Production, Properties And Cloning Of An Alkaline Protease From Bacillus Subtilis I15

Medium composition and culture conditions for the alkaline protease production by Bacillus subtilis 115 were optimized, with mono-factor experiment. The activity of alkaline protease is determined by Folin phenol method. Effect of different factors and parameters on protease production revealed that the maximum secretion(3384 U/mL)occurred as follows:corn starch 1.2% as carbon source,soybean cake powder0.3% as nitrogen source,pH 8.0, fermentation temperature 37℃,fermentation time16 h. Proteolytic activity of the culture supernatant showed maximum activity at 65℃and pH 8.0.The enzyme showed good thermostability and pH stability. The protease activity was stable over a pH range from 7.0 toll.0,retaining more than 82% of its activity after 24 h of incubation at 25℃and high activity was detected after incubation of the culture supernatant at various temperatures (40 to 50℃),The half-live was 2.5 h at 70℃.Ca2+ can promote Proteolytic activity. However,the activity of the culture supernatant was inhibited almost by PMSF, indicating serine protease activity. In addition, it showed excellent stability and compatibility with surfactant and detergent.The alkaline protease gene(sap gene) was cloned from the total DNA of Bacillus subtilis 115 by PCR and its nucleotide sequence was determined.Gene bank accession number is GQ339614.1.Multiple alignments of the alkaline protease amino acid sequences from different reported alkaline protease is done by BLAST program and its homology is 99%.It confirmed our earlier speculation:the alkaline protease is belong to serine alkaline protease.The signal peptide and tertiary structure of the alkaline protease was predicted by network resources.The result showed that the Front-end 29 amino acids may be the signal peptide.We successfully constructed the expression vector pET-25b-sap,but the host bacteria E.coli BL 21 was no activity both in the skim milk plate and the supernatant.