Characteristic Studies On The PSL Of Xylanase Xyn11B From Sorangium Cellulosum So0157-2

Sorangium cellulosum can grow with crystal cellulose (filter paper) as the sole carbon source. The efficient degradation of filter paper relies on the contact with bacteria cells. A epothilone producing strain S. cellulosum So0157-2 was isolated previously which can grow with filter paper or xylan as the only carbon source. The genome of S. cellulosum So0157-2 contains a series of genes encoding xylanases from different GH (glucoside hydrolase) families.S. cellulosum So0157-2 Xyn11B, which belongs to GH11 family, contains a unique polyserine(PSL) region between the lipoprotein signal peptide and the catalytic domain. While proteins containing PSLs are rare among prokaryotes, their hosting bacteria show difference in habitats and taxonomic positions, suggesting that the existence of PSLs are not accidental but to some extent a general phenomenon. On the other hand, these PSLs share significant similarities in amino acid sequence and composition despite their difference in lengths, suggesting the PSLs are conservative regions with significant functions. PSLs are predicted to be disordered and thus susceptible to protease attack, requring some protection against proteolysis. Since the PSL tract of honeybee AmVg is phosphorylated and resistent to trypsin and chymotrypsin digestion and the O-glycosylation of C.fimi Cex Pro-Thr linker also makes this region resistent to protease attack, similar modifications can be expected on Xyn11B PSL region.Expression vectors pET-22b-Xyn11B and pGEX-6P1-Xyn11B were constructed and tranformed into E.coli BL21(DE3). Phosphorylation was not detected on Xyn11B expressed in E. coli, possibly due to the lack of specific kinases in E.coli compared to S. cellulosum So0157-2. Degradation bands of Xyn11B were detected by Western Blot when expressed in pET-22b, which might be caused by protease attacks on disordered PSL region. Removal of GST tag from GST-Xyn11B caused degradation of Xyn11B, while GST-Xyn11B-C (PSL region deleted, only catalytic domain of Xyn11B left) released both GST and Xyn11B-C bands, suggesting PSL may exert some influence on the two potential cleavage sites in the catalytic domain.The PSL region of Xyn11B was randomly truncated at seven sites and expressed in pGEX-6P-1. Removal of GST tag of the seven truncated proteins by PPase showd that S48 was degraded while other proteins with shorter PSLs were not, suggesting that longer PSL might cause degradation more easily than shorter ones.Secreted expression of Xyn11B in Pichia pastoris GS115 was performed and Xyn11B was purified from the supertant in a glycosylated form. Removal of N-linked glycosylation with PNGaseF generated a smaller band but still much larger than the expected molecular weight, suggesting the existence of O-linked glycosylation, which might occur on the PSL region. Limited proteolysis raleased only a small amount of catalytic domain, with most of Xyn11B uncleaved, supporting the view that O-linked glycosylation protects protein from proteolysis.