瘤胃細菌纖維及蛋白質分解酵素之生產與利用

Production and utilization of cellulytic enzyme and protease from rumen bacteria

王翰聰(Han-Tsung Wang)

指導教授 ： 徐濟泰

國立臺灣大學/生物資源暨農學院/畜產學研究所/博士(2004年)

https://doi.org/10.6342/NTU.2004.00983

摘要

第一部份：瘤胃細菌纖維素分解酵素複合體

瘤胃中纖維分解菌Fibrobacteria succinogenes S85於含瘤胃液之初始培養基（ATCC medium 1715E）中添加0.2% 之纖維雙糖，在39℃下進行培養48至96小時，可獲得最佳之生長及纖維素分解酵素外泌表現。但是培養基中含過高之纖維雙糖（1.0%），並無助於細菌之纖維素分解酵素之生產表現。轉換培養基為不含瘤胃液之酵素生產培養基時，添加濾紙為碳源，可於培養後72小時達最佳生產效果。生產纖維素分解酵素時，收集F. succinogenes S85之細胞外泌液，經離子交換法及膠體過濾法進行純化後，在外泌液中，可得到兩群具有較高纖維素分解酵素活性之蛋白質，分子量分別為60及120 kDa。利用蔗糖溶液進行回收細胞表面樣品時，酵素活性及回收量均較利用Triton X-100者為佳。取細胞表面樣品進行純化，可較外泌液多獲得一群分子量約40 至50 kDa之纖維素分解酵素。為取得聚木醣分解酵素，本試驗利用離子交換法與膠體過濾法對Ruminococcus albus 7之細胞外泌液進行純化後，所得之聚木醣分解酵素分子量約為62 kDa，比活度為94 μg xylose/min/mg。進行纖維素結合蛋白質收集時，F. succinogenes S85之纖維素結合蛋白質以結晶性纖維進行吸附後，配合含有5% SDS之回收緩衝液進行溶離，可得較佳之回收效果。纖維素結合蛋白質之添加效果顯示，R. albus 7之聚木醣分解酵素與F. succinogenes S85之纖維素分解酵素，在加入纖維素結合蛋白質後可有效提升其酵素作用之特異性1.1至8.3倍，顯示纖維素結合蛋白質除了可以提升同一細菌來源的纖維素分解酵素活性外，亦可作用於不同菌株之不同性質纖維分解酵素，提升其酵素之效力。

第二部分：利用瘤胃蛋白分解菌生產飼料用蛋白質分解酵素

本試驗以瘤胃中蛋白分解菌Prevotella ruminicola 進行蛋白質分解酵素之生產與特性探討。以蔗糖為培養碳源，配合蛋白腖為氮源，可得最高增殖效率，蔗糖及葡萄糖均有助於刺激蛋白質分解酵素之分泌。氮源利用上則以酪蛋白及蛋白腖（peptone）之效果較佳。最佳酵素收取條件為0.5％葡萄糖或蔗糖，配合0.1％氯化銨加上0.2％ 蛋白腖，於39℃培養18至20小時。酵素活性分布上，有65％活性存於外泌液中，細胞表面活性占18％，細胞間質及胞內液活性較低。酵素最適作用條件在溫度40℃，pH 6.8，當作用溫度改變為30或50℃時，酵素活性約為最佳活性之50％，而pH值於6至8之間，均可維持酵素活性。經蛋白質分解酵素抑制劑測試，得知酵素粗萃液具有絲胺酸型、天門冬酸型及金屬型蛋白質分解酵素之特性。以魚粉、血粉、羽毛粉、乳清粉及大豆粕為基質，進行酵素分解能力測定，酵素粗萃液對於乳清粉、魚粉及羽毛粉之分解活性( 2.39 , 2.60 and 1.76 μmol amino acid/mg/hr )優於大豆粕及血粉( 1.11 and 1.09 μmol amino acid/mg/hr )。

第三部分：填充物對酵素製劑之活性保護效果

本部分試驗以Aspergillus niger 之纖維素分解酵素、Bacillus sp.之

關鍵字

瘤胃細菌 ； 纖維分解酵素 ； 蛋白質分解酵素 ； 酵素保護

並列摘要

Part I

The best growth performance and cellulase production were obtained as the ruminal cellulolytic bacterium Fibrobacteria succinogenes S85 was incubated in ATCC medium 1715E with 0.2% cellobiose for 48 to 96 hr at 39℃. However, higher level of cellobiose (1.0%) had no benefit on bacterial cellulase production. The use of filter paper as a carbon source in enzyme production medium without rumen fluid resulted in the best enzyme production of F. succinogenes S85 after 72 hr of incubation. After passing through ion exchange and gel filtration columns, the extra cellular fraction (ECF) of F. succinogenes S85 yielded two groups of cellulase with molecular weights of 60 and 120 kDa. Sucrose wash incurred higher cellulase activity and more protein than Triton X-100 extraction for recovering cell surface associated enzymes from F. succinogenes S85. Groups of proteins of 40 to 50 kDa molecular weight with cellulase activity was purified from cell surface sample of F. succinogenes S85. Xylanase of 62 kDa molecular weight was purified from Ruminococcus albus 7 ECF by using ion-exchange and gel filtration column, and the specifity activity of the xylanase was 94 μg xylose/min/mg. Cellulose binding protein (CBP) from F. succinogenes S85 was collected by adherence to Avicel PH 101 followed by 5% SDS buffer elution. Specific activities of cellulase from F. succinogenes S85 and xylanase from R. albus 7 were increased by 1.1 to 8.3 times in the presence of CBP. The result showed that CBP can effectively support non-cellulase fibrolytic enzyme from other rumen fibrolytic bacterium.

Part 2
In this study, Prevotella ruminicola 23 (ATCC 19189), a ruminal proteolytic bacterium, was used to examine the optimal condition for protease production. The carbon and nitrogen sources for the maximum growth of bacteria were sucrose and peptone. Both sucrose and glucose could stimulate protease production. Casein and peptone were the better nitrogen sources for protease production. The best enzyme production condition was 18 to 20 hours incubation at 39℃in the broth of　5% glucose or sucrose as carbon source with 0.1% ammonium chloride plus 0.2% peptone as nitrogen sources. Most of the protease activity was secreted into broth (65%) and 18% on cell surface, protease activity was low in periplasmic and intracellular portions. The optimal condition for protease reaction were 40℃and pH 6.8. The crude extract maintained 50% of its protease activity at 30 and 50℃, and its protease activity was stable between pH 6 to 8. The protease inhibitor test showed that serine, aspartic acid and metallo-protease inhibitors could cause the inhibition of proteolysis. Protein feedstuff degradation test showed that protease in crude extract had high degradation ability on fish meal, whey, and feather meal ( 2.39 , 2.60 and 1.76μmol amino acid/mg/hr ) in comparison with soybean meal and blood meal ( 1.11 and 1.09μmol amino acid/mg/hr ) .

Part 3

Cellulase from Aspergillus niger,

並列關鍵字

rumen bacteria ； cellulytic enzyme ； protease ； enzyme protection

參考文獻

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