Construction Of The Penicillin G Acylase-producing Bacillus Subtilis Strain And Optimization Of Fermentation Conditions

In this paper, we obtained recombinant strains with high enzyme PGA by gene engineering method, Then optimized the medium culture and fermentation conditions of the recombinant strains, in order to gain the efficient production of PGA enzyme and become more suitable for industrial application. In the construction of the recombinant strain, PGA gene from Bacillus megaterium was cloned into the expression vector pMA5 of Bacillus subtilis, and then making it expression highly. To optimization of culture conditions of recombinant strain, after single factor test about medium and fermentation conditions of recombinant strain, we used response surface to gain the best medium and the optimum fermentation conditions of recombinant strains.The results were as follows:(1)After making the standard curve of PGA,we determined the PGA enzyme of initial bacterial strain.wth the fermenting of Bacillus megaterium and Bacillus subtilis, the activity of PGA were 5.41U/mL and 6.84U/m L.(2)On the basis of Bacillus subtilis expression shuttle vector pMA5, the recombinant plasmid pMA5-PGA and strain of Bacillus subtilis were successfully constructed.(3)The fermentation supernatant were analyzed by SDS-PAGE. The results showed that pMA5-PGA was successfully expressed in Bacillus subtilis 10397. the size of protein is 28 kDa. The activity of PGA of the recombinant Bacillus subtilis can be reached 12.43U/m L, which was the 2.29 times than in Bacillus megaterium and 1.82 times than in Bacillus subtilis.(4)The optimum medium of the recombinant Bacillus subtilis were consisted of glucose 20.51g/L, tryptone 16.14g/L, NaCl 3.02g/L.(5)The optimum fermentation conditions of the recombinant Bacillus subtilis were confirmed through response surface: cultivated temperature 36.83℃, pH 7.04, shaking speed 220.78r/min, cultivated time 48.18 h.(6)With the best culture conditions, the activity of PGA was reached 13.97U/mL, which was the 1.12 times than it in initial fermentation conditions of recombinant strain.