Improvement Of The Thermostability Of Xylanase By C-terminus Replacement

Xylanases are glycosidases that degrade xylan to xylooligosaccharide and xylose.Xylanase have been successfully introduced to a wide range of industrial applications and processes,including the paper,food,feed and bioconversion industries.With high catalytic activity,our enzyme Xyn A can not work well in t he engineering environment involving high temperature,so the transformation of improving thermal stability is making a great sence.The structural description of the Xyn A is a ?-jelly-roll architecture in the Protein Data Bank.The ?-jelly-roll architecture can be regarded as three components: the upstream of the ?-helix,the ?-helix and the downstream of the ?-helix.The ?-jelly-roll architecture is classic and common in varieties of Glycoside Hydrolases family,and it is highly conserved.The glycanase(Glu)that we preserved has the same architecture with Xyn A,and its theromostability is optimistic.Considering that many hydrogen bonds between the ?-strands stabilize the framework,the mutant enzyme(Xyn?-?x-Glu?)is obtained by means of replacing the downstream of the ?-helix from Glu into the Xyn A.The replacement should be possible to improve the theromostability of enzyme molecular.Obtained results are as follows:(1)The recombinant plasmid(pET20b-Xyn?-?x-Glu?)builds by a new way that gene connected with vector successfully.In this way,fusion gene and template have some complementary sequences.They were connected to line by PC R.The recombinant vectors were obtained by way of T4 DNA ligase cyclizing the line fragment.(2)The result display that expression level is extremely low.The level of protein expression was improved strikingly by means of codon optimization from Top-Ten amino acid of the recombinant gene sequences.However,most of the proteins existed as inclusion bodies.(3)Many attempts have been made to remove inclusion bodies in this paper.By connecting recombinant gene(Xyn?-?x-Glu?)with pel B leader from p ET-20 b,the secretory plasmid was constructed,and the extracellular soluble proteins were produced successfully.The extracellular enzyme activity increased significantly until 6.5U/ m L,at the time of 18 h.(4)The p H optimum(pHopt)of extracellular recombinant protein Xyn?-?x-Glu? is 5.2,significantly improved than the native Xyn(p Hopt 3.8).The optimal activity temperature for Xyn?-?x-Glu? is 55?,whereas the Topt for Xyn activity is 47?,which indicates that Topt improved obviously after the transformation.The t1/2 of recombinant protein is 27 min at 55?,increased 19 min than the native Xyn's 8 min.It can be seen that after the replacement of Xyn's downstream ?-sheet with the same structure from Glu according to the homology and conservative of spatial structure,the mutant enzymes' s Topt and t1/2 at 55? are greatly improved,compared with the native Xyn.These provide new research ideas for enzyme molecular modification based on structure.