Research On The Depolymerase Enzyme And Function Genes From Klebsiella Phage P13

Bacteriophage is a virus able to infect bacteria and ubiquitous in the nature witha biomass of over1031. Because of the small size of the unity and genome, it isrelatively easy for the research on the bacteriophage. The property of infectingbacteria specifically provides high application value for the research on the phages.Klebsiella K13has the property of synthesize and secrete exopolysaccharide, whichrequire its phages release the depolymerase enzyme to degrade the capsularpolysaccharide before infect bacteria. In this study, the research on the depolymeraseenzyme and some function genes from Klebsiella phage P13was carried on, includingthe characterization and purification of the enzyme, structural analysis of enzymaticproducts, and the analysis of genes encoding depolymerase enzyme, endolysin andholin.Firstly, the phage-borne depolymerase enzyme has been purified andcharacterized. After purification by acetone precipitation and ultrafiltrationcentrifugation, the enzyme is separated from the phage particles and furtherpurification is performed by Q-Sepharose Fast Flow, showing two components withenzyme activity, which indicates that the depolymerase enzyme of phage P13has twoforms, combined with phage particles and dissociative in the solvent. The solubleenzyme has an optimum degradation activity at48C and pH6.5. The cations Fe2+、K+has slight promoting effects on the enzyme activity while Na+, Mg2+and Ca2+restrains the enzyme activity slightly, Al3+、Zn2+、Cu2+suppresses the enzyme activitystrongly, especially the cation Cu2+; EDTA has a slight suppress effect on enzymeactivity, the Km and Vmax are4.66mg/mL and6.42U/(mL·min), respectively, whichare obtained by calculating.After the capsular polysaccharide is degraded by depolymerase enzyme fromphage P13, the Klebsiella K13cells are exposed in the environment, which increasesthe possibilities of destruction by harmful factors. In this study, high temperature,high osmotic pressure solvent and sanitizer are added to the bacteria cells suspensionand the survival bacterial was accounted. The results showed that after theexopolysaccharide was removed, bacterial was hard to survive when faced with undesirable elements, especially the sanitizer.Klebsiella K13has the property of synthesize and secrete exopolysaccharide. Inthis text, the structure of the exopolysaccharide from Klebsiella K13was determined.The monosaccharide constituents of the exopolysaccharide were identified usingHPLC. For the glycoside sequence analysis, the phage-borne depolymerase enzymewas prepared to degrade the exopolysaccharide. Bio-Gel P4and Bio-Gel P6wereused to purify the oligosaccharides. The purified oligosaccharides were thencharacterized by mass spectroscopy, infrared spectroscopy and1H-and13C-NMR，2D-NMR spectroscopy. The result indicated that the exopolysaccharide is composedof a pentasaccharide repeating unit with a definite structure which is different withpreviously published structure of the pentasaccharide repeating unit ofexopolysacchride from Klebsiella K13.The genes encoding depolymerase and endolysin, which both have degradeactivities during the lytic cycle of phage P13, are analyzed. According to the result ofphage P13genome analysis, gene49and50are likely to be genes encodingdepolymerase and gene50is further analyzed by PSI-BLAST. The result proves thatgene50is the gene encoding depolymerase enzyme. Meanwhile, the genes encodingendolysin and holin have also been analyzed by PSI-BLAST and FASTA. The resultindicated that gene36encodes the endolysin of phage P13with the active ofglucosidase and is able to pass through the cell membrane with the presence of holinwhich is encoded by gene39.