Blue Multicopper Oxidases Research Articles

Overexpression of SmLAC25 promotes lignin accumulation and decreases salvianolic acid content in Salvia miltiorrhiza

Laccase (LAC) is a blue multicopper oxidase that contains four copper ions, which is involved in lignin polymerization and flavonoid biosynthesis in plants. Although dozens of LAC genes have been identified in Salvia miltiorrhiza Bunge (a model medicinal plant), most have not been functionally characterized. Here, we explored the expression patterns and the functionality of SmLAC25 in S. miltiorrhiza. SmLAC25 has a higher expression level in roots and responds to methyl jasmonate, auxin, abscisic acid, and gibberellin stimuli. The SmLAC25 protein is localized in the cytoplasm and chloroplasts. Recombinant SmLAC25 protein could oxidize coniferyl alcohol and sinapyl alcohol, two monomers of G-lignin and S-lignin. To investigate its function, we generated SmLAC25-overexpressed S. miltiorrhiza plantlets and hairy roots. The lignin content increased significantly in all SmLAC25-overexpressed plantlets and hairy roots, compared with the controls. However, the concentrations of rosmarinic acid and salvianolic acid B decreased significantly in all the SmLAC25-overexpressed lines. Further studies revealed that the transcription levels of some key enzyme genes in the lignin synthesis pathway (e.g., SmCCR and SmCOMT) were significantly improved in the SmLAC25-overexpressed lines, while the expression levels of multiple enzyme genes in the salvianolic acid biosynthesis pathway were inhibited. We speculated that the overexpression of SmLAC25 promoted the metabolic flux of lignin synthesis, which resulted in a decreased metabolic flux to the salvianolic acid biosynthesis pathway.

Isolation, Screening and Production of Fungal Laccases

Abstract: Laccase (E.C. 1.10.3.2) are enzyme belonging to the group of blue multi-copper oxidases. Laccases catalyze the oxidation of variety of phenolic compounds, diamines and aromatic amines. Fungal isolate producing laccase was screened on Potato Dextrose Agar medium enriched with 0.02% guaiacol from soil as well as decaying wood samples collected from farm/forest site. Confirmation of laccase production was done by Bavendam test which is a plate assay technique using 3mM guaiacol. The isolate was identified and found to belong genera Aspergillus and was tested for producing laccase enzyme. An attempt was made to isolate, screening and production of laccase enzyme produced from Aspergillus niger. Isolate giving best result was processed further for production of laccase using Submerged fermentation. Guaiacol and sodium citrate buffer were used to assay laccase production. Characterization of fungal isolate was done by morphological analysis. This study showed that enzyme production can be increased by media manipulation in the fungal cultures. Optimization of the media condition can maximize the desired enzyme production. Application or Dye decolorization assay was been performed using 0.1ml crystal violet solution, where dye decolorization efficiency showed the ability of laccase to decolorize dyes. It is present in Ascomycetes, Deuteromycetes and Basidiomycetes and abundant in lignin degrading white / brown rot fungi. In the recent years, these enzymes have gained application in the field of textile, pulp and paper and food industries. Recently, it is also used in the design of biosensors, biofuel cells as a medical diagnostics tool and bioremediation agent to clean up herbicides, pesticides and certain explosive in soil. Laccases have received attention of researchers in the last few decades due to their ability to oxidize both phenolic and non phenolic lignin related compound as well as highly recalcitrant environmental pollutants.

Successful Production and Ligninolytic Activity of a Bacterial Laccase, Lac51, Made in Nicotiana benthamiana via Transient Expression.

