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Applied Biochemistry and Biotechnology

, Volume 189, Issue 4, pp 1274–1290 | Cite as

Cloning, Purification, and Characterization of Recombinant Thermostable β-Xylanase Tnap_0700 from Thermotoga naphthophila

  • Attia Hamid
  • Muhammad Nauman AftabEmail author
Article

Abstract

The gene of a β-xylanase (Tnap_0700) was cloned from a hyperthermophilic Thermotoga naphthophila strain ATCC BAA-489 and expressed in Escherichia coli BL21 (DE3) via pET-21a (+) as an expression vector. The growth steps were upgraded for highest β-xylanase expression via several factors, for example, IPTG concentration, time of induction, pH, and temperature. The pH and temperature optima for the extreme expression of β-xylanase were 7.0 pH and 37 °C, correspondingly. Recombinant enzyme purified by heat treatment process, then later by immobilized metal ion affinity chromatography. Molecular mass of the purified β-xylanase was 38 kDa observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was stable at room temperature for 30 days. It exhibited high stability over wide series of temperature 50–90 °C and pH 4.0–9.0 upon the addition of 1 mM Ca+2 and reduced in the existence of Cu+2 and EDTA. The addition of about 10–30% different organic solvents have no considerable effect on enzyme. However, SDSF and urea acting as an inhibitor leads to decrease in the enzyme activity. The β-xylanase enzyme was active to hydrolyze xylan from beechwood forming xylose. Thermostable β-xylanase causes the breakdown of complex carbohydrates into monosaccharide components. This thermostable β-xylanase revealed remarkable properties, which make it an encouraging candidate for various industrial applications especially in the alteration of renewable biomaterials into ethanol production, and biofuels from lignocellulosics has acknowledged much devotion subsequently in the last decade.

Keywords

Cloning Expression Thermostability Saccharification 

Notes

Acknowledgments

The manuscript is thoroughly read and corrected for English language by Prof. Dr Stephen J. Perkins, Professor Emeritus, Department of Structural and Molecular Biology, Darwin Building, Gover Street, University College of London, UK.

Funding Information

This work was supported by the Higher Education Commission Pakistan under grant no. and GC University Lahore Ministry of Science and Technology, Government of Pakistan under grant no. 5535 project “Cost effective pilot scale production of bioethanol.”

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. 1.Institute of Industrial BiotechnologyGovernment College UniversityLahorePakistan

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