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Laccases are unable to oxidize the non-phenolic components of complex lignin polymer due to their less redox potential (E0). Catalytic efficiency of laccases relies on the mediators that potentiates their oxidative strength; for breaking the recalcitrant lignin. Laccase from Bacillus sp. SS4 was evaluated for its compatibility with natural and synthetic mediators. (2 mM). It was found that acetosyringone, vanillin, orcinol and veratraldehyde have no adverse effect on the laccase activity up to 3 h. Syringaldehyde, p-coumaric acid, ferulic acid and hydroquinone reduced the enzyme activity ≥50% after 1.0 h, but laccase activity remained 100 to ~120% in the presence of synthetic mediators HBT (1-Hydroxylbenzotrizole) and ABTS. (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) after 3 h. MgSO4 and MnSO4 (40 mM) increased the enzyme activity 3.5 fold and the enzyme possessed ≥70% activity at a very high concentration. (2 M) of NaCl. The enzyme retained 40-110% activity in the presence of 10% DMSO (dimethylsulfoxide), acetone, methanol and ethyl acetate. On the other hand, CuSO4 (100 μM) induced the laccase production 8.5 fold without increasing the growth of bacterial cells. Laccase from SS4 appropriately decolorized the indigo carmine (50 μM) completely in the presence of acetosyringone (100 μM) within 10 min and 25% decolorization was observed after 4 h without any mediator.

Laccases are multicopper oxidases known to catalyze the transformation of a wide range of phenolic and non-phenolic substrates using oxygen as electron acceptor and forming water as the only by product. Their potential relevance in several industries requires the constant search for novel laccases. Positive outcome of the isolation of laccase producing bacteria depends on the nature and concentration of media constituents. Several attempts to isolate laccase producing bacteria failed when the phosphate-containing M9 minimal medium was used. Shift to phosphate-less M162 medium led to successful isolations. Seven bacterial isolates belonging to genera Bacillus, Lysinibacillus, Bhargavaea and Rheinheimera were used to study the effect of medium constituents on laccase production. Inorganic phosphate (≥50 mM) was found to regulate laccase synthesis negatively though no inhibitory effect of phosphate (10-500 mM) was seen on laccase activity. All isolates ceased laccase synthesis when grown in the presence of tryptone (0.2-1%), with R. tangshanensis as an exception, or yeast extract (1.5-2%) as the only C/N source in M162 medium. Supplementation upto 0.1% of glucose in basal M162 medium increased laccase production in five isolates but decreased at higher concentrations. The influence of medium components on laccase synthesis was further affirmed by zymographic studies. These observations offer possibilities of isolating promising laccase producers from diverse environmental sources. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:541-548, 2017.

A putative multicopper oxidase, encoded as CopA in the proteome of Acinetobacter baumannii 19606, and designated as AbMCO, was expressed heterologously in E. coli (pET-28a) and purified by Ni-NTA affinity chromatography. The purified AbMCO exhibited in vitro oxidase activities upon exogenous addition of ≥1 μM copper ions. Kinetic studies revealed its phenol oxidase activity as it could catalyze the oxidation of substrates viz. 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), guaiacol, pyrogallol and catechol. Additionally, AbMCO displayed siderophore oxidase activity which depicted its role in metal homeostasis and protection from the toxic redox states of copper and iron. Importantly, expression of abMCO increased manifold upon challenge with high concentrations of copper sulphate (CuSO4, 1.5 mM) and sodium chloride (NaCl, 700 mM) which suggested its protective role in stress adaptation and management. Intra-macrophage assay of abMCO-expressing and abMCO-non expressing cells depicted no significant change in the survival rate of A. baumannii inside the macrophages. These findings indicate that A. baumannii encodes a multicopper oxidase, conferring copper tolerance and survival under stress conditions but had no role in virulence of this pathogen.

The acquisition of metal ions such as iron, copper and manganese is essential for the survival of microorganisms as these are constituents of metalloproteins including enzymes, storage proteins, structural elements, transcription factors and antimicrobial factors in various biological processes. However, excess of these metal ions is associated with significant toxicity due to spontaneous redox cycling of ions and obstruction of normal metabolic pathways. To overcome this, microbes have developed a variety of metal regulatory systems allowing them to adapt to the changing biotic and abiotic environments. Multi-copper oxidases (MCOs) such as ceruloplasmins, ferroxidases, laccases and nitrite reductases are such regulatory systems employed by microbes to resist the toxicity of metal ions by controlling their oxidation states under aerobic conditions. MCOs help pathogens survive during an infection by evasion of the toxic environment generated by the host immune system and thus are considered necessary determinants of virulence. This review summarizes the role of MCOs in metal homeostasis under stressful conditions and the extent to which these MCOs contribute to microbial virulence within the host that might prove as an esteemed avenue for the development of novel antimicrobial therapies.