Giant panda could have bamboo as their exclusive diet for about 2 million years because of the contribution of numerous enzymes produced by their gut bacteria, for instance laccases. Laccases are blue multi-copper oxidases that catalyze the oxidation of a broad spectrum of phenolic and aromatic compounds with water as the only byproduct. As a “green enzyme,” laccases have potential in industrial applications, for example, when dealing with degradation of recalcitrant biopolymers, such as lignin. In the current study, a bacterial laccase, Lac51, originating from Pseudomonas putida and identified in the gut microbiome of the giant panda’s gut was transiently expressed in the non-food plant Nicotiana benthamiana and characterized. Our results show that recombinant Lac51 exhibits bacterial laccase properties, with optimal pH and temperature at 7–8 and 40°C, respectively, when using syringaldazine as substrate. Moreover, we demonstrate the functional capability of the plant expressed Lac51 to oxidize lignin using selected lignin monomers that serve as substrates of Lac51. In summary, our study demonstrates the potential of green and non-food plants as a viable enzyme production platform for bacterial laccases. This result enriches our understanding of plant-made enzymes, as, to our knowledge, Lac51 is the first functional recombinant laccase produced in plants.

Eminent Industrial and Biotechnological Applications of Laccases from Bacterial Source: a Current Overview.

Laccases are blue multicopper oxidases that oxidize a wide range of phenolic as well as non-phenolic substrates in the presence or absence of mediators. They occur in various species of bacteria, fungi, insects, and plants; bacterial laccases show high substrate specificity. Bacteria produce these enzymes either extracellularly or intracellularly and exhibit stability to a wide range of pH and temperature. Therefore, they are suitable for various industrial processes such as food, textile, and paper and pulp industry. They are also valuable for producing biofuels, pharmaceuticals, biosensors, and degradation of various environmental pollutants and xenobiotics compounds. Since bacterial laccases are more versatile in the sense of nutritional needs and ecological factors, their use can provide a promising solution to various problems related to industry and the field of biotechnology. However, there is a need for a thorough understanding of the chemistry and activity of bacterial laccases to enable their full potential use in bioremediation and biofuel production.

Function and versatile location of Met-rich inserts in blue oxidases involved in bacterial copper resistance

Cuproxidases form a subgroup of the blue multicopper oxidase family. They display disordered methionine-rich loops, not observable in most available crystal structures, which have been suggested to bind toxic Cu(I) ions before oxidation into less harmful Cu(II) by the core enzyme. We found that the location of the Met-rich regions is highly variable in bacterial cuproxidases, but always inserted in solvent exposed surface loops, at close proximity of the conserved T1 copper binding site. We took advantage of the large differences in loop length between cold-adapted, mesophilic and thermophilic oxidase homologs to unravel the function of the methionine-rich regions involved in copper detoxification. Using a newly developed anaerobic assay for cuprous ions, it is shown that the number of Cu(I) bound is nearly proportional to the loop lengths in these cuproxidases and to the number of potential Cu(I) ligands in these loops. In order to substantiate this relation, the longest loop in the cold-adapted oxidase was deleted, lowering bound extra Cu(I) from 9 in the wild-type enzyme to 2–3 Cu(I) in deletion mutants. These results demonstrate that methionine-rich loops behave as molecular octopus scavenging toxic cuprous ions in the periplasm and that these regions are essential components of bacterial copper resistance.

Novel Transaminase and Laccase from Streptomyces spp. Using Combined Identification Approaches

