HPLC法定量测定酵母发酵上清液中重组新蛭素的含量

摘要

目的 建立HPLC技术定量测定发酵上清液中重组新蛭素(EH)含量的方法,用于监测发酵过程中EH的表达量变化与诱导时间的关系,使EH产量最大化.方法 利用HPLC技术建立EH的检测方法,通过对该方法的灵敏度、精密度和加标回收率实验的考察,确立该方法的可行性;利用该方法对EH样品进行稳定性考察,并对酵母发酵上清液进行跟踪检测.实现了对发酵过程中发酵上清液中EH的实时监测.结果 EH在214nm处有较强的特异性吸收峰,其吸收峰面积与含量存在很好的线性关系,r2=0.9995,EH线性浓度范围在0.012 ～ 4.8 mg/ml.该测定方法具有很好的精密度,样品多次重复测定的RSD值为1.4227％;加标回收率在95％ ～ 98％.应用该方法对EH样品的稳定性测定结果显示,样品4℃保存稳定性较好,24 h内样品主峰面积百分比＞95％,在20℃条件下,8h内样品稳定性良好,主峰面积百分比＞95％.该方法准确度高、重复性好,进行测定时,可根据EH的特异吸收峰,对发酵过程中发酵上清液中EH含量进行实时定量测定.测定结果显示,在一定时间范围内,EH的表达量与诱导时间成正相关.结论 本研究建立了HPLC技术检测EH含量的方法,该方法可用于发酵过程中EH的实时定量监测,使EH的产量达到最大值,为EH的临床样品制备提供有力支持.%Objective To develop a practical method for the determination of recombinant neorudin (referred to as the EH) in yeast fermentation supematants by HPLC,in order to monitor of the relationship between the expression of recombinant EH and induction time during the fermentation process to optimize the induction time for maximizing the yield of EH.Method The relationship between the EH and ultraviolet absorption was detected by HPLC to establish a method to determine the quantity of EH protein.The feasibility of the method was further validated with regard to the sensitivity,precision and recovery rate.The stability of EH and the trace EH in yeast fermentation supernatants were examined using the method.Results A strong absorption peak at 214 nm was found for EH,having high sensitivity with RSD value of 1.4227％.Good linearity in the EH concentration range of 0.012-4.8 mg/ml with r2 =0.9995 was observed.Standard addition recovery rate was between 95％ and 98％.The result of the EH stability showed that the sample had a good preservation stability,with main peak area percentage being over 95％ at 4 ℃ for 24 h or at 20 ℃ for 8 h.The expression of EH in yeast fermentation supematants was related to the induction time of methanol.Conclusion The method for detecting the contents of EH by HPLC is established.Real-time monitoring of EH in yeast fermentation supematants to get the maximum production of EH will be achieved.