Production Process Optimization And Stability Study Of Recombinant Neorudin

A method of quantitative determination of recombinant neorudin(EH)in yeast fermentation supernatant was established by HPLC technology.Real-time monitoring the expression of EH in fermentation was realized,and the fermentation conditions of EH were optimized through the above method.Moreover,the ultrafiltration of fermentation supernatant was improved.As a result,the production efficiency of EH was increased with the increase of the EH expression level and the decrease of time and cost.The quality of EH produced by new process was in line with the formulated quality standard.In addition,the influencing factors and long-term stability of EH protein were studied.A method for quantitative determination of EH content in yeast fermentation supernatant by HPLC method was studied.EH has a strong specific absorption peak at 214 nm.There is a good linear relationship between the absorption peak area and the content of EH,r~2=0.9995,and the linear concentration range of EH is 0.012~4.8mg/mL.The analytical method has high accuracy and precision.The RSD value of the sample is 1.4227%and the recovery rate is 95%~98%.Because of high accuracy and reproducibility,the method can be used to determine the content of EH in the fermentation supernatant in real time,according to the specific absorption peak of EH.Therefore,the method is used for real-time quantitative monitoringEH in the fermentation process,so as to optimize the fermentation.The determination results of EH in fermentation supernatant showed that the expression level of EH was positively correlated with the methanol induction time in a certain time range.The fermentation conditions of EH were optimized by pH and methanol induction.After the optimization,the new fermentation is that the pH increase from3.0 to 5.85 after methanol induction,and the initial rate of methanol addition was3.6mL/h per liter of the initial fermentation liquid,and then 7.3 mL/h 4h later to maintain the dissolved oxygen level at 4-8%.The induction results showed that the expression of EH was positively correlated with the amount of methanol addition in a certain amount of methanol.At the later stage of fermentation,the content of EH in the fermentation supernatant was detected regularly by HPLC method.Once there was a downward trend or no obvious upward trend,the fermentation were immediately ended.Ultrafiltration membrane was replaced by hollow fiber columns in the ultrafiltration technology of EH.After centrifugation,the fermentation supernatant was filtered through a 50kD hollow fiber to remove the yeast fragments and macromolecular impurities and then was concentrated through a 3kD hollow fiber.The ultrafiltration time was shortened greatly compared with the ultrafiltration membrane with higher production efficiency and lower production cost.The result of quality measurement of EH produced with optimized fermentation showed that the new fermentation did not reduce the the quality of EH.The influence factors and long-term stability of EH protein were studied including impact of light,high temperature,high humidity,and 18 months long-term storage.The results showed that the solution of EH was sensitive to temperature and light,so the solution of EH should be preserved in lower temperature and light-free.The freeze-dried powder of EH was not sensitive to light and high humidity,but degraded under high temperature.In summary,the original solution of EH can be stored at-80℃and-20℃for 18 months,and the preparation of EH can be stored at-20℃and 4℃for 18 months.