摘要
研究了菌株RC-3产硫酸软骨素酶的最适发酵条件及酶的分离纯化,分别优化了碳源种类、碳源浓度、氮源种类、氮源浓度和NaCl浓度等条件,在最适发酵条件下对RC-3进行发酵培养,收集菌体进行细胞破碎制备粗酶液。粗酶液依次经过Q-Sepharose Fast Flow(QFF)阴离子交换层析与Sephacryl S-1000凝胶过滤层析和高效液相色谱(HPLC)进行逐级分离纯化,并利用聚丙烯酰胺凝胶电泳(PAGE)的方法验证纯化酶活力。结果表明,最优碳源为0.5%的岩藻多糖(Fucoidan)加乳糖、最优氮源为1.0%的牛肉膏、NaCl的最优浓度为0.3%,在上述条件下发酵48h,获得的菌体生物量最高,酶活力较优化前提高3倍,达1066.89 U/mL。粗酶经过QFF离子交换层析和Sephacryl S-1000葡聚糖凝胶纯化后获得相对较纯的酶,该酶经过HPLC检测表明主要有2个峰,并用聚丙烯酰胺凝胶电泳证实了此二峰均具有酶活力。
This paper studies the strain RC-3 of chondroitinase producing the optimal fermentation conditions and enzyme separation and purification were optimized carbon source,carbon source concentration,nitrogen source,nitrogen source concentration and NaCl concentration conditions.In the optimum fermentation conditions of RC-3 fermentation,collected thalli cell broken preparation of crude enzyme solution.Fast Flow Q-Sepharose（QFF） anion exchange chromatography and S-1000 Sephacryl gel filtration chromatography and HPLC were used for the separation and purification of the crude enzyme,and the activity of the purified enzyme was verified by polyacrylamide gel electrophoresis（PAGE）.Results show that optimal carbon source for 0.5% of fucoidan polysaccharide and lactose,and the best nitrogen source for the best concentration of 1.0% beef extract,NaCl is 0.3%,under the above conditions fermentation for 48 h,the mycelium biomass of the highest enzyme activity than that before optimization improve 3 times,up to 1066.89 U/mL.After purification of QFF ion exchange chromatography and S-1000 Sephacryl,the enzyme was purified to a relatively pure enzyme,and the enzyme was detected by HPLC,which showed that there were two peaks and two peaks were confirmed by polyacrylamide gel electrophoresis.
出处
《食品科技》
CAS
北大核心
2016年第4期7-12,共6页
Food Science and Technology
基金
山东省高等学校科技计划项目(J15LE13)
青岛农业大学高层次人才启动基金项目(631347)
