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bioRxiv · 10.1101/2024.09.06.611769

3D modeling of thermostable xylanase from Thermotoga naphthophila a member of GH10 family: characterization studies of recombinant xylanase

Waris, A.·Awan, A. R.·Wasim, M.·Hashmi, A. S.·Rashid, N.·Firyal, S.·Khalid, A.·Tayyab, M.
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Abstract

The current study was planned keeping in view the significance, industrial impact and import of xylanase to Pakistan. In this study, a thermostable recombinant xylanase from Thermotoga naphthophila was produced and characterized. The PCR product (1.1 kb) was purified, ligated in the pTZ57R/T and was used for transformation of DH5 cells. The presence of the gene in the recombinant pTZ57R/T was confirmed by restriction analysis. The gene was sub-cloned in pET21a and expression was examined using BL21 CodonPlus (DE3) cells. The recombinant xylanase was expressed as an intracellular soluble protein. SDS-PAGE demonstrated the purified recombinant xylanase as 37 kDa protein. Xylanase showed its optimal activity at 90{degrees}C and pH 7. The enzyme was found thermostable and retained 67% activity after an incubation of 1.5h at 90{degrees}C in the presence of Mn2+. The xylanase activity was enhanced in the presence of Triton X-I00 while the presence of SDS, Tween 20 and Tween 80 showed a declined impact on the activity. Kinetics studies showed the Vmax and Km values of 2313 mol/mg/min and 3.3 mg/ml respectively. The 3D structure analysis demonstrated the presence of a conserved active site comprised of two glutamate and substrate accommodate sites comprised of + 1, +2 and -1, -2 xylose binding sites in the structure of xylanase. The ability of this thermostable xylanase to work at a wide range of temperatures and pH makes it a suitable candidate for industrial applications.

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Waris, A., Awan, A. R., Wasim, M., Hashmi, A. S., Rashid, N., Firyal, S., Khalid, A., Tayyab, M.. 2024-09-07. 3D modeling of thermostable xylanase from Thermotoga naphthophila a member of GH10 family: characterization studies of recombinant xylanase. https://doi.org/10.1101/2024.09.06.611769

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