利用人造油體系統進行聚木糖酶及聚葡糖酶之固定化與純化

Abstract

由於聚木糖酶（xylanase）與聚葡醣酶（β-glucanase）對木質纖維素之水解能力，現已為工業上使用之兩大主要酵素族群。而近年來發展出來的人造油體（artificial oil body）系統，可同時視為一種酵素之固定化技術，及一種酵素的純化方法。因此，本研究擬嘗試利用此系統進行聚木糖酶與聚葡糖酶的固定化及純化。實驗中所使用之聚木糖酶與聚葡糖酶基因為xynCDBFV及1,3-1,4-β-D-glucan 4-glucanohydrolase，分別分離自瘤胃真菌Neocallimastix patriciarum及瘤胃細菌Fibrobacter succinogenes。首先，聚木糖酶與聚葡糖酶基因主要以intein為連接區域，融合至oleosin基因的胺基端，隨後誘導大腸桿菌過量表達此二融合蛋白質，並利用重組人造油體的過程進行酵素之固定化。然後再藉由反應曲面法（response surface methodology）及序列二次規畫法（sequential quadratic programming），獲得人造油體固定化聚木糖酶及聚葡糖酶之最適作用pH及溫度分別為pH 6.0、59℃及pH 8.8、39℃。在熱穩定性方面，人造油體固定化聚木糖酶於59℃培養120分鐘後，其活性仍維持在起始活性的60%；而人造油體固定化聚葡糖酶於40℃培養120分鐘後，其活性仍維持在起始活性的67%，但培養於50℃時，在20分鐘後活性就降至49%，120分鐘後則只剩10%。在重複使用性方面，人造油體固定化聚木糖酶使用9次後，活性為起始活性的70%；人造油體固定化聚葡糖酶在使用10次後，活性為起始活性的60%。另外，人造油體在加入1,4-dithiothreitol（DTT）後，確實可以誘導intein進行自我裂解，釋放出聚木糖酶及聚葡糖酶，且經活性電泳證實人造油體系統可純化出具有活性的聚木糖酶及聚葡糖酶。

Xylanases and cellulases are the two major groups of industrial enzymes due to their fibrolytic functions and the potential applications to a big range of industrial processes. These enzymes are produced by immobilization or purification techniques. One of the commendable techniques is called artificial oil body system, also called AOB system. During the previous years, the AOB system had been established and has provided as a novel method for enzyme immobilization and purification. The objective of this study is to investigate the efficiency of immobilization and purification of the xylanase and β-glucanase by using the AOB system. The two target enzyme genes used in this study are xyn-CDBFV and 1,3-1,4-β-D-glucan 4- glucanohydrolase gene. The xyn-CDBFV is a xylanase gene from rumen fungus, Neocallimastix patriciarum, and the 1,3-1,4-β-D-glucan 4-glucanohydrolase gene is from rumen bacterium, Fibrobacter succinogenes. Both of the enzyme genes were first overexpressed in Escherichia coli as recombinant proteins fused to the N terminus of oleosin with intein as a linker. These two recombinant enzymes were then immobilized on AOB through the process of AOB reconstitution. Afterward, response surface methodology (RSM) was applied to identify the most optimal reaction condition of the AOB-immobilized xylanase and AOB-immobilized β-glucanase. As the results, the optimal reaction condition for the highest AOB-immobilized xylanase activity (3.93 U/mg of total protein) was observed at pH 6 and 59℃, whereas it for the highest AOB-immobilized β-glucanase activity (6.9 U/mg of total protein) was observed at pH 8.8 and 39℃. Additionally, AOB-immobilized xylanase retained 60% of its maximal activity after 120 minutes at 59℃; AOB-immobilized β-glucanase retain 67% of its maximal activity after 120 minutes at 40℃, but only retain 10% at 50℃. They could be recycled by brief centrifugation. After reusing nigh times, AOB-immobilized xylanase still retained more than 70% of the original activity; while the activity of AOB-immobilized β-glucanase dropped to 60% for reusing 10 times. In terms of purification, xylanase and β-glucanase could be released from AOB by inducing self-splicing of intein and centrifuging. In this study, the results show that AOB system is an applicable method on immobilizing and purifying recombinant rumen microbial xylanase and β-glucanase. Through the system, the two enzymes could then possibly be applied to industrial purpose.