Distribution Of Xylanases In Sorangium Cellulosum So0157-2and Heterologous Expression,Characterization Of Xyn11C

S. cellulosum So0157-2is rich in xylan degrading enzymes. Its genome has at least five GH11xylanase genes and some other xylanase genes belonging to GH8, GH10, GH43, and GH52as well. Research shows that Xyn11A and Xyn11B have different characteristics. In this paper xyn11C was cloned into pET-30a (+) to construct pET-30a (+)-xynllC. E. coli BL21(DE3) was used as the host of the expression. The recombinant protein rXyn11C was mainly expressed as inclusion body. The inclusion body was resolved and purified with Ni-affinity chromatography. Dialysis was done to recover the activity.RXyn11C showed activity only on xylans, especially on xylan from beechwood. The Km was4.030mg/ml and Vmax was0.533mmolmin-1mg-1used xylan from beechwood as substrate, while using xylan from birch wood as substrate the Km was3.403mg/ml and Vmax was0.276mmolmin-1mg-1.The optimum pH and temperature of the purified enzyme rXynllC were7.0and45℃respectively. After the preincubation in the buffer with pH10.5for30minutes,50%of the activity was retained. Approximate60%activity was lost after30-min preincubation at45℃,and no activity was detected after preincubation at50℃.The activity of rXyn11C was enhanced by the presence of Tween20. But the chemicals left and most of the metal ions detected could inhibit the activity of rXyn11C except Li+、K+. RXyn11C could degrade xylan from beechwood to different kinds of xylooligosaccharides. It could also degrade xylotetraose, xylopentaose, and xylohexaos to xylooligosaccharides with lower polymerization degree. And at the same time xylooligosaccharides with higher polymerization degree could also be detected by TLC. RXyn11C showed both degrading activity and synthesizing activity using xylooligosaccharides as the substrates. And the synthesizing activity could be enhanced when adding xylotriose to the reaction system of xylotetraose, while rXyn11C showed no activity on xylotriose.S. cellulosum So0157-2can use crystal cellulose (filter paper) as the sole carbon source, and it shows different ability of using other carbohydrates. So0157-2grew well when using Stach from potato、L-Arabinose、Dextrin、D-Glucose or Xylan from birch wood as the only carbon source, and it could not use D-Xylose、 Carboxymethylcellulose (CMC) or Chitosan as the the sole carbon source to grow. So0157-2grew best in M26, better in0.75%glucose and0.75%xylan, and reached its maximum biomass after being cultured for1to2days.All of the extracellular fractions, membrane fractions and intracellular soluble fractions from So0157-2cultured in M26, CNST-G and CNST-X showed xylanase activity and carboxymethyl cellulase (CMCase) activity. The xylanase activities of these three membrane fractions were more than50%of the total. It seems like that the membrane fraction plays an important role in the process of xylan degradation. The xylanase activity of these fractions was examined by zymograms. Intense but not well-defined xylanase activity bands were found in the zymograms of the extracellular fractions, indicating that So0157-2in liquid culture might secrete xylanases out of the cells. The activity bands of the extracellular fractions obtained from M26and CNST-X were obviously more than those of CNST-G. The activity profiles obtained for the intracellular soluble fractions were very similar, revealing three well-defined activity bands owing to the same xylanases. The activity profile of the membrane fractions obtained from So0157-2cultured in CNST-X presented an intense and well-defined xylanase activity band at230kDa above, and the lower bands were almost the same as those obtained from So0157-2cultured in M26and CNST-G. These differences may be due to the different carbohydrates in culture mediums. These fractions were also compared by zymograms for CMCase activities, and the activity bands were detected approximately between25and100kDa. There were many differences among the CMCase activitiy profiles of the fractions obtained from So0157-2cultured in different mediums, but each fraction revealed more bands than for the xylanases. The Western-blotting result showed that the polyclonal antibody reacted to the expressed rXynllA. Several proteins of So0157-2could also react to the polyclonal antibody, but only the intracellular soluble fractions and the extracellular fraction obtained from CNST-G got two close bands of similar theoretical molecular weight. To research the localization of Xyn11A, plasmolysed cells of So0157-2from CNST-filter medium were harvested after being resuspended with25%sucrose. We chose two strains, E. coli BL21(DE3)-pET-22b induced by IPTG and E. coli BL21(DE3)-pET-22b-xyn11A induced by IPTG, separately as negative and positive controls. Ultrathin sections were prepared. Specifically bound anti-rXyn11A antibodies used to probe the XynllA in the thin sections are labeled with anti-rabbit IgG conjugated with10-nm gold particles, which can be enlarged by the silver and then detected via transmission electron microscopy. The location of the gold spheres in relation to the cellular substructures demonstrated that XynllA was mostly distributed in the cytoplasm of So0157-2, while the outer membrance, the periplasm and the cytoplasmic membrane irregularly carried no significant amount of label.Further studies of these xylanases on their enzymatic properties and distribution in So0157-2will help us to understand their different physiological functions, lay the foundation for exploring their physiological and coordinating roles, and find xylanases with potential application value as well.