Study On The Improvement Of Thermal Stability Of Aspergillus Niger ?-1,4-Endo-xylanase By Introducing N-Glycosylation Sites

Objective:xylananse as one of the most widely used industrial enzymes in the industry,it can be used in food,paper,feed and other fields.In industrial,there are large demand for high temperature and alkali resistance enzymes,but natural xylanases are mostly medium temperature enzymes and are not stable enough in an alkaline environment to meet the needs of industrial production,which making xylanases became bottleneck in the industrial field by widely used.Thence improving the thermal stability of xylanase has become a research hotspot in the field of application.N-glycosylation is one of the most important post-translational modifications in eukaryotic cells.It not only facilitates the exchange of information between cells and the recognition of proteins and proteins,maintains the structural integrity of proteins,promote the formation of disulfide bonds and protein secretion,but aslo plays an important role in maintaining the stability of protein structure and improving the stability of proteins in polar environments.In this study,Aspergillus niger IA-001?-1,4-endo-xylan?xyn B?was consider as the modification object.Based on computer introduced N-glycosylation site for xyn B by the rational design.During the eukaryotic expression process,xyn B will automatically introduce sugar chain molecules,to explore the feasibility of N-glycosylation modification to improve the thermal stability of the protein.And quest a new strategy for molecular thermodynamic modification of proteins.Method:?1?Using the Discovery Studio 4.5 to homologously model the?-1,4-endo-xylanase xyn B and optimize the initial model to obtain xyn B the three-dimensional structure of the highly reliable;?2?Determine the xyn B active site key amino acids by molecular docking;?3?Analysis of the secondary structure of the xyn B protein three-dimensional model,to finding the?-turn and oscillating Loop region located on the surface of the protein which was as a candidate sequence for mutation;?4?avoiding key amino acids to constructed Asn-X-Thr mutant by amino acid virtual mutation,and the spatial rationality of glycosylation at the mutation site was evaluated by Gly Prot.At last,using the kinetic simulation to optimize the mutant.?5?Finally,the mutants was expressed recombinantly in Pichia pastoris SMD1168 and analysis for enzymatic properties.Result:in this study,four mutants were selected by homology modeling,amino acid virtual mutation and kinetic simulation.xyn B^G48>T,xyn B^G66>N,A68>T,xyn B^{G66>F,D67>N,G69>T},xyn B^{D130>N,N132>T} and xyn B^A92>N,D94>T,recombinant expression and enzymatic properties were analyzed.In the thermal stability analysis,the protease was essentially inactivated after incubation with wild-type protease at 80°C for 30min,while the mutant xyn B^G66>N,A68>T and xyn B^{G66>F,D67>N,G69>T}were kept at 80°C for 30 min,and the residual enzyme activity was increased by more than 30%than the wild type.The mutant xyn B^A92>N,D94>T can be 60-80°C.The residual enzyme activity in the 30 min incubation period was higher than that in the wild type,but the degree of change was not significant.The mutant xyn B^{D130>N,N132>T} had no significant change compared with the wild type.In the optimum p H analysis,the optimum p H of xyn B^{G66>F,D67>N,G69>T} was 6.0,which was shifted from the wild type.In the p H stability analysis,the residual enzyme activity of the wild type decreased after p H value above 7.0,xyn B^G66>N,A68>T and xyn B^{G66>F,D67>N,G69>T} at p H8.0-10.0.The high residual enzyme activity remained in the range and showed an increasing trend,indicating that the mutant had better stability in an alkaline environment.The kinetic parameter analysis of the enzyme showed that xyn B^{G66>F,D67>N,G69>T}Michaelis constant?Km?was three times that of wild type,while mutants xyn B^A92>N,D94>T and xyn B^{D130>N,N132>T}the reaction rate?Vmax?was increased by a factor of 2 compared to the wild type.Conclusion:Introduction of N-glycosylation at the?-turn and Loop regions of the protein surface can improve the enzymatic thermal stability of Aspergillus niger?-1,4-endo-xylan,which molecular modification strategy can provide a meaningful reference for thermal stability modification of other enzymes.