Analysis Of Binding Sites Architecture In CBMs And Study Of Protein Thermostability In TrCel12A

The efficient conversion and utilization of cellulosic biomass is an important subject to solve the problems of resource misallocation,environmental pollution and energy shortage.Most of the natural cellulose degrading enzymes have catalytic modules(CDs)and carbohydrate binding modules(CBMs).CBMs have the function of recognition and binding of substrates to assist CDs on the hydrolysis of substrates.There are two strategies to improve the catalysis efficiency of cellulose.One approach is improve the binding force of enzyme on the cellulose substrate by increasing the number of CBMs modules,or improving CBMs' and CDs' binding force;Besides,to improve the thermostability of enzyme.The biorefinery industry endures a high temperature environment during production that accelerates reaction rates and reduces contamination by killing miscellaneous bacteria,thereby thermostable enzyme are needed to improve degradation efficiency and reduce production costs.TrCel12A of GH12 family consists of only a CD module and 234 amino acids,is a mesophilic endocellulase,its structure is rather simple,and it has great room for improvement.In view of the uncertainty of directional evolution and long time consumption,bioinformatics and molecular dynamics stimulation have been used in this paper,studying the binding modes of the different CBMs families by functional structure analysis,and to screen a proper CBMs module to build a recombination with TrCel12A.Molecular dynamics stimulation has been used on the investigation of thermostable mechanism of TrCel12A,located potential mutation targets on N-Terminal.Combination of computing methods and experimental methods can greatly reduce the workload of experiments.The main contents and results are as follows:(1)CBMs can fold and function itself.In the CAZy database,the CBMs with sequence similarity above 30%is attributed to the same CBMs family and has similar structural characteristics.The analysis of functional architecture of 7 CBMs families is of great importance to study the functional hybridity and to design mutations to improve binding force of CBMs rationally.This study found that the CBMs functional architecture have preference of aromatic amino acids(W,Y)and polar amino acids(Q,R,N).CBMs with different binding patterns have different preferences for acid-base amino acids,which is the molecular basis of recogniting different substrates;CBMs in different families maintain capacity of binding celluose by increase the number of subunits or increase the number of amino acids on each units;Functional architecture sequence spectrum showed that different families have different subsites to bind substrates and a relatively small number of conserved amino acids,usually aromatic amino acids(W,Y)and weakly polar amino acids(N,Q),most of which are distributed in type A CBMs;Conserved amino acids recognize and binding to certain oxygen atoms of sugar rings,thus CBMs can identify substrates with similar structure partially,which may be the structural basis for non specificity of CBMs families.(2)Analysis of thermostability of TrCel1 A by molecular dynamics simulation.In this paper,Gromacs software was used to simulate the solvent environment,and three temperature gradients(300K,350K,400K?500K)were set up.Analysis of RMSF showed that the distribution of flexibility on protein was uneven,and the N-terminal and active site of protein were highly flexible sites;The simulation results of four N-terminal mutations located on different secondary structures indicate that extending the side chain of amino acids(G41F)and introducing charged groups(Q6K)can enhance the internal interaction of N-terminal,thereby enhancing the overall structural stability of protein;S25P reduced the flexibility of a-helix by adding an amide ring,but the stability of single secondary structure did not improve the thermostability of the overall protein.(3)Effect of N-terminal mutations on thermostability of TrCel 1 A.Four mutants were expressed via Escherichia coli expression system and their catalysis activities had been measured at 50?,55? and 60?.The catalysis activity of G41F and Q6K of 55 ? and 60? was higher than that of wild type.The Tm values of Q6K and G41F measured by DSC were higher than those of wild type,which showed that G41F and Q6K improved the thermostability of proteins.All in all,stabilizing the N-terminal structure is an effective way to improve the thermostability of cellulose,and the accuracy and reliability of the molecule dynamic simulation has been proved.