A Novel Two-Domain Laccase with Middle Redox Potential: Physicochemical and Structural Properties.

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 Km and kcat of the enzyme for ABTS were 0.1 mM and 20 s-1, respectively. The Km and kcat 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.

Multifunctional Enzyme with Endoglucanase and Alginase/Glucuronan Lyase Activities from Bacterium Cellulophaga lytica.

Cellulophaga lytica is a Gram-negative aerobic bacterium in the genome of which there are many genes encoding polysaccharide degrading enzymes. One of the enzymes named ClGP contains a glycoside hydrolase domain from the GH5 family and a polysaccharide lyase domain from the PL31 family. The enzyme also contains the TAT signaling peptide and the TIGR04183 domain that indicates extracellular nature of the enzyme. Phylogenetic analysis has shown that the enzymes most closely related to ClGP and containing all four domains (TAT, GH5, PL31, TIGR04183) are widespread among bacterial species belonging to the Flavobacteriaceae family. ClGP produced by the recombinant strain of E. coli was purified and characterized. ClGP exhibited activity of endoglucanase (EC 3.2.1.4) and catalyzed hydrolysis of ß-D-glucan, carboxymethyl cellulose sodium salt (CMC-Na), and amorphous cellulose, but failed to hydrolyze microcrystalline cellulose and xylan. Products of CMC hydrolysis were cellobiose and cellotriose, whereas ß-D-glucan was hydrolyzed to glucose, cellobiose, cellotetraose, and cellopentaose. ClGP was more active against the poly-ß-D-mannuronate blocks than against the poly-α-L-glucuronate blocks of alginic acid. This indicates that the enzyme is a polyM lyase (EC 4.2.2.3). ClGP was active against polyglucuronic acid, so it displayed a glucuronan lyase (EC 4.2.2.14) activity. The enzyme had a neutral pH-optimum, was stable in the pH range 6.0-8.0, and displayed moderate thermal stability. ClGP effectively saccharified two species of brown algae, Saccharina latissima and Laminaria digitata, that suggests its potential for use in the production of biofuel from macroalgae.

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.