一种pH稳定的黄色漆酶的快速纯化和性质特征

A pH-stable Yellow Laccase from White Rot Fungus Trametes sp. SQ01: Rapid Purification and Characterization

摘要:

通过丙酮沉淀和 DEAE- cellulose DE52 柱层析, 快速、有效地从一株白腐菌 Trametes sp. SQ01 的发酵液中纯化了漆酶。纯化的漆酶并非传统漆酶那样呈现蓝色, 而是一种黄色蛋白。以 ABTS 为底物时, 该酶的最适 pH 和温度分别是 pH 4.5 和 70°C, Km 为 0.029 mmol/L。T. SQ01 漆酶在 pH 3.0~11.0时, 酶活相对稳定, 在 pH 5.0 时最为稳定, 是目前报道的 pH 稳定性最好的漆酶。低浓度的金属离子(1 mmol/L) Cu2+、Mg2+ 、Ca2+ 和Co2+ 对漆酶有促进作用, 而高浓度(5 mmol/L)的Co2+、Zn2+、 Mn2+、Mg2+ 却抑制漆酶酶活。SDS 对该酶有激活作用, 当其浓度为1 mmol/L时, 漆酶相对酶活达到128%。DTT对漆酶强烈抑制, 即使是浓度为1 mmol/L, 亦可完全抑制漆酶酶活。纯化后的漆酶对亮蓝(RBBR) (100 mg/L)的脱色能力显著, 0.5 U/mL 的漆酶在 10 min内即可达到 80%的脱色率。T. sp. SQ01 漆酶的快速纯化以及高效脱色的能力表明该酶在染料脱色降解方面有着广阔的应用前景。

Abstract:

The white rot fungus Trametes sp. SQ01 secretes a high level of laccase in the basal liquid medium without induction. The laccase has been purified to homogeneity through acetate acetone precipitation and DEAE-cellulose 52 anion-exchange chromatography with a final purification fold of 15.4 and an overall yield of 43.6%. The purified enzyme was identified with a molecular mass of 62 kD by SDS gel electrophoresis. The purified enzyme was not blue like the typical laccase but yellow, and had not the basic spectroscopic features of a typical blue laccase. The enzyme oxidized a series of diphenol, creosol and non-phenol, including 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonate) (ABTS), catechols, hydropuinone, 2,6-dimethoxyphenol (DMP), and guaiacol. With ABTS as a substrate, the optimum pH and temperature for the purified laccase were 4.5 and 70°C, respectively. The enzyme was highly stable in the pH range 3~11, and the most stable under the pH 5.0. The enzyme was stable up to 50°C, and had half-life of 30 min at 60°C. The susceptibility of laccase towards several surfactants, inhibitors and metal cations was also assessed. The enzyme activity was completely inhibited by DTT at the concentration of 1 mmol/L, but 1 mmol/L of SDS activated the laccase activity by 128%. Laccase activity was also inhibited by several metal cations at a 5 mmol/L of concentration, especially Mn2+. The purified enzyme efficiently decolorized Remazol Brilliant Blue R (RBBR) in the absence of added redox mediators. The high production of Trametes. sp. SQ01 laccase as well as its decolorization ability demonstrated its potential application on dye decolorization.