Three Streptomyces sp. strains with a multitude of target enzymatic activities confirmed by functional screening, namely BV129, BV286 and BV333, were subjected to genome sequencing aiming at the annotation of genes of interest, in-depth bioinformatics characterization and functional expression of the biocatalysts. A whole-genome shotgun sequencing followed by de novo genome assembly and annotation was performed revealing genomes of 6.4, 9.4 and 7.3 Mbp, respectively. Functional annotation of the proteins of interest resulted in between 2047 and 2763 putative targets. Among the various enzymatic activities that the three Streptomyces strains demonstrated to produce by functional screening, we focused our attention on transaminases (TAs) and laccases due to their high biocatalytic potential. Bioinformatics search allowed the identification of a putative TA from Streptomyces sp. BV333 as a potentially novel broad substrate scope TA and a putative laccase from Streptomyces sp. BV286 as potentially novel blue multicopper oxidase. The two sequences were cloned and overexpressed in Escherichia coli and the two novel enzymes, transaminase Sbv333-TA and laccase Sbv286-LAC, were characterized. Interestingly, both enzymes resulted to be exceptionally thermostable, Sbv333-TA showing a melting temperature (TM = 85 °C) only slightly lower compared to the TM of the most thermostable transaminases described to date (87–88 °C) and Sbv286-LAC being even thermoactivated at temperature >60 °C. Moreover, Sbv333-TA showed a broad substrate scope and remarkably demonstrated to be active in the transamination of β-ketoesters, which are rarely accepted by currently known TAs. On the other hand, Sbv286-LAC showed an improved activity in the presence of the cosolvent acetonitrile. Overall, it was shown that a combination of approaches from standard microbiological and biochemical screens to genome sequencing and analysis is required to afford novel and functional biocatalysts.

Autotolerant ceruloplasmin based biocathodes for implanted biological power sources

High-performance autotolerant bioelectrodes should be ideally suited to design implantable bioelectronic devices. Because of its high redox potential and ability to reduce oxygen directly to water, human ceruloplasmin, HCp, the only blue multicopper oxidase present in human plasma, appears to be the ultimate biocatalyst for oxygen biosensors and also biocathodes in biological power sources. In comparison to fungal and plant blue multicopper oxidases, e.g. Myrothecium verrucaria bilirubin oxidase and Rhus vernicifera laccase, respectively, the inflammatory response to HCp in human blood is significantly reduced. Partial purification of HCp allowed to preserve the native conformation of the enzyme and its biocatalytic activity. Therefore, electrochemical studies were carried out with the partially purified enzyme immobilised on nanostructured graphite electrodes at physiological pH and temperature. Amperometric investigations revealed low reductive current densities, i.e. about 1.65 µA cm−2 in oxygenated electrolyte and in the absence of any mediator, demonstrating nevertheless direct electron transfer based O2 bioelectroreduction by HCp for the first time. The reductive current density obtained in the mediated system was about 12 µA cm−2. Even though the inflammatory response of HCp is diminished in human blood, inadequate bioelectrocatalytic performance hinders its use as a cathodic bioelement in a biofuel cell.

Temperature depending bioelectrocatalysis current of multicopper oxidase from a hyperthermophilic archaeon Pyrobaculum aerophilum

The thermostable blue multicopper oxidase is stable at high temperature, however, because of this structural stability this enzyme has low activity. In this study, high electrochemical catalytic activity was achieved by immobilizing a thermostable blue multicopper oxidase from hyperthermophilic archaeon Pyrobaculum aerophilum (McoP) onto a single-walled carbon nanotube (SWCNT) electrode. The both results of electrochemical investigation for the McoP immobilized the SWCNT and spectroscopic measurements for the McoP solution suggested that a structural transition occured around 40 °C.

The catalytic properties of Thermus thermophilus SG0.5JP17-16 laccase were regulated by the conformational dynamics of pocket loop 6

BackgroundLaccase is one member of the blue multicopper oxidase family. It can catalyze the oxidation of various substrates. The Thermus thermophilus SG0.5JP17-16 laccase (lacTT) is thermostable, pH-stable, and high tolerance to halides, and can decolorize the synthetic dyes. In lacTT, the function of the loop 6 constructing the substrate-binding pocket wasn't clear. MethodsThe residues Asp394 and Asp396 located in loop 6, and were used to probe how the loop 6 influenced catalytic properties of the laccase. Site-directed mutagenesis was performed for two amino acids. Kinetic assay was utilized to characterize the catalytic efficiency of mutants. Mutants with different catalytic activities were used to decolorize the synthetic dyes to clarify the relationship between the catalytic efficiency and dye decolorization. Redox potential, structural and spectral analyses were performed to explain the differences in laccase activity between wild type and mutant enzymes. ResultsD394M, D394E and D394R mutants with the lower laccase activity displayed a decreased decolorization efficiency, while D396A, D396M and D396E mutant enzymes with higher catalytic efficiency decolorized the synthetic dye more efficiently than the wild type enzyme. ConclusionsThe pocket loop 6 might experience a conformational dynamics. The D394 residue controlled this conformation change by amino acid interaction networks containing the D396 residue at the entrance of substrate channel. General significancesThese studies may provide clues to improve the activity of the laccase for the better use in industrial applications, and/or contribute to further understanding the mechanism of laccase oxidation on the substrate.

