Cloning and characterization of a new laccase from Bacillus licheniformis catalyzing dimerization of phenolic acids

A new laccase gene (cotA) was cloned from Bacillus licheniformis and expressed in Escherichia coli. The recombinant protein CotA was purified and showed spectroscopic properties, typical for blue multi-copper oxidases. The enzyme has a molecular weight of ~65 kDa and demonstrates activity towards canonical laccase substrates 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), syringaldazine (SGZ) and 2,6-dimethoxyphenol (2,6-DMP). Kinetic constants K _M and k _cat for ABTS were of 6.5 ± 0.2 μM and 83 s⁻¹, for SGZ of 4.3 ± 0.2 μM and 100 s⁻¹, and for 2,6-DMP of 56.7 ± 1.0 μM and 28 s⁻¹. Highest oxidizing activity towards ABTS was obtained at 85°C. However, after 1 h incubation of CotA at 70°C and 80°C, a residual activity of 43% and 8%, respectively, was measured. Furthermore, oxidation of several phenolic acids and one non-phenolic acid by CotA was investigated. CotA failed to oxidize coumaric acid, cinnamic acid, and vanillic acid, while syringic acid was oxidized to 2,6-dimethoxy-1,4-benzoquinone. Additionally, dimerization of sinapic acid, caffeic acid, and ferulic acid by CotA was observed, and highest activity of CotA was found towards sinapic acid.     

Substrate and dioxygen binding to the endospore coat laccase from Bacillus subtilis

The CotA laccase from the endospore coat of Bacillus subtilis has been crystallized in the presence of the non-catalytic co-oxidant 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS), and the structure was determined using synchrotron radiation. The binding site for this adduct is well defined and indicates how ABTS, in conjunction with laccases, could act as an oxidative mediator toward non-phenolic moieties. In addition, a dioxygen moiety is clearly defined within the solvent channel oriented toward one of the T3 copper atoms in the trinuclear center.     

Crystal structure of a bacterial endospore coat component. A laccase with enhanced thermostability properties

Endospores produced by the Gram-positive soil bacterium Bacillus subtilis are shielded by a proteinaceous coat formed by over 30 structural components, which self-assemble into a lamellar inner coat and a thicker striated electrodense outer coat. The 65-kDa CotA protein is an abundant component of the outer coat layer. CotA is a highly thermostable laccase, assembly of which into the coat is required for spore resistance against hydrogen peroxide and UV light. Here, we report the structure of CotA at 1.7-A resolution, as determined by x-ray crystallography. This is the first structure of an endospore coat component, and also the first structure of a bacterial laccase. The overall fold of CotA comprises three cupredoxin-like domains and includes one mononuclear and one trinuclear copper center. This arrangement is similar to that of other multicopper oxidases and most similar to that of the copper tolerance protein CueO of Escherichia coli. However, the three cupredoxin domains in CotA are further linked by external interdomain loops, which increase the packing level of the structure. We propose that these interdomain loops contribute to the remarkable thermostability of the enzyme, but our results suggest that additional factors are likely to play a role. Comparisons with the structure of other monomeric multicopper oxidases containing four copper atoms suggest that CotA may accept the largest substrates of any known laccase. Moreover, and unlike other laccases, CotA appears to have a flexible lidlike region close to the substrate-binding site that may mediate substrate accessibility. The implications of these findings for the properties of CotA, its assembly and the properties of the bacterial spore coat structure are discussed.     

