蜜环菌胞外漆酶的合成、纯化及性质研究

研究了蜜环菌胞外漆酶合成条件和酶学性质。实验表明，培养基初始pH5.5、培养温度25℃有利于菌株产酶；与麦芽糖、山梨糖和半乳糖相比，纤维二糖和棉子糖作为碳源时漆酶产量更高；有机氮源比无机氮源有利于漆酶合成。泥炭提取液可显著诱导漆酶生成，当其含量为50%时，菌株漆酶最高产量是对照组的7倍。在蜜环菌发酵上清液中检测到3个漆酶同功酶组分，其主要活性（约占75%）组份漆酶A经 (NH₄)₂SO₄沉淀、制备级PAGE电泳和阴离子交换柱层析被分离纯化至电泳均一，SDSPAGE法测得酶亚基分子量59kD，凝胶过滤色谱法测定活性酶分子量58kD。纯化的漆酶A等电点pI为4.0，氧化愈创木酚的最适反应pH为5.6，最适温度为60℃，在60℃和65℃时半衰期分别为45min和36.8min，在pH5.2～7.2范围内稳定性较好。100mmol/L Cl^-对该酶有显著抑制作用，1mmol/L SO^2-₄ 对漆酶有激活作用，1mmol/L NaN₃可完全抑制酶活性，10 mmol/L EDTA对漆酶活没有明显影响，1mmol/L Cu²⁺对漆酶有激活作用。以愈创木酚为底物时，测得酶的K_m=1.026mmol/L，V_max=5μmol/(min·mg)；以ABTS为底物时，测得其K_m=0.22mmol/L，V_max=69μmol/(min·mg)。

Studies on Production, Purification and Partial Characteristics of the ExtraceHular Laccase from Armilliria mellea

The production conditions of extracellular laccase from Armilliria mellea and the characteristics of the enzyme were studied. The experiment proved that initial pH5.5 of the culture medium and temperature at 25 degrees C were favorable for laccase synthesis. As carbon resources, cellobiose and raffinose were better in terms of productivity than maltose, sorbose and galactose. Organic nitrogen source was more suitable for Armilliria mellea to synthesize laccase than inorganic nitrogen source. Peat extract (PE) enhanced notably the yield of laccase; the maximal yield was 7 times as much as that of the control when PE concentration was 50%. Three isozymes were detected in culture supernatant named A, B and C respectively after their mobility on PAGE. After concentrated by (NH4)2SO4 precipitation, laccase A was further purified to homogeneity by preparative native PAGE and anion exchange column chromatography. The native enzyme was a single polypeptide with a molecular mass of approximately 59 kD estimated by SDS-PAGE, while 58 kD by gel filtration chromatography under non-denaturing conditions. Determined by IEF its isoelectric point was 4.0. The optimal pH value and temperature were 5.6 and 60 degrees C respectively in catalytic reaction of oxidizing guaiacol. At 60 degrees C and 65 degrees C, half-lives of laccase A were 45 min and 36.8 min, respectively. Enzyme activity was inhibited with 100 mmol/L Cl-, but was activated with 1 mmol/L SO4(2-). However, if the concentration of NaN3 was only 1 mmol/L, laccase A lost its activity completely. 10 mmol/L EDTA had no effect on laccase A, while 1 mmol/L Cu2+ could enhance its activity. Laccase A showed a good stability when the pH of the buffer varied from 5.2 to 7.2. Using guaiacol as the substrate, the Km was 1.026 mmol/L and the Vmax was 5 mumol/(min.mg); using ABTS instead, the Km was 0.22 mmol/L and Vmax was 69 mumol/(min.mg).