在品質較差而價格較低廉的草料中，往往含有較多難以被酵素分解的聚木糖與木質素等成分，這些成分的存在，間接影響了牛隻的飼料使用效率；然而，台灣水牛(Bubalus bubalus carabanesis)為一沼澤生活型的牛種，具有極高抵抗環境壓力與適應粗草料的特性。本論文從台灣水牛的瘤胃真菌Neocallmastix patriciarum S20中，選殖出一個具有雙功能的聚木糖分解酵素基因，名為xynS20E，並利用此酵素進行了以下的研究：

第一部分 – 從瘤胃真菌Neocallmastix patriciarum S20 cDNA庫中選殖具有雙功能聚木糖分解酵素的基因

本研究從N. patriciarum cDNA庫中選殖出一條具有乙醯化聚木糖酯酶活性與聚木糖酶活性的酵素基因，命名為 xynS20E。此基因具有一個2,016鹼基對的完整序列，其中包含162鹼基對的5’端非轉譯區與243鹼基對的3’端非轉譯區。此基因可轉譯出671個胺基酸的蛋白質，分子量約為72.4 kDa。其胺基酸序列中， N端具有一個碳水化合物酯酶第1族(carbohydrate esterase family 1)的功能性區域，而C端具有醣苷水解酶第11族(glycosyl hydrolase family 11)的功能性區域。此外，在這兩個功能性區域之間，則含有兩個真菌搭載結合蛋白第1型(fungal dockerin domain type I)之序列。後續實驗中，利用基因工程技術，將xynS20E選殖於pET29a表現載體，並利用大腸桿菌大量表達此帶有6個組胺酸之重組酵素，並藉由鎳離子親和性管柱純化，以製備XynS20E酵素。

第二部分 – 利用反應曲面法最佳化XynS20E之乙醯化聚木糖酯酶與雙功能聚木糖酶

反應曲面法搭配複合式中心設計法與統計迴歸分析可以有效率地得知酵素最佳反應活性的溫度與酸鹼值。利用此方法，得知XynS20E的乙醯化聚木糖酯酶在58°C與pH 8.2具有最佳比活性，為873.1±18.0 U/mg；XynS20E的聚木糖酶在49°C與pH 5.8達到最佳比活性，為128.7±32.9 U/mg。另外，乙醯化聚木糖酯酶功能性區域被獨立選殖於表現載體pET29a中，命名為AxeS20E。AxeS20E經由反應曲面法分析，於54.6°C與pH 7.8時具有最佳比活性；在80°C加熱120分鐘後，仍可保有85%以上的酵素活性，展現良好的耐熱穩定性。

第三部分 – 利用嵌合體酵素分解木質纖維素

XynCDBFV唯一已知具有高活性之聚木糖酶。應用反應曲面法更進一步地得知此酵素的最佳反應條件為55.3°C與pH 5.3，此反應條件下可測得比活性為9543.6±434.9 U/mg；進一步利用基因工程技術，分別將AxeS20E融合於XynCDBFV的N端與C端，並在兩個酵素之間設計了二個重複的GGGGS胺基酸鏈作為連結，產生兩個不同的嵌合體酵素(分別命名為AxeS20E-XynCDBFV和XynCDBFV-AxeS20E)。以反應曲面法分析此二嵌合體酵素，可得AxeS20E-XynCDBFV的最佳反應條件為62°C與pH 5.4，最佳比活性為6225.4±528.5 U/mg； XynCDBFV-AxeS20E的最佳反應條件為60°C與pH 5.4，最佳比活性為3945.6±154.1 U/mg。AxeS20E-XynCDBFV和XynCDBFV-AxeS20E在耐熱穩定性實驗中，在60°C環境中處理120分鐘後，分別保有60%與80%以上的原始活性；此雙功能融合酵素在協同效益的測試中，聚木糖分解能力較單一酵素單獨作用降低了8% ~ 30%酵素活性；將天然稻桿以酵素處理48小時後，以AxeS20E-XynCDBFV和XynCDBFV-AxeS20E處理分別較單一酵素處理時提高了1.3和1.2倍的還原糖濃度。
本研究為第一個從瘤胃真菌中發現具有乙醯化聚木糖酯酶與聚木糖酶的雙功能聚木糖分解酵素。結果亦顯示，反應曲面法搭配複合式中心設計法與統計迴歸分析提供了一個尋求反應條件最佳化的有利策略。研究並證實，AxeS20E具有良好的耐熱穩定性，此一特點將是應用於工業發酵中相當受到重視的一環；此外，人造雙功能嵌合體酵素展現了高於單一酵素的木質纖維素分解效率。