Purification and characterization of two laccase isoenzymes from a ligninolytic fungus Trametes gallica

Constant laccase activities were detected in culture supernatant of newly isolated basidiomycete Trametes gallica. Tryptone and glucose have great effects on the production of laccase. Two laccase isoenzymes (Lac I and Lac II) produced by T. gallica were purified to homogeneity (51- and 50-fold, respectively) by gel filtration chromatography, anion exchange chromatography, and improved native PAGE, with an overall yield of 24.8%. Lac I and Lac II from this fungus are glycoproteins with 3.6% and 4% carbohydrate content, the same molecular masses (by SDS-PAGE) of 60 kDa, and the pI of 3.1 and 3.0, respectively. Native gel electrophoresis indicates that the two laccases have different migration ratios. Lac I and Lac II have the same optimal pH of 3.0 on 2,6-dimethoxyphenol (DMP), pH 2.2 on 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and of pH 4.0 on guaiacol. The highest rate of ABTS oxidation for both laccases was reached at 70 degrees C. Both laccases are stable from pH 6 to 9, retaining 88-90% activity after 24 hr incubation, and show good stability when incubated at temperatures lower than 40 degrees C. The Km values of Lac I for ABTS, DMP, and guaiacol are 0.118 x 10(-2), 0.420, and 0.405 mM, respectively; the Km values of Lac II for ABTS, DMP, and guaiacol are 0.086 x 10(-2), 0.41, and 0.40 mM, respectively. Their N-terminal sequences are determined and show strong similarity with those from other basidiomycetes. Graphite-furnace atomic absorption analysis revealed that both laccases have four copper atoms per protein molecule, but they have no type I copper signal at around 600 nm and a type III copper signal near 330 nm. Cyanide, azide, and halides completely inhibit the enzyme activity, whereas EDTA has less inhibition.     

Enhanced expression of a recombinant bacterial laccase at low temperature and microaerobic conditions: purification and biochemical characterization

Laccases (benzenediol oxygen oxidoreductase; EC 1.10.3.2) have many biotechnological applications because of their oxidation ability towards a wide range of phenolic compounds. Within recent years, researchers have been highly interested in the identification and characterization of laccases from bacterial sources. In this study, we have isolated and cloned a gene encoding laccase (CotA) from Bacillus sp. HR03 and then expressed it under microaerobic conditions and decreased temperature in order to obtain high amounts of soluble protein. The laccase was purified and its biochemical properties were investigated using three common laccase substrates, 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), syringaldazine (SGZ) and 2,6-dimethoxyphenol (2,6-DMP). K _M and k _cat were calculated 535 μM and 127 s⁻¹ for ABTS, 53 μM and 3 s⁻¹ for 2, 6-DMP and 5 μM and 20 s⁻¹ for SGZ when the whole reactions were carried out at room temperature. Laccase activity was also studied when the enzyme was preincubated at 70 and 80°C. With SGZ as the substrate, the activity was increased three-fold after 50 min preincubation at 70°C and 2.4-fold after 10 min preincubation at 80°C. Preincubation of the enzyme in 70°C for 30 min raised the activity four-fold with ABTS as the substrate. Also, l-dopa was used as a substrate. The enzyme was able to oxidize l-dopa with the K _M and k _cat of 1,493 μM and 194 s⁻¹, respectively.     

Laboratory evolution of laccase for substrate specificity

A laccase, CotA, from Bacillus subtilis was engineered using a combination of rational and directed evolution approaches. CotA is a generalist, an enzyme with broad specificity, and it was optimized to be a specialist, an enzyme with narrowed specificity. Wild-type CotA oxidizes ABTS (ABTS = diammonium 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) and SGZ (SGZ = 4-hydroxy-3,5-dimethoxy-benzaldehyde azine), and it was engineered for increased specificity for ABTS. Based on the ABTS-bound crystal structure of CotA, 19 amino acids are within 6 Å of ABTS, and they were simultaneously randomized. A mutant was identified that was 132 times more specific for ABTS. Unexpectedly, the variant was found to acquire enhanced thermal stability. The half-life for the heat inactivation (t_1/2) at 80 °C was increased by 62 min for the mutant. Laccases have several applications in biotechnology, which include pulp bleaching, biosensors, bioremediation, and biofuel cells. The substrate specificity of CotA is moldable and the enzyme can be tailored to oxidize a variety of target molecules for specific purposes.     

