A fusion was constructed between xyn3A and xyn4B genes, each encoding a catalytic domain of XYN3 and XYN4 bifunctional endoxylanases of Neocallimastix frontalis. The XYN3A4 chimeric enzyme, as XYN3, XYN3A and XYN4 recombinant xylanases, were expressed in the Escherichia coli host system with a C-term (His)(6)-tag. Overexpression was closely examined to enhance the solubility of recombinant enzymes. Best conditions determined by optimization experiments were found as growth and induction temperature of 37 degreesC, with the addition of 1 M sorbitol and 2.5 mM glycyl-betaine to the culture medium. Enzymes were purified to homogeneity by chromatography on SP-sepharose, Cu-IDA and TSK-gel consecutively, and SDS-PAGE analysis of pure enzyme fractions gave molecular masses of 62, 32, 20 and 59 kDa respectively for XYN3, XYN3A, XYN4 and XYN3A4. These values were in agreement with those obtained by the exclusion chromatography. Despite the fact that XYN3A4 showed the same values of temperature (50 degreesC) and pH (7) for maximum activity than XYN3 and XYN3A, the chimeric enzyme XYN3A4 exhibited an improved affinity with a better rate of hydrolysis toward the xylan substrate. However, regarding to the enzyme stability, XYN3A4 (t(0.5) = 52 min) was almost twice less stable than XYN3 (t(0.5) = 90 min) at the operating temperature of 50 degreesC. Another difference was observed with the activation energy values which were estimated in the temperature interval 20-50 degreesC and were shown to be approximately 100, 70 and 50 kJ.mole(-1) for XYN3, XYN3A and XYN3A4 respectively.