ISOLATION, PURIFICATION, AND CHARACTERIZATION OF THERMOPHILIC T₈₀ ISOENZYME OF XYLOSE ISOMERASE FROM THE XEROPHYTE Cereus pterogonus

Abstract

A thermostable isoenzyme (T₈₀) 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 T₈₀ xylose isomerase isoenzyme were investigated. The molecular mass of the purified T₈₀ isoenzyme was 68 kD determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Polyclonal antibodies against the purified T₈₀ 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 T₈₀ 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 Mn²⁺ and Co²⁺ 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.

Keywords:

• Cereus pterogonus
• characterization
• purification
• thermophilic
• xylose isomerase

ACKNOWLEDGMENTS

The authors acknowledge the UGC, India, for its financial support under number F3-31/2004 (SR) Dt.14 Jan 2004. The technical help and facility of Prof. S. Sambasiva Rao, Head, Dept. of Chemistry, for EPR studies, and the help received from Dr. K. Manja and Prof. Nambinarayanan, Dept. of Physics, for the DSC work are gratefully acknowledged. The present address for Sambandam Ravikumar is School of Chemical Engineering & Bioengineering, University of Ulsan, Ulsan 680-749, Korea.

Notes

Note. Enzyme activity was assayed with xylose as a substrate.

^a Protein concentrations were determined by the Bradford assay, using BSA as a standard.

^b One unit (1 U) of isomerase activity was defined as the amount of enzyme required to produce 1 µmol of product per minute under assay condition.

Note. Enzyme activities were determined as described in the Materials and Methods section. These data correspond to an experiment representing a total of three independent experiments closely coincident.