Purification and characterisation of a novel laccase from the ascomycete Melanocarpus albomyces

A novel laccase from the ascomycete Melanocarpus albomyces was purified and characterised. The enzyme was purified using anion exchange chromatography, hydrophobic interaction chromatography and gel filtration, and the purified laccase was biochemically characterised. It had activity towards typical substrates of laccases including 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate), dimethoxyphenol, guaiacol, and syringaldazine. The laccase showed good thermostability and it had a pH optimum at neutral pH, both unusual properties for most known fungal laccases. The activity of the laccase from M. albomyces was highest at 60-70 degrees C. With guaiacol and syringaldazine the pH optima were rather broad: 5-7.5 and 6-7, respectively. It retained 50% of its activity after 5 h incubation at 60 degrees C. The molecular weight of the laccase was about 80 kDa and the isoelectric point 4.0. The ultraviolet-visible absorption and electron paramagnetic resonance spectra of the purified laccase indicated that the typical three types of copper were present.     

Kraft pulp biobleaching and mediated oxidation of a nonphenolic substrate by laccase from Streptomyces cyaneus CECT 3335

A new laccase (EC 1.10.3.2) produced by Streptomyces cyaneus CECT 3335 in liquid media containing soya flour (20 g per liter) was purified to homogeneity. The physicochemical, catalytic, and spectral characteristics of this enzyme, as well as its suitability for biobleaching of eucalyptus kraft pulps, were assessed. The purified laccase had a molecular mass of 75 kDa and an isoelectric point of 5.6, and its optimal pH and temperature were 4.5 and 70 degrees C, respectively. The activity was strongly enhanced in the presence of Cu(2+), Mn(2+), and Mg(2+) and was completely inhibited by EDTA and sodium azide. The purified laccase exhibited high levels of activity against 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) and 2,6-dimethoxyphenol and no activity against tyrosine. The UV-visible spectrum of the purified laccase was the typical spectrum of the blue laccases, with an absorption peak at 600 nm and a shoulder around 330 to 340 nm. The ability of the purified laccase to oxidize a nonphenolic compound, such as veratryl alcohol, in the presence of ABTS opens up new possibilities for the use of bacterial laccases in the pulp and paper industry. We demonstrated that application of the laccase from S. cyaneus in the presence of ABTS to biobleaching of eucalyptus kraft pulps resulted in a significant decrease in the kappa number (2.3 U) and an important increase in the brightness (2.2%, as determined by the International Standard Organization test) of pulps, showing the suitability of laccases produced by streptomycetes for industrial purposes.     

Purification,molecular characterization and reactivity with aromatic compounds of a laccase from basidiomycete <Emphasis Type="Italic">Trametes</Emphasis> sp. strain AH28-2

A recently isolated basidiomycete, Trametes sp. strain AH28-2, can be induced to produce a high level of laccases when grown on a cellobiose-asparagine liquid medium. After induction by kraft lignin, two major isozymes were detected in the fermentation supernatant of the fungus. The principal component laccase A, which accounts for about 85% of the total activity, can be purified to electrophoretic homogeneity by three chromatographic steps: DEAE-Sepharose FF, Superdex-200 and Mono-Q. The solution containing purified laccase is blue in color, and the ratio of absorbance at 280 nm to that at 600 nm is 22. The molecular mass of laccase A is estimated to be 62 kDa by SDS-PAGE, 57 kDa by FPLC, and measured as 58522 Da by MALDI mass spectrum. Laccase A is a monomeric glycoprotein with a carbohydrate content of 11-12% and an isoelectric point of 4.2. The optimum pH and temperature for oxidizing guaiacol are 4.5 and 50 degrees C, respectively. The half-life of the enzyme at 75 degrees C is 27 min. The enzyme shows a good stability from pH 4.2 to pH 8.0. The K(m) values of the enzyme toward substrates 2,2'-azino-bis (3-ethylbenzothazoline-6-sulfonate) (ABTS), guaiacol and 2,6-dimethoxyphenol are 25, 420 and 25.5 microM, respectively, and the corresponding V(max) values are 670, 66.8, and 79 microM min(-1) x mg(-1), respectively. Laccase A activity is strongly inhibited by 0.1 mM NaN(3) or 0.1 mM cyanide. Two units of laccase A alone is able to completely oxidize 100 micromol 2,6-chlorophenol in 6 h. In the presence of 1 mM ABTS and 1-hydroxybenzotriazole, 15.0 U laccase A is able to oxidize 45% and 70% of 50 micromol fluorene in 12 and 18 h, respectively. The laccase A gene was cloned by a PCR method, and preliminary analysis of its sequence indicates 87.0% similarity to the corresponding segment in the phenoloxidase gene from Coriolus hirsutus.     

