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 Co²âº) showed stimulatory effects of both catalytic activities, where as in the presence of Hg²âº, Cd²âº, Cu²âº 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.

Isolation, purification, and characterization of two thermostable endo-1,4-ß-D-glucanase forms from Opuntia vulgaris.

Four endoglucanase temperature isoforms (T (30), T (50), T (70), and T (90)) were identified and purified from the cladodes of the xerophytic plant Opuntia vulgaris. These isoforms exhibited optimum catalytic activity at 30 °C, 50 °C, 70 °C, and 90 °C and yielded an apparent molecular mass of 150, 20, 74, and 45 kDa, respectively, on gel filtration chromatography. These isoforms were purified 24-, 25-, 29-, and 27-fold with a yield of 15%, 12%, 17%, and 19% and having a specific activity of 120, 125, 144, and 136 U/mg, respectively. The thermostable T (70) and T (90) isoforms exhibited optimum activity at pH 4.5 and 7 and also yielded a molecular weight of 66 and 36 kDa, respectively, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The T (70) had a K (m) of 43 mM and a V (max) of 12.5 µmol min(-1) µg(-1) of protein, and the T (90) isoform had a K (m) of 40 mM, with an apparent V (max) of 10 µmol min(-1) µg(-1) of protein. Western blot, immunodiffusion, and in vitro inhibition assays established the reactivity of the T (90) isoform with polyclonal anti-T (90) antibody raised in rabbit. Cross-reactivity of this antibody with the T (70) endoglucanase isoform was also noted.

Purification and characterization of a novel thermostable xylose isomerase from Opuntia vulgaris mill.

Thermophilic xylose isomerase from the xerophytic eukaryote Opuntia vulgaris can serve as a good alternate source of enzyme for use in the production of high fructose corn syrup. The existence of two temperature stable isoforms having optimal activity at temperatures 70 °C (T(70)) and 90 °C (T(90)), respectively, is reported here. These isoforms were purified to homogeneity using column chromatography and SDS-polyacrylamide gel electrophoretic techniques. Only the T(90) isoform was subjected to full biochemical characterization thereafter. The purified T(90) isoform was capable of converting glucose to fructose with high efficiency under the assay conditions. The enzyme at pH 7.5 exhibited a preference to yield the forward isomerization reaction. The melting temperature of the native enzyme was determined to be 90 °C employing differential scanning colorimetery. Thermostability of the enzyme protein was established through temperature-related denaturation kinetic studies. It is suggested that the thermostability and the wide pH activity of this eukaryotic enzyme will make it an advantageous and dependable alternate source of catalytic activity for protected use in the high fructose corn syrup sweetener industry.

Purification and characterization of thermophilic xylanase isolated from the xerophytic-Cereus pterogonus sp.

A thermo stable xylanase was purified and characterized from the cladodes of Cereus pterogonus plant species. The enzyme was purified to homogeneity by ammonium sulfate (80%) fractionation, ion exchange and size exclusion chromatography. The enzyme showed a final specific activity of 216.2 U/mg and the molecular mass of the protein was 80 KDa. The optimum pH and temperature for xylanase activity were 5.0 and 80 °C, respectively. With oat spelt xylan as a substrate the enzyme yielded a Km value of 2.24 mg/mL and a Vmax of 5.8 µmol min(-1) mg(-1). In the presence of metal ions (1 mM) such as Co(2+),Mn(2+), Ni(2+), Ca(2+) and Fe(3+) the activity of the enzyme increased, where as strong inhibition of the enzyme activity was observed with the use of Hg(2+), Cd(2+), Cu(2+), while partial inhibition was noted with Zn(2+) and Mg(2+). The substrate specificity of the xylanase yielded maximum activity with oat spelt xylan.