Purification and biochemical characterization of two major thermophilic xylanase isoforms (T70 and T90) from xerophytic Opuntia vulgaris plant spp. |
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Authors: | Jeyaraman Vikramathithan Sambandam Ravikumar Pandurangan Muthuraman Gali Nirmalkumar Sivalingam Shayamala Kotteazeth Srikumar |
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Institution: | 1. Department of Biochemistry and Molecular Biology, School of Life Sciences, Pondicherry University, Kalapet, Puducherry, 605014, India 2. Department of Chemical Engineering, University of Ulsan, Ulsan, South Korea 3. Department of Animal Sciences, Chonbuk National University, Chonju, South Korea
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Abstract: | Thermostable xylanase isoforms T70 and T90 were purified and characterized from the xerophytic Opuntia vulgaris plant species. The enzyme was purified to homogeneity employing three consecutive steps. The purified T70 and T90 isoforms yielded a final specific activity 134.0 and 150.8 U mg?1 protein, respectively. The molecular mass of these isoforms was determined to be 27 kDa. The optimum pH for the T70 and T90 xylanase isoforms was 5.0 and the temperature for optimal activity was 70 and 90 °C, respectively. The Km value of T70 and T90 enzyme isoforms was 3.49, 2.1 mg ml?1, respectively when oat spelt xylan was used as a substrate. The T70 had a Vmax of 10.4 μmol min?1 mg?1, and T90 had a Vmax of 8.9 μmol min?1 mg?1, respectively. In the presence of 10 mM Co2+, and Mn2+ the activity of T70 and T90 isoforms increased, where as 90 % inhibition was noted with of the use 10 mM Hg2+, Cd2+, Cu2+, Zn2+ while partial inhibition was observed in the presence of Fe3+, Ni2+, Ca2+and Mg2+. The T70 and T90 isoforms retained nearly 50 % activity in the presence of 2.0 M urea, while use of 40 mM SDS lowered the activity nearly 38–41 %. The substrate specificity of both T70 and T90 isoforms showed maximum activity for oat spelt xylan. Western blot, immunodiffusion, and in vitro inhibition assays confirmed reactivity of the T90 isoform with polyclonal anti-T90 antibody raised in rabbit, as well as cross-reactivity of the antibody with the T70 xylanase isoform. |
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