University of SaskatchewanHARVEST
  • Login
  • Submit Your Research
  • 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

      Investigation of Inositol dehydrogenase-related enzymes

      Thumbnail
      View/Open
      AAMUDALAPALLI-THESIS.pdf (4.579Mb)
      Date
      2013-02-25
      Author
      AAMUDALAPALLI, HARI BABU
      Type
      Thesis
      Degree Level
      Masters
      Metadata
      Show full item record
      Abstract
      Inositol dehydrogenase (IDH) catalyzes the oxidation of myo-inositol to scyllo-inosose using NAD+ as the coenzyme. IDH-related genes (Lp_iolG1 to Lp_iolG4) from Lactobacillus plantarum WCSF1 and (Lc_iolG1 and Lc_iolG2) from Lactobacillus casei BL23 were cloned into the vector pQE-80L, expressed in E. coli host cells and the proteins were purified to homogeneity. IDH activity of the purified enzymes was explored with myo-inositol and other structurally related compounds. It was found that IDH-related enzymes from L. plantarum WCSF1 did not exhibit any activity with tested substrates but, LcIDH1 and LcIDH2 from L. casei BL23 showed activity with myo-inositol and other related compounds. pH-rate profile studies have demonstrated the optimum pH for the reactions catalyzed by the active enzymes. Steady-state kinetics of the active enzymes was performed as with IDH from Bacillus subtilis (BsIDH), revealing that LcIDH1 is a myo-inositol dehydrogenase and LcIDH2 is a scyllo-inositol dehydrogenase. Both LcIDH1 and LcIDH2 are observed to be NAD+-dependent. Kinetic isotopic effect experiments for LcIDH1 have demonstrated that the chemical step in the reaction is partly rate-limiting. Substrate spectrum of LcIDH1 and LcIDH2 was explored and compared to BsIDH. Finally, a multiple sequence alignment of IDH-related enzymes was performed and the proposed consensus sequence motifs were considered to understand the activity differences between these enzymes.
      Degree
      Master of Science (M.Sc.)
      Department
      Chemistry
      Program
      Chemistry
      Supervisor
      Palmer, David
      Committee
      Sanders, David; Gravel, Michel; Yang, Jian
      Copyright Date
      January 2012
      URI
      http://hdl.handle.net/10388/ETD-2012-01-314
      Subject
      Inositol
      Inositol dehydrogenase(IDH)
      Lactobacillus plantarum WCFS1
      Lactobacillus casei BL23
      gene cloning
      gene expression
      protein purification
      and enzyme kinetics.
      Collections
      • Graduate Theses and Dissertations
      University of Saskatchewan

      University Library

      © University of Saskatchewan
      Contact Us | Disclaimer | Privacy