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        Investigation of Inositol dehydrogenase-related enzymes

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        AAMUDALAPALLI-THESIS.pdf (4.579Mb)
        Date
        2013-02-25
        Author
        AAMUDALAPALLI, HARI BABU
        Type
        Thesis
        Degree Level
        Masters
        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.
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