Taguchi Optimization, Purification, and Characterization of laccase enzyme from Aspergillus niger

Laccases are oxidoreductases enzymes that belong to blue multi-copper oxidases. In the recent years, laccases have gained application in the field of textile, pulp and paper, and food industries. In this work, optimization of cultural conditions of the fungal species Aspergillus niger was performed by Taguchi orthogonal array statistical method to enhance laccase production. The crude extract was precipitated by ammonium sulphate, purified by ion exchange column of sephadex G-100. The molecular weight of the purified laccase identified by SDS-PAGE was 135 kDa. The highest activity of purified laccase was obtained at enzyme concentration of 0.054 mg/ ml, substrate concentration of 2 mmol/L, temperature of 30?C and pH of 5.6. In addition, copper sulfate exhibited the highest significant promoted effect on laccase activity, while zinc sulfate caused almost its complete inhibition.

Bilirubin oxidase oriented on novel type three-dimensional biocathodes with reduced graphene aggregation for biocathode

Aggregation of reduced graphene oxide (RGO) due to π-π stacking is a recurrent problem in graphene-based electrochemistry, decreasing the effective working area and therefore the performance of the RGO electrodes. Dispersing RGO on three-dimensional (3D) carbon paper electrodes is one strategy towards overcoming this challenge, with partial relief aggregation. In this report, we describe the grafting of negatively charged 4-aminobenzoic acid (4-ABA) onto a graphene functionalized carbon paper electrode surface. 4-ABA functionalization induces separation of the RGO layers, at the same time leading to favorable orientation of the blue multi-copper enzyme Myrothecium verrucaria bilirubin oxidase (MvBOD) for direct electron transfer (DET) in the dioxygen reduction reaction (ORR) at neutral pH. Simultaneous electroreduction of graphene oxide to RGO and covalent attachment of 4-ABA are achieved by applying alternating cathodic and anodic electrochemical potential pulses, leading to a high catalytic current density (Δjcat:193 ± 4 μA cm−2) under static conditions. Electrochemically grafted 4-ABA not only leads to a favorable orientation of BOD as validated by fitting a kinetic model to the electrocatalytic data, but also acts to alleviate RGO aggregation as disclosed by scanning electron microscopy, most likely due to the electrostatic repulsion between 4-ABA-grafted graphene layers. With a half-lifetime of 55 h, the bioelectrode also shows the highest operational stability for DET-type MvBOD-based bioelectrodes reported to date. The bioelectrode was finally shown to work well as a biocathode of a membrane-less glucose/O2 enzymatic biofuel cell with a maximum power density of 22 μW cm−2 and an open circuit voltage of 0.51 V.

"Yellow" laccase from Sclerotinia sclerotiorum is a blue laccase that enhances its substrate affinity by forming a reversible tyrosyl-product adduct.

Yellow laccases lack the typical blue type 1 Cu absorption band around 600 nm; however, multi-copper oxidases with laccase properties have been reported. We provide the first evidence that the yellow laccase isolated from Sclerotinia sclerotiorum is obtained from a blue form by covalent, but nevertheless reversible modification with a phenolic product. After separating the phenolics from the extracellular medium, a typical blue laccase is obtained. With ABTS as model substrate for this blue enzyme, a non-natural purple adduct is formed with a spectrum nearly identical to that of the 1:1 adduct of an ABTS radical and Tyr. This modification significantly increases the stability and substrate affinity of the enzyme, not by acting primarily as bound mediator, but by structural changes that also alters the type 1 Cu site. The HPLC-MS analyses of the ABTS adduct trypsin digests revealed a distinct tyrosine within a unique loop as site involved in the modification of the blue laccase form. Thus, S. sclerotiorum yellow laccase seems to be an intrinsically blue multi-copper oxidase that boosts its activity and stability with a radical-forming aromatic substrate. This particular case could, at least in part, explain the enigma of the yellow laccases.

