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<p>A cellulase-free xylanase gene of 927&#xa0;bp size (Xyn-GM) was isolated from the metagenomic library of the Tapovan Hot Spring in Uttarakhand, India. This gene encodes a 308-amino acid xylanase enzyme classified under the glycoside hydrolase family 10 (GH-10). The Xyn-GM gene was introduced into the pET28a ( +) vector and expressed in host cells of <i>Escherichia coli</i> BL21 (DE3). The recombinant xylanase (rXyn-GM), with a molecular weight ~ 32.5&#xa0;kDa, was isolated through a one-step purification process using Ni<sup>2</sup>⁺-NTA affinity chromatography. The purified enzyme exhibited broad thermostability (50–100&#xa0;°C) and pH stability (4.0–11.0), with optimal activity at 70&#xa0;°C and pH 9.0. Its activity increased by 67% in the presence of 1&#xa0;mM Mn<sup>2</sup>⁺. rXyn-GM retained ~ 65% activity after 2&#xa0;h at 50&#xa0;°C and 60&#xa0;°C and ~ 75% activity at pH 9.0 after 3&#xa0;h. It showed a preference for beechwood xylan, with kinetic parameters Km 20.9&#xa0;mg/mL and Vmax 156.25&#xa0;µmol/mg/min. Furthermore, rXyn-GM catalysed the production of xylo-oligosaccharides from beechwood xylan, suggesting its potential utility as prebiotics in the food and pharmaceutical industries.</p>

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Cloning and Heterologous Expression of a Novel Thermo-Alkalistable GH-10 Xylanase (rXyn-GM) Retrieved from Tapovan Hot-Spring Soil Metagenome and its Characterization for Kinetic Parameters

  • 期刊论文

  • 发表日期：

    2025年4月30日

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  • Garima Chauhan,
  • Nivedita Bisht,
  • Praveen Gautam,
  • Meghna Arya,
  • Asha Kumari,
  • Digvijay Verma,
  • Monica Sharma

摘要

A cellulase-free xylanase gene of 927 bp size (Xyn-GM) was isolated from the metagenomic library of the Tapovan Hot Spring in Uttarakhand, India. This gene encodes a 308-amino acid xylanase enzyme classified under the glycoside hydrolase family 10 (GH-10). The Xyn-GM gene was introduced into the pET28a ( +) vector and expressed in host cells of Escherichia coli BL21 (DE3). The recombinant xylanase (rXyn-GM), with a molecular weight ~ 32.5 kDa, was isolated through a one-step purification process using Ni2⁺-NTA affinity chromatography. The purified enzyme exhibited broad thermostability (50–100 °C) and pH stability (4.0–11.0), with optimal activity at 70 °C and pH 9.0. Its activity increased by 67% in the presence of 1 mM Mn2⁺. rXyn-GM retained ~ 65% activity after 2 h at 50 °C and 60 °C and ~ 75% activity at pH 9.0 after 3 h. It showed a preference for beechwood xylan, with kinetic parameters Km 20.9 mg/mL and Vmax 156.25 µmol/mg/min. Furthermore, rXyn-GM catalysed the production of xylo-oligosaccharides from beechwood xylan, suggesting its potential utility as prebiotics in the food and pharmaceutical industries.