Cloning And Expression Of Thermostable Neutral Protease Gene From Bacillus Licheniformis XJT9503

Neutral proteases are a commercially important group of enzymes.It has been apply universally to industry.But the generic neutral proteases are not thermostable,it is very easy to lost their catalyse activity in application.The thermostable neutral proteases producing by thermophilic microorganism have more thermostable characteristic.Now,we have found that the most thermostable neutral protease is belong to metalloproteinase.A gene fragment,named dmp,was amplified by PCR from Bacillus licheniformis XJT9503.The result of sequence and alignment analysis indict that dmp contain a open frame(ORF),encoded 313 amino acids,DNA sequence and amino acids sequence encoded by ORF shared 98%and 99%homology with reported mpr gene of metalloproteinase in bacillus subtilis.The tnp fragment was obtained by digested dmp with Pstâ… ,Hindâ…¢.The recombinant plasmids,named pPL-tnp,was obtained by ligated the tnp with pPL608 vector and eletroporated into bacillus subtilis strains AB97013,it was shown that tnp gene can express in bacillus subtilis strains AB97013 and its product have similar characters in optimum catalytic pH and temperature of enzymology with extracellular product of XJT9503.It proved the tnp gene is aim gene,the thermostable neutral proteases gene.In order to make the tnp gene to express in Pichia pastoris,designed three pairs of primers to change the sequence of tnp.The new gene fragment was digested with EcoRâ… ,Notâ… ,ligated with pPIC9K vector,and then obtained recombinant plasmids,named pPIC9K-tnp. pPIC9K-tnp was digested with Sacâ… and electroporated into Pichia pastoris strain GS115, obtained recombinant strain PIC-5.The tnp gene can express in recombinant strain PIC-5,and the product of tnp has similar characters with extracellular product of XJT9503 in enzymology.