Gene Cloning, Expression And Characterization Of Xylanase From Clostridium Clariflavum DSM 19732

Clostridium clariflavum DSM 19732 is an anaerobic, cellulosome-forming thermophile, secreting a large number of lignocellulose degradation enzymes, including high hemicelluloses degrading ones. However, there are few reports on the properties of these enzymes in detail. In our previous study, a microbial consortium RXS was contructed in which C. clariflavum was characterized as one of the key microbes. In this study, two xylanase genes(xyn2083 and xyn2441 from C. clariflavum DSM 19732) were amplified from the total genomic DNA of RXS and expressed in Escherichia coli BL21(DE3).The recombinant xylanases were purified and characterized.The activity of intracellular recombinant xylanase rXyn2083(78.5 ku) was 1.46 U·m L-1(2.84 U·mg-1) under the optimal inducing condition of OD600 1.1, IPTG 0.4 mmol·L-1 and 9 hours under temperature 20℃. The activity of recombinant xylanase rXyn2441(88.7 ku) was 23.2 U·m L-1(34.4 U·mg-1) under the optimal inducing condition of OD600 1.2, IPTG 0.5 mmol·L-1 and 7 hours under temperature 25℃.After purification by Ni2+-NTA affinity chromatography, the activity of r Xyn2441 was 715.6 U·mg-1. SDS-PAGE showed three bands mainly, and MALDI-TOF-MS analysis indicated that these three bands were all agreed with target protein. The activity of rXyn2441 was 1305.7 U·mg-1 after further purification by gel filtration G200.The optimal temperature and pH of rXy2083 were 65℃ and 6~6.5. rXy2083 had a wide pH tolerance and was stable under 60℃. MgSO4, CoCl2, 5 mmol·L-1 NiSO4 and 1 mmol·L-1 CuSO4 promoted the activity of rXy2083 towards xylan; 5 mmol·L-1 FeSO4 and CuSO4 inhibited the activity strongly. Products of rXyn2083 towards xylan were xylose and xylobiose which was not able to be futher degraded.The optimal temperature and pH of rXy2441 were 70℃, 6.5. rXy2441 was stable in neutral and alkaline pH, and deactivated beyond 50℃ quickly. 1 mmol·L-1 MgSO4, CoCl2 and CuSO4 promoted the activity of rXy2441 towards xylan; 5 mmol·L-1 FeSO4 and CuSO4 also inhibited the activity strongly. rXyn2441 was a typical endo-xylanase, degrading xylan to xylobiose, xylotriose and xylotetraose, but not xylose.