探討聚木醣酶XynR8(N58D)形成酵素交聯聚集體的方法及對酵素生化特性的影響

Investigation on the method of forming cross-linked enzyme aggregates for xylanase XynR8(N58D) and the biochemical properties of the resulting aggregates

徐瑋涏(Hsu Wei-Ting)

指導教授 ： 鄭雪玲

國立屏東科技大學/農學院/生物科技系/碩士(2021年)

https://doi.org/10.6346/NPUST202100475

摘要

本團隊於前期研究，由台灣水牛瘤胃真菌選殖出一株高活性聚木醣酶並成功改造之，稱之XynR8(N58D)。本研究嘗試以酵母菌Pichia pastoris 胞外表現XynR8(N58D)，並以硫酸銨沉澱下來再以glutaraldehyde進行交聯製作成酵素交聯聚集體(cross-linked enzyme aggregates ；CLEA)。將XynR8(N58D)的未交聯酵素(free enzyme)及CLEA進行生化特性之比較。結果發現，以2.5% glutaraldehyde反應2小時製作成CLEA為最適條件。CLEA的最佳反應溫度及pH值，分別為65℃及pH 7.0；free enzyme則為55℃及pH 6.0。於受質濃度為20 mg/mL時，CLEA及free enzyme的比活性分別為6917.0 units/mg及12649.2 units/mg。CLEA明顯比free enzyme耐熱，可耐熱至80℃。CLEA也明顯比free enzyme耐酸鹼。另外，CLEA對SDS及對有機溶劑(甲醇及乙醇)的耐受性，明顯比free enzyme佳。再者，CLEA於重複使用5次後，仍能保持73.1  2.6%的活性；重複使用8次後，活性為38.2  0.5%。所以研究證實，製作成CLEA能改善XynR8(N58D)的穩定性。結論是，XynR8(N58D)製作成CLEA，除了能維持高活性外，明顯提升其穩定性及重複利用性，使之更有商業利用的潛力。

關鍵字

台灣水牛 ； 瘤胃真菌 ； 聚木醣酶 ； 酵素交聯聚集體

並列摘要

A highly active xylanase XynR8 was subcloned from rumen fungi of Bubalus bubalis. The enzyme was engineered to further elevate its catalytic activity by site-directed mutagenesis. The mutant was designated XynR8(N58D). In this study, XynR8(N58D) was expressed in Pichia pastoris extracellularly, precipitated with ammonia sulfate, and cross linked by glutaraldehyde to produce cross-linked enzyme aggregates (CLEA). The biochemical properties of XynR8 (N58D) free enzyme and CLEA were then compared. The optimal condition for generating CLEA was found to be 2.5% glutaraldehyde reacted for 2 hours. The optimal reaction temperature and pH for CLEA were 65°C and pH 7.0, and 55°C and pH 6.0 for the free enzyme. The specific activity of CLEA and free enzyme were 6917.0 units/mg and 12649.2 units/mg, respectively, when substrate concentrate was 20 mg/mL. CLEA was obviously more thermo-stable than free enzyme that CLEA can tolerate up to 80°C. CLEA was also more stable in extreme pH than free enzyme. Moreover, CLEA was obviously more tolerant to SDS, methanol and ethanol than free enzyme. Furthermore, CLEA maintained 73.1  2.6% activity after repeatedly used for 5 times, and 38.2  0.5% activity after 8 times of repected use. Therefore, this study confirmed that the CLEA of XynR8 (N58D) is more stable than its free enzyme. The CLEA of XynR8 (N58D) still possess a high activity are more stable than free enzyme, and can be used repeatedly, leading to elevated potential of XynR8(N58D) in commercial application .

並列關鍵字

Bubalus bubalis ； rumen fungi ； xynR8 ； cross-linked enzyme aggregates

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