Shandong Provincial Key Laboratory of Energy Genetics, Key Laboratory of Biofuels, Qingdao Institute of BioEnergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189 Songling Road, Qingdao 266101, China.
Appl Biochem Biotechnol. 2012 Oct;168(3):708-17. doi: 10.1007/s12010-012-9811-8. Epub 2012 Jul 29.
Cis-epoxysuccinic acid hydrolase (CESH) is an enzyme that catalyzes cis-epoxysuccinic acid to produce enantiomeric L(+)-tartaric acid. The production of tartaric acid by using CESH would be valuable in the chemical industry because of its high yield and selectivity, but the low stability of CESH hampers its application. To improve the stability of CESH, we fused five different carbohydrate-binding modules (CBMs) to CESH and immobilized the chimeric enzymes on cellulose. The effects of the fusion and immobilization on the activity, kinetics, and stability of CESH were compared. Activity measurements demonstrated that the fusion with CBMs and the immobilization on cellulose increased the pH and temperature adaptability of CESH. The chimeric enzymes showed significantly different enzyme kinetics parameters, among which the immobilized CBM30-CESH exhibited twofold catalytic efficiency compared with the native CESH. The half-life measurements indicated that the stability of the enzyme in its free form was slightly increased by the fusion with CBMs, whereas the immobilization on cellulose significantly increased the stability of the enzyme. The immobilized CBM30-CESH showed the longest half-life, which is more than five times the free native CESH half-life at 30 °C. Therefore, most CBMs can improve enzymatic properties, and CBM30 is the best fusion partner for CESH to improve both its enzymatic efficiency and its stability.
顺式-环氧琥珀酸水解酶（CESH）是一种酶，能够催化顺式-环氧琥珀酸生成对映体 L(+)-酒石酸。由于产率和选择性高，使用 CESH 生产酒石酸在化学工业中具有重要价值，但 CESH 的低稳定性限制了其应用。为了提高 CESH 的稳定性，我们将五个不同的碳水化合物结合模块（CBMs）融合到 CESH 中，并将嵌合酶固定在纤维素上。比较了融合和固定化对 CESH 的活性、动力学和稳定性的影响。活性测量表明，CBM 融合和纤维素固定化提高了 CESH 的 pH 和温度适应性。嵌合酶显示出明显不同的酶动力学参数，其中固定化 CBM30-CESH 的催化效率比天然 CESH 提高了两倍。半衰期测量表明，CBM 融合略微提高了游离酶的稳定性，而纤维素固定化则显著提高了酶的稳定性。固定化 CBM30-CESH 的半衰期最长，在 30°C 时是游离天然 CESH 半衰期的五倍多。因此，大多数 CBM 可以改善酶的性质，而 CBM30 是与 CESH 融合以提高其酶效率和稳定性的最佳伴侣。
