Screening Of The Alkaliphilic Pectinase-Producting Strain And Gene Cloning, Heterogeneous Expression Of Alkaline Pectate Lyases

Alkaline pectinases can be used in food industry, such as tea fermentation and plant oil extraction, and textile, and paper making. Construct a alkaline pectinase-producting engineering bacteria by using molecular biological methods to improve the yield of pectinase. As the most widely used heterologous expression systems, construction a Escherichia coli engineering bacteria can brought a efficient, stable and secure production of alkaline pectinase.In this study,47strains alkaline pectinase-producing strain was selected by isolation medium from5samples of Daqing saline, Daqing wastewater etc, under the condition of pH10.0by DNS. Amplification the conserved region of pectin depolymerase gene from47strain, we selected3strains with more novel pectinases gene, Ochrobactrum anthropi IE1, Stenotrophomonas maltophilia IE3and Bacillus subtilis IF7, from Daqing saline.Three pectate lyase gene complete sequence was amplified by TA1L-PCR from1E1、1E3and IF7chromosomal DNA. And the IE1pectate lyase gene sequence was1050bp, codes348amino acids, molecular weight was49.6kDa, with an isoelectric point of7.66:the IE3pectate lyase gene sequence was984bp, encoded340amino acids, molecular weight was34.2kDa. with an isoelectric point of9.15; the IF7pectate lyase gene sequence was1263bp. encoded420amino acids, molecular weight was45.5kDa,with a signal peptide and an isoelectric point of7.76.The three full-length genes were cloned from IE1、IE3、IF7and expressed heterologously. The pectate lyase pel-BslF7genes from Bacillus subtilis IF7was expressed in E. coli, getting a gene engineering strain E.coli BL21/pET-pel-BslF7. The influence of induction temperature and induction time on the recombinant production was examined. When induction time was1h, the recombinant production was expressed at30℃and37℃, the production was stable under overnight at30℃Analysed the pel-BslF7, and13functional domains was found, containing a beta spiral parallel series (PbH1). Builded a tertiary structure by homology modeling method, got a credible model and its consistency was94.99%, beta helix barrel structure.The recombinant pectinase was purified by ultrasonic crushing method and nickel ion affinity chromatography, and detected using SDS-PAGE. Then its characterization was analyzed. The results showed that its specific activity was11998U/mg. Its optimal temperature and pH were45-55℃and9.0-9.5. It was stable at pH7.0-10.0. When the recombinant pectinase incubated for30min at60℃, the relative activity was still approximately40%. Different metal ions revealed different effects on recombinant pectinase. Ca2and Co2could activate recombinant pectinase while Fe2could inhibit recombinant pectinase.