Optimization Of Fermentation Conditions Of Bacillus Subtilis Expression System And Related Modification

Bacillus subtilis is an important producer of industrial enzymes due to its huge capacity to secrete proteins directly into the growth medium.A great number of foreign protein genes from different organisms have been cloned and expressed in B.subtilis,but the low yield of the secreted proteins has not been solved.To enable B.subtilis to be an efficient secretion host for heterologous proteins,here several strategies,i.e.directed evolution,optimization of fermentation condition and chassis cell transformation,were applied to improve the production of heterologous proteins.Two kinds of protein a-amylases(AmyL and AmyS)and D-psicose3-epimerase,encoding by amyl,amys and rdpe,are from Bacillus licheniformis,Bacillus stearothermophilus and Ruminococcus sp.respectively.The a-amylases(AmyL and AmyS)and RDPE were successfully expressed in B.subtilis.Furthermore,directed evolution was performed on AmyL.Especially after H133YA209V double mutation,the effect is obvious that its resistance to acid and heat is improved.The optimum pH value was decreased to 6.0～6.5,and the relative enzyme activity reached nearly 80%at pH 5,and the optimum temperature increased to 100℃,and the residual enzyme activity reached 93%when it was treated in 90℃ by 2 h.Even it was treated in 100℃ by 1h,the a-amylases still shown activity(6%).The growth and fermentation environment of microorganism are important influence factors of its expression and sercetion of target protein.In this study,through fermentation optimization of the a-amylase in shaking flask,we obtained that the optimum carbon source was starch,the optimum nitrogen source was peptone,the optimal temperature was 37 degrees and optimum initial pH value is 7.0.The highest enzyme activity reached 571 U/mL by Shake flask.Through optimization of alpha amylase in fermentation tank,it is concluded that the optimum fermentation conditions for dissolved oxygen was 30%～40%,temperature was 37℃ and pH maintained 7.0.Under the most suitable fermentation conditions,the strain 1A751L and 1A751S were fermented,and the highest activity of a-amylase was reached at 72 h,respectively,810 U/mL and 2812 U/mL.And it were higher 40%and 100.8%,respectively,compared with the shake flask.Rare sugars have a wide range of functions and applications.Because they have many advantages,such as low heat,low absorption.In the food,health,medicine and other fields they have an important functional role.It is feasible that using DTEase family enzymes to produce high concentration of D-psicose.By optimizing the fermentation conditions,we find a kind of sub-type fermentation strategy based on the regulation of pH value in this study.The high level expression and secretion of RDPE in Bacillus subtilis expression system was achieved by the method that cell growth and protein expression were separated.In a 7.5L fermentor,and the protein yield reached 2.2 g/L and the activity reached 74 U/mL in the fermentation broth after 72 h.This provided an important role for the industrial production of D-psicose by fructose.Bacillus in the latter part of the growth will generate autolytic cleavage phenomenon,freeing the large amounts of by-products into the culture medium,and even affect the expression of target protein,and subsequent purification process.In this study,the genes associated with the dissolution of the Bacillus subtilis were knocked out.On the basis of the knocking out of gene lytC,the gene xpf and spbC were knocked out.After removing the gene lytC,xpf and spbC,the growth of strain was greatly improved,greatly eased the problem of autolytic cleavage in the late atage of fermentation.Strain does not go into logarithmic phase and shortly stable phase,then directly into the decline phase,but experiences a long term stable phase after enters the stable period.