Fermentation Optimization And Fermentation Kinetics Of Nattokinase Produced By Pichia Pastoris LNF013

Nattokinase is a serine protease with excellent thrombolytic activity and is widely used in the treatment of cardiovascular and cerebrovascular diseases and daily health care.Microbial fermentation is the main method for the industrial production of nattokinase.With the development of genetic engineering technology,nattokinase can be expressed in engineering bacteria.However,the currently developed prokaryotic nattokinase-producing bacteria ferment and produce nattokinase with many heteroproteins,and the product is difficult to separate and purify.The eukaryotic expression system has high yield and less miscellaneous protein,but there are disadvantages such as poor fermentation conditions,long fermentation cycle,and high fermentation cost.In fact,there are a lot of problems to be solved about nattokinase from microbial fermentation production to separation and purification applications.This paper mainly carried out research on the fermentation production of nattokinase with Pichia pastoris LNF013 as the strain and the industrial grade medium as the raw material,in order to reduce the fermentation cost,shorten the fermentation time and increase the production of nattokinase.In the shake flask stage,various methods such as single factor optimization and response surface optimization were used to obtain the optimal medium formula and some fermentation parameters;the fermentation process was further optimized through the 10 L fermenter,and the kinetic model of each stage of the 10 L fermenter was established,and A 100 L fermenter was used to verify the optimization results;the fermentation product was separated and purified to study the enzymatic properties of nattokinase.The main research content and results are as follows:The composition of the basal medium for nattokinase fermentation production was established by single-factor analysis in the shake flask stage:glycerol was used as the initial carbon source,and peptone and yeast extract powder were used as the nitrogen source（the ratio of the two was 1:1）.Through single factor experiment and response surface optimization analysis test,the optimal inorganic salt formula is determined.The optimum medium formula（g/L）and fermentation process parameters determined by shake flask and 10L fermenter were:glycerin 40.00,yeast extract powder 20.00,peptone 20.00,Mg SO₄.7H₂O 20.00,K₂SO₄ 9.00,Ca SO₄0.84;（0～24h）temperature28°C,transition period（24～28h）temperature 24°C,fermentation late stage（28～96h）temperature 24°C;feeding rate of glycerin in the early stage of fermentation is 5 g/L,glycerol 8 g/L,methanol 2 g/L in the transition period,the feeding rate of methanol in the later stage of fermentation was 4 g/L;the inoculum size was 1.0%,the p H was 6～7during the transition period and later stage of fermentation,the aeration rate was 3L/min,and the stirring speed was 180r/min.Under this condition,the activity of nattokinase was 357.69 Fu/m L,which was 41.46%higher than before optimization.On the basis of obtaining the optimal fermentation parameters,a 10 L fermenter was used for further exploration,and the fermentation time was further shortened to84h.Using 100L fermenter to scale up and verify the optimized scheme,and properly adjust the rotation speed and ventilation rate,the activity of nattokinase can reach362.31 Fu/m L,which is equivalent to the fermentation result of 10 L fermenter.With the help of Matlab software and the fermentation data of 10 L fermenter,the mathematical models of glycerol batch fermentation stage,glycerol fed batch fermentation stage and methanol fed batch fermentation stage were established.The mathematical models of each stage can simulate the fermentation process well.Further scale-up test to produce nattokinase has certain guiding significance.Through centrifugation,ammonium sulfate precipitation,Sephadex-75 gel chromatography,freeze-drying and other methods,the freeze-dried powder of nattokinase was obtained in the form of white powder at room temperature,and the recovery rate was 52.84%.The enzymatic properties and thrombolytic performance of the nattokinase were studied.The results showed that the optimal storage temperature of nattokinase was-20°C or 4°C,the Km value of TAME as substrate was 35.73mmol/L,and the enzyme activity was retained in different degrees in a wide range of temperature and p H,and had good thrombolytic activity,metal ion and chemical reagent verification experiments showed that the nattokinase is a serine protease.In addition,compared with the freeze-dried nattokinase powder obtained by traditional fermentation methods on the market,the freeze-dried nattokinase powder fermented in this experiment has higher thrombolytic activity per unit mass.The above research work shows that the activity and thrombolytic performance of the target product,nattokinase,can be improved by optimizing the fermentation of Pichia pastoris LNF013.The experimental results obtained are of great significance for further in-depth research on the fermentation mechanism of nattokinase and industrial fermentation production.More meaningfully,the successful optimization of the expression of nattokinase in Pichia pastoris laid the foundation for the further development and utilization of nattokinase.