Orientation of Cel5A and Xyn10B in a fusion construct is important in facilitating synergistic degradation of plant biomass polysaccharides

Abstract

One approach to achieve efficient and economical saccharification of plant biomass would be using thermostable and multifunctional enzymes from hyperthermophiles such as Thermotoga maritima. Thus, the bifunctional constructs, Cel5A-Xyn10B and Xyn10B-Cel5A, were produced by fusing cellulase Cel5A at the N- and C-terminals of xylanase Xyn10B, respectively. The Cel5A-Xyn10B fusion construct showed cellulase activity of 1483 Uμmol-1 against carboxymethyl cellulose, which was nearly the same as that of Cel5A in the free form. However, xylanase activity of this construct increased by 2-fold against beechwood xylan as compared to that of Xyn10B in free form. The synergistic effect between Cel5A and Xyn10B in the form of Cel5A-Xyn10B fusion resulted an overall increase in the release of reducing sugars. However, Xyn10B-Cel5A showed about 60% decrease in activities of both the component enzymes as compared to their activities in the free form. Both the fusion constructs were active in a wide range of pH from 4.0 to 9.0 and temperatures from 50 to 90°C. Nearly 80% of cellulase and xylanase activities were retained in Cel5A-Xyn10B fusion after incubation at 60°C for 1h. Secondary structures of the component enzymes were retained in the Cel5A-Xyn10B fusion as observed by circular dichroism spectroscopy. Docking and simulation studies suggested that the enhanced xylanase activity in Cel5A-Xyn10B was due to the high binding energy, favorable orientation of the active sites, as well as relative positioning of the active site residues of Cel5A and Xyn10B in closer vicinity, which facilitated the substrate channeling.