Knockout Of Polyglutamate Gene From Bacillus Natto And Separation And Purification Of Nattokinase

Nattokinase is a thrombolytic enzyme produced by Bacillus natto.Compared with thrombolytic drugs on the market,it has fewer side effects and is not harmful to the human body.The production efficiency of nattokinase is low,mainly because the polyglutamic acid(γ-PGA)produced during liquid fermentation makes the fermentation broth viscous,which makes the separation and purification of nattokinase difficult.Although there have been many studies on the separation and purification of nattokinase,these methods are often costly and the separation steps are cumbersome,which limits their large-scale production.Therefore,the innovative idea of knocking out the viscous genes of Bacillus natto from the genetic level is proposed,which reduces the viscous substances produced during the fermentation process,thereby reducing the viscosity of the fermentation broth,reducing the difficulty of nattokinase separation and purification,and improving The separation and purification efficiency of nattokinase.In this paper,using pCBs as a vector,using the method of homologous recombination and double exchange,knock out the key gene pgsB for the synthesis of γ-PGA in Bacillus natto,reduce the content of γ-PGA in the fermentation broth and the viscosity of the fermentation broth,and improve the subsequent nattokinase Separation and purification efficiency.The results showed that the pgsB gene-deficient strain had a significant decrease in the yield of γ-PGA in the fermentation broth compared with the wild-type strain.Theγ-PGA content decreased by 57.9% and the viscosity decreased by 15% at 24 hours of fermentation.There was no significant difference in cell density,but the enzyme activity was increased by 15% compared to the wild-type strain.It shows that the deletion of pgsB gene can significantly reduce the viscosity of the fermentation broth and has no obvious effect on the growth of the bacteria.In the process of precipitating nattokinase with ammonium sulfate,the recovery rate of nattokinase of the gene-deficient strain was increased by 18.14% compared with the wild type.In the process of purifying nattokinase by ultrafiltration,the recovery rate of nattokinase from the gene-deleted strain was 19.2%higher than that of the wild type.Therefore,after knocking out the pgsB gene,the separation and purification efficiency of nattokinase has been improved,which provides a new method for the industrial production of nattokinase.In addition,the flocculation method is used to pretreat the fermentation broth of Bacillus natto and optimize the flocculation conditions.In the process of separation and purification of nattokinase,the fermentation broth is often processed by centrifugation to obtain crude nattokinase enzyme solution.However,centrifugation is a method with high energy consumption,and has a small processing capacity,which requires high instrumentation,and is not suitable for industrialized mass production.Compared with centrifugation,the flocculation method adopted in this paper has lower energy consumption,relatively simple operation,large processing volume,low instrument requirements,and also reduces the cost required for production.It is a kind of pretreatment suitable for industrial production.method.In this paper,the recovery rate,filtration flux,light transmittance and flocculation rate of nattokinase under different conditions were first investigated with different single-factor variables.Finally,orthogonal analysis was carried out.The study concluded that the best flocculation conditions were: Sugar 0.5 g/L,coagulant trehalose 0.1g/L,pH 6.0,dilution factor 1.0,flocculation temperature between 20-40 ℃,the recovery rate of enzyme activity after flocculating the fermentation broth under this condition It is71.73±1.41 %,the recovery rate of enzyme activity is between 5000 g and 6000 g,but the processing cost is greatly reduced.Finally,due to the short storage time of nattokinase,and the commonly used method of lyophilization to extend the storage period of nattokinase,the optimization of the lyophilized protective agent was carried out.Single-factor experiments with different types and concentrations of protective agents were carried out to investigate the freeze-drying effects.The main indicator is the retention rate of enzyme activity.Then,the orthogonal experiment was conducted according to the single-factor experiment effect,and the ratio of the freeze-dried protective agent with the highest retention rate of nattokinase enzyme activity was selected: skimmed milk powder 5%,sucrose 15%,lactose 5% and ascorbic acid 1.0%,natto The highest kinase recovery rate reached 96.08±1.54%.