| Sorangium cellulosum is a kind of myxobacteria that can grow on mineral salt medium containing crystal cellulose filter paper or xylan as sole carbon source.S.cellulosum So0157-2 is an epothilone producing strain isolated and extensively studied in our lab.Its genome sequencing revealed numerous genes coding xylanase related to xylan,the key component of hemicelluloses,degrading.Bioinformatics analysis of the xylanase in S.cellulosum So0157-2 genome revealed that one of the xylanases,Xyn11B,is unique in some conserved domains/modules compared to the common xylanase.In addition to the conserved domain of the glycoside hydrolase 11 family(GH11),Xyn11B possess some specific regions or sequences.First of all,there is a N-terminal typical lipoprotein signal peptide(type ?signal peptide)which will sort protein to anchor cell membrane or outer membrane,rather than a conventional type ? signal peptide leading protein secreted out of cell.Secondly,there is a polyserine linker(PSL)between the signal peptide and the GH11 catalytic domain,the number of serine residues reaches a high level of 86%(51/58).Thirdly,compared to the conventional GH11 catalytic domain,two additional insertions appear in the catalytic domain,and there is no carbohydrate-binding module(CBM)as in the general endo-xylanase.So,Xyn11B is an important representative of the diverse xylanase in nature as the result of long evolution history.We focus on it to reveal the function and mechanism of these particularities.Previous study showed that Xyn11B was degraded when expressed in multiple vectors of Escherichia coli,and the target protein could not be obtained.Only when the catalytic domain was expressed,not only the target protein but also the enzyme activity was obtained,indicating that the existence of PSL had effect on protein stability.So the PSL truncations were constructed to further study the effect of PSL length and sequence on the target protein.So we constructed 8 PSL truncations random and expressed in pGEX-6P-1.When remove the GST tag by Ppase,except for S48 and S61,which the target protein was degraded,Other truncations could be obtained for different degrees of stability of the target protein.Found that the more complete retention of PSL,the more easily the target protein degradation,carrying the shorter PSL,the more stable the purpose of protein.It is proved that the presence of PSL and its different length affect the stability of the target protein.And found that when the presence of PSL,xylanase could resistant to acidic conditions.But it is not possible to establish the relationship between sequence characteristics and protein stability.By analyzing the enzymatic properties of PSL truncated mutants,it was found that the presence of PSL could tolerate strong acidity.The results provide a basis for further analysis of the existence,stability and function of the protein in the original strain S.cellulosum So0157-2,which provides the basis for obtaining the stable protein which can be used for research and application.When constructing the PSL truncations and measuring its enzymatic properties,it was found that the amino acid sequence which located at the N-terminus of the catalytic domain could significantly increase the optimum temperature of the enzyme.The optimal temperature of D111 is 65?,while the optimum temperature of Xyn11B-C(T131)is 40?,they differ by 20 amino acids,but the optimum temperature difference is 25 ?.There are many factors that affect the optimum temperature,including hydrogen bonds,ionic bonds,hydrophobic ring hydrophobic,disulfide bonds and so on.By analyzing the three-dimensional structure diagram of Xyn11B,it is found that there is a cross-staggered hydrogen bond between Y123-T131,which can not be accurately mapped to one or several amino acids.And there are three tyrosine residues in this amino acid sequence,respectively Y123,Y126,Y130,suggesting that the interaction between the benzene ring leads to a significant increase in the optimum temperature.Using the method of site-directed mutagenesis,it is further determined that the interaction between Y126 and the W346 in the catalytic domain(Y126 and W346)has a significant effect on the optimum temperature.By measuring the temperature tolerance of the mutant,Xyn11B-S and Y126A/Y130A were able to withstand high temperatures and still survived after treatment at 100?for 60 min.And Y130A could not tolerate high temperature,and the enzyme activity was significantly decreased after treatment at 70? for 20 min.The W346A mutant could not tolerate high temperature,too.After 5min treatment at 60?,the enzyme activity was almost completely lost,indicating that the thermal stability of Y130 and W346 had an important impact.The amino acid sequence between Y123 and T131 significantly increased the effect of the optimum temperature.Whether it was universal we selected Xyn11C from S.cellulosum So0157-2 as the modified object with the optimum temperature of 40?and belonged to medium temperature xylanase.The sequences between Y123 and T131 were added to the mature protein dlp-Xyn11C and the catalytic domain Xyn11C-C,respectively.It was found that the optimum temperature was increased by 6?and 8?,respectively,which indicated that the optimum temperature could be improved by adding the sequence directly to the whole sequence or the catalytic domain.But the determination of its temperature tolerance found,still can not tolerate high temperatures.In this paper,the method of truncated or site-directed mutagenesis was used to show that the interaction between aromatic rings played a significant role in the optimum temperature of the protein.So as to provide some theoretical basis and ideas for the optimum temperature and heat resistance of the enzyme and adapt to the corresponding application target.Previous experiments had shown that the expressed Xyn11B catalytic domain(Xynl 1B-C)acted on birch xylan when the product is only xylose.When analyzed the Xyn11B-C crystal structure,it was found that the two insert sequences in the Xyn11B catalytic domain were located near the "thumb" of the enzyme structure,named loopland loop2,compared with Trichoderma reesei Xylanase XYNII.Because the "thumb"of GH11 xylanase is thought to play a key role in the catalytic reaction,it is presumed that these two loops are the cause of Xyn11B-C enzymatic hydrolysis of xylan products as xylan.Previously,loop1 was reduced to four GH11 family xylanase shared amino acids,and the enzyme activity was completely lost,which was not related to the product of xylan disaccharide.In this study,loop2 was deleted from Xyn11B-C to construct the mutant protein Xynl 1B-S2,TLC product analysis found that the product is no longer xylose,and a series of oligosaccharides.Showing typical characteristics of endo-xylanase.The results indicate that the presence of loop2 is the cause of the Xyn11B product as a single xylose.The results of this study provide a way for the transformation of xylanases,the production of specific products of the spectrum of oligosaccharides. |
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