Transformation of low molecular compounds and soil humic acid by two domain laccase of Streptomyces puniceus in the presence of ferulic and caffeic acids.

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.