活性污泥多源基因體中新穎酯酶基因之探勘

Abstract

廢水處理設施中之活性污泥含有複雜的微生物聚落。以BLASTN方法分析活性污泥中16S rRNA基因庫得知，大部分的細菌(90%)是屬於未培養者。因此，利用多源基因體學方式，從活性污泥樣品中篩選出新穎生物催化劑基因的機率將頗高。本研究採直接萃取法，將DNA從活性污泥樣品中分離純化，並利用質體系統建立多源基因庫。基因庫中重組質體之平均插入片段大小約5.1 kb。利用三丁酸甘油酯平板分析，總共發現12個具有脂解活性的株系，其篩選機率高達0.31%。經由分子分析，12個株系中含有16個可能的脂解基因ORFs，與資料庫的蛋白質序列比對分析後發現，大部分ORFs與最相近蛋白質之相同度介於28–55%。簡而言之，由本研究結果得知，活性污泥為一個可從中篩選新穎脂解基因之理想生物資源。由於其中兩個酯酶基因est6與est13在胺基酸序列上具有高新穎性，因此進行深入的研究分析。est6基因長729 bp可轉譯出242個胺基酸的蛋白質稱為Est6。而est13基因長1326 bp可轉譯出441個胺基酸的蛋白質稱為Est13。經BLASTP比對分析，大部分與Est6和Est13相近的蛋白質都是未經特性分析，且從微生物全基因組定序資料所推測轉譯出來的。從多重序列分析與類緣遺傳分析得知，Est6是屬於第六類之脂解酵素，Est13則屬於第二類脂解酵素之新成員。est6與est13基因可在大腸桿菌中表現，且所表現出來的可溶性產物可藉由親和性管柱純化成無多餘標籤的同質狀態酵素。純化的Est6不管是溶在pH 7或pH 8緩衝液中，皆以單體的狀態存在並具活性。以分光光度分析法測試，Est6對受質具有明顯的位置選擇性。然純化的Est13溶在pH 8緩衝液中，則以12倍體的狀態存在並具活性。Est6在pH 8和45°C狀態下具有最佳的活性表現；Est13則在pH 8和35°C狀態下，具有最佳的活性表現，且其在5－20°C低溫範圍內具有45－72%最大活性。此兩種酯酶在廣大的溫度與pH範圍活性穩定，且在有機溶劑、金屬離子和清潔劑環境中具有活性。綜而言之，兩種酯酶皆具有值得注意的生化特性，因此具有潛力可被開發以應用於生物技術產業。

Activated sludge in a swine wastewater treatment facility comprises a complicated microbiological community. Based on BLASTN analysis of cloned 16S rRNA genes, we found that most of these communities (90%) were of uncultivated bacteria. It is highly possible that we may to discover new biocatalyst genes from activated sludge samples collected from such facilities using the metagenomic approach. The metagenomic library was constructed using a plasmid vector and DNA extracted directly from samples of activated sludge. The average insert size was approximately 5.1 kb. A total of 12 unique and lipolytic clones were obtained using the tributyrin plate assay. The rate of discovering lipolytic clones in this study was as high as 0.31%. Molecular analysis revealed that most of the 16 putative lipolytic enzymes showed 28–55% identity with non-redundant protein sequences in the database. Briefly, our study demonstrated that activated sludge was an ideal bioresource for isolating new lipolytic enzymes. Two esterase genes est6 and est13 were studied in depth for their highly novel amino acid sequences compared to the database. The est6 gene with 729-bp DNA encoded a 242-amino acid protein (designated Est6), and the est13 gene with 1326-bp DNA encodes a 441-amino acid protein (designated Est6). Most of the closely related proteins of both esterases were uncharacterized and conceptually translated from whole genome sequencing data of microorganisms. Multiple sequence alignments and phylogenetic analysis of Est6 and Est13 showed that Est6 and Est13 belonged to family VI and family II esterases/lipases, respectively. In addition, both esterase genes could be over expressed in their soluble form in Escherichia coli and then purified to a tag-free, homogeneous form by affinity chromatography. The purified Est6 in pH 7.0 or pH 8.0 buffers was active as a monomer, but the purified Est13 in Tris–HCl buffer (pH 8.0) was active as a dodecamer. Est6 showed significant regioselectivity in spectrophotometric analysis. The optimum temperature and pH for Est6 and Est13 were pH 8.0, 45°C, and pH 8.0, 35°C, respectively, when using p-nitrophenyl acetate as a substrate. In particular, the Est13 esterase was active with 45%－72% of maximal activity across low temperature ranges of 5°C－20°C. Both esterases were stable over wide temperature and pH ranges, and they exhibited activity in the presence of organic solvents, cations, or detergents. Because Est6 and Est13 possess noteworthy properties, they have the potential to be developed for biotechnological applications.