Localization Of Xylanases In Sorangium Cellulosum So0157-2

The aerobic gram-negative bacteria Sorangium cellulosum is the few group in Myxobacteria which can degrade cellulose and hemicellulose efficiently. Strain So0157-2can degrade filter paper (crystalline cellulose) efficiently,showing the degradating characteristics of contacting dependency.While it can grown in medium where xylan (main composition of hemicellulose) as the sole carbon source.Gene sequence analysis shows that So0157-2contains complete xylanolytic enzyme system including the endo-xylanase, xylosidase, esterase, and other hydrolases.There are at least22endo-xylanases from different glycoside hydrolase family,these xylanases homodisperse in the genome.Analysis on the motif character showing that the model of xylan degradation exhibit novel properties, which is different from the cellulosome strategy and the free enzyme strategy.Lipoprotein accounts for a high proportion.These would reflect the uniqueness of the process of xylan degradation of S.cellulosum.So0157-2grow in three culture media with different carbon sources (M26, G-CNST, X-CNST),we separate the total protein to three components, getting the extracellular fractions, membrane fractions and intracellular soluble fractions.All these fractions show xylanase activity while membrane fractions accounted for more than50%of the proportion, playingan important role in xylan degration.As to proteins from extracellular fractions,we attempt identificating the exist of xylanase through active electrophoresis.We concentrate proteins to run active electrophoresis,and use2D zymography,but failed to identificate the target protein; finally we choose toammonium sulfate precipitation method and after active electrophoresis,we get4objective proteins, three of them are endo-xylanases, and a CBM. In the M26medium, with the same method,we identify2endo xylanase from ntracellular fractions. Notably,the xylanase7384has been repeatedly identified, playing a crucial role in the degradation of xylan.Due to the high proportion of lipoprotein in the enzyme system, we choose Xyn11B for studying.Xyn11B has the typical characteristics of lipoprotein signal peptide, according to reports in the literature,Xyn11B is likely localized in the inner or outer membrane.To avoid the immune cross interference in origin bacteria Sorangium cellulosum, we will make the heterologously expression in E.coli.We express the full-length sequence in BL21-pET-22b-Xyn11B, and the sequence without signal peptide in BL21-pET-22b-Xyn11B-dlp.From detecting the xylanase activity and Western blot,we confirm the two protein expressed successfully.The two recombination strain provides the basis for following study of protein localization. Sorangium cellulosum is rich in xylanase.study of the distribution of these xylanases at protein level,will help us to understand the method of So0157-2using xylan, lay the foundation for exploring their new strategy in degrading cellulose and hemicellulose.