【目的】研究木质素降解子囊菌Paraconiothyrium variabile GHJ-4分泌的漆酶(laccase)、锰过氧化物酶(MnP)和木质素过氧化物酶(LiP)3种木质素降解酶的酶活性变化规律及其酶学性质,为利用该菌株进行木质素降解酶的工业化生产和应用提供依据。【方法】分别以愈创木酚、2,6-二甲基苯酚和黎芦醇为底物测定laccase,MnP和LiP的酶活性,研究3种酶的最适反应温度和pH、温度和pH稳定性及金属离子对其活性的影响。【结果】GHJ-4菌株发酵18,15和21天后,分别获得laccase,MnP和LiP最高酶活为1 390.3,30.3和52.5 U·mL^-1。laccase的最适反应温度为55℃左右,最适反应pH为5.5左右,在55℃以下、pH 4.0-7.0的范围内较为稳定;MnP的最适反应温度为60℃左右,最适反应pH为5.0左右,在55℃以下、pH 4.0-9.0的范围内较为稳定;LiP的最适反应温度为40℃左右,最适反应pH为3.0左右,在40℃以下、pH 2.0-4.0的范围内较为稳定。Mg^2+,Zn^2+,Cu^2+,K^+对laccase起促进作用,Na^+和Zn^2+对LiP起促进作用,Mn^2+对MnP起激活作用,而Fe^3+,Ca^2+,Pb^2+,Co^2+,Al^3+对3种酶均起抑制作用,其中尤以5 mmol·L^-1Fe^3+对3种酶的抑制作用最强。【结论】温度、pH及金属离子对Paraconiothyrium variabile GHJ-4 3种木质素降解酶的酶学特性均有着不同程度的影响。
【Objective】The production and enzymological characteristics of ligninolytic enzymes,including laccase,manganese peroxidase(MnP) and lignin peroxidase(LiP) produced by Paraconiothyrium variabile GHJ-4 were investigated.This study could provide theoretical data for commercial process and application of Paraconiothyrium variabile GHJ-4 ligninolytic enzymes.【Method 】Laccase,MnP and LiP activity were measured with guaiacol,2,6-Dimethylphenol and veratryl alcohol as substrate respectively.Effects of temperature,pH value and metal irons on three ligninolytic enzymes were assayed.【Result】Laccase,MnP and LiP activity reached the peak value of 1 390.3 U·mL^-1,30.3 U·mL^-1 and 52.5 U·mL^-1 respectively on the 18 th,15thand 21 stday during the fermentation.The optimum temperature and pH of laccase were 55 ℃ and 5.5 respectively,and the enzyme activity was stable under 55 ℃ and pH4.0-7.0.The optimum temperature and pH of MnP were 60 ℃ and 5.0 respectively,and the enzyme activity was stable under 55 ℃ and pH 4.0-9.0.The optimum temperature and pH of LiP were 40 ℃ and 3.0 respectively,and the enzyme activity was stable under 40 ℃ and pH 2.0-4.0.Laccase activity was enhanced by the metal ions Mg^2+,Zn^2+,Cu^2+,K^+,LiP activity was enhanced by Na+and Zn^2+,and MnP activity was enhanced by Mn^2+.Whereas three ligninolytic enzymes were inhibited by Fe^3+,Ca^2+,Pb^2+,Co^2+ and Al^3+,especially the effect of Fe^3+ was the strongest.【Conclusion】The effects of temperature,pH and metal ions on activities of ligninolytic enzymes from Paraconiothyrium variabile GHJ-4 were different.