title: Isolation, purification, and characterization of thermophilic T80 isoenzyme of xylose isomerase from the xerophyte Cereus pterogonus.
abstract: A thermostable isoenzyme (T(80)) of xylose isomerase from the eukaryote xerophyte Cereus pterogonus was purified to homogeneity by precipitation with ammonium sulfate and column chromatography on Dowex-1 ion exchange, with Sephadex G-100 gel filtration, resulting in an approximately 25.55-fold increase in specific activity and a final yield of approximately 17.9%. Certain physiochemical and kinetic properties (K(m) and V(max)) of the T(80) xylose isomerase isoenzyme were investigated. The molecular mass of the purified T(80) isoenzyme was 68 kD determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Polyclonal antibodies against the purified T(80) isoenzyme recognized a single polypeptide band on Western blots. The activation energy required for the thermal denaturation of the isoenzyme was determined to be 61.84 KJ mol(-1). The use of differential scanning calorimetry established the melting temperature of the CPXI isoenzyme to be 80°C, but when studied with added metal ions, melting temperature increases to more than the normal. Fluorescence spectroscopy of T(80) isoenzymes yielded an emission peak with λ(em) at 320 nm and 340 nm, respectively, confirming the presence of Trp residue in these proteins. Electron paramagnetic resonance (EPR) analysis at liquid nitrogen temperature established the presence of Mn(2+) and Co(2+) associated with each isoenzyme. These enzyme species exhibited different thermal and pH stabilities compared to their mesophilic counterparts and offered greater efficiency in functioning as a potential alternate catalytic converter of glucose in the production of high-fructose corn syrup (HFCS) for the sweetener industry and for ethanol production.
authors: Department of Biochemistry and Molecular Biology, School of Life Sciences, Pondicherry University, Puducherry, India.
authors: Sambandam
authors: S
authors: Ravikumar
authors: Sivalingam
authors: S
authors: Shyamala
authors: Pandurangan
authors: P
authors: Muthuraman
authors: Kotteazeth
authors: K
authors: Srikumar
mesh_terms: D019747:Aldose-Ketose Isomerases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D029421:Cactaceae / Q000737:chemistry; D002152:Calorimetry, Differential Scanning; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003035:Cobalt / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008345:Manganese / Q000737:chemistry; D008970:Molecular Weight; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: D007527:Isoenzymes; D010940:Plant Proteins; D003035:Cobalt; D008345:Manganese; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase