PURIFICATION AND CHARACTERIZATION OF AN EXTRACELLULAR MN(II)-DEPENDENT PEROXIDASE FROM THE LIGNIN-DEGRADING BASIDIOMYCETE, PHANEROCHAETE-CHRYSOSPORIUM
PURIFICATION AND CHARACTERIZATION OF AN EXTRACELLULAR MN(II)-DEPENDENT PEROXIDASE FROM THE LIGNIN-DEGRADING BASIDIOMYCETE, PHANEROCHAETE-CHRYSOSPORIUM
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DOI：
10.1016/0003-9861(85)90217-6
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发表时间：
1985-01-01
影响因子：
3.9
通讯作者：
GOLD, MH
中科院分区：
文献类型：
--
作者：
GLENN, JK;GOLD, MH
在白色腐真菌黄孢原毛平革菌木质素降解培养物的胞外培养基中发现了一种Mn（II）依赖的过氧化物酶，该酶经DEAE-Sepharose离子交换层析、Blue Agarose层析和Sephadex G-100凝胶过滤纯化。十二烷基硫酸钠凝胶电泳表明，均匀的蛋白质具有46，000的Mr。酶的吸收光谱表明存在血红素辅基。吡啶血色素的吸收光谱表明，该酶含有一个血红素分子的铁原卟啉IX。天然酶的最大吸收（406 nm）在还原酶中移动到433 nm，在还原-CO复合物中移动到423 nm。CN-和N3-都很容易与天然酶结合，表明存在一个可用的配位位点，并且血红素铁是高自旋的。H2 O2酶复合物的吸收光谱（最大值在420 nm处）与辣根过氧化物酶化合物II的吸收光谱相似。黄孢原毛平革菌过氧化物酶活性依赖于Mn（II），在100 μ M以上达到最大活性。该酶也被α-β-葡萄糖苷酶不同程度地刺激。羟基酸（例如，苹果酸，乳酸）和蛋白质（例如，明胶、白蛋白）。过氧化物酶能够氧化NADH和多种染料，包括Poly B-411和Poly R-481。几种底物（靛蓝三磺酸盐、NADH、Poly B-411、Variamine蓝RT盐和Poly R-481）被这种Mn（II）依赖性过氧化物酶氧化的速率比辣根过氧化物酶催化的速率快得多。该酶迅速氧化锰（II）为锰（III），后者是由其焦磷酸盐复合物的特征吸收光谱检测。抑制氧化的底物二铵2，2-连氮基-双（3-乙基-6-苯并噻唑啉磺酸盐）（ABTS）由焦磷酸钠表明，锰（III）在酶的机制中发挥作用。
A Mn(II)-dependent peroxidase found in the extracellular medium of ligninolytic cultures of the white rot fungus, Phanerochaete chrysosporium, was purified by DEAE-Sepharose ion-exchange chromatography, Blue Agarose chromatography, and gel filtration of Sephadex G-100. Sodium dodecyl sulfate-gel electrophoresis indicated that the homogeneous protein has an Mr of 46,000. The absorption spectrum of the enzyme indicates the presence of a heme prosthetic group. The pyridine hemochrome absorption spectrum indicates that the enzyme contained one molecule of heme as iron protoporphyrin IX. The absorption maximum of the native enzyme (406 nm) shifted to 433 nm in the reduced enzyme and to 423 nm in the reduced-CO complex. Both CN- and N3- readily bind to the native enzyme, indicating an available coordination site and that the heme iron is high spin. The absorption spectrum of the H2O2 enzyme complex, maximum at 420 nm, is similar to that of horseradish peroxidase compound II. P. chrysosporium peroxidase activity is dependent on Mn(II), with maximal activity attained above 100 .mu.M. The enzyme is also stimulated to varying degrees by .alpha.-hydroxy acids (e.g., malic, lactic) and protein (e.g., gelatin, albumin). The peroxidase is capable of oxidizing NADH and a wide variety of dyes, including Poly B-411 and Poly R-481. Several of the substrates (indigo trisulfonate, NADH, Poly B-411, variamine blue RT salt, and Poly R-481) are oxidized by this Mn (II)-dependent peroxidase at considerably faster rates than those catalyzed by horseradish peroxidase. The enzyme rapidly oxidizes Mn(II) to Mn(III); the latter was detected by the characteristic absorption spectrum of its pyrophosphate complex. Inhibition of the oxidation of the substrate diammonium 2,2-azino-bis(3-ethyl-6-benzothiazolinesulfonate) (ABTS) by Na-pyrophosphate suggests that Mn(III) plays a role in the enzyme mechanism.