Wei Yuxia, Zhang Xian, Hu Mengkai, Shao Yu, Pan Shan, Fujita Morihisa, Rao Zhiming
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China.
Sheng Wu Gong Cheng Xue Bao. 2021 Dec 25;37(12):4303-4313. doi: 10.13345/j.cjb.210005.
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.
D-阿洛酮糖-3-表异构酶（DPEase）是将D-果糖异构化为D-阿洛酮糖的关键酶。为了提高其热稳定性，将短亲水性肽（SAP）融合到DPEase的N端。SDS-PAGE分析表明，在枯草芽孢杆菌中异源表达的DPEase折叠正确，蛋白大小为33 kDa。在40℃孵育48小时后，SAP1-DSDPEase的残余酶活性为58%。为了使重组枯草芽孢杆菌菌株可重复使用，用海藻酸钠（SA）和二氧化钛（TiO2）的复合载体固定细胞。结果表明，2%的SA、2%的CaCl2、0.03%的戊二醛溶液以及TiO2与SA的比例为1:4是固定化的最佳条件。在这些条件下，固定化细胞的活性最高可保留82%。与游离细胞相比，固定化细胞的最佳反应温度保持在80℃不变，但热稳定性提高。连续10个循环后，机械强度保持不变，同时可保留58%的酶活性，转化率达到28.8%。本研究展示了一种利用SAP提高重组酶热稳定性的简单方法。此外，在固定化过程中向SA中添加TiO2可提高机械强度并减少细胞泄漏。
