Construction And Optimization Of Fermentation Conditions Of A Dual-promoter Subsystem For High Expression Of Nattokinase In Pichia Pastoris X33

Nattokinase(NK)is a protease that can dissolve blood clots and prevent the formation of blood clots in the body.In this study,Pichia pastoris X33 was used as the eukaryotic expression host,the plasmid pGAPZ?-A-NKt preserved in our laboratory was used as a template to construct a dual-promoter expression vector to increase the expression level and enzyme activity of nattokinase and prepare polyclonal antibodies,identify the properties and functions of the target protein.By using the response surface optimization method to optimize fermentation conditions on the basis of single factor,the protein expression and enzyme activity was further improved on the basis of recombinant strain fermentation.The main research results are as follows:1.The recombinant strains X33-FLD1,X33-GAP-GAP and X33-GAP-FLD1 were successfully constructed.Previous studies have shown that two kinds of promoters can coexist in a single expression vector without affecting the expression efficiency.It is predicted that the tandem promoter could enhance the expression of nattokinase in Pichia pastoris X33.The GAP promoter gene fragment was amplified by PCR using the plasmid pGAPZ?-A-NKT stored in our laboratory and the FLD1 promoter gene fragment synthesized by Jinkairui Biological Company.Based on the plasmid pGAPZ?-A-NKT constructed by predecessors in our laboratory,the recombinant vectors pGAPZ?-FLD1-NKt,pGAPZ?-GAP-GAP-NKt and pGAPZ?-GAP-FLD1-NKt were electrically transfected into Pichia pastoris X33 respectively.The nattokinase expressed by the original strain and the three recombinant strains were named RNKT1,RNKT2,RNKT3 and RNKT4,respectively.2.Test the properties of the target protein expressed by Pichia pastoris X33,and identify the protein in the fermentation broth of X33.WB was used to verify the properties of the homemade nattokinase antibody and expressed protein.The results showed that there was a specific band with the same size as the SDS-PAGE result,which was about 50 KD,indicating that the homemade antibody was effective and the expressed protein was nattokinase.The BCA protein detection method showed that the expression level of RNKT3 was significantly higher than that of other recombinant proteins,the expression level was 4.461 ± 0.254 ?g/?L,which was17.8% higher than the expression level of RNKT1.The enzyme activity of RNKT3 was 355.83 ± 0.564 FU/mL,which was 61.7% higher than that of RNKT1,according to the enzyme activity determination method of Japan Nattokinase Association.3.The single-factor experiment method was used to screen the factors that affect the fermentation expression of Pichia pastoris X33.The results showed that the three factors of glycerol concentration,methanol concentration and initial pH of the medium had a great influence on the fermentation expression.In view of this,we used the Design-expert software and the response surface method to analyze the optimal fermentation conditions.The results showed that the strain X33-GAP-GAP containing the pGAPZ?-GAP-GAP-NKt recombinant vector was expressed under the optimized fermentation conditions.After expression,the enzyme activity reached485.37 ± 8.19 FU/mL,which was 36.4% higher than that before optimization.The comprehensive results showed that the promoter series could significantly increase the expression level and enzyme activity of nattokinase.The optimization of fermentation conditions were optimized to further improve the enzyme activity of nattokinase,which laid a foundation for the application of nattokinase in large-scale industrial production in the future.