Characterization of a low redox potential laccase from the basidiomycete C30.

A new exocellular laccase was purified from the basidiomycete C30. LAC2 is an acidic protein (pI = 3.2) preferentially produced upon a combined induction by copper and p-hydroxybenzoate. The spectroscopic signature (UV/visible and EPR) of this isoform is typical of multicopper oxidases, but its enzymatic and physico-chemical properties proved to be markedly different from those of LAC1, the constitutive laccase previously purified from the same organism. In particular, the LAC2 kcat values observed for the oxidation of the substrates syringaldazine (kcat = 65 600 min-1), ABTS (2,2-azino-bis-[3-ethylthiazoline-6-sulfonate] (kcat = 41 000 min-1) and guaiacol (kcat = 75 680 min-1) are 10-40 times those obtained with LAC1 and the redox potential of its T1 copper is 0.17 V lower than that of LAC1 (E degrees = 0.73 V). This is the first report on a single organism producing simultaneously both a high and a low redox potential laccase. The cDNA, clac2, was cloned and sequenced. It encodes a protein of 528 amino acids that shares 69% identity (79% similarity) with LAC1 and 81% identity (95% similarity) with Lcc3-2 from Polyporus ciliatus (AF176321-1), its nearest neighbor in database. Possible reasons for why this basidiomycete produces, in vivo, enzyme forms with such different behaviors are discussed.     

CotA,a Multicopper Oxidase from Bacillus pumilus WH4, Exhibits Manganese-Oxidase Activity

Multicopper oxidases (MCOs) are a family of enzymes that use copper ions as cofactors to oxidize various substrates. Previous research has demonstrated that several MCOs such as MnxG, MofA and MoxA can act as putative Mn(II) oxidases. Meanwhile, the endospore coat protein CotA from Bacillus species has been confirmed as a typical MCO. To study the relationship between CotA and the Mn(II) oxidation, the cotA gene from a highly active Mn(II)-oxidizing strain Bacillus pumilus WH4 was cloned and overexpressed in Escherichia coli strain M15. The purified CotA contained approximately four copper atoms per molecule and showed spectroscopic properties typical of blue copper oxidases. Importantly, apart from the laccase activities, the CotA also displayed substantial Mn(II)-oxidase activities both in liquid culture system and native polyacrylamide gel electrophoresis. The optimum Mn(II) oxidase activity was obtained at 53°C in HEPES buffer (pH 8.0) supplemented with 0.8 mM CuCl₂. Besides, the addition of o-phenanthroline and EDTA both led to a complete suppression of Mn(II)-oxidizing activity. The specific activity of purified CotA towards Mn(II) was 0.27 U/mg. The K_m, V_max and k_cat values towards Mn(II) were 14.85±1.17 mM, 3.01×10⁻⁶±0.21 M·min⁻¹ and 0.32±0.02 s⁻¹, respectively. Moreover, the Mn(II)-oxidizing activity of the recombinant E. coli strain M15-pQE-cotA was significantly increased when cultured both in Mn-containing K liquid medium and on agar plates. After 7-day liquid cultivation, M15-pQE-cotA resulted in 18.2% removal of Mn(II) from the medium. Furthermore, the biogenic Mn oxides were clearly observed on the cell surfaces of M15-pQE-cotA by scanning electron microscopy. To our knowledge, this is the first report that provides the direct observation of Mn(II) oxidation with the heterologously expressed protein CotA, Therefore, this novel finding not only establishes the foundation for in-depth study of Mn(II) oxidation mechanisms, but also offers a potential biocatalyst for Mn(II) removal.     

