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

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。

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 ℃ 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(TiO;). The results showed that 2% SA, 2% CaCl;, 0.03% glutaraldehyde solution and a ratio of TiO;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 ℃ 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 TiO;into SA during immobilization was demonstrated to increase the mechanical strength and reduce cell leakage.