纖維素水解酶 C7(-105)形成酵素交聯聚集體的研究

團隊於前期研究，分析一段選殖自台灣黃牛瘤胃真菌Orpinomyces sp. Y102的外切型纖維素水解酶基因，轉譯的酵素稱之為C7。經過研究後將C7改造成N端truncated form，稱為C7(-105)。本研究的目的是嘗試以酵母菌Pichia pastoris胞外表現C7(-105)，然後以硫酸銨進行沉澱，再以glutaraldehyde進行交聯製作成酵素交聯聚集體(cross-linked enzyme aggregates；CLEA)。分析C7(-105) CLEA形成的條件及其生化特性，並與原酵素 (soluble enzyme)比較穩定性。結果發現，以連續式沉澱法先用49.85 %硫酸銨飽和濃度進行第一次沉澱去除部分雜蛋白，再用76.11 %硫酸銨飽和濃度進行第二次沉澱將大部分C7(-105)沉澱下來。接著以4.5 % glutaraldehyde反應2小時進行交聯，是製作C7(-105) CLEA的適當條件。CLEA的最適反應溫度及pH值分別為50 ℃及pH 6.5；soluble enzyme則為45 ℃及pH 6.0。於受質barley β-glucan濃度為10 mg/mL時，CLEA及soluble enzyme的比活性分別為663.8 ± 36.9 units/mg及1026.7 ± 110.2 units/mg。CLEA於60 ℃仍維持80 %以上的活性，soluble enzyme則是45 ℃；CLEA於pH 4.5~11.0能維持80 %以上的活性，soluble enzyme則於pH 5.0~6.5能維持80 %以上的活性。另外，CLEA對介面活性劑 (SDS)和有機溶劑 (甲醇與乙醇)的耐受性皆明顯比soluble enzyme佳；CLEA於4 ℃保存8天可維持50 %以上的活性，soluble enzyme則是4天。最後，CLEA於重複使用7次後，仍有66.3 %活性。綜合這些結果，認為C7(-105) 製作成CLEA能提高C7(-105)的穩定性及保存期限，雖然活性約為原酵素的65 %，在外切型纖維素水解酶中仍是高活性，使之更具有工業應用的潛力。

關鍵字

瘤胃真菌；畢赤酵母菌；外切型纖維素水解酶；酵素交聯聚集體

並列摘要

C7(-105) is an N-terminal truncated cellobiohydrolase originated from the rumen fungus Orpimonyces sp. Y102. C7(-105) is highly active but unstable. The purpose of this study was to develop an appropriate protocol for producing the cross-linked enzyme aggregates (CLEA) of C7(-105), and to examine whether the CLEA of C7(-105) are more stable than its soluble form. C7(-105) was over-expressed in the extracellular space of Pichia pastoris. Ammonium sulfate was added into the crude enzyme solution (the medium of P. pastoris) to reach 49.85% saturation to precipitate some unwanted proteins, followed by adjusting ammonium sulfate to 76.11% saturation to precipitate most C7(-105) from the solution. C7(-105) in the pellet was subsequently cross-linked with 4.5% glutaraldehyde for 2h. The resulting product is CLEA of C7(-105). The optimum reaction temperature and pH for C7(-105) CLEA are 50℃ and pH 6.5, respectively；they are 45℃ and pH 6.0 for soluble C7(-105), respectively. The specific activity of CLEA was 663.8 ± 36.9 units/mg at 10 mg/mL substrate (barley β-glucan) concentration；that of soluble enzyme was 1026.7 ± 110.2 units/mg. CLEA maintained over 80% activity when pre-heated at 60°C；that for soluble enzyme was 45℃. CLEA kept over 80% activity in pH 4.5 to 11.0, whereas soluble enzyme maintained over 80% activity in pH 5.0 to 6.5. The tolerance of CLEA to SDS, ethanol, and methanol was significantly better than that of soluble enzyme. When stored in 4℃, CLEA remained over 50% activity on Day 8th；that for soluble enzyme was Day 4th. After recycled use for 7 times, the remaining activity of CLEA was 66.3%. Cross linking can improve the stability and shelf life of C7(-105). Although the activity of CLEA is 65% of that of soluble enzyme, C7(-105) CLEA is still highly active among exoglucanase. Therefore, the CLEA of C7(-105) is highly potential for industrial applications.

並列關鍵字

rumen fungi ； Pichia pastoris ； cellobiohydrolase ； cross-linked enzyme aggregates

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