Isolation and characteristics of micromycetes--producers of neutral phenol oxidase from trophic soil with a high level of dioxins

Samples of South Vietnamese soils intensely treated with Agent Orange defoliant were tested for the presence of fungi and actinomycetes with elevated phenol oxidase activity. As a result, fast-growing non-sporulating strain producing neutral phenol oxidases was isolated and identified as Mycelia sterilia INBI 2-26. The strain formed extracellular phenol oxidases during surface growth on liquid medium in the presence of guayacol and copper sulfate, as well as during submerged cultivation in liquid medium containing wheat bran and sugar beet pulp. Isoelectric focusing of cultural liquid has revealed two major catechol oxidases (PO1 and PO2) with pI 3.5 and 8, respectively. The enzymes were purified by ultrafiltration, ion exchange chromatography and exclusion HPLC. Both were stable between pH 3 and 8. At pH 8 and 40 degrees C they retained at least 50% of activity after incubation for 50 h. At 50 degrees C PO2 was more stable and retained 40% of activity after 50 h, whereas PO1 was inactivated in 3-6 h. The pH optimums for PO1 and PO2 towards catechol were equal to 6 and 6.5, and the Km values were 1.5 +/- 0.35 and 1.25 +/- 0.2 mM, respectively. PO1 and PO2 most optimally oxidized 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) at pH 3 with Km values 1.6 +/- 0.18 and 0.045 +/- 0.01 mM, respectively, but displayed no activity towards tyrosine. The PO2 absorbance spectrum had a peak at 600 nm, thus indicating the enzyme to be a member of the laccase family.     

Purification and characterization of laccase from the white rot fungus Trametes versicolor

Laccase is one of the ligninolytic enzymes of white rot fungus Trametes versicolor 951022, a strain first isolated in Korea. This laccase was purified 209-fold from culture fluid with a yield of 6.2% using ethanol precipitation, DEAE-Sepharose, Phenyl-Sepharose, and Sephadex G-100 chromatography. T. versicolor 951022 excretes a single monomeric laccase showing a high specific activity of 91,443 U/mg for 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as a substrate. The enzyme has a molecular mass of approximately 97 kDa as determined by SDS-PAGE, which is larger than those of other laccases reported. It exhibits high enzyme activity over broad pH and temperature ranges with optimum activity at pH 3.0 and a temperature of 50 degrees C. The Km value of the enzyme for substrate ABTS is 12.8 micrometer and its corresponding Vmax value is 8125.4 U/mg. The specific activity and substrate affinity of this laccase are higher than those of other white rot fungi, therefore, it may be potentially useful for industrial purposes.     

Biochemical characterization and molecular evidence of a laccase from the bird's nest fungus Cyathus bulleri

Cyathus bulleri, a bird's nest fungus, known to decolorize polymeric dye Poly R-478, was found to produce 8 U ml(-1) of laccase in malt extract broth. Laccase activity appeared as a single band on non-denaturing gel. Laccase was purified to homogeneity by anion exchange chromatography and gel filtration. The enzyme was a monomer with an apparent molecular mass of 60 kD, pI of 3.7 and was stable in the pH range of 2-6 with an optimum pH of 5.2. The optimal reaction temperature was 45 degrees C and the enzyme lost its activity above 70 degrees C. Enzyme could oxidize a broad range of various phenolic substrates. K(m) values for ABTS, 2,6-dimethoxyphenol, guaiacol, and ferulic acid were found to be 48.6, 56, 22, and 14 mM while K(cat) values were 204, 180, 95.6, and 5.2, respectively. It was completely inhibited by KCN, NaN(3), beta-mercaptoethanol, HgCl(2), and SDS, while EDTA had no effect on enzyme activity. The N-terminal amino acid sequence of C. bulleri laccase showed close homology to N-terminal sequences of laccase from other white-rot fungi. A 150 bp gene sequence encoding copper-binding domains I and II was most similar to the sequence encoding a laccase from Pycnoporus cinnabarinus with 74.8% level of similarity.     

