融合卷曲螺旋结构域的蔗糖异构酶活性包涵体异源表达及其酶学性质

Heterologous expression and enzymatic properties of active inclusion bodies of sucrose isomerase fused with coiled-coil domain

目的 融合表达源自细胞表面蛋白tetrabrachion的四聚化卷曲螺旋结构域(tetrameric coiled-coil domain of the cell-surface protein tetrabrachion,TdoT)的蔗糖异构酶(sucrose isomerase,SI),并研究其重组酶的酶学性质。方法 分别将TdoT与SI的N/C-端融合,构建重组表达载体pET-24a-TdoT-SI和pET-24b-SI-TdoT,转化E.coli BL21(DE3),进行诱导表达,纯化重组酶后进行酶学性质表征和产物特异性研究。结果 TdoT-SI和SI-TdoT表达为具有催化活性的包涵体,而未融合TdoT的SI包涵体则无催化活性。酶学性质表征结果显示,TdoT-SI和SI-TdoT活性包涵体的最适反应温度均为40℃,最适反应pH分别为5.5和5.0。TdoT-SI活性包涵体的动力学常数K_(m)为(103.9±9.5)mmol/L,k_(cat)/K_(m)为(0.06±0.002)L/(mmol·s);SI-TdoT活性包涵体的动力学常数K_(m)为(54.4±6.6)mmol/L,k_(cat)/K_(m)为(0.03±0.002)L/(mmol·s)。产物特异性分析结果显示,随着反应温度升高,产物中异麦芽酮糖含量并未出现较大变化,而产物中海藻酮糖含量随转化反应温度升高而降低,同时单糖的含量随转化反应温度升高而增加。结论 通过融合表达技术成功制备出融合卷曲螺旋结构域的SI活性包涵体,其作为一种新型自固定化酶,具有同步实现表达和固定化的优势,为实现重组SI的规模化制备和高效利用提供了新策略。

Objective To express the sucrose isomerase(SI) fused with the tetrameric coiled-coil domain of the cell surface protein tetrabrachion(TdoT),and study the enzymatic properties of the recombinant enzymes.Methods The gene of SI fused with TdoT at the N/C terminus was cloned into the expression vectors respectively to construct the recombinant expression vectors pET-24a-TdoT-SI and pET-24b-SI-TdoT,which were transformed into E.coli BL21(DE3) and induced to express recombinant enzymes.The enzymatic properties and product specificity of the purified recombinant enzymes were studied.Results TdoT-SI and SI-TdoT were expressed as inclusion bodies with catalytic activity,while SI inclusion bodies without TdoT showed no catalytic activity.The results of enzymatic property analysis showed that the optimum reaction temperature for TdoT-SI and SI-TdoT active inclusion bodies was 40 ℃,and the optimum reaction pH was 5.5 and 5.0,respectively.The K_(m) of TdoT-SI active inclusion bodies was(103.9±9.5) mmol/L and the k_(cat)/K_(m) was(0.06±0.002) L/(mmol·s),while the K_(m) of SI-TdoT active inclusion bodies was(54.4±6.6) mmol/L and the k_(cat)/K_(m) was(0.03±0.002) L/(mmol·s).The results of product specificity analysis exhibited that the proportion of isomaltulose in the product did not change significantly,while the proportion of trehalose decreased,and the proportion of monosaccharides increased with increasing reaction temperature.Conclusion The active inclusion bodies of SI fused with coiled-coil domain were successfully prepared by fusion expression technology.As a novel self-immobilized enzyme,it has the advantage of simultaneous expression and immobilization,which provides a new strategy for large-scale preparation and efficient utilization of recombinant SI.