Molecular cloning, overexpression and purification of Paenibacillus sp. isolate BL11 xylanase from black liquor

• Main Authors: Chung-Hung Tsai, 蔡忠宏
• Other Authors: 柯淳涵
• Format: Others
• Language: en_US
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/82320215872283439918

碩士 === 國立臺灣大學 === 森林學研究所 === 93 === Paenibacillus sp. BL11 was isolated by screening bacterial strains from a thermo-alkaline black liquor (58°C, pH 9.83). The black liquor was collected from Hsinying Paper Mill of Taiwan Pulp and Paper Company and contained large amounts of hemicellulose, lignin and small fiber fragments. Strain BL11 exhibited strong xylanase activity and its phylogenetic relationship with other bacteria was identified by 16S rDNA sequencing. The xylanase of strain BL11 has been cloned and analyzed. The cloned BL11 xylanase is composed of 1,131 nucleotides and can encode a protein of 41 kDa. The N-terminal of the enzyme contains a deduced signal peptide of 39 amnio acids in length. Zymographic analyses of the BL11 xylan-degrading system revealed that cloned BL11 xylanase was secreted into the extracellular medium of the bacterium. Optimum temperature and pH for crude BL11 xylanase activity were 60°C and pH 7, respectively. The activity was 23 IU/mg. The cloned BL11 xylanase was fused to a His-tag for purification by gene manipulation. The engineered BL11 xylanase was induced with 0.1 mM of IPTG at 28°C in an E. coli host. The induced xylanase was purified with Ni-NTA agarose by bio-affinity. The optimal temperature and pH for the purified BL11 xylanase were still the same of original ones. The purified BL11 xylanase activity was 2392 IU/mg under optimal condition. At pH 11, the activity was still as high as 517 IU/mg