器壁固定化脂肪酶Pseudomonas cepacia lipase的非水活性与稳定性

许多脂肪酶在有机体系中表现出催化作用，可用于绿色有机合成. 但其催化活性和稳定性明显低于水/油（有机相）界面上的表现. 为了提高脂肪酶在有机反应体系中的活性和稳定性，依据脂肪酶的界面活化机制，以水为酶蛋白构象优化剂、羧甲基纤维素为赋形剂，通过物理吸附的方式，将典型的假单胞菌脂肪酶（Pseudomonas cepacia lipase）固定在锥形瓶的内壁上，形成简易的生物反应器. 为方便检测器壁固定化酶促反应动力学，选择特征吸收为640 nm的生化指示剂2,6-二氯靛酚为反应底物,乙酸乙烯酯为酰化试剂,丙酮为溶剂. 光谱检测表明，催化反应0.5 h后，器壁固定化脂肪酶转化底物的能力是脂肪酶粉的6倍. 在每次催化5 h共10次的循环催化中，器壁固定化脂肪酶的催化活性平均每次仅降低3.2%，而酶粉降低11.8%. 结果表明,该器壁固定化脂肪酶的活性和稳定性相对于酶粉明显提高，这将为通过固定化有效提高脂肪酶的非水催化作用提供重要的参考.

Nonaqueous Activity and Stability of Wall-immobilized Pseudomonas Cepacia Lipase

Lipases usually catalyze reactions in organic phases and being used for green organic synthesis. However, the catalytic activity and stability of lipases are lower as compare to that in water/oil environment. To overcome this problem, immobilization on the inner wall of conical flask with carboxymethyl cellulose as recipient for water absorption is considered to process Pseudomonas cepacia lipase, a typical lipase, and form a simple bioreactor. For conveniently tracing the kinetics of reaction catalyzed by such surface-immobilized lipase, a biochemical indicator 2, 6-dichloroindophenol with 640 nm characteristic absorption can be employed as the substrate to react with acylation reagent vinyl acetate in acetone. The results of spectral analysis indicated that immobilized lipase transformed 6 folds as much substrate as native lipase was capable with in 0.5 hours. The deterioration of the activity was only 3.2%, much lower than 11.8% in average of native lipase during 5-hour of 10 catalytic recycles. Our data demonstrated that the wall-immobilized lipase improved the activity and stability over the native lipase.