Electrochemical characterization of purified Rhus vernicifera laccase: voltammetric evidence for a sequential four-electron transfer

Rhus vernicifera (Rv) laccase was purified to electrophoretic homogeneity by hydrophobic interaction chromatography. A comprehensive study of the direct electrochemistry of Rv laccase covalently immobilized at a gold electrode using alkanethiol monolayers was undertaken. The observed midpoint potential was 410 mV versus the normal hydrogen electrode (NHE), consistent with reduction potentials obtained by potentiometric titration for the T1 copper site. Evidence is presented for a concerted 4-electron reversible process at slow scan rates (v) on the basis of peak current ratios (i(pa)/i(pc)). Catalytic currents were observed in the presence of the biological substrate oxygen, indicating that laccase activity is retained throughout the immobilization process. Electrochemical characteristics of the immobilized laccase were essentially invariant across the pH range 5.5-8.5 and the temperature range 5-35 degrees C. The purified enzyme displayed a pH optimum of 9.0, when assayed spectrophotometrically with syringaldazine as a substrate. Inhibition of the laccase activity with azide or fluoride showed an I(50)(NaN(3)) of 2.5 mM and an I(50)(NaF) of 18.5 mM. Electrochemistry in the presence of azide reduces the anodic current by ca. one-half, consistent with the 4-electron process decreasing to a 2-electron process. However, fluoride has no effect on anaerobic electrochemistry. These electrochemical results suggest that the pH dependence of laccase activity is related to the effects of pH on the structure or binding of the substrate.     

Characterisation of a novel white laccase from the deuteromycete fungus Myrothecium verrucaria NF-05 and its decolourisation of dyes

A novel 'white' laccase was purified from the deuteromycete fungus, Myrothecium verrucaria NF-05, which was a high laccase-producing strain (40.2 U·ml(-1) on the thirteenth day during fermentation). SDS-PAGE and native-PAGE revealed a single band with laccase activity corresponding to a molecular weight of approximately 66 kDa. The enzyme had three copper and one iron atoms per protein molecule determined by ICP-AES. Furthermore, both UV/visible and EPR spectroscopy remained silence, indicating the enzyme a novel laccase with new metal compositions of active centre and spectral properties. The N-terminal amino acid sequence of the purified protein was APQISPQYPM. Together with MALDI-TOF analysis, the protein revealed a high homology of the protein with that from reported M. verrucaria. The highest activity was detected at pH 4.0 and at 30°C. The enzyme activity was significantly enhanced by Na(+), Mn(2+), Cu(2+) and Zn(2+) while inhibited by DTT, NaN(3) and halogen anions. The kinetic constant (Km) showed the enzyme was more affinitive to ABTS than other tested aromatic substrates. Twelve structurally different dyes could be effectively decolourised by the laccase within 10 min. The high production of the strain and novel properties of the laccase suggested its potential for biotechnological applications.     

Selective induction, purification and characterization of a laccase isozyme from the basidiomycete Trametes sp. AH28-2

