Design Of Stable Hybrid Xylanase Based On Structural Bioinformatics Methods

Zymin is the cornerstone of the rapid development of industrial biotechnology.Designing an efficient and stable zymin is a key scientific problem that needs to be overcome in the era of industrial biotechnology.In recent years,the development of protein engineering technology has facilitated diverse designing strategies.However,the inadequate understanding of the relationship between structures and functions of enzymes often fails to produce the expected results.The emergence of biological big data has promoted the birth and development of a new rational design strategy of enzymes by the guidance of structural bioinformatics.This strategy starts with the analysis of protein families,comprehensively considers the relationship between structures and functions,effectively reduces the search strength of sequence space,and then realizes the construction of a "small and smart" library.In this paper,based on the structural bio informatics,the sequences and structures of the alkali-resistant/halotolerant xylanases of GH11(Glucoside Hydrolase)family were further excavated and analyzed,and the surface characteristics of these enzymes were explored.In combination with the mapping of sequence profiles of surface chargedresidues,nine surface charged residues of 77XynA(Thermomyces lanuginosus Xylanase A)were specifically localized,and alkali-resistance and halotolerance of TlXynA were successfully improved by different numbers mutations.Finally,the application of related strategies was verified in the GH12 family.The major results of the dissertation are as follows:1.Structural bioinformatics analysis of alkali-resistant/halotolerant enzymes of GH11 familyThe function of the key residues in the active architecture of enzyme determines the efficiency of binding and catalytic cleavage.However,the surface charged residues also have important effects on the properties of enzyme,including predominantly halotolerance,pH resistance and ionic denaturant tolerance,etc.In this dissertation,the structural bioinformatics analysis of the alkali-resistant/halotolerant enzymes of GH11 family revealed a significant difference between the surface characteristics of TlXynA and these enzymes.Further sequence alignment revealed that the surface charged residues of TlXynA mostly correspond to neutral uncharged residues such as N/Q/S/T of these enzymes,which may lead to functional differences such as alkali-resistance/halotolerance.Therefore,by mapping the surface sequence profiles and analysing the mutation frequency of the surface functional residues of the enzyme,nine charged residues on the surface of TlXynA were selected as mutation sites,and different numbers of combinatorial mutations were identified,which indicated the direction of experimentation.2.Qualitative characterization of series alkali-resistant mutants of TlXynAThe surface charged residues,D/E,of TlXynA were selected and mutated to neutral ones belong to the alkaline enzymes of the same family.Through the construction of E.coli heterologous expression system,the heterologous expression of related proteins were successfully realized.Through the determination of enzyme activity,kinetic parameters,DSC,and alkali-resistance,we found these surface charged residues can affect the expression level,enzyme activity,thermostability,alkali-resistance of TlXynA,and different numbers of combinatorial mutations have different effects on properties of enzyme.Too much negative residues of TlXynA may be detrimental to its alkali-resistance,which can be improved by purposeful mutation.Mutant aM3(E63N-E109N-E31S-D143S)showed the most significant improvement in alkali-resistance,with an increase of 55%at pH 11.0.3.Qualitative characterization of series halotolerant mutants of TlXynACombined with structural bioinformatics analysis,the K/R residues on the surface of TlXynA were mutated to be electronegative ones in order to improve its halotolerance.Experimental determinations endorsing the adverse effect of surface positively charged residues of TlXynA on halotolerance,and changing them to negative or polar residues can significantly improve its halotolerance.The halotolerance of mutant hMl(R116D-R161S)is most significantly improved and it can be tolerant of 5M NaCl and 3M Na2SO4.However,changing only two surface charged residues did not improve TlXynA’s tolerance to ionic denaturants SDS.4.Application promotion of alkali-resistance design strategy in GH12 familyGH12 family is a cellulase family with the simplest domains and belongs to GH-C superfamily with GH11 family.Therefore,in order to verify the universality of alkali-resistance designing strategy of TlXynA,the cellulase TrCel12A of GH12 family was used as material to construct two alkali-resistant mutants.It was determined that the both mutants reduced activity largely and alkali-resistance failed to be increased.However,the mutations caused changes in their acid-resistance and product spectrum.The related data indirectly indicate that changes in the surface charge characteristics can affect the properties of the enzymes.In future studies,searching for richer reference information and identifying more hot spot residues may improve the alkali-resistance of TrCel12A.