G. K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences (IBPhM RAS), Pushchino, Russia.
Faculty of Soil Science, Lomonosov Moscow State University, Moscow, Russia.
PLoS One. 2020 Sep 18;15(9):e0239005. doi: 10.1371/journal.pone.0239005. eCollection 2020.
The two-domain bacterial laccases oxidize substrates at alkaline pH. The role of natural phenolic compounds in the oxidation of substrates by the enzyme is poorly understood. We have studied the role of ferulic and caffeic acids in the transformation of low molecular weight substrates and of soil humic acid (HA) by two-domain laccase of Streptomyces puniceus (SpSL, previously undescribed). A gene encoding a two-domain laccase was cloned from S. puniceus and over-expressed in Escherichia coli. The recombinant protein was purified by affinity chromatography to an electrophoretically homogeneous state. The enzyme showed high thermal stability, alkaline pH optimum for the oxidation of phenolic substrates and an acidic pH optimum for the oxidation of K4[Fe(CN)6] (potassium ferrocyanide) and ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt). Phenolic compounds were oxidized with lower efficiency than K4[Fe(CN)6] and ABTS. The SpSL did not oxidize 3.4-dimethoxybenzoic alcohol and p-hydroxybenzoic acid neither in the absence of phenolic acids nor in their presence. The enzyme polymerized HA-the amount of its high molecular weight fraction (>80 kDa) increased at the expense of low MW fraction (10 kDa). The addition of phenolic acids as potential mediators did not cause the destruction of HA by SpSL. In the absence of the HA, the enzyme polymerized caffeic and ferulic acids to macromolecular fractions (>80 kDa and 10-12 kDa). The interaction of SpSL with HA in the presence of phenolic acids caused an increase in the amount of HA high MW fraction and a two-fold increase in the molecular weight of its low MW fraction (from 10 to 20 kDa), suggesting a cross-coupling reaction. Infrared and solution-state 1H-NMR spectroscopy revealed an increase in the aromaticity of HA after its interaction with phenolic acids. The results of the study expand our knowledge on the transformation of natural substrates by two-domain bacterial laccases and indicate a potentially important role of the enzyme in the formation of soil organic matter (SOM) at alkaline pH values.
两域细菌漆酶在碱性 pH 下氧化底物。天然酚类化合物在酶氧化底物中的作用还知之甚少。我们研究了阿魏酸和咖啡酸在低分子量底物和土壤腐殖酸 (HA)转化中的作用，所用的酶是一株紫色链霉菌（Streptomyces puniceus）的两域漆酶（SpSL，以前未描述过）。从紫色链霉菌中克隆了一个编码两域漆酶的基因，并在大肠杆菌中过表达。通过亲和层析将重组蛋白纯化至电泳均一状态。该酶表现出高热稳定性、酚类底物氧化的碱性 pH 最优值和 K4[Fe(CN)6]（亚铁氰化钾）和 ABTS（2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt）氧化的酸性 pH 最优值。与 K4[Fe(CN)6]和 ABTS 相比，酚类化合物的氧化效率较低。SpSL 既不能在没有酚酸的情况下也不能在有酚酸的情况下氧化 3.4-二甲氧基苯甲酸和对羟基苯甲酸。该酶聚合 HA-其高分子量部分（>80 kDa）的量增加，而低 MW 部分（10 kDa）的量减少。添加潜在的介体酚酸并没有导致 SpSL 破坏 HA。在没有 HA 的情况下，该酶将阿魏酸和咖啡酸聚合到高分子量部分（>80 kDa 和 10-12 kDa）。在有酚酸的情况下，SpSL 与 HA 的相互作用导致 HA 高分子量部分的量增加，其低 MW 部分（从 10 到 20 kDa）的分子量增加一倍，表明发生了交叉偶联反应。红外和溶液 1H-NMR 光谱显示，HA 与酚酸相互作用后芳香度增加。该研究结果扩展了我们对两域细菌漆酶转化天然底物的认识，并表明该酶在碱性 pH 值下形成土壤有机质 (SOM) 中可能具有重要作用。