The white-rot fungus Trametes sp. AH28-2 can synthesize extracellular laccase by induction in cellobiose-based liquid culture medium. Both yields and composition of laccase isozymes, produced by Trametes sp. AH28-2, would be quite different with induction by different small-molecule aromatic compounds, o-toluidine, guaiacol and 3,5-dihydroxytoluene, which affected microbial growth and the synthesis of laccase isozymes differentially. Higher concentrations of the three inducers could considerably increase laccase isozymes yields but not change the laccase composition. Coculturing of Trametes sp. AH28-2 with either Aspergillus oryzae or Gloeophyllum trabeum showed a few effects on laccase production. Laccase isozyme, laccase B, was selectively induced by 3,5-dihydroxytoluene and purified to homogeneity by two-step chromatography. Purified laccase B appeared as blue, with a broad peak at about 600 nm and a shoulder peak at about 330 nm. The ratio of absorbance at 280 nm to that at 600 nm was 21. Every molecule of laccase B had approximately four copper atoms. Molecular mass of laccase B was estimated to be 74 kDa on SDS-PAGE, 72 kDa by FPLC and was determined to be 71?454 Da by mass spectrum. After being treated with N-glycosidase F, laccase B lost 25% of its molecular mass. The isoelectric point of laccase B was 4.0. Its optimal pH and temperature for oxidizing guaiacol were respectively 4.7 and 45 C. The half-life of the enzyme at 60 C was 14.0 min. The enzyme showed a good stability in a range of pH value of 3.5-7.5. The K(m) values of the enzyme toward substrates syringaldazine, guaiacol, ABTS, and DMOP were respectively 28.0, 1249.0, 177.0 and 109.8 μM. The corresponding V(max) are 504.0, 1910.0, 117.4 and 159.0 μM min(-1) mg(-1). In addition, activity of laccase B was inhibited strongly by sodium azide and cyanide, mildly by SDS and trifluoroacetic acid, but only weakly by dimethyl sulfoxide.     

Bilirubin oxidase activity of Bacillus subtilis CotA

The spore coat protein CotA from Bacillus subtilis was previously identified as a laccase. We have now found that CotA also shows strong bilirubin oxidase activity and markedly higher affinity for bilirubin than conventional bilirubin oxidase. This is the first characterization of bilirubin oxidase activity in a bacterial protein.     

Production of two novel laccase isoforms by a thermotolerant strain of Pycnoporus sanguineus isolated from an oil-polluted tropical habitat

A thermotolerant and halotolerant strain of Pycnoporus sanguineus was isolated from an oil-polluted site in a tropical area located in Veracruz, Mexico. This strain was able to grow at 47 degrees C and in culture medium containing 500 mM NaCl. The strain was also tolerant to the presence of 30,000 ppm of crude Maya oil. A 68-kDa protein purified from submerged cultures exhibited laccase activity towards 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), guaiacol, syringaldazine, and o-dianisidine, for which it presented the highest affinity (Km = 43 microM). Two-dimensional gel electrophoresis analysis showed that, unusual for laccases, the enzyme has two active isoforms, with isoelectric points of 7.00 and 7.08. The purified enzyme showed high thermostability, retaining 40% of its original activity after 3 h at 60 degrees C. This property seems to correlate with a long "shelf-life," given that at 40 degrees C enzyme activity was only gradually lost over a 5-day period incubation. Both the fungus and its laccase are likely to have high potential for biotechnological applications.     

Purification and characterization of laccase produced by a white rot fungus Pleurotus sajor-caju under submerged culture condition and its potential in decolorization of azo dyes

An extracellular laccase was isolated and purified from Pleurotus sajor-caju grown in submerged culture in a bioreactor, and used to investigate its ability to decolorize three azo dyes. The extracellular laccase production was enhanced up to 2.5-fold in the medium amended with xylidine (1 mM). Purification was carried out using ammonium sulfate (70% w/v), DEAE-cellulose, and Sephadex G-100 column chromatography. The enzyme was purified up to 10.3-fold from the initial protein preparation with an overall yield of 53%. The purified laccase was monomeric with an apparent molecular mass of 61.0 kDa. The purified enzyme exerted its optimal activity with 2,2-azino–bis(3-ethylbenzo-thiazoline-6-sulfonate (ABTS) and oxidized various lignin-related phenols. The catalytic efficiencies k _cat/K _m determined for ABTS and syringaldazine were 9.2×10⁵ and 8.7×10⁵, respectively. The optimum pH and temperature for the purified enzyme was 5.0 and 40 °C, respectively. Sodium azide completely inhibited the laccase activity. The absorption spectrum revealed type 1 and type 3 copper signals. The purified enzyme decolorized azo dyes such as acid red 18, acid Black 1, and direct blue 71 up to 90, 87, and 72%, respectively. Decolorization ability of P. sajor-caju laccase suggests that this enzyme could be used for decolorization of industrial effluents.