Heterologous expression and enzymatic properties of active inclusion bodies of sucrose isomerase fused with coiled-coil domain
10.13200/j.cnki.cjb.003957
- VernacularTitle: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
- Author:
MA Zhiyu
- Publication Type:Journal Article
- Keywords:
Sucrose isomerase(SI);
Self-immobilized enzyme;
Coiled-coil domain;
Enzymatic properties
- From:
Chinese Journal of Biologicals
2023;36(7):793-799
- CountryChina
- Language:Chinese
-
Abstract:
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.
