Tuisel H, Sinclair R, Bumpus J A, Ashbaugh W, Brock B J, Aust S D
Biotechnology Center, Utah State University, Logan 84322-4430.
Arch Biochem Biophys. 1990 May 15;279(1):158-66. doi: 10.1016/0003-9861(90)90476-f.
The wood-destroying fungus Phanerochaete chrysosporium secretes extracellular enzymes known as lignin peroxidases that are involved in the biodegradation of lignin and a number of environmental pollutants. Several lignin peroxidases are produced in liquid cultures of this fungus. However, only lignin peroxidase isozyme H8 has been extensively characterized. In agitated nutrient nitrogen-limited culture, P. chrysosporium produces two lignin peroxidases in about equal proportions. The molecular weights of these two major proteins (H2 and H8) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 38,500 (H2) and 42,000 (H8). The isoelectric points of these enzymes were 4.3 for H2 and 3.65 for H8. All subsequent experiments in this study were performed with H2 as it contributed the most (42%) to total activity and had the highest specific activity (57.3 U/mg). The Km values of lignin peroxidase H2 for H2O2 and veratryl alcohol were calculated to be 47 microM and 167 microM at pH 3.5, respectively. The pH optima for veratryl alcohol oxidase activity were pH 2.5 at 25 degrees C, pH 3.0 at 35 degrees C, and pH 3.5 at 45 degrees C. In the same manner the temperature optimum shifted from 25 degrees C at pH 2.5 to 45 degrees C at pH 3.5 and approximately 45-60 degrees C at pH 4.5. During storage the resting enzyme was relatively stable for 48 h up to 50 degrees C. Above this temperature the enzyme lost all activity within 6 h at 60 degrees C. At 70 degrees C all activity was lost within 10 min. The resting enzyme retained approximately 80% of its initial activity when stored at 40 degrees C for 21 h at a pH range of 4.0-6.5. Above pH 7.5 and below 4.0, the enzyme lost all activity in less than 5 h. During turnover the enzyme remained active at pH 5.5 for over 2 h whereas the enzyme activity was lost after 45 min at pH 2.5. The oxidation of veratryl alcohol was inhibited by EDTA, azide, cyanide, and by the catalase inhibitor 3-amino-1,2,4-triazole, but not by chloride. In the absence of another reducing substrate incubation of lignin peroxidase H2 with excess H2O2 resulted in partial and irreversible inactivation of the enzyme. The spectral characteristics of lignin peroxidase H2 are similar to those of other peroxidases. The suitability of lignin peroxidases for industrial applications is discussed.
破坏木材的真菌黄孢原毛平革菌会分泌一种名为木质素过氧化物酶的胞外酶，这种酶参与木质素和多种环境污染物的生物降解。该真菌在液体培养中会产生几种木质素过氧化物酶。然而，只有木质素过氧化物酶同工酶H8得到了广泛的表征。在搅拌的营养氮限制培养中，黄孢原毛平革菌产生两种比例大致相等的木质素过氧化物酶。通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳测定，这两种主要蛋白质（H2和H8）的分子量分别为38,500（H2）和42,000（H8）。这些酶的等电点H2为4.3，H8为3.65。本研究中所有后续实验均使用H2进行，因为它对总活性的贡献最大（42%）且比活性最高（57.3 U/mg）。木质素过氧化物酶H2对过氧化氢和藜芦醇的米氏常数在pH 3.5时分别计算为47 microM和167 microM。藜芦醇氧化酶活性的最适pH在25℃时为pH 2.5，35℃时为pH 3.0，45℃时为pH 3.5。同样，最适温度从pH 2.5时的25℃转变为pH 3.5时的45℃，在pH 4.5时约为45 - 60℃。在储存过程中，静止酶在高达50℃的温度下48小时内相对稳定。高于此温度，在60℃时酶在6小时内失去所有活性。在70℃时，所有活性在10分钟内丧失。当在40℃、pH范围为4.0 - 6.5的条件下储存21小时时，静止酶保留了其初始活性的约80%。高于pH 7.5和低于4.0时，酶在不到5小时内失去所有活性。在周转过程中，酶在pH 5.5时保持活性超过2小时，而在pH 2.5时45分钟后酶活性丧失。藜芦醇的氧化受到EDTA、叠氮化物、氰化物以及过氧化氢酶抑制剂3 - 氨基 - 1,2,4 - 三唑的抑制，但不受氯离子抑制。在没有另一种还原底物的情况下，木质素过氧化物酶H2与过量过氧化氢孵育会导致酶的部分不可逆失活。木质素过氧化物酶H2的光谱特征与其他过氧化物酶相似。文中讨论了木质素过氧化物酶在工业应用中的适用性。