Partial purification and characterization of the novel halotolerant and alkalophilic laccase produced by a new isolate of Bacillus subtilis LP2

Laccases (benzenediol: oxygen oxidoreductases, [EC1.10.3.2] are mostly known as members of the blue multicopper oxidase family that are used in very different industrial applications: textile, pulp and paper, food, cosmetics industries, bioremediation process, biosensor, biofuel and organic synthesis. Stability against the extreme conditions is an important property and it makes laccase suitable for several industrial processes. Laccase should have salt resistance to be used in textile dye degradation because the textile wastewaters include dyes with high concentrations of salts, especially NaCl. Bacterial laccases are preferable to be used for bioremediation process due to their high stability to extremely salt contaminated and alkalophilic environment. Bacillus subtilis LP2 was identified as a source of alkali-tolerant, salt resistant laccase. Laccase showed activity over a wide pH (4–10) and temperature (30–80 °C) range. Maximum laccase activity was observed as 140.4 U/mg (umol/min*mg) at pH 8 and 50 °C with the substrate guaiacol. Stability of laccase was determined as 60% and 20% after incubation of the enzyme for different time intervals of 20 and 40 min at 50 °C and pH 8. SDS (10 mM) and EDTA (5 mM) decreased laccase activity from 100% to 0% and 56%, respectively. Despite the other inhibitors, NaCI increased the activity of laccase to 167% at 500 mM concentration. Laccase from Bacillus subtilis LP2 barely showed the activity on the substrates vanillin and L-tyrosine. These results clearly show that laccase from Bacillus subtilis LP2 has high potential to be used for several applications in textile industry.

Laccases with Variable Properties from Different Strains of Steccherinum ochraceum: Does Glycosylation Matter?

Laccases are blue multi-copper oxidases with an extensive number of actual and potential industrial applications. It is known that laccases from different fungal strains may vary in properties; however, the reason of this remains unclear. In the current study we have isolated and characterized seven laccases from different strains of Steccherinum ochraceum obtained from regions of central Russia. Although all seven laccases had the same primary sequences, there was a little variation in their molecular weights and thermostabilities. Moreover, statistically significant differences in laccases’ catalytic parameters of oxidation of phenolic substrates and ABTS were observed. After the deglycosylation of four selected laccases by Endo H and PNGase F, their affinities to pyrocatechol and ABTS became the same, suggesting a substantial role of N-linked glycosylation in moderation of enzymatic properties of laccases.

Mode of Action, Properties, Production, and Application of Laccase: A Review.

Background and Source: Laccase belongs to the blue multi-copper oxidases, which are widely distributed in fungi and higher plants. It is present in Ascomycetes, Deuteromycetes, and Basidiomycetes and found abundantly in white-rot fungi. Applications: Laccase enzymes because of their potential have acquired more importance and application in the area of textile, pulp and paper, and food industry. Recently, it is being used in developing biosensors for detection and removal of toxic pollutants, designing of biofuel cells and medical diagnostics tool. Laccase is also being used as a bioremediation agent as they have been found potent enough in cleaning up herbicides pesticides and certain explosives in soil. Because of having the ability to oxidize phenolic, non-phenolic lignin-related compounds and highly fractious environmental pollutants, laccases have drawn the attention of researchers in the last few decades. Commercially, laccases have been used to determine the difference between codeine and morphine, produce ethanol and are also being employed in de-lignify woody tissues. We have revised patents related to applicability of laccases. We have revised all the patents related to its wide applicability. Conclusion: For fulfillment of these wide applications, one of the major concerns is to develop a system for efficient production of these enzymes at a broad scale. Research in the field of laccases has been accelerated because of its wide diversity, utility, and enzymology. This paper deals with recent trends in implementation of the laccases in all practical possibilities with the help of optimizing various parameters and techniques which are responsible for mass production of the enzyme in industries.

