酶进行β-（2，6）果聚糖的高效合成。以分子动力学模拟的方式筛选出具有潜在高热稳定性的β-（2，6）果聚糖蔗糖酶。将目的酶在大肠杆菌Escherichia coli BL21（DE3）中进行重组及诱导表达，并通过镍柱亲和层析进行纯化。在不同温度下进行保温实验以验证LS的热稳定性，并通过条件优化获得产物的高转化率。Cedi-LS在65 ℃时显示出最高活性，远高于已鉴定的其他LS。同时，在45 ℃下保温72 h，重组酶Cedi-LS能够保留90%以上的相对活性；在55 ℃下保温60 h，其保留活性仍可达到初始活性的60%以上，表现出优异的热稳定性。在反应过程中，Cedi-LS可同时生成低聚糖、低相对分子质量的β-（2，6）果聚糖和高相对分子质量的β-（2，6）果聚糖。当温度从65 ℃降至35 ℃时，Cedi-LS倾向于产生HMW-levan，其相对分子质量可达8.4×10⁶。在pH 5.5和35 ℃的条件下，以质量分数30%的蔗糖为底物，加酶量定20 μg/mL，蔗糖转化为β-（2，6）果聚糖的平衡转化率为41.4%。作者鉴定了一种具有高热稳定性的LS，并且这种LS能够高效生产一系列具有不同相对分子质量的果聚糖。

Abstract:

β-(2,6) levansucrase (LS) with high thermostability was identified and the efficient synthesis of levan by levansucrase was studied. High-temperature molecular dynamics simulation was used to screen the LS with potential high thermostability. The target enzyme was heterogeneously expressed in Escherichia coli BL21(DE3) and purified through one-step Ni²⁺-affinity purification. The thermostability of LS was determined by incubating the enzyme at different temperatures, and the highest conversion was obtained through the optimization of conditions. The Cedi-LS showed the highest activity at 65 ℃, which was much higher than the other identified LSs. Meanwhile, Cedi-LS remained more than 90% active when incubated at 45 ℃ for 72 h, and it also remained more than 60% of its initial activity at 55 ℃ for 60 h, exhibiting excellent thermostability. The Cedi-LS produced oligosaccharides, low molecular weight (LMW) β-(2,6) levan, and high molecular weight (HMW) β-(2,6) levan simultaneously during reaction. When the temperature decreased from 65 to 35 ℃, Cedi-LS tended to produce HMW-levan with a Mw of 8.4×106 Da. At pH 5.5 and 35 ℃, the equilibrium conversion ratio from sucrose to levan was 41.4% with 30% sucrose and 20 μg/mL enzyme. In summary, an extreme thermostable LS was screened which could efficiently produce levan with different molecular weights.