University of SaskatchewanHARVEST
  • Login
  • Submit Your Work
  • About
    • About HARVEST
    • Guidelines
    • Browse
      • All of HARVEST
      • Communities & Collections
      • By Issue Date
      • Authors
      • Titles
      • Subjects
      • This Collection
      • By Issue Date
      • Authors
      • Titles
      • Subjects
    • My Account
      • Login
      JavaScript is disabled for your browser. Some features of this site may not work without it.
      View Item 
      • HARVEST
      • Electronic Theses and Dissertations
      • Graduate Theses and Dissertations
      • View Item
      • HARVEST
      • Electronic Theses and Dissertations
      • Graduate Theses and Dissertations
      • View Item

      Cloning, expression, and characterization of lactic acid bacteria recombinant prolidases

      Thumbnail
      View/Open
      yang_si.pdf (1.287Mb)
      Date
      2007
      Author
      Yang, Soo In
      Type
      Thesis
      Degree Level
      Masters
      Metadata
      Show full item record
      Abstract
      Lactobacillus plantarum (Lb. plantarum) NRRL B4496 and Lactococcus lactis (Lc. lactis) NRRL B1821 prolidase genes were isolated, cloned, and sequenced. The sequence-confirmed genes were subcloned into the expression systems. The recombinant prolidases from the pKK223-3 systems were purified through ammonium sulphate precipitation and anion-exchange column chromatography. Recombinant Lb. plantarum prolidase, however, demonstrated a loss of activity during the purification. The following characterization work was performed on purified recombinant Lc. lactis prolidase. The mass spectroscopic result and the molecular modelling suggested a 80 kDa homodimer with two metal cations at the catalytic centre of the prolidase. The optimum temperature was 50 ºC and showed more than 50% activities between 40 and 55 ºC. The enzyme was most stable at 30 ºC and withstood 20 min of heat-treatment up to 60 ºC, however, lost activity over 70 ºC. Circular dichroism indicated a denaturation temperature of 67 ºC. The optimum pH was 6.5 for hydrolyzing Leu-Pro and the enzyme did not display any activity below pH 5.5 nor above pH 7 with this peptide. However, Phe-Pro was hydrolyzed the fastest at pH 7 and Arg-Pro had a maximum rate at pH 9. This metallopeptidase exhibited a broad range of metal cation preference, hydrolyzing Leu-Pro with Mn++, Co++, Zn++, Ca++, and Mg++. Further kinetic analysis showed unusual allostery of the enzyme (Hill coefficient: 1.3). The unique substrate intakes onGlu-Pro and tripeptides were observed while Val-Pro was not hydrolyzed. The molecular modelling of this prolidase suggested a difference in the substrate specificity resulting from a loop structure, L33 to R40, near the substrate binding site.
      Degree
      Master of Science (M.Sc.)
      Department
      Applied Microbiology and Food Science
      Program
      Applied Microbiology and Food Science
      Supervisor
      Tanaka, Takuji
      Committee
      Shand, Phyllis J.; Korber, Darren R.
      Copyright Date
      2007
      URI
      http://hdl.handle.net/10388/etd-04142007-123803
      Subject
      Lactic acid bacteria
      PepQ
      prolidase
      Collections
      • Graduate Theses and Dissertations
      University of Saskatchewan

      University Library

      © University of Saskatchewan
      Contact Us | Disclaimer | Privacy