高活力5′-磷酸二酯酶制备及水解RNA的研究

目的:获得高活力5′-磷酸二酯酶液,提高核酸RNA酶解效率。方法:采用超滤和盐析技术对从麦芽根浸提液中纯化5′-磷酸二酯酶工艺进行研究,采用单因子试验法优化酶解工艺条件。结果:浸提液依次经过5万Da超滤膜浓缩、40%饱和度硫酸铵盐析、5万Da超滤膜脱盐后,酶活力可达1 500U/ml;第1次超滤膜透过液可作为浸提液循环使用,酶活力是水浸提的1.15倍;第2次超滤膜透过液浓缩5倍后,可回收56.46%硫酸铵,浓缩母液可按1∶2比例循环使用;在底物浓度5.8%、酶用量8%、反应时间2h条件下,RNA酶解率可达95%。结论:初步建立了适合工业化规模的核苷酸生产新工艺。

Studies on the Preparation of High Activity 5'-phosphodiesterase and Its Appplication in the RNA Digestion

Objective: Obtained the high activity 5' - phosphodiesterase liquid, and raised the enzymatic hydrolysis yield of RNA. Method: The purification technology and enzyme hydrolysis condition were studied by ultrafiltration, salting out technique and individual factor experiment. Result: The best technology was concentrating by 50 000 Da ultrafiltration membrane, salting out with 40% saturation (NH_4)_2SO_4 ,and desalination by 50 000 Da ultrafiltration membrane in turn, so that the liquid of 1500 U/ml activity could be obtained;the filtrate of first ultrafiltration could be reused as new extract liquid, and its enzyme activity was 1. 15 times than the water extract; the filtrate of second ultrafiltration was concentrated to l/5 volume, then 56.46% (NH_4)_2SO_4 could be harvest, and the mother liquor could be reused in the ratio of 1:2; under the condition of 5.8% RNA,8% enzyme concention,and 2 hours reaction time, the 95% enzymatic hydrolysis yield of RNA could be reached. Conclusion: The technology of this paper could be used in nucleotide manufacture on industry scale.