Secreting Of Penicillin G Acylase In Tat Pathway From Bacillus Subtilis

Industry ofβ-lactam antibiotics production is undergoing a huge reformation.Because the biocatalyzed processes are replacing the chemical synthesis processes. Penicillin G Acylase(EC.3.5.1.11, PGA) now plays an important role in the production ofβ-lactam antibiotics.At present,a mass of proteins were expressed in package form,difficult comeback,the secretive expression of protein is attented accordingly.The heterogenous protein may be secreted when it was cloned following the signal peptide,and the signal peptide was cleaved in course of transforming.Bacillus subtilis includes two secretive pathway:the Sec-dependent pathway and twin-arginine-dependent pathway or Tat pathway.A striking feature of Tat pathway discovered translacaton system is its ability to transport folded proteins eventually bound to a cofactor before export.In contrast,by the sec mechanism, exported proteins are transported in an unfolded state.1.The Tat signal peptide detecting vector of PGA were constructed. The pga gene(Genbank accession numble is BMU07682) from B.megaterium(ATCC 14945) is cloned. We applied the promoter p43 and 720 bp gene to construct the vector. Used levansucrase signal peptide SacB secreted PGA,which improved effect of the vector .The result indicated the detecting vector is avaible.2 . The tweenty-four Tat signal peptides were cloned from the B.subtilis 168 genome(Genbank accession numble is NC 000964),and constructed 24 expressive vectors respectively.We studied Tat the signal peptides on whether transported the PGA into periplasm of WB700.By the method of NIPAB filter paper,the result indicated the signal peptides of AlbB,YmzC,AmyX,and LipA gene could direct PGA protein into the periplasm of WB700.Enzyme activeties are 0.21U·ml^-1,0.26 U·ml^-1,0.11 U·ml^-1,0.08 U·ml^-1 respectively in 36h fermentation.It proved the appropriate signal peptide may assist in transported heterogenous protein in B.subtilis.3.We studid on the pga gene was expressed under the control of Pglv promoter and p43 promoter differently.Their effects on PGA expression were imported to testify secretion overloading or not.Apparently,in the result,the enzyme activety of PGA under control of Pglv promotermore than p43 promoter ,making clear no overloading phenomena.