磁性壳聚糖微球固定化（S）-立体专一酰胺酶的研究

发布时间：2018-06-19 06:46

  本文选题：磁性壳聚糖微球 + 固定化酶　； 参考：《河北大学》2014年硕士论文

【摘要】：哌嗪-2-羧酸作为非蛋白组成成分的氨基酸，是重要的手性药物中间体，其不同的单一对映体（S型和R型）衍生物具有不同的药理活性。本实验室筛选得到的副球菌(Paracoccous sp.) W10173产生的(S)-酰胺酶能够立体选择性水解(S)-哌嗪-2-甲酰胺生成重要医药中间体(S)-哌嗪-2-羧酸的单一对映体。由于固定化酶与游离酶相比具有便于回收、稳定性高、操作连续可控、工艺简便等一系列的优点，故对(S)-酰胺酶进行固定化研究对其应用于工业生产有重要意义。 本研究首先对实验室保藏的副球菌W10173经超声波破壁和分级盐析得到实验用粗酶。超声波破壁条件为功率400W，工作时间3S，间歇时间6S，循环240次；并确定分级盐析的最佳硫酸铵饱和度为20%-50%，且重复三次。 以化学共沉淀法制备的Fe3O4磁性粒子为磁核，液体石蜡为分散介质，Span-80为乳化剂，，戊二醛为交联剂，采用反相悬浮交联法合成磁性壳聚糖微球。以此为载体，通过吸附-交联联合固定化方法制备固定化(S)-酰胺酶，并探讨了酶给予量、吸附时间、交联时间、戊二醛浓度等因素对固定化效果的影响。最终确定(S)-酰胺酶的最佳固定条件为：10mL0.95mg/mL粗酶液/50mg载体吸附2h、戊二醛浓度1%交联4.5h。 对固定化(S)-酰胺酶酶学性质进行研究结果表明，固定化(S)-酰胺酶的最佳温度为32℃；最佳pH为11，比游离酶最佳pH向碱性移动2个单位。同时还发现该固定化酶具有良好的热稳定性，60℃处理4h后仍能保留约88%活性。获得的固定化(S)-酰胺酶的活力为2.3U/mg载体，酶活回收率为36.9%。固定化酶连续反复使用10批次后，酶活仍保留71%以上，具良好的操作稳定性。(S)-酰胺酶被固定化后，与游离酶相比，其贮存稳定性也有所提高。
[Abstract]:Piperazine-2-carboxylic acid is an important chiral drug intermediate as an amino acid of non-protein component. Its different single enantiomers S-type and R type) derivatives have different pharmacological activities. Paracoccous sp.) W10173 can stereoselective hydrolyze the single enantiomer of the important pharmaceutical intermediates, namely, Schi-piperazine-2-carboxylic acid. The monozyme can be stereoselective hydrolyzed by the thiosamine-piperazine-2-formamide. Because the immobilized enzyme has a series of advantages such as easy recovery, high stability, continuous and controllable operation, simple process and so on, it is of great significance for the application of immobilized enzyme to industrial production. In this study, the experimental crude enzymes were obtained by ultrasonic wave breaking and salting out. The conditions of ultrasonic wall breaking are power 400W, working time 3s, intermittent time 6S, cycle 240, and the optimum saturation of ammonium sulfate for grading salting out is determined to be 20 ~ 50 and repeated three times. Magnetic chitosan microspheres were synthesized by reverse phase suspension crosslinking method using Fe _ 3O _ 4 magnetic particles prepared by chemical coprecipitation as magnetic nucleus, liquid paraffin as dispersing medium Span-80 as emulsifier and glutaraldehyde as crosslinking agent. In this paper, the immobilized Scu-actamases were prepared by the method of adsorption-crosslinking and immobilization, and the effects of enzyme dosage, adsorption time, crosslinking time and glutaraldehyde concentration on the immobilization effect were discussed. The optimal immobilization conditions were determined as follows: 1 10mL0.95mg / mL crude enzyme solution / 50mg carrier adsorbed for 2 h, glutaraldehyde 1% crosslinked for 4.5h. The results showed that the optimum temperature and pH were 32 鈩

本文编号：2038978