Purification and biochemical characterization of a bacteriophage lyase specific to cleave the capsular polysaccharides of PLA Klebsiella pneumoniae NTUH-K2044

• Main Authors: Wei-Lin Tu, 涂瑋霖
• Other Authors: Shih-Hsiung Wu
• Format: Others
• Language: en_US
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/45253945105138408744

碩士 === 國立臺灣大學 === 生化科學研究所 === 100 === Klebsiella pneumoniae is one of the microorganisms causing pyogenic liver abscess (PLA) in Asian populations over two decades. This kind of PLA is associated with significant mortality because of developing into sepsis, bacteremia, metastatic meningitis or endophthalmitis. The clinic investigation showed that K. pneumonia serotypes K1 and K2 are predominant among capsular serotypes of PLA strains, around 50-85％. The major purpose of this study is to construct a capsular polysaccharide (CPS)-protein conjugate vaccine for prevention of K. pneumonia PLA. Since CPS is too large to be conjugated to carrier proteins, the depolymerization of CPS will increase the coupling efficiency to the carrier protein. According to our previous study, the chemical cleavage will destruct the CPS modifications including acetylation and pyruvation and, meanwhile, abolish the immune responses. In the present study, the K1 lyase from bacteriophage was cloned, expressed and purified by the traditional methods. The relative K1 lyase activity can be measured by UV absorbance at 232 nm. The optimal pH and temperature of K1 lyase is 7.5 and 30℃, respectively. The magnesium ion is essential for the activity of K1 lyase, but, to some extent, can be replaced by other metal ions such as cobalt and nickel. We also constructed a mutant of K1 lyase which could cleave the CPS more stably and efficiently. Based on the analysis of MS, acetylation and pyruvation in CPS remain intact after enzymatic cleavage. Capillary electrophoresis was also used to kinetically detect the fragmentation of CPS.