源自活性污泥多源基因體新穎酯酶Est03之蛋白純化與特性分析

Purification and characterization of a novel esterase Est03 derived from an activated sludge metagenome

黃子瑄(Tzu-Hsuan Huang) ； 廖仁寶(Ren-Bao Liaw)

《畜產研究》 54卷4期 (2021/12) Pp. 273-281

https://doi.org/10.6991/JTLR.202112_54(4).0006

摘要

本研究目的為針對從活性污泥多源基因體篩選而得的新穎酯酶基因est03進行次選殖、表現、純化及特性分析，以期供後續研究與應用。est03基因長762bp，可轉譯為含253個胺基酸的酯酶，分子大小約為28.8kDa，稱為Est03。將新穎酯酶基因est03次選殖於表現載體pPAL7上，轉形於表現宿主大腸桿菌（Escherichia coli BL 21（DE3））中。轉形株經IPTG誘導大量表現標的蛋白質後，利用套組純化並切除後成為與原始胺基酸序列相同的純化酵素。經特性分析後發現，Est03對於短鏈脂肪酸受質有較高喜好度，尤其對二碳受質（C2）活性顯著高於其他（P＜0.001），在溫度試驗中顯示它在35－50℃具有較佳活性，最適溫度為45℃，比活性為12.15 unit/mg，k_(cat)/K_m為1.58×10^(-3) s^(-1)．μM^(-1)。Est03在pH6.5至pH9.0間，相較於在其他pH值有較佳活性，且於pH8.0時活性最高。同時，Est03在常用種類與濃度之有機溶劑、金屬離子及介面活性劑環境中皆具有活性。綜而論之，Est03具有值得關注的生化特性，期能進一步開發應用於生物技術產業。

關鍵字

活性污泥多源基因體 ； 新穎酯酶 ； 特性分析

並列摘要

The purpose of this research was to perform sub-cloning, expression, purification, and characterization of the novel esterase gene est03 selected from the activated sludge metagenome for subsequent research and application. The est03 gene is 762 bp in length and can be translated into an esterase, namely Est03, which contains 253 amino acids with a molecular mass of about 28.8 kDa. The est03 gene was sub-cloned on the expression vector pPAL7 and transformed into the expression host Escherichia coli BL21 (DE3). After the transformant was induced by IPTG to express a large number of target proteins, the expressed esterase was purified and excised using the purification kit to become a purified enzyme with the same amino acid sequence of the original esterase. It was found that Est03 had a higher preference for short-chain fatty acid substrates, and particularly the two-carbon substrate (C2) activity was significantly higher than others (P ＜ 0.001). The temperature test showed that the esterase expressed higher activity between 35-50℃, with the optimal temperature of 45℃. The specific activity and k_(cat)/K_m of Est03 was 12.15 unit/mg and 1.58 × 10^(-3) s^(-1)．μM^(-1), respectively. Est03 had greater than 50% activity between pH 6.5 and pH 9.0, with the highest activity at pH 8.0. At the same time, Est03 was active in common types and concentrations of organic solvents, metal ions, and surfactants. In summary, Est03 has noteworthy biochemical properties and the potential for development in the applications of the biotechnology industry.

並列關鍵字

Activated sludge metagenome ； Novel esterase ； Characterization