5L发酵罐发酵壳聚糖酶的条件研究

摘要

从实验室产壳聚糖酶菌株出发,研究壳聚糖酶的发酵培养基配方及发酵条件.结果表明,最佳的碳源是壳聚糖10 g/L、葡萄糖6g/L,最佳的氮源是硫酸铵20g/L,最佳初始pH为7.0.摇瓶最佳培养条件是温度32℃,转速150r/min,装液量300/500mL.5L发酵罐培养结果表明,在搅拌速度为100r/min、通风比为0.75条件下,培养66h,可以得到酶活12.5 U/mL的酶液.酶液最佳的反应条件是pH 5.6、温度50℃,酶液长期存储需要采用-20℃冷冻,3天内短期存储可以4℃冷藏,酶活可以保留90％以上.壳聚糖酶的动力学分析表明,反应符合米氏方程,米氏常数Km为0.247g/L,最大反应速率Vm为10.65 μmol/min.%The research on optimal fermentation mediums and conditions of a chitosanase producing bacterium was studied.The results showed that the best carbon source were powder chitosan 10 g/L,glucose 6 g/L,and the best nitrogen source was (NH4) 2SO420g/L,and the optimal initial pH was 7.0.The optimal conditions of culture shaking were at 32℃,150r/min shaking speed and shaking 300mL medium in 500mL flask.The optimal conditions of 5L fermenter were:fermenter agitation 100 r/min and ventilation amount 0.751/(1 · min).Under the optimized conditions,the chitosanase activity could reach to 12.5U/mL after incubated for 66 hours.The optimum pH and temperature of the chitosanase were 5.6 and 50 ℃.The chitosanase can maintain 90％ activity within 30 days under-20 ℃,or within 3 days of 4℃ cold storage.The Michaelis-Menten parameters (Km),maximum velocity (Vmax) of the enzyme were 0.247g/L,10.65 μmol/min at 50 ℃ and pH5.6.