Study On DNA Shuffling Of Extracellular Neutral Metalloprotease Of Bacillus Subtilis

Neutral proteases produced by Bacillus subtilis have been widely used in abstergent, tanning,silk processing,food production and many other fields as important industrial products.In this study,the mutated nprE gene by DNA shuffling was obtained and a high-yield neutral protease-producing Bacillus subtilis strain was got by screening.At the same time, an expression vector used as comparison was constructed.The main work as following:1.The gene of strong promoter P43 of Bacillus subtilis and the full-length gene of nprE had been cloned by using PCR.These two genes were also cloned into pMD18-T vector and sequenced.As result,nprE was totally the same compared with published data.2.11 strains of Bacillus subtilis with good performance in producing neutral proteases were identified.There were 8 of their nprE genes were cloned.3.An expression vector pNP43-nprE was successfully constructed.The recombinant plasmid was transformed into b.s168 strain and nprE gene was expressed.The 42kDa enzyme was identified by 12%SDS-PAGE.Under a significent antibiotics selection press, the max yield of nprE showed up while OD₆₀₀=0.8,while the activity of nprE is 3 times than the type strain.But the yield of nprE decreased while there was no antibiotics selection press.4.The nprE gene of type strain and wild-type strain were used as initiate stuff.Through one cycle of error prone PCR and one cycles of DNA shuffling,the activity of neutral protease was promped over 126%,based on the first cycle,it was promped of 89%in the second circle of DNA shuffling.These were caused by the changes of the base pair of nprE gene.Compared these two ways,it was found that the DNA shuffling method would show more benefits than using expression vector.