d-阿洛酮糖3-差向异构酶重组枯草芽孢杆菌构建及固定化应用

Construction and immobilization of recombinant Bacillus subtilis with d-allulose 3-epimerase

摘要:

d-阿洛酮糖3-差向异构酶 (d-allulose-3-epimerase) 是异构化d-果糖生成d-阿洛酮糖 (d-allulose) 的关键酶。为提高d-阿洛酮糖3-差向异构酶的热稳定性并获得可重复使用的d-阿洛酮糖3-差向异构酶重组枯草芽孢杆菌固定化细胞，N端融合双亲短肽，通过聚丙烯酰胺凝胶电泳 (SDS-PAGE) 分析，异源d-阿洛酮糖3-差向异构酶在枯草芽孢杆菌中正确折叠，蛋白大小为33 kDa。40 ℃孵育48 h，SAP1-DSDPEase残余酶活仍保持在58%。固定化细胞最优条件为海藻酸钠浓度2%、二氧化钛添加量1︰4 (二氧化钛︰海藻酸钠)、氯化钙溶液浓度2%、戊二醛0.02%作为交联剂。该条件下固定化细胞酶活回收率高达82%，固定化细胞与游离细胞相比，最适反应温度不变均为80 ℃，热稳定性提高，连续10次操作使用，酶活回收率仍保留58%，机械强度仍保持100%，转化率仍保持在28.8%，残余酶活保持在70.5%。在海藻酸钠溶液中加入二氧化钛可减少固定化细胞的细胞泄露，增大了机械强度。

Abstract:

d-allulose-3-epimerase (DPEase) is the key enzyme for isomerization of d-fructose to d-allulose. In order to improve its thermal stability, short amphiphilic peptides (SAP) were fused to the N-terminal of DPEase. SDS-PAGE analysis showed that the heterologously expressed DPEase folded correctly in Bacillus subtilis, and the protein size was 33 kDa. After incubation at 40 °C for 48 h, the residual enzyme activity of SAP1-DSDPEase was 58%. To make the recombinant B. subtilis strain reusable, cells were immobilized with a composite carrier of sodium alginate (SA) and titanium dioxide (TiO2). The results showed that 2% SA, 2% CaCl2, 0.03% glutaraldehyde solution and a ratio of TiO2 to SA of 1:4 were optimal for immobilization. Under these conditions, up to 82% of the activity of immobilized cells could be retained. Compared with free cells, the optimal reaction temperature of immobilized cells remained unchanged at 80 °C but the thermal stability improved. After 10 consecutive cycles, the mechanical strength remained unchanged, while 58% of the enzyme activity could be retained, with a conversion rate of 28.8% achieved. This study demonstrated a simple approach for using SAPs to improve the thermal stability of recombinant enzymes. Moreover, addition of TiO2 into SA during immobilization was demonstrated to increase the mechanical strength and reduce cell leakage.