Redox Potential‐Dependent Formation of an Unusual His–Trp Bond in Bilirubin Oxidase

Bilirubin oxidase (BOD) belongs to the family of blue multicopper oxidases, and catalyzes the concomitant oxidation of bilirubin to biliverdin and the reduction of molecular oxygen to water via a four-electron reduction system. The active sites of BOD comprise four copper atoms; type I copper (T1Cu) forms a mononuclear site, and a cluster of three copper atoms forms a trinuclear center. In the present study, we determined the high-resolution crystal structures of BOD from the fungus Myrothecium verrucaria. We investigated wild-type (WT) BOD and a BOD mutant called Met467Gln, which is inactive against bilirubin. The structures revealed that a novel post-translational crosslink between Trp396 and His398 is formed in the vicinity of the T1Cu site in WT BOD, whereas it is absent in the Met467Gln mutant. Our structural and computational studies suggest that the His-Trp crosslink adjusts the redox potential of T1Cu to that of bilirubin to efficiently abstract electrons from the substrate.

Deciphering the effect of salts on Bilirubin Oxidases activity

Bilirubin Oxidases (BODs) and laccases belong to the Blue Multicopper Oxidases family (MCOs). Both enzymes couple the oxidation of substrates to the reduction of O2 to H2O. In contrast to laccases, BODs display high activity and stability at neutral pH and are more tolerant to chloride [1]. These unique features make BODs useful in a large number of biotechnological applications such as biosensors for jaundice detection, dyes degradation, and as a cathodic enzyme in enzymatic biofuel cells [2]. Glucose/oxygen enzymatic biofuel cells use the energy of redox reactions catalyzed by enzymes to produce electrical energy. Glucose oxidizing enzymes such as Glucose Oxidase (GOX) and oxygen reducing enzymes such as BODs are used at the anode and at the cathode respectively. Such biofuel cells are good candidates to be implanted in the human body of diabetic people and would be the only in vivo power source able to power glucose sensors. To be implanted, cathodic and anodic enzymes have to be enough stable and resistant to physiological salts to be efficient several weeks or months in biological fluids.Here, I will focus my presentation on the enzymatic cathodic part. BODs and laccases are well known to be sensitive to high salt concentrations [3] and their stability on the electrode needs to be improved. Efforts in this direction allowed our team to identify the BOD from Bacillus pumilus with interesting features as a cathodic enzyme [4]. By using a sequence analysis approach, a new BOD was identified with very interesting features compared to other BODs which makes it of great interest to be used as a cathodic enzyme in biofuel cells.Particularly, in contrast to most BODs described, this new BOD is not inhibited but activated by several salts (up to 1,7 times). This characteristic makes this BOD very interesting considering the fact that all biological fluids contain relatively high levels of salts (such as 140mM NaCl in the blood for example). For this reason, we have compared the effect of various salts and their mechanisms on the enzymatic activity of BOD from Bacillus pumilus and the new BOD in solution and immobilized on electrode. Molecular modeling studies were also performed on both enzymes to decipher the mechanisms of activation/inhibition of different salts and more precisely the sodium chloride effects at the atomic level.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

New efficient laccase immobilization strategy using ionic liquids for biocatalysis and microbial fuel cells applications

