Expression of a copper activated xylanase in yeast: Differential properties of C-tagged or untagged recombinant xylanases

Background: Endo-xylanase (EC 3.2.1.8), which hydrolyze the backbone structure of β-1,4-xylans, can be used together with laccase (activated by copper) to hydrolyse lignocellulosic and hemicellulosic materials in bioethanol production. A previous study described the cloning of the xylanase gene from Aspergillus niger US368 and obtaining the recombinant His-tagged protein using E. coli expression system. This recombinant xylanase was activated by copper which is a strong inhibitor for xylanases activities. The activation of the enzyme toward copper is due to the 36 extra amino acids provided by the vector pET28a. However, the expression of this xylanase was relatively low and intracellular that limits its potential industrial application. Hence, we aimed to over-express this interesting enzyme using the Pichia pastoris system for use in biofuel production. Results: A copper activated xylanase produced by E. coli BL21 was expressed in Pichia pastoris using the pGAPZαB expression vector. Two recombinant xylanase forms were obtained (Non-C-terminal tagged-His-tagged r-XAn11 and C-terminal tagged-His-tagged r-XAn11). The findings revealed that the two recombinant xylanases displayed different behaviors toward the copper. In the presence of 3 mM Cu 2+ , the relative activity of the Non-C-terminal tagged-His-tagged r-XAn11 was enhanced by about 52%. However, the xylanase activity of the C-terminal tagged one was strongly inhibited by copper. The Non-C-terminal tagged recombinant enzyme was more thermostable, in the presence of 3 mM Cu 2+ , than the C-terminal tagged one with a half-life of 10 min at 60 °C. 3D models of the two recombinant forms were constructed. The results showed that the created copper site in the Non-C terminal tagged protein was loosed in the C terminal tagged protein due to the high fluctuation and probably new interactions among the C and N-terminal amino acids. Conclusion: The present work reports the expression and purification of two recombinant xylanases forms (non C-terminal tagged and C-terminal tagged His-tagged r-XAn11).