Engineering Of Promoter In Bacillus Subtilis And Its Application In Nattokinase Expression

Bacillus subtilis is a kind of gram positive bacteria which has been identified as GRAS?Generally regarded as safe?by American FDA.It has the advantage of good protein secretion ability,and the fermentation conditions are simple,so it is widely used in the production of industrial and food enzyme preparations.Although on many industrial,food enzymes have been implemented recombinant protein expression in B.subtilis,the existing system,which has a heterologous protein production capacity,can not meet the needs of large-scale production.The promoter element in the B.subtilis expression plays a key role in the system,most of the B.subtilis promoters exist the problem of low activity.In this paper,we studied B.subtilis strong promoter activity,molecular modification,fermentation level in the production of a variety of recombinant proteins in the system,and optimized the fermentation medium and the ratio of key components,to achieve an efficient secretory expression system of recombinant nattokinase.The specific contents of this study are as follows:?1?Construction of recombinant expression vectors of Bacillus subtilis.First,the expression vector was constructed with gfp as the reporter gene,and the expression activity of P_HpaII,P_srfA,P_p43,P_veg and P_sigw were compared and analyzed.The results showed that there were differences in the activity and expression level of the promoters,among which the P_srfA activity was the highest.?2?The ideal design for the strong promoter P_srfA.?a?The upstream promoter sequnences were deleted,the promoter activity increased slightly;?b?The core sequence of high activity promoter is mutated into a conserved one,among which the mutated promoters,P10 is the highest,compared with the original promoter activity increased 150%;?c?The ribosome binding site RBS of the P10 promoter has been designed with different initial translation efficiency,but promoter activity decreased.?d?The expression of the constructed expression system was successfully used in the expression of aminopeptidase,and the enzyme activity of the promoter was increased by 360%.?3?Construction of recombinant nattokinase expression system of Bacillus subtilis.The constructed expression system was used for the expression of nattokinase.At the same time,through the selection of signal peptide,the modification of the promoter,the selection of the host further improved the efficiency of the expression of nattokinase,the level of enzyme production in shake flask fermentation reached 290 FU·mL-1.?4?Establishment of amplification culture process of Nattokinase 5L fermentor.This study takes NK as the nattokinase production from the medium components?nitrogen source,glucose concentration,the ratio of carbon and nitrogen?and culture conditions?temperature,inoculation amount?of recombinant bacteria fermentation to optimize,the optimum fermentation conditions: soybean peptone was the best nitrogen source;glucose concentration was 10 g·L-1;the best ratio of carbon and nitrogen was 1:4;the optimum culture temperature was 37?;the best inoculation amount was 4%.The fermentation conditions were 5 L fermentation experiment,the biomass concentration was OD₆₀₀=41.36,the expression of recombinant nattokinase was 375.0 FU· mL-1.