Model Analysis Of Xylanases From Sorangium Cellulosum And Gene Cloning, Expression And Characterization Of Xyn11A/B From So0157-2

Sorangium cellulosum can grow under the condition of crystal cellulose (filter paper) as the sole carbon source. Its efficient degradation relies on the contact between cells and the substrate. The feature of cellulose degradation is the material basis of the growth and the production of active substances. This characteristic is the foundation of the purification and taxonomic of S. cellulosum. However, there are few reports on the cellulose degradation of S. cellulosum.The previous work has realized the cellulose degradation process relies highly on the contact between the bacteria and the substrate. The endoglucanase and xylanase activity could be detected in the washing solvent of the cell. This supposed the enzyme components were combined with the cell. However, the organization of these enzyme components has not been resolved. Moreover, it becomes a tough work to separate the single component because of the combination of the enzyme and the cell. This limits the research of the biochemical characteristic of the enzyme. Our work based on the genome sequencing of S. cellulosum So0157-2, and the publish genome of S. cellulosum So ce56. We analyzed the motif character of the xylanases from these two strains in order to conjecture the model of xylan degradation and the physiology function of xylanase. These would reflect the uniqueness of the process of xylan degradation of S. cellulosum. We cloned, expressed and characterized the xylanase. In the mean time, we could prepare the antibody of the xylanase in order to localize the protein in the wild strain of S. cellulosum So0157-2.First of all, we analyzed the motif character of the xylanases from S. cellulosum So0157-2 and So ce56. No dockerin was detected. And no scaffold or cohesin from scaffold was detected neither. These supposed that the degradation was not the same as the cellulosome. Additionally, most of the xylanases did not contain CBM. The CBM contained xylanses were belonged to GH 43 family. GH10 and GH11, the most distributed and characterized xylanase, contained no CBM. Only the xylanase contig00901-2 contained the CBM. And contig00016, contig00028 contained a RICIN domain. These were different from degradation strategy with the dissociate enzymes. Another feature was the two strains all contained an unexpected numbers of predicted lipoprotein. Considered the research of S. cellulosum in our lab and the feature of celluase, we concluded that S. cellulosum had its own strategy in xylan and cellulose degradation, not the same as the well characterized dissociate enzyme strategy or cellulosome.Two genes, coding GH11 xylanases from the genome of S. cellulosum So0157-2, were cloned. We named them Xyn11A and Xyn11B. They were expressed and purified. The biochemical feature was characterized using the active protein.Xynll A was cloned into pET-22b to construct pET-22b-Xyn11 A. E. coli BL21 (DE3) was used as the host of the expression. The recombinant protein was expressed as inclusion body. The inclusion body was resolved and purified with Ni affinity chromatography. Dialysis was done to recover the activity. The acitive recombinant protein was named r-Xyn11A. Superdex 200 10/300 GL was used for the further purification. The optimal pH of r-Xyn11A was between 6.5 and 7.0. The optimal temperature was 45℃. After the preincubation in the buffer with pH 10.5,95% of the activity was retained. r-Xyn11A could not function under high temperature. No activity was detected under 60℃. Most of the metal ions detected could inhibit the activity of r-Xyn11A. Li+, K+, Mg2+ could slightly enhance the activity.0.1%SDS could almost double the activity (189.5%). r-Xyn11A could function with the presence of 1%(36.7mM) SDS. However, most protein would denature under the presence of 7mM SDS. r-Xyn11A could only degrade three kinds of xylan from different sources. The Km was 1.27mg/ml and Vmax was 0.27mmolmin-1mg-1 used xylan from birch wood as substrate. r-Xyn11A could degraded xylan (birch wood) to different kinds of xylooligosaccharides. r-Xyn11A was an alkaline stability and SDS resistence endoxylanase. The antibody of r-Xyn11A was also prepared for the further research.The C terminal of Xyn11B (Xyn11B-C), containing the whole catalytic domain, was cloned into pGEX-6P-1. The recombinant plasmid was named pGEX-6P-1-Xyn11B-C. The recombinant protein could expressed in soluble and active form. The protein was purified with the scission of the GST tag. The recombinant protein was named Xyn11B-C. The optimal pH was 7.0. The optimal temperature was 40℃. It could retain 20% of the activity under 100℃. After preincubated in the buffer with pH 10.5, 90% of the activity was retained. The thermal stability of Xyn11B-C was specific. After preincubated under 60℃or 70℃for 10min to lhr, no activity was detected. However,30% of the activity was detected after preincubated under 90℃for lhr.20% of the activity was detected after preincubated under 100℃for 30min. Most of the mental ions and chemical compounds detected could inhibit the activity. Xyn11B-C could only degrade the three xylans from different resources. No activity toward other polysaccharides used in this experiment was detected. Xyn11B-C could degrade xylan from birchwood to xylobiose. This supposed Xyn11B-C as an exo-acting xylanase. Y215 was supposed to be the subsite of Xyn11B-C. And we constructed a mutation with Y215 to A. The mutation (Xyn11B-C-Y-A) was expressed and purified. The hydrolysis product toward xylan (birchwood) was detected. Different kinds of xylooligosaccharides could be detected. The enzyme was changed into an endo-acting mode. Not only degradation activity but also synthesize activity were detected using xylooligosaccharide as the substrate.