Analysis Of Lignocellulose Degrading Enzymes And Expression Of Xylanase In Microbial Consortium RXS

Microbial consortium RXS can secreted variety of lignocellulose degradation enzymes. The studies on these enzymes properties will facilitate the efficiency of cellulose degradation and investigate the new enzyme species.The key enzymes of lignocellulose degradation enzymes in RXS were analyzed in this paper, then the purification of xylanase, the expression of xylanase gene of Clostridium clariflavum DSM 19732 in E. coli and the degradation of multienzyme on cassava residue were investigated. The main results are as follows:1. The effects of metal ions, Cu2+,Fe2+,Co2+,Mn2+,Zn2+ and Mg2+ on FPAase, CMCase and xylanase were studied and the experimental conditions were optimized. The results showed that the activities of these three enzymes reached the maximum value, 0.017 U·m L-1, 0.038 U·m L-1 and 0.373 U·m L-1, when RXS was cultivated for 48 h. At p H 6.0 and temperature of 55℃, Cu2+ inhibited the FPAase and xylanase activity effectively, and the weight loss rate of cassava residue is only 0.62%; Co2+ and Zn2+ inhibited these three enzymes activities to some extent, and the weight loss rates were only 3.29% and 4.7% respectively; Fe2+ and Mg2+ inhibited FPAase activity, CMCase and xylanase degraded cassava residue with the weight loss rate of 12.77% and 15.81% respectively which indicated that the cooperation of CMCase and xylanase played a major role in cassava residue degradation.2. The purification of xylanase was carried out by 40-60% ammonium sulfate precipitation, DEAE, Q and G200 chromatography. The dockerin-like protein in Clostridium clariflavum DSM 19732 detected by mass spectrometry indicated that the enzyme from C. clariflavum DSM 19732 participated in cellulosome formation.3. The xylanase gene of Clostridium clariflavum DSM 19732 was expressed in E. coli. The optimization of expression conditions were OD600 1.25, IPTG concentration of 0.1 m M, 23℃ and cultured for 6 h, the xylanase activity was 15.15 U·mg-1.4. Addition of recombinant xylanase to RXS, the degradation of cellulose and hemicellulose in cassava residue were enhanced, the weight loss rate of cassava residue reach to 28.78% with cellulose and hemicellulose content decreasing to 10.62% and 5.77%, which indicated synergistic effect of the recombinant xylanase and cellulase was obvious.