Lígia O. Martins

Enzymatic biotransformation of the azo dye Sudan Orange G with bacterial CotA-laccase

In the present study we show that recombinant bacterial CotA-laccase from Bacillus subtilis is able to decolourise, at alkaline pH and in the absence of redox mediators, a variety of structurally different synthetic dyes. The enzymatic biotransformation of the azo dye Sudan Orange G (SOG) was addressed in more detail following a multidisciplinary approach. Biotransformation proceeds in a broad span of temperatures (30-80 degrees C) and more than 98% of Sudan Orange G is decolourised within 7h by using 1 U mL(-1) of CotA-laccase at 37 degrees C. The bell-shape pH profile of the enzyme with an optimum at 8, is in agreement with the pH dependence of the dye oxidation imposed by its acid-basic behavior as measured by potentiometric and electrochemical experiments. Seven biotransformation products were identified using high-performance liquid chromatography and mass spectrometry and a mechanistic pathway for the azo dye conversion by CotA-laccase is proposed. The enzymatic oxidation of the Sudan Orange G results in the production of oligomers and, possibly polymers, through radical coupling reactions. A bioassay based on inhibitory effects over the growth of Saccharomyces cerevisiae shows that the enzymatic bioremediation process reduces 3-fold the toxicity of Sudan Orange G.

Combined effect of the growth temperature and salinity of the medium on the accumulation of compatible solutes by Rhodothermus marinus and Rhodothermus obamensis

Abstract In this study we propose revised structures for the two major compatible solutes of Rhodothermus marinus. We have also examined the accumulation of compatible solutes by the type strains of the slightly halophilic and thermophilic species Rhodothermus marinus and Rhodothermus obamensis at several growth temperatures and salinities. The major solutes of R. marinus were identified as α-mannosylglycerate (α-MG) and α-mannosylglyceramide (α-MGA), whereas R. obamensis accumulated only α-mannosylglycerate. The total osmolyte content was higher during the early exponential phase and decreased abruptly as growth continued into the stationary phase. At low growth temperatures, R. marinus responded to water stress by accumulation of α-mannosylglycerate and its amide, in addition to low levels of trehalose, glutamate, and glucose. At the highest growth temperature, α-mannosylglycerate was the major compatible solute and α-mannosylglyceramide was not detected. When both compounds were present, an increase in the salinity of the growth medium favored the accumulation of α-mannosylglyceramide over α-mannosylglycerate. The absence of α-mannosylglyceramide in R. obamensis at all growth temperatures and salinities constituted the most pronounced difference in the profiles of compatible solute accumulation by the two strains. Trehalose was also a prominent solute in this organism. Both organisms accumulated higher levels of α-mannosylglycerate as the temperature was raised. The importance of the two compounds in the mechanisms of thermoadaptation and osmoadaptation is discussed.

Biosynthesis of Mannosylglycerate in the Thermophilic Bacterium Rhodothermus marinus BIOCHEMICAL AND GENETIC CHARACTERIZATION OF A MANNOSYLGLYCERATE SYNTHASE

The biosynthetic reaction scheme for the compatible solute mannosylglycerate in Rhodothermus marinusis proposed based on measurements of the relevant enzymatic activities in cell-free extracts and in vivo 13C labeling experiments. The synthesis of mannosylglycerate proceeded via two alternative pathways; in one of them, GDP mannose was condensed withd-glycerate to produce mannosylglycerate in a single reaction catalyzed by mannosylglycerate synthase, in the other pathway, a mannosyl-3-phosphoglycerate synthase catalyzed the conversion of GDP mannose and d-3-phosphoglycerate into a phosphorylated intermediate, which was subsequently converted to mannosylglycerate by the action of a phosphatase. The enzyme activities committed to the synthesis of mannosylglycerate were not influenced by the NaCl concentration in the growth medium. However, the combined mannosyl-3-phosphoglycerate synthase/phosphatase system required the addition of NaCl or KCl to the assay mixture for optimal activity. The mannosylglycerate synthase enzyme was purified and characterized. Based on partial sequence information, the corresponding mgs gene was identified from a genomic library of R. marinus. In addition, the mgs gene was overexpressed inEscherichia coli with a high yield. The enzyme had a molecular mass of 46,125 Da, and was specific for GDP mannose andd-glycerate. This is the first report of the characterization of a mannosylglycerate synthase.

