漂前黑液內Paenibacillus sp. isolate BL11之木聚醣酶分生選殖、大量表現與純化

Abstract

Paenibacillus sp. isolate BL11 是由高溫高鹼的製漿漂前黑液中 (58℃, pH 9.83) 所分離出的菌種。本研究的黑液是由台灣紙業新營廠所收集而來，其成分包含大量的半纖維素、木質素、和部分細小纖維。經由活性測試，BL11具有很強的木聚醣酶活性，並利用16S rDNA 定序鑑定出與其他菌種的親緣關係。 由Paenibacillus sp. isolate BL11 所選殖(cloning)出的木聚醣酶基因(xylX)是由1,131個核苷酸所組成，經由轉錄(transcription)及轉譯(translation)可得到大小為41kDa的木聚醣酶。分析結果顯示出選殖得到的木聚醣酶其N端胺基酸序列具有一長度為39個胺基酸的信息胜肽(signal peptide)，和木聚醣酶的分泌有關。 BL11菌種本身木聚醣酶最佳反應條件為pH 7和60℃，活性為23 IU/mg。本研究為了方便木聚醣酶的純化，將選殖出來的木聚醣酶的C端胺基酸序列接上His-tag，並在0.1 mM IPTG和 28℃培養下，大量誘導。誘導出的木聚醣酶利用與Ni-NTA agarose 的親和力，加以純化。純化後的木聚醣酶最佳反應條件仍為pH 7和60℃，而活性可高達2392 IU/mg。在pH 11的條件下，木聚醣酶活性可達517 IU/mg。

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