白腐真菌 Cerrena sp. WR1 漆化酵素之生化特性及染劑脫色效果與其在菸草內表現之研究

Abstract

漆化酵素 (laccase) 被視為是一個廣泛應用在許多工業上的綠色酵素，可應用在生物製漿，生物復育和人工染料的脫色。本研究的目標是探討一個分離自台灣本土的白腐真菌 Cerrena sp. WR1 所純化得到的漆化酵素之生化特性及其對於一般廣泛應用在紡織工業上具毒性合成染料之脫色效率。我們利用40-60%的硫酸銨沉澱法和 Q-Sepharose 陽離子交換膠體管柱層析法純化得到漆化酵素，發現其為一醣蛋白質，分子量約為62.6 kDa。利用 LC-ESI tandem mass spectrometry 分析其胺基酸序列，發現此酵素是由我們研究先前從 Cerrena sp. WR1 所分離出的 laccase 3基因所表達，因而命名此酵素為 laccase 3。由光譜分析顯示，laccase 3為一典型的攜銅漆化酵素。laccase 3在 pH 8-10的鹼性環境下具有較高的穩定性，並且在 pH 6.0和50℃的環境下放置10小時仍能維持50%的酵素活性。在酵素動力學分析上，以 ABTS、2,6-dimethoxyphenol、guaiacol 及 catechol 為反應基質，laccase 3的催化效率 (kcat/Km) 分別為521 s-1 μM-1，1.11 s-1 μM-1，0.88 s-1 μM-1以及0.13 s-1 μM-1。此外，laccase 3在50 mM 檸檬酸緩衝液 (pH 3.0)、25℃和1.2 U/mL 酵素活性的反應條件下對於工業染料 Acid Blue 80 (AB80) 的脫色效果可達到99%；而對於 Acid Red 37 (AR37)、Remazol Brilliant Blue R (RBBR)、以及 Direct Red 24 (DR24) 在24 U/mL 酵素活性下的脫色效果分別是達到73%、91%與71%；對於 Direct Blue 71 (DB71) 的脫色效果在12 U/mL 酵素活性時可達到65%。此研究並利用 initial rate kinetics 和等溫滴定微量熱法 (isothermal titration calorimetry, ITC) 分析 laccase 3對於這些染劑化合物的動力學特性。由一般動力學分析結果顯示，laccase 3對 AB80、AR37、RBBR、以及 DR24 的催化效率分別為13.4 s-1 μM-1，0.24 s-1 μM-1，0.18 s-1 μM-1以及0.013 s-1 μM-1。而利用 ITC 分析的結果，對 AB80 及 RBBR 的催化效率分別為4.1 s-1 μM-1及0.08 s-1 μM-1。我們也利用結構模型分析來模擬 laccase 3的蛋白質立體結構及其和不同染劑化合物彼此間可能的氫鍵分子鍵結。其結果顯示有一個、三個、兩個氫鍵分別在 AB80 和 His454 之間，在 RBBR 和 Asp206 或 Asn264 之間，還有在 AR37 和 Arg157 之間形成。此外，植物生長之毒性試驗結果顯示，本實驗所使用的工業染料對水稻、阿拉伯芥與菸草種子發芽後植物幼苗與根系的生長有不同程度之抑制作用，然而這些化合物經 laccase 3處理後相對毒性顯著減弱。在另一方面，本篇研究也嘗試探討真菌漆化酵素在植物系統內的表達能力。利用農桿菌轉殖法獲得一些可能帶有 laccase 3基因的轉殖菸草植株，然而 laccase 3在菸草內的表現量和功能仍需進一步分析與確認。

Laccase (ρara-diphenol:dioxygen oxidoreductase, EC 1.10.3.2) is considered to be a green enzyme widely used in various industrial applications, such as biopulping, bioremediation, and synthetic dyes decolorization. The objective of this study was to characterize a native laccase purified from Cerrena sp. WR1, a locally identified white-rot fungus for its biochemical properties and degradation efficiency on toxic synthetic dyes popularly used in textile industry. The purified laccase (designated laccase 3) from Cerrena sp. WR1 was obtained by 40-60% ammonium sulfate precipitation and Q-Sepharose fast flow column chromatography. Laccase 3 protein was shown glycosylated and corresponding to the laccase 3 gene isolated in our laboratory, as verified by LC-ESI tandem mass spectrometry. The UV-Visible spectral analysis indicated that the typical type 1 copper absorption spectrum in laccase 3. Laccase 3 exhibited a higher stability at alkaline pH range of 8-10, and retained 50% of its activity after a 10-h incubation at 50℃ and pH 6.0. The catalytic efficiencies (kcat/Km) of laccase 3 determined for ABTS, 2,6-dimethoxyphenol, guaiacol and catechol as the substrates, were 521 s-1 μM-1, 1.11 s-1 μM-1, 0.88 s-1 μM-1, and 0.13 s-1 μM-1, respectively. A 99% decolorization efficiency on Acid Blue 80 (AB80) (with 1.2 U/mL laccase 3), 73% on Acid Red 37 (AR37) (with 24 U/mL laccase 3), 91% on Remazol Brilliant Blue R (RBBR) (with 24 U/mL laccase 3), 71% on Direct Red 24 (DR24) (with 24 U/mL laccase 3), and 65% on Direct Blue 71 (DB71) (with 12 U/mL laccase 3) was observed under 50 mM citrate buffer (pH 3.0) and 25℃ for 1-h. The kinetic properties of laccase 3 reacted with various dyes were analyzed using initial rate kinetics and isothermal titration calorimetry (ITC). The catalytic efficiencies (kcat/Km) of laccase 3 determined by initial rate kinetics on AB80, AR37, RBBR, and DR24 were 13.4 s-1 μM-1, 0.24 s-1 μM-1, 0.18 s-1 μM-1, and 0.013 s-1 μM-1, respectively, while kcat/Km of AB80 and RBBR determined by ITC were 4.1 s-1 μM-1 and 0.08 s-1 μM-1, respectively. Structural modeling analysis suggested that one, three, and two hydrogen bonds can be formed between AB80 and His454, between RBBR and Asp206, or Asn264, and between AR37 and Arg157, respectively. Phytotoxicity studies indicated that the toxicity of selected dyes on the growth of seedling and root of germinated rice, Arabidopsis and tobacco seeds can be effectively attenuated by laccase 3 treatment. On the other hand, this study also intended to study the expression efficiency of the fungal laccase in plant system. Some putative transgenic tobacco plants were obtained after agrobacterium-mediated laccase 3 gene transformation. The expression and function of the transformed fungal laccase 3 in transgenic tobacco plants, however, are remained to be further studied.