Background: Two-domain laccases are copper-containing oxidases found in bacteria in the beginning of 2000ths. Two-domain laccases are known for their thermal stability, wide substrate specificity and, the most important of all, their resistance to so-called «strong inhibitors» of classical fungal laccases (azides, fluorides). Low redox potential was found to be specific for all the two-domain laccases, due to which these enzymes lost the researchers’ interest as potentially applicable for various biotechnological purposes, such as bioremediation. Searching, obtaining and studying the properties of novel two-domain laccases will help to obtain an enzyme with high redox-potential allowing its practical application. Methods: A gene encoding two-domain laccase was identified in Catenuloplanes japonicus genome, cloned and expressed in an Echerichia coli strain. The protein was purified to homogeneity by immobilized metal ion affinity chromatography. Its molecular properties were studied using electrophoresis in native and denaturing conditions. Physico-chemical properties, kinetic characteristics, substrate specificity and decolorization ability of laccase towards triphenylmethane dyes were measured spectrophotometrically. Results: A novel two-domain recombinant laccase CjSL appeared to be a multimer with a subunit molecular mass of 37 kDa. It oxidized a wide range of phenolic substrates (ferulic acid, caffeic acid, hydroquinone, catechol, etc.) at alkaline pH, while oxidizing of non phenolic substrates (K₄[Fe(CN)₆], ABTS) was optimal at acidic pH. The UV-visible absorption spectrum of the purified enzyme was specific for all two-domain laccases with peak of absorption at 600 nm and shoulder at 340 nm. The pH optima of CjSL for oxidation of ABTS and 2, 6-DMP substrates were 3.6 and 9.2 respectively. The temperature optimum was 70 °C. The enzyme was most stable in neutral-alkaline conditions. CjSL retained 53% activity after pre-incubation at 90 °C for 60 min. The enzyme retained 26% activity even after 60 min of boiling. The effects of NaF, NaN₃, NaCl, EDTA and 1,10-phenanthroline on enzymatic activity were investigated. Only 1,10-phenanthroline reduced laccase activity under both acidic and alkaline conditions. Laccase was able to decolorize triphenylmethane dyes and azo-dyes. ABTS and syringaldehyde were effective mediators for decolorization. The efficacy of dye decolorization depended on pH of the reaction medium.

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.

Laccase belongs to the family of copper-containing oxidases. A study was made of the mechanism that sustains the incorporation of copper ions into the T2/T3 centers of recombinant two-domain laccase ...

The gene for a previously unexplored two-domain laccase was identified in the genome of actinobacterium Streptomyces carpinensis VKM Ac-1300. The two-domain laccase, named ScaSL, was produced in a heterologous expression system (Escherichia coli strain M15 [pREP4]). The enzyme was purified to homogeneity using affinity chromatography. ScaSL laccase, like most two-domain laccases, exhibited activity in the homotrimer form. However, unlike the most two-domain laccases, it was also active in multimeric forms. The enzyme exhibited maximum activity at 80°C and was thermally stable. Half-inactivation time of ScaSL at 80°C was 40 min. The laccase was able to oxidize a non-phenolic organic compound ABTS at a maximum rate at pH 4.7, and to oxidized a phenolic compound 2,6-dimethoxyphenol at a maximum rate at pH 7.5. The laccase stability was observed in the pH range 9-11. At pH 7.5, laccase was slightly inhibited by sodium azide, sodium fluoride, and sodium chloride; at pH 4.5, the laccase was completely inhibited by 100 mM sodium azide. The determined K_m and k_cat of the enzyme for ABTS were 0.1 mM and 20 s^–1, respectively. The K_m and k_cat for 2,6-dimethoxyphenol were 0.84 mM and 0.36 s^–1, respectively. ScaSL catalyzed polymerization of humic acids and lignin. Redox potential of the laccase was 0.472 ± 0.007 V. Thus, the ScaSL laccase is the first characterized two-domain laccase with a middle redox potential. Crystal structure of ScaSL was determined with 2.35 Å resolution. Comparative analysis of the structures of ScaSL and other two-domain laccases suggested that the middle potential of ScaSL may be associated with conformational differences in the position of the side groups of amino acids at position 230 (in ScaSL numbering), which belong to the second coordination sphere of the copper atom of the T1 center.