Cloning Of A Pectinase Genetic Fragment And Construction Of The Expression Vector

Bacteria pectinase could be widely used in plant bio-degumming, coffee and tea fermentations, wastewater treatment, paper making, and other industrial areas. To build high pectinase engineering bacteria by genetic engineering had far-reaching significance for industrial development. In this paper a pelB gene amplified from of Bacillus subtilis S-1genomic DNA and another DNA fragment amplified from Acinetobacter junii S-3DNA were studied and analysed. The pelB gene was cloned into pET32a, and recombined expression plasmid pET32a-pe/B was constructed. The main results were as follows:1. The pelB gene was amplified from B. subtilis S-1DNA, and then was cloned into pMD20-T to recombine a pMD20-T-pe/B plasmid and sequenced. The result showed that pelB was1033bp and NJ phylogenetic tree was analyzed. The pelB was similar to pectate lyase. The identity of pelB with pel-15is40.98%and39.20%with pelA from Bacillus sp., and40.32%with pelB from Bacillus pumilus. And pelB was similar to pectate lyase.2. The predicted results of the property of the putative protein of pelB gene were as follows:its formula was C1671H2704N490O493S25, Molecular weight was38,348.3Da, Theoretical pI was8.88, The N-terminal of the sequence considered was G (Gly), The putative protein was unstable, hydrophilic, and had25.3%Helix,14.0%Strand,60.7%Loop.3. The pelB gene was cloned into pET32a, and then the combined plasmid pET32a-pelB was transformed into E.coli BL21and induced with0.5mmol/L IPTG for4hours. The SDS-PAGE electrophoresis showed that the expressed target protein was about40kD.4. A DNA fragment was amplified from A. junii S-3DNA and sequenced; it was917bp, contained a747bp open reading frame. The Formula of the pupative protein was C1223H1962N336O376S7, its Molecular weight was27,613.4Da, Theoretical pI was5.61, The N-terminal of the sequence considered was M (Met), and the protein was stable, hydrophilic, and had39.76%Helix,18.88%Strand, and41.37%Loop, Signal anchor probability0.117, two transmembrane helices.