Impact Of Lipoprotein Signal Peptide On The Location Of Xylanase Xyn11B From Sorangium Cellulosum So0157-2

S.cellulosum So0157-2 has the ability to grow on inorganic plate with filter paper or xylan as the sole carbon souce. Because of the heterogeneity and complexity of celluloses, they need many kinds of cellulase working in coorperation to be degradated. And this suggests that S.cellulosum So0157-2 is rich in cellulases and semi-cellulases which can be used to utilize the cellulose and semi-cellulose resource.Utilization of insoluble polysaccharide subtrates by S.cellulosum is a cell-contact manner. Cellulases and xylanases activity detection showed that the enzyme activities were cell contacted and extracellular enzyme activities were extremely low. The above evidences made us speculate that the regarding enzymes could be binding to the cells. Analysis of So0157-2 genome showed that: Firstly, there were no I signal peptide which guided the proteins excret extracellularly in many polysaccharide degrading enzymes. This agreed with the phenomenon that there were low enzyme activities outside the cells, which illustrated that polysaccharide degrading enzymes weren’t excreted extracellularly to work in cooperation; Secondly, no homologous sequences to scoffdin and dockrin of cellulosome were found in So0157-2 genome, which implied that there was no cellulosome in So0157-2. Therefore, S.cellulosum So0157-2 may employ a new pattern different from the two classic microorganism cellulose degradating systems of cellulose degradation.Previous studies of Xyn11B showed that it included three parts:lipoprotein signal peptide, polyserine linkers and catalytic domain belonging to GH11. The fisrt two characteristics were rare in polysaccharide degrading enzymes, which helped draw our attention of Xyn11B. Active expression of catalytic domain of Xyn11B proved that corresponding enzyme activity was also existed in original bacteria So0157-2. Catalytic domain of Xyn11B possessed exo-xylanase properties and catalyzed xylan to xylobiose, which was not been reported in GH11. The subcellular localization of Xyn11B guided by lipoprotein signal peptide and sorting sequence properties are closely related to the substrate degradation properties and physiological functions, also are one of the basis to solve its degradation stragy. Lipoprotein can guide lipoprotein localized to inner or outer membrane, and the amino acid sequence after cleavage site make the final decision. Over 50％xylanase activity was spotted in the membrane of So0157-2 based on previous studies, illustrating that xylanases in the membrane played a vital role. In this study, we hope to obtain an evdience for conjecturing S.cellulosum So0157-2 cellulase degradation system through exploring the impact of lipoprotein signal peptide on localization of Xyn11B.In this study, the N-terminal lipoprotein signal peptide and amino acid sequence following the cleavage site were undergone bioinformatic analysis. Bioinformatic analysis of the 237 glycoside hydrolases in So0157-2 indicated that there were 35 lipoproteins, in which 9 turned out to be xylanses. And in family GH11 from bacteria, there were 19 lipoproteins with 5 belonging to S.cellulosum. The bioinformatic analysis above indicated that lipoproteins shared certain propotion in glycoside hydrolases and they may play an important role in cellulose utilization.We chose heterologous expression in myxobacteria DK1622 which were closely related phylogenetically with S.cellulosum and E.coli which was also gram-negative due to the genetic operation limitations in indigenous microorganism. And two parallel experimenal groups were set up incluing the whole Xyn11B gene and the Xyn11B-dlp without lipoprotein signal peptide.pZJY41 was chosen as the expression vector in myxobacteria DK1622, and two reconstruction vectors were built named pZJY41-X11B and pZJY41-X11B-dlp. We could not determine the expression of target protein through xylanase activity measurement with 1％xylan from beech wood. The reason may be xylan from beechwood is not the optimum substrate for Xyn11B. With anti-Flag monoclonal antibody blotting, target protein has been defined to expressed. Extracellular components concentration, whole cell lysis, cytoplasmic components and membrane components were obtained from component fractionation. Western blot detection of bacteria with reconstruction plasmid pZJY41-X11B showed that strong positive signal were around 25kDa and 33kDa localizing mainly in cytoplasmic components, which represented the catalytic domain of Xyn11B and Xyn11B without lipoprotein signal peptide respectively. Western blot detection of bacteria with reconstruction plasmid pZJY41-X11B-dlp showed that the only strong positive signal was around 25kDa, localing in the cytoplasmic components. The above results indicated that the expressed proteins were truncated from N-terminal no matter with or without lipoprotein signal peptide sequence in gene. The lipoprotein signal peptide could not lead its role, so interest proteins were cytoplasmic retention. 4%paraforrmaldehyde-0.5％ glutaraldehyde was testied to be feasible for bacteria fixation before ultrathin section. Immunogold was able to label bacteria specifically after using 10％ H2O2to etch ultrathin section. TEM pictures after immunogold label declared that gold particles were specifically distributed in cytoplasmic, which matched with the western blot detection. The targeting proteins localized intracellularly because of undifinited reasons causing degradation or slicing, so we could not spectulate the impact of lipoprotein signal peptide on Xyn11B location.Then Xyn11B was expressed in E.coli BL21(DE3). pET-22b was chosen as the expression vector. Two reconstruction expression vectors were constructed named as pET-22b-X11B and pET-22b-X11B-dlp. The interest protein was expressed using xylan from birchwood as substrates to detect enzyme activity. However, the quantity of expressed target protein was low. Through components fractionation, we gained extracellular components, whole cell components, cytoplasmic components, periplasmin components and membrane components. P-galactosidase was selected as the marker enzyme of cytoplasmic components. Measurements of β-galactosidase in every components determined the interference of cytoplasmic components to other componets was insignificant. Xylanase activity detection of E.coli (X11B) revealed that over 80％xylanase activity belonged to the membrane components, while that of the E.coli (X11B-dlp) distributed in cytoplasmic components and periplasmic components equally. Western Blot detection of components of E.coli (X11B) showed that strong signals were existed in membrane components, while that of E.coli (X11B-dlp) were existed in periplasmic and cytoplasmic components. However, immunogold labeling showed some boundedness with no observation of specific immunogold in membrane of E.coli (X11B) and perisplamic space of E.coli (X11B-dlp) with TEM pictures.Our results suggested that in bacteria with the insertion of whole Xyn11B gene, lipoprotein signal peptide may help Xyn11B localize to the membrane of BL21(DE3). However, bacteria with the insertion of Xyn11B-dlp, periplasmic localization was emerged. And this kind of periplasmic localization without guidance of signal peptide may result from the particular N-terminal amino acid sequence.