The Thermostability And Its Thermostable Mechanism Of Xylanase Xyn10A With High Temperatures Tolerance

To investigate the thermostability and its thermostable mechanism of Xyn10A from Thermotoga thermarum DSM 5069,protein structure modeling and site-directed mutagenesis were carried out in this study to find out the domain involved in the Ca²⁺binding in the Xyn10A.Then we tried to obtain mutants with better stability by mutating some amino acids of the Ca²⁺binding domain.Furthermore,the catalytic residues were also confirmed by the site-directed mutagenesis of key amino acids in the catalytic domain of Xyn10A.The main results are as follows:?1?The recombinant plasmid p ET-20b-xyn-T-E was constructed by inserting the gene fragments of T2A?a peptide with cleavage function?and e GFP?enhanced green fluorescent protein?in plasmid p ET-20b-xyn10A.Then the recombinant plasmid was transferred into E.coli BL21?DE3?for expression.The bacteria showed green fluorescence after induction,and there was still a band near 130 k Da on SDS-PAGE.This results indicate that the plasmid p ET-20b-xyn10A could successfully express and that T2A also have the function of self-cleavage in E.coli.?2?Glutamic acid at position 600 and 707 were separately mutated to alanine to construct mutations E600A and E707A.The original xylanase?Xyn10A?and mutant enzymes were characterized for their enzymatic properties.The specific activity of Xyn10A was 100 U/mg,but the two mutant enzymes both had no activity,indicating that both the two sites(E⁶⁰⁰ and E⁷⁰⁷)are the catalytic residues of the xylanase Xyn10A.?3?Protein structure modeling and site-directed mutagenesis were carried out to find out the domain involved in the Ca²⁺binding in the Xyn10A,and the thermostable mechanism of Xyn10A was discussed.The results of structural simulation and superposition of Xyn10A showed that the conservation of protein structure was much higher than that of amino acid sequence.The local loop region from 712 to 724(⁷¹²IYRDNATKYEIPP⁷²⁴)was proven to be closely associated with Ca²⁺binding in Xyn10A.The multi-point substitution and deletion mutation were introduced into the loop region(⁷¹²IYRDNATKYEIPP⁷²⁴)to construct the mutations Catb1,Catb2 and Catb3.Then the enzymatic properties of three mutants were characterized.Compared to original xylanase?Xyn10A?,all mutants showed poorer thermostability,even lost their ability to bind with Ca²⁺.This indicates that the thermostability of Xyn10A mainly depends on the affinity and interaction between the loop region(⁷¹²IYRDNATKYEIPP⁷²⁴)and Ca²⁺.We speculate that the side chains and backbone of loop region(⁷¹²IYRDNATKYEIPP⁷²⁴)interact with Ca²⁺by forming a stable coordination complex,and the resultant greatly restricts the flexibility and degree of freedom of the catalytic domain of Xyn10A.Therefore,the thermostability and the catalytic ability of Xyn10A could be effectively maintained at the higher temperature.?4?Arginine at position 714,alanine at position 717 and lysine at position 719 were separately mutated to aspartic acid to construct mutations R714D,A717D and K719D.The enzymatic properties of mutant enzymes were characterized.The results showed that the half-life of the mutant R714D was prolonged by 26 min at 90°C compared with original enzyme Xyn10A,indicating that the thermostability of the mutant R714D was improved.It is presumed that it forms a local microenvironment with more negative charge,when the arginine at position 714was converted into negatively charged aspartic acid,together with the tyrosine containing phenolic hydroxyl group at position 713 and the aspartic acid at position 715.Then it enhanced the interaction with Ca²⁺,resulting in increased thermostability of the mutant R714D.