Cloning And Expression Of Bacterial Peptidases DNA Fragments From Impure Culture

Peptidases play important roles in both cellular metabolic process and industrial community. Lots of their genes have been found and cloned. Our lab has successfully cloned peptidase-coding genes with fibrinolytic, dehairing or collagenlytic activity, etc. In order to construct more recombinant strains producing peptidases, as well as to make directed evolution of peptidase genes, whose products are expected to show some new properties, the cloning of peptidase DNA fragments from impure culture were carried out.Subtilisins of Serine Peptidase Faimly S8 were selected according to amino acid multiple alignment in database MEROPS and their DNA came from GenBank. Ten primers were designed and synthesized, aiming at coding sequences or at mature peptide DNA of those peptidases.Twenty-one samples rich in microorganisms were collected from different places. Bacteria producing extracellular peptidases were enriched on defatted milk plates, and then cultured together for total DNA extraction. Twelve DNAs were prepared, each one was used for touchdown PCR (TD-PCR) using 6 pairs of primers. There were total 47 DNA fragments obtained and 19 with 800-1,000bp in length were selected for sequence analysis. Among them, 8 fragments are peptidase DNAs, 3 have sequences highly similar to those but not peptidase genes in GenBank, other 8are not similar to any known sequence.Further analysis showed that the 8 peptidase fragments belong to 4 genes and difference between two nucleotide sequences amplified by the same primers reaches to 32%, which proves the feasibility of cloning new peptidase-coding genes from impure culture merely by PCR.G2 fragment is almost identical to CDS of alkaline protease E (GenBank No., AJ539133), and has the most similarity of 94% to other homologous genes in nucleotides, and at least difference of 20 amino acid residues. No report yet was on the characteristics of its product. G2 fragment was expressed using pTWINl in Escherichia coli ER2566 and the results were shown as follows: final concentration of IPTG needed for induction was 0.3mmol/L; product was mainly inclusion bodies either at 37℃ or at 28 ℃, while soluble and active after overnight induced at 18℃. Active products were lethal to E.coli, secreted into the medium. Because the band of the mature enzyme was not clear on the silver-stained polyacrylamide gel, and the enzyme produced a big hydrolyzed zone on the defatted milk plate, AprE seemed to have a strong proteolytic activity.