AbstractBACKGROUNDLaccases belong to the family of blue multicopper oxidases, these enzymes are efficient in many applications, including in biofuel cells to produce electricity through chemical reactions. Several laccase immobilization methods have been elucidated, such as covalent link and entrapment.RESULTSThe present work describes how, for the first time, laccase was successfully immobilized by entrapment into polymer inclusion ionic liquid membranes (PILMs) based on ionic liquids and polyvinylchloride. The immobilization rate was very high in all cases (99.2 ± 0.6%), so that the enzyme entrapment strategy described can be considered as successful and one of the best methods for laccases immobilization that have been reported to date. The assayed membranes were evaluated as performing carrier for laccase by means of the ABTS oxidation. Four screened ionic liquids were used for the first time for this purpose: [OMIM][NTf2], [EMIM][NTf2], [Epy][NTf2] and [Chol][NTf2]. This work demonstrated the efficiency in terms of the activity rate and stability of the enzyme versus IL concentrations. The most suitable IL as regards activity [Chol][NTf2]. However, the reuse assays showed that the most stable PILM was the one constituted by 50% [Epy][NTf2]. For physical resistance and water insolubility, the [OMIM][NTf2] based PILM was selected for microbial fuel cell (MFC) application.CONCLUSIONThis is the first report describing this original entrapment strategy for laccase immobilization into PILM. The application of this laccase‐membrane in MFC showed high stability during almost one week of use and good efficiency in producing bio‐energy and removing COD from industrial wastewater. © 2017 Society of Chemical Industry

The StLAC2 gene is required for cell wall integrity, DHN-melanin synthesis and the pathogenicity of Setosphaeria turcica

Laccases are blue multicopper oxidases, play important roles in various biological processes. These processes include fungal dihydroxynaphthalene (DHN)-melanin biosynthesis and pathogenicity, cellular growth, morphogenesis, and differentiation. This study investigated functions of the laccase gene StLAC2 in Setosphaeria turcica. The Δlac2 mutant colony color was distinct from that of the S. turcica wild-type (WT) isolate, and the mutants exhibited defective conidial formation. In contrast to the WT, the mutants exhibited a lighter color on the 2, 2-azino-di-[3-ethylbenzo-thia-zolin-sulphonate] (ABTS) plates, and the intracellular laccase activity was lower. Notably, StLAC2 gene loss correlated with decreased DHN-melanin biosynthesis and affected the integrity of the cell wall, where the StLAC2 gene mutants showed thinner, more transparent walls with a higher number of mitochondria than the WT. The Δlac2 mutants also lost their pathogenicity in maize. The results indicated that the StLAC2 gene involved in cell wall integrity, melanin biosynthesis and appressorial and conidial formation.

Substrate specificity and copper loading of the manganese-oxidizing multicopper oxidase Mnx from Bacillus sp. PL-12.

Manganese(ii) oxidation in the environment is thought to be driven by bacteria because enzymatic catalysis is many orders of magnitude faster than the abiotic processes. The heterologously purified Mn oxidase (Mnx) from marine Bacillus sp. PL-12 is made up of the multicopper oxidase (MCO) MnxG and two small Cu and heme-binding proteins of unknown function, MnxE and MnxF. Mnx binds Cu and oxidizes both Mn(ii) and Mn(iii), generating Mn(iv) oxide minerals that resemble those found on the Bacillus spore surface. Spectroscopic techniques have illuminated details about the metallo-cofactors of Mnx, but very little is known about their requirement for catalytic activity, and even less is known about the substrate specificity of Mnx. Here we quantify the canonical MCO Cu and persistent peripheral Cu bound to Mnx, and test Mnx oxidizing ability toward different substrates at varying pH. Mn(ii) appears to be the best substrate in terms of kcat, but its oxidation does not follow Michaelis-Menten kinetics, instead showing a sigmoidal cooperative behavior. Mnx also oxidizes Fe(ii) substrate, but in a Michaelis-Menten manner and with a decreased activity, as well as organic substrates. The reduced metals are more rapidly consumed than the larger organic substrates, suggesting the hypothesis that the Mnx substrate site is small and tuned for metal oxidation. Of biological relevance is the result that Mnx has the highest catalytic efficiency for Mn(ii