Biochemical and biophysical characterization of purified thermophilic xylanase isoforms in Cereus pterogonus plant spp.
Two thermostable xylanase isoforms T₆₀ and T₈₀ were purified to homogeneity from the cladodes of the xerophytic Cereus pterogonus plant species. After three consecutive purification steps, the specific activity of T₆₀ and T₈₀ isoforms were found to be 178.6 and 216.2 U mg⁻¹ respectively. The molecular mass of both isoforms was determined to be 80 kDa. The optimum temperature for T₆₀ and T₈₀ xylanase isoforms were 60 and 80 °C respectively. The pH was 5.0 for both isoforms. The presence of divalent metal ions (10 mM Co²⁺) showed stimulatory effects of both catalytic activities, where as in the presence of Hg²⁺, Cd²⁺, Cu²⁺ showed inhibitory effect on these activities at all concentrations studied. The thermodynamic analysis of xylanase activity using denaturation kinetics and the presence divalent cations at 30-100 °C, showed lower ΔH, ΔS, and ΔG values at all the temperatures investigated. The melting temperature of purified T₈₀ xylanase isoform as determined by TG/DTA analysis and it showed the unfolding temperature was 80 °C. The g value and hyperfine (A) value purified xylanase T₈₀ isoform was 2.017 and 10.80 respectively. Immunoblot analysis with antiserum raised against the purified T₈₀ xylanase isoforms revealed single immunolgically related polypeptides of 80 kDa, identical with the polypeptide band produced on SDS-PAGE. The results of double immunodiffusion against the T₈₀ isoforms showed a single precipitin line indicating that the serum used was specific to these xylanase isoforms. The kinetic and thermodynamic properties suggested that xylanase from C. pterogonus may have a potential usage in various industries.
Department of Biochemistry and Molecular Biology, School of Life Sciences, Pondicherry University, Kalapet, Puducherry, India.
Jeyaraman
J
Vikramathithan
Pandurangan
P
Muthuraman
Sambandam
S
Ravikumar
Shivalingam
S
Shayamala
Gali Nirmal
GN
Kumar
Kotteazeth
K
Srikumar
D000918:Antibody Specificity; D001665:Binding Sites; D029421:Cactaceae / Q000201:enzymology*; D002104:Cadmium / Q000494:pharmacology; D003035:Cobalt / Q000378:metabolism; D003300:Copper / Q000494:pharmacology; D043364:Endo-1,4-beta Xylanases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008628:Mercury / Q000494:pharmacology; D008970:Molecular Weight; D009994:Osmolar Concentration; D010940:Plant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011489:Protein Denaturation; D013818:Thermogravimetry; D014990:Xylans / Q000378:metabolism
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D010940:Plant Proteins; D014990:Xylans; D002104:Cadmium; D003035:Cobalt; D003300:Copper; D043364:Endo-1,4-beta Xylanases; D008628:Mercury