Display Of Functionally Active Lipases On The Escherichia Coli Cell Surface

Cell-surface display technology was used widely in the filed of high throughput screening of a mutant library, which promoted the development of protein engineering. As a kind of the industrial biocatalysts, LipA from Bacillus subtilis displayed unique3D structure and enzymatic characterization and used widely in many fields, including polymer synthesis, et al. In this dissertation, LipA from Bacillus subtilis was fuctionally displayed on the cell surface of Escherichia coli.Firstly, the lip A gene was directly cloned from Bacillus subtilis, and then sequenced. The sequence of the lipA gene was submitted to NCBI nucleic acid database, and the accession number is JX048066. The full length of the lip A gene was639bp, which coded212amino acid residues. The sequence of the lip A gene was also aligned with other homologous gene sequences, which displayed high similarity. The catalytic triad of LipA consisted of Ser108、 Asp164and His187, and the Ser108was in the conservative peptide fragment, Ala-His-Ser-Met-Gly.Secondly, the recombinant LipA was purified to homogeneity. The lipA gene fragment coding the mature peptide of LipA was inserted into the expression plasmid pET22b, which resulted in pET22b-lipA. The pET22b-/ipA was then transformed into E.coli BL21(DE3), and then the lip A gene was induced expression by IPTG. The recombinant LipA was purified to homogeneitywith HisTrapTM HP chromatography.Thirdly, the expression plasmid for cell surface display, pHBHE and pBCMB-X2, was constructed and the LipA from Bacillus subtilis was fuctionally displayed on the cell surface of Escherichia coli. The lip A gene was fused with the gene fragment of estA coding the carboxyl terminal domain of EstA from Pseudomonas aeruginosa by overlap extension PCR. The PCR fragment was inserted into pACYC vector, which resulted in the pBCMB-X2plasmid. The recombinant pBCMB-X2plasmid was then transformed into E. coli JK321and E. coli UT5600, respectively. The lipA gene was induced expression by IPTG and the recombinant LipA was functionally displayed on the cell surface of E. coli JK321and E. coli UT5600, respectively. The the whole cell lipase activity was qualitatively assayed on the testing plate and quantitatively assayed with colorimetric method. The hydrolysis activity of the whole cell LipA was2.8±0.1U/OD600 and2.6±0.06U/OD600, respectively.