Dioxygen reduction by multi-copper oxidases; a structural perspective.

The multi-copper oxidases oxidise substrate molecules by accepting electrons at a mononuclear copper centre and transferring them to a trinuclear centre. Dioxygen binds to the trinuclear centre and, following the transfer of four electrons, is reduced to two molecules of water. The precise mechanism of this reduction has been unclear, but recent X-ray structural studies using the CotA endospore coat protein from Bacillus subtilis have given further insights into the principal stages. It is proposed that the mechanism involves binding of the dioxygen into the trinuclear centre so that it is sited approximately symmetrically between the two type 3 copper ions with one oxygen atom close to the type 2 copper ion. Further stages involve the formation of a peroxide intermediate and following the splitting of this intermediate, the migration of the hydroxide moieties towards the solvent exit channel. The migration steps are likely to involve a movement of the type 2 copper ion and its environment. Details of a putative mechanism are described herein based both on structures already reported in the literature and on structures of the CotA protein in the oxidised and reduced states and with the addition of peroxide and the inhibitor, azide.

Proximal mutations at the type 1 copper site of CotA laccase: spectroscopic, redox, kinetic and structural characterization of I494A and L386A mutants.

In the present study the CotA laccase from Bacillus subtilis has been mutated at two hydrophobic residues in the vicinity of the type 1 copper site. The mutation of Leu(386) to an alanine residue appears to cause only very subtle alterations in the properties of the enzyme indicating minimal changes in the structure of the copper centres. However, the replacement of Ile(494) by an alanine residue leads to significant changes in the enzyme. Thus the major visible absorption band is upshifted by 16 nm to 625 nm and exhibits an increased intensity, whereas the intensity of the shoulder at approx. 330 nm is decreased by a factor of two. Simulation of the EPR spectrum of the I494A mutant reveals differences in the type 1 as well as in the type 2 copper centre reflecting modifications of the geometry of these centres. The intensity weighted frequencies , calculated from resonance Raman spectra are 410 cm(-1) for the wild-type enzyme and 396 cm(-1) for the I494A mutant, indicating an increase of the Cu-S bond length in the type 1 copper site of the mutant. Overall the data clearly indicate that the Ile(494) mutation causes a major alteration of the structure near the type 1 copper site and this has been confirmed by X-ray crystallography. The crystal structure shows the presence of a fifth ligand, a solvent molecule, at the type 1 copper site leading to an approximate trigonal bipyramidal geometry. The redox potentials of the L386A and I494A mutants are shifted downwards by approx. 60 and 100 mV respectively. These changes correlate well with decreased catalytic efficiency of both mutants compared with the wild-type.

Mechanisms underlying dioxygen reduction in laccases. Structural and modelling studies focusing on proton transfer

BackgroundLaccases are enzymes that couple the oxidation of substrates with the reduction of dioxygen to water. They are the simplest members of the multi-copper oxidases and contain at least two types of copper centres; a mononuclear T1 and a trinuclear that includes two T3 and one T2 copper ions. Substrate oxidation takes place at the mononuclear centre whereas reduction of oxygen to water occurs at the trinuclear centre.ResultsIn this study, the CotA laccase from Bacillus subtilis was used as a model to understand the mechanisms taking place at the molecular level, with a focus in the trinuclear centre. The structures of the holo-protein and of the oxidised form of the apo-protein, which has previously been reconstituted in vitro with Cu(I), have been determined. The former has a dioxygen moiety between the T3 coppers, while the latter has a monoatomic oxygen, here interpreted as a hydroxyl ion. The UV/visible spectra of these two forms have been analysed in the crystals and compared with the data obtained in solution. Theoretical calculations on these and other structures of CotA were used to identify groups that may be responsible for channelling the protons that are needed for reduction of dioxygen to water.ConclusionsThese results present evidence that Glu 498 is the only proton-active group in the vicinity of the trinuclear centre. This strongly suggests that this residue may be responsible for channelling the protons needed for the reduction. These results are compared with other data available for these enzymes, highlighting similarities and differences within laccases and multicopper oxidases.

Cutinase unfolding and stabilization by trehalose and mannosylglycerate.

The unfolding of cutinase at pH 4.5 was induced by increasing the temperature and guanidine hydrochloride concentration in the presence of potassium chloride, trehalose, and mannosylglycerate potassium salt. Protein thermal unfolding approached a two‐state process, since the unfolding transitions were coincident within experimental error when assessed by near‐ultraviolet (UV) difference, tryptophyl, and 8‐anilino‐1‐naphthalene sulfonic acid (ANS) fluorescence spectroscopy. Trehalose at 0.5 M increased the temperature at which 50% of cutinase is unfolded by 3°C. Unfolding induced by guanidine hydrochloride is clearly a non‐two‐state process. The presence of a stable intermediate was detected because unfolding assessed by near‐UV difference spectroscopy occurs earlier than unfolding assessed by tryptophyl fluorescence. The intermediate is molten globule in character: the ANS fluorescence is higher than in the presence of the folded or unfolded state, showing native‐like secondary structure and losing many tertiary interactions of the folded state, i.e., those surrounding the tyrosyl microenvironment. The stabilization effect of trehalose and mannosylglycerate was quantified by fitting the unfolding transitions to a model proposed by Staniforth et al. ( Biochemistry 1993;32:3842–3851 ). This model takes into consideration the increase in solvation energies of the amino acid side‐chains as the denaturant concentration was increased and the fraction of amino acid side‐chains that become exposed in the unfolded structure of cutinase. Trehalose and mannosylglycerate stabilize the folded state relative to the intermediate by 1.4–1.6 and 1.6 kcal/mol and the intermediate relative to the unfolded state by 1.0 and 1.5 kcal/mol, respectively. Proteins 2001;42:542–552.

Synergistic action of azoreductase and laccase leads to maximal decolourization and detoxification of model dye-containing wastewaters.

The azoreductase PpAzoR from Pseudomonas putida shows a broader specificity for decolourization of azo dyes than CotA-laccase from Bacillus subtilis. However, the final products of PpAzoR activity exhibited in most cases a 2 to 3-fold higher toxicity than intact dyes themselves. We show that addition of CotA-laccase to PpAzoR reaction mixtures lead to a significant drop in the final toxicity. A sequential enzymatic process was validated through the use of 18 representative azo dyes and three model wastewaters that mimic real dye-containing effluents. A heterologous Escherichia coli strain was successfully constructed co-expressing the genes coding for both PpAzoR and CotA. Whole-cell assays of recombinant strain for the treatment of model dye wastewater resulted in decolourization levels above 80% and detoxification levels up to 50%. The high attributes of this strain, make it a promising candidate for the biological treatment of industrial dye containing effluents.

Methyl syringate: an efficient phenolic mediator for bacterial and fungal laccases.

The aim of the present work is to provide insight into the mechanism of laccase reactions using syringyl-type mediators. We studied the pH dependence and the kinetics of oxidation of syringyl-type phenolics using the low CotA and the high redox potential TvL laccases. Additionally, the efficiency of these compounds as redox mediators for the oxidation of non-phenolic lignin units was tested at different pH values and increasing mediator/non-phenolic ratios. Finally, the intermediates and products of reactions were identified by LC-MS and (1)H NMR. These approaches allow concluding on the (1) mechanism involved in the oxidation of phenolics by bacterial laccases, (2) importance of the chemical nature and properties of phenolic mediators, (3) apparent independence of the enzymes properties on the yields of non-phenolics conversion, (4) competitive routes involved in the catalytic cycle of the laccase-mediator system with several new C-O coupling type structures being proposed.

Expression system of CotA-laccase for directed evolution and high-throughput screenings for the oxidation of high-redox potential dyes

Laccases are useful biocatalysts for many diverse biotechnological applications. In this study we have established efficient and reliable expression systems and high-throughput screenings for the recombinant CotA-laccase from Bacillus subtilis. The expression levels of cotA-laccase were compared in five different Escherichia coli host strains growing in 96-well microtiter plates under different culture conditions. Lower coefficients of variance (around 15%) were achieved using crude cell lysates of BL21 and KRX host strains growing under microaerobic conditions. Reproducible high-throughput screenings for the decolorization of high redox potential azo and anthraquinonic dyes were developed and optimized for identification of variants with increased redox potential. The enzymatic assays developed were tested for the screening of one mutant library from CotA-laccase created by error-prone PCR.