Paenibacillus campinasensis BL11纖維素酵素基因之分子選殖、大量表現與純化

Abstract

Paenibacillus campinasensis BL11是由高溫高鹼的製漿漂前黑液中 (58°C, 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。BL11具有分泌多種多醣類水解酵素之能力。經由SDS-PAGE與zymogram活性測試得知，BL11具有分泌3種纖維素酵素之能力，其酵素分子量分別約為42 kDa、57 kDa和87 kDa。其中一種纖維素酵素之DNA序列在此研究中被選殖出並分析之。 由Paenibacillus campinasensis BL11所選殖 (clone)出的纖維素酵素基因 (PcBC56)乃利用質粒pBCKS(+)為載體，轉型至Escherichia coli (DH5α)中，並利用含有甲基羧基纖維素 (CMC)之固態培養基篩選後得到之。所選殖出的纖維素酵素基因是由1,473個核苷酸所組成，但缺少末端的DNA序列，因此PcBC56的末端是與pBCKS(+)上部份MCS融合後才得到一完整的ORF，經由轉錄 (transcription)及轉譯 (translation)可得到大小為56 kDa的纖維素酵素。分析結果顯示此纖維素酵素其N端胺基酸序列具有一長度為29個胺基酸的信息序列 (signal peptide)，和纖維素酵素的胞外分泌有關。同時也具有一個屬於家族5的醣類水解酵素區 (glycosyl hydrolase domain; catalytic domain)和C端一個屬於家族3的碳水化合物結合區 (carbohydrate binding module)。 本研究為了利於纖維素酵素的純化，將選殖得到的纖維素酵素C端胺基酸序列接上His-tag，並在0.1 mM IPTG和28oC培養下，誘導酵素大量表現。誘導得到的纖維素酵素利用與Ni-NTA agarose的親和力加以純化，但是卻無法成功純化此纖維素酵素。另一方面，PcBC56b所表現出的纖維素酵素在zymography的分析中顯示出兩個不同纖維素分解活性的位置，分別是56 kDa和35 kDa。胞外纖維素酵素和胞內纖維素酵素前驅物 (precursor)之間的分子量大小差異，扣除信息胺基酸 (約3 kDa)後大約有18 kDa左右，會產生此一現象可能是因為蛋白質轉譯後修飾 (post-translational modification)的結果。因此或許纖維素酵素的C端因而被切斷，造成His-tag的喪失，無法以Ni-NTA agarose完成純化。 誘導後的粗纖維素酵素液最佳反應條件為70oC和pH 6，酵素活性可達170.97 IU/mg。相信在完成純化之後必會得到更高的酵素活性，在工業應用上也更具潛力。

Paenibacillus campinasensis BL11 was isolated by screening bacterial strains from thermo-alkaline black liquor (58oC, pH 9.83). The black liquor was collected from Hsinying Paper Mill of Taiwan Pulp and Paper Company. The collected sample contained large amounts of hemicellulose, lignin and small fiber fragments. Strain BL11 could exhibit many kinds of polysaccharide hydrolases. Strain BL11 exhibited strong cellulase activity and possessed three different kinds of cellulases. According to the result of SDS-PAGE and zymogram the cellulases are 42 kDa, 57 kDa and 87 kDa in molecular mass, respectively. One of the cellulases has been cloned and analyzed. The cellulase gene (PcBC56) was cloned in Escherichia coli (DH5α) with pBCKS(+). The cellulase-related plasmid, pBCCMC1, was isolated by screening a library of randomly cloned BL11 DNA fragments on carboxylmethylcellulase (CMC) indicator plates. The cloned BL11 cellulase gene is composed of 1,473 bp nucleotides that is a C-terminal-lost DNA sequence. PcBC56 was fused with 75 bp of MCS of pBCKS(+) and became a new ORF (PcBC56b) which encode a protein of 56 kDa. The N-terminal of the cellulase contains a deduced signal peptide of 29 amino acids in length and a glycosyl hydrolase domain (catalytic domain) in family 5. There is a carbohydrate binding module in family III at C-terminal of this. The cloned BL11 cellulase was fused with a His-tag at its C-terminal for purification by gene manipulation. The engineered BL11 cellulase was induced with 0.1 mM IPTG at 28oC in an E. coli host for overexpression. The induced cellulase was purified with Ni-NTA agarose by bio-affinity but without success. In the other hand, zymographic analysis of the PcBC56b gave two cellulase activities whose molecular weights were 56 kDa and 35 kDa. The apparent ca. 18 kDa different between extracellular cellulase and its precursor form minus the signal peptide (ca. 3 kDa) could be due to one of several post-translational modifications. The C-terminal segment of the cellulase might be removed so that the purification would be fail because of the loss of His-tag. The optimal temperature and pH of the induced crude cellulase were 70oC and pH 6. The induced crude cellulase activity was 170.97 IU/mg. It would be mush better after purification and have potential for further industrial application.