环氧交联剂和氨基载体固定化海洋假丝酵母脂肪酶*

游离酶经过固定化后,稳定性和环境耐受性得到提高,在食品、医药、化工、环境和皮革等领域可以很好的提高酶的利用率并降低生产成本,具有极大的应用潜力。新型交联剂在固定化酶工艺的应用极大推进了固定化酶研究的深入。借助新型交联剂聚乙二醇二缩水甘油醚(PEGDGE),利用氨基载体LX-1000HA固定化海洋假丝酵母脂肪酶,结合单因素和正交试验优化得到交联及固定化条件为:交联温度30℃,交联2h,交联剂浓度0.75%,pH7.0,加酶量800U,载体量0.5g,固定化2h,固定化温度45℃。根据上述最佳固定化工艺,制备得到固定化酶LX-1000HA-PEGDGE-CRL在最适条件下测得酶活达到160.81U/g,约为此前制备的固定化酶LX-1000HA-GA-CRL(由LX-1000HA和戊二醛交联脂肪酶得到)和LX-1000EA-PEGDGE-CRL(由短链氨基载体LX-1000EA和PEGDGE交联脂肪酶得到)酶活的2倍,发现固定化酶LX-1000HA-PEGDGE-CRL的最适反应温度相比于游离酶提高15℃;在70℃的环境中3h后酶活仍存留70%;循环使用6次后残留65%左右的酶活;酸碱耐受性和储存稳定性也表现良好,4℃保存30天后剩余约70%的初始酶活。同时,将制备的固定化酶LX-1000HA-PEGDGE-CRL与游离酶、固定化酶LX-1000HA-GA-CRL、固定化酶LX-1000EA-PEGDGE-CRL进行了比较,发现固定化酶LX-1000HA-PEGDGE-CRL在温度耐受性和重复使用性等方面具有更好的使用效果。

Immobilization of Marine Candida Lipase Using Novel Epoxy Cross-linker and Amino Carrier

After immobilization, the stability and environmental tolerance of the free enzyme were optimized, which can improve the utilization rate of enzymes and reduce the production costs in diverse fields such as food, medicine, chemical industry, environment and leather, and thus possesses great application potentials. The utilization of novel crosslinking agents in immobilized enzyme process has greatly advanced the study of immobilized enzymes.The optimized crosslinking and immobilization conditions were obtained by using the new crosslinking agent polyethylene glycol diglycidyl ether (PEGDGE), which was immobilized with the amino carrier LX-1000HA, and by using single factor and orthogonal experiment optimization: crosslinking temperature 30℃, crosslinking time 2h, crosslinking agent concentration 0.75%, pH 7.0, enzyme amount 800U, carrier volume 0.5g, immobilized time 2h, immobilized temperature 45℃. Using the optimized immobilization process, the immobilized enzyme LX-1000HA-PEGDGE-CRL was prepared to achieve 160.81U/g which was about twice of the activities of immobilized enzyme LX-1000HA-GA-CRL(obtained by LX-1000HA and glutaraldehyde crosslinked lipase) and LX-1000EA-PEGDGE-CRL(obtained by short-chain amino carrier LX-1000EA and PEGDGE crosslinked lipase). The optimal reaction temperature of immobilized enzyme LX-1000HA-PEGDGE-CRL was found to be 15℃ higher than that of free enzyme; the immobilized enzyme could still remain 70% of its original activity after incubation at 70℃ for 3h; the immobilized enzyme could remain about 65% of its original activity after recycling for 6 times; the immobilized enzyme also behaved good tolerance to acid and alkali condition and good storage stability, the immobilized enzyme could remain about 70% of the initial enzyme activity after storage at 4℃ for 30 days. After the comparation of immobilized enzyme LX-1000HA-PEGDGE-CRL with free enzyme, immobilized enzyme LX-1000HA-GA-CRL and immobilized enzyme LX-1000EA-PEGDGE-CRL, the immobilized enzyme LX-1000HA-PEGDGE-CRL behaved better application effect in thermo-tolerance and reusability.