代谢物组学研究组知识库

顺式环氧琥珀酸水解酶（CESH）可以催化顺式环氧琥珀酸生成L(+)-酒石酸，但是该酶稳定性差，目前还只能采用整菌催化方式生产L(+)-酒石酸。固定化酶在提高酶的稳定性方面有其独特的优势，能避免生产周期长、生成的副产物影响目的产物分离纯化等缺点，因此可以对CESH进行固定化以提高其稳定性。

纤维素价格低廉，性质稳定，生物相容性好，能作为酶固定化的良好载体。碳水化合物结合模块（CBM）是存在于多种降解木质纤维素酶中的非催化模块，它能特异识别纤维素底物，可用于介导酶在纤维素基载体上的固定化。本研究选择来源不同的五个CBM分别融合在CESH的N端进行表达，采用微晶纤维素作为载体制备固定化酶。研究了原酶、游离融合酶和固定化融合酶的基本酶学性质，并验证了融合CBM及通过CBM固定化到纤维素后对酶的稳定性提升的效果。

通过酶活力测定表明，游离的和固定化的融合酶比原酶有更宽的适宜pH、适宜温度范围。游离形式、固定化形式的融合酶与原酶的动力学参数显著不同，其中经固定化的CBM30-CESH的催化效率k_cat/k_m为236.47 mM^-1·min^-1，是原酶（97.81 mM^-1·min^-1）的2.3倍。融合CBM后的游离酶稳定性稍有提高，但经纤维素固定化后酶的稳定性有不同程度的提高。其中固定化的CBM30-CESH的稳定性显著提高，在30℃下半衰期达210 min，是原酶半衰期（46.2 min）的4.5倍。其它固定化融合酶在30℃条件下的半衰期也延长到原酶的3-4倍。当温度升高至55℃时，固定化对酶稳定性的提高作用明显下降。

融合CBM拓宽了CESH的最适pH和最适温度范围，通过亲和固定化提高了酶的稳定性。而且不同来源的CBM对酶稳定性提高的程度不同，这与CBM本身性质有关，而与来源菌株的嗜热性质无关。融合CBM30的固定化酶表现出最高的催化效率和最长的半衰期，因此CBM30可以作为良好的融合标签对CESH进行融合表达和一步纯化固定制备固定化酶，提高酶的稳定性。

Cis-epoxysuccinic acid hydrolase (CESH) is an enzyme that catalyzes cis-epoxysuccinic acid to produce enantiomeric L(+)-tartaric acid. However, CESHs are unstable in their purified form. At present, the method of fermentation bacteria was commonly used to produce L(+)-tartaric acid. Taking into account that the immobilized enzyme has its unique advantages: increasing enzyme stability, avoiding the long production cycle, reducing the impact of the byproducts on the subsequent separation and purification of the target product. Therefore, to improve the enzyme stability, immobilizing CESH is necessary.

Carbohydrate, especially cellulose, is inexpensive, inert, biocompatible, thus it can serve as a good carrier of immobilization. Carbohydrate-binding modules (CBMs) are widespread non-catalytic components of various polysaccharide-degrading enzymes that can recognize various types of carbohydrate. In our study, five CBMs from different source were fused at the N-terminal of CESH. Then the free CBM-CESHs were immobilized on the microcrystalline cellulose (Avicel). We studied the enzymatic properties of the native CESH， free and immobilized CBM-CESHs. Besides, the effects of fusion and immobilization on enzymatic stability improving were verified.

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 2.3-fold catalytic efficiency（k_cat/k_m 236.47 mM^-1·min^-1） compared with the native CESH（97.81 mM^-1·min^-1）.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 increased the stability of the enzyme to different degrees. The immobilized CBM30-CESH showed the longest half-life at 30 ºC (210 min) which was 4.5-fold compared with that of CESH（46.2 min）.And the half-lives of other immobilized fusing enzymes were more than 3-4 times for the free CESH half-life at 30 ºC. 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. With temperature rose to 55 °C, the degree of enzyme stability increased by immobilization was gradually decreased.

Fusing CBM could broaden the pH and temperature adaptability of CESH, and affinity immobilization with the interaction between CBM and cellulose could improve the stability of CESH. Because of the various degrees of enzymatic stability improvement, the source of CBM from thermophilic bacterium or not appears to be unimportant to the stability of the immobilized chimeric proteins. The stabilization effects can be attributed mainly to the intrinsic nature of CBMs. Among the five CBM-fused enzymes, immobilized CBM30-CESH showed the highest catalytic efficiency and the longest half-life. Therefore, CBM30 could be a good candidate for one step purification-immobilization of CESH to improve its stability.