Expression of a Copper Activated Xylanase in Yeast: Location of the His-Tag in the Protein Significantly Affects the Enzymatic Properties.

Abstract

A copper activated xylanase produced by E. coli BL21 was expressed in Pichia pastoris using the pGAPZαB expression vector. Two recombinant GH11 xylanase forms were obtained (N-His-rXAn11 and N-C-His-rXAn11). The findings revealed that the two recombinant xylanases displayed different behaviors toward the copper. In the presence of 3-mM Cu2+, the relative activity of the N-His-rXAn11 was enhanced by about 52%. However, the xylanase activity of the N- and C-terminal tagged one (N-C-His-rXAn11) was strongly inhibited by copper. In the presence of 3-mM Cu2+, the N-His-rXAn11 revealed to be thermostable at 60°C with a half-life of 10min. However, the N-C-His-rXAn11 was noted to be unstable since it was inactivated after 15min of incubation at 55°C. 3D models of the two recombinant forms showed that the created copper site in the N-His-rXAn11 was loosed in the C-terminal tagged protein. The C-terminal tag could trigger some structural changes with a notable displacement of secondary structures leading to great hindrance of the active site due to high fluctuations and probably new interactions among the N- and C-terminal amino acids.