摘要
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 2 5℃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 5 0 %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) 2 SO4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 5 9kD ,凝胶过滤色谱法测定活性酶分子量 5 8kD。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 6 0℃ ,在 6 0℃和6 5℃时半衰期分别为 45min和 36 8min ,在pH5 2～ 7 2范围内稳定性较好。 10 0mmol LCl- 对该酶有显著抑制作用 ,1mmol LSO2 -4 对漆酶有激活作用 ,1mmol LNaN3 可完全抑制酶活性 ,10mmol LEDTA对漆酶活没有明显影响 ,1mmol LCu2 +对漆酶有激活作用。以愈创木酚为底物时 ,测得酶的Km =1 0 2 6mmol L ,Vmax=5 μmol (min·mg) ;以ABTS为底物时 ,测得其Km=0 2 2mmol L ,Vmax=6 9μmol (min·mg)。
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℃ 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 (NH 4) 2SO 4 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 59kD estimated by SDS-PAGE, while 58kD 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℃ respectively in catalytic reaction of oxidizing guaiacol. At 60℃ and 65℃, half-lives of laccase A were 45min and 36.8min, respectively. Enzyme activity was inhibited with 100mmol/L Cl -, but was activated with 1mmol/L SO 2- 4. However, if the concentration of NaN 3 was only 1mmol/L, laccase A lost its activity completely. 10 mmol/L EDTA had no effect on laccase A, while 1mmol/L Cu 2+ 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 K m was 1.026mmol/L and the V max was 5μmol/(min·mg); using ABTS instead, the K m was 0.22mmol/L and V max was 69μmol/(min·mg).
出处
《生物工程学报》
CAS
CSCD
北大核心
2002年第4期457-462,共6页
Chinese Journal of Biotechnology
基金
安徽省自然科学基金资助项目 (No .982 12 411)
教委自然科学基金资助项目 (No .2 0 0 0J10 13)~~
关键词
蜜环菌
胞外漆酶
合成
纯化
发酵条件
酶学性质
Armilliria mellea , extracellular laccase, fermentation conditions, enzyme characteristics