The plant cell walls of the low-quality forage usually contain a high percentage of ligin and xylan, hence, are difficult to be degraded enzymatically. Taiwanese water buffalo, Bubalus bubalus carabanesis, belongs to the swamptype and can adapt to the environment stress and graze on the low-quality pasturelands. Taiwanese water buffalo is known to be able to digest crude fiber in a better way than the cattle. In this study, a bifunctional xylanolytic enzyme gene (xynS20E) was isolated from the cDNA library of the rumen fungus Neocallmastix patriciarum S20 established from the Taiwanese water buffalo. The whole study of this dissertation is divided into three parts as follows:

Part I - Cloning of a bifunctional xylanolytic enzyme gene from the ruminal fungus Neocallmastix patriciarum S20

The cDNA library of N. patriciarum was constructed and used to screen the xylanolytic enzyme. A gene encoding a bifunctional acetylxylan esterase-xylanase, named XynS20E, was cloned. The cDNA sequence of xynS20E was found to contain a complete open reading frame (ORF) of 2,016 bp with 5’ and 3’ untranslated regions of 162 and 243 bp, respectively. Translation of the open reading frame of xynS20E revealed a protein of 671 amino acids with a predicted molecular weight of 72.4 kDa. According to the sequence-based classification, a putative conserved domain of family 1 carbohydrate esterase (CE) was observed at the N-terminus of XynS20E and a putative conserved domain of glycosyl hydrolase (GH) family 11 was found at the C-terminus of XynS20E. Two putative conserved fungal dockerin domains (FDDs) type I were found between the N-terminal family 1 CE catalytic domain and the C-terminal family 11 GH catalytic domain of XynS20E. To examine the activity of the gene product, xynS20E gene was cloned into the pET-29a expression vector and expressed in E. coli as recombinant His6 fusion proteins. The purified XynS20E-His6 fusion proteins were obtained after purification by the immobilized nickle ion-affinity chromatography.

Part II – Statistical optimization of acetylxylan esterase and xylanase activities of the bifunctional xylanolytic enzyme XynS20E

Response surface methodology (RSM), with central composite design (CCD), and regression analysis were successfully applied to obtain the optimal temperature and pH conditions of the recombinant XynS20E. The optimal condition for the highest enzyme activity of CE1 domain of XynS20E toward acetylxylan was observed at 58°C and pH 8.2 with specific activity of 873.1±18 U/mg. The optimal condition of GH11 domain toward oat spelt xylan was observed at 49°C and pH 5.8 with the specific activity of 128.7±32.9 U/mg. Further, the DNA sequence encoding CE1 domain of XynS20E gene, named axeS20E, was cloned into the pET29a expression vector and purified as recombinant AxeS20E-His6 fusion proteins. The optimal conditions for the highest activity of AxeS20E were observed at 54.6 °C and pH 7.8. This enzyme retained more than 85% of the initial activity after 120 minutes of heating at 80°C.

Part III – Application of chimeric enzyme in lignocellulose degradation

XynCDBFV is an alkalophilic endo--1,4-xylanase with high activity. The optimal conditions for the highest activity of XynCDBFV were observed at 55.3 °C and pH 5.3 with specific activity of 9543.6±434.9 U/mg. Further, AxeS20E was fused to the N-terminus of XynCDBFV, named AxeS20E-XynCDBFV, with (GGGGS)2 peptide as linker. And another chimeric enzyme was AxeS20E fused to the C-terminus of XynCDBFV, named XynCDBFV-AxeS20E, with the same linker. These two optimal conditions for highest activity of AxeS20E-XynCDBFV and XynCDBFV-AxeS20E were observed at 62°C, pH 5.4 and at 60 °C, pH 5.4 with specific activity of 6225.4±528.5 and 3945.6±154.1 U/mg, respectively. In the thermostability assay, AxeS20E-XynCDBFV and XynCDBFV-AxeS20E were retained more than 60% and 80% of initial activity after 120 min of heating at 60 °C, respectively. In lignocellulosic biomass conversion assay, the reducing sugars released in AxeS20E-XynCDBFV and XynCDBFV-AxeS20E treatment were 1.3 and 1.2 folds higher than that in XynCDBFV treatment after 48 h, respectively.
This is the first report of a bifunctional xylanolytic enzyme with acetylxylan esterase and xylanase activities from the rumen fungus. The results suggested that RSM combined with CCD and regression analysis were effective in determining optimized temperature and pH conditions for the enzyme activity. The results also proved AxeS20E was thermo-tolerant and might be a good candidate for various biotechnological applications. Last, the constructed chimeric enzyme, AxeS20E-XynCDBFV and XynCDBFV-AxeS20E, showed a higher xylanase activities than the parental enzymes toward natural xylans of rice straw as substrates.

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