Association Study Of Thermal Stability And Structural Characteristics Of The .11 Family Xylanase

The protein structure determintes its function.Some structural basis like disulfide bond,ion pairs,hydrophobic effect may affect the protein thermostability.As the protein interior hydrophobic region of thermostable proteins and mesostable homologues are both packed almost as efficiently as possible,mutation in the protein interior hydrophobic region are often destabilizing,most current stabilization strategies are directed on the protein surface.In the presented researches,by using directed evolution in combination with site-directed mutagenesis,we identified a point-mutation G201C taken place in protein interior hydrophobic region in a family 11 xylanase(XynC) from Neocallimastix patriciarum.G201C is hydrophobically associated with C50 rather than the formation of a new disulfide bond between C50 and C201.RosettaDesign and point mutation using residues with different hydrophobic nature demonstrate that the thermostability of the variant is correlation with hydrophobicity of the residues in site 50 and 201.Two mutants,G201C and C60A-G201C,were identifed greatly increased thermostability than the wild-type. Both in site 50 and 201 of these two mutants are cys,which is considered as the most hydrophobicity residue.The far-ultraviolet circular dichroism signal showed that the transition temperature(T_m) of the mutant G201C is about 9℃higher than that of the wild-type,while the C60A-G201C about 12℃higher.This paper first found a hydrophobic interaction between two cysteines,this interaction stabilizes the protein by decreasing the entropy of the unfolded state.Accompanied with the increased thermostability,these two mutants also possess higher special activity than their parent at all tested pHs and temperatures.This suggests that cysteine could stabilize the protein not only by the formation of disulfide bond,but also for its strong hydrophobicity in the protein interior hydrophobic region.The obtained mutants with higher thermostability and special activity are implied their potential applications in industrial processes.Our results suggest that we could improve the protein thermostability through modifing the residues in protein inner hydrophobic region.On the other hand,extrinsic factors such as substrate molecules have long been known to stabilize the enzymes.Many thennostable enzymes have been improved intrisincally by site-directed mutagenesis,but increasing the thermostability of the enzymes in the presence of substrate by such mutation method is rarely been reported. Here,we report a mutant(D57N) of XynC in the active site could be stabilized by the substrates while the wild-type not.Despite the thermostability of these two enzymes are almost identical in the absence of the substrate,the mutant enzyme displays an increased optimal temperature.The potential hydrogen bond between N57.E202 of the mutant and the substrate molecule may account for this stabilization.These results suggest that enzyme thermostablity engineering processes should consider the stabilization effect by the substrate.The 'thermophilic' mutants which could be stabilized by the substrate from such engineering processes will deepen our understanding about the binding mechanism between the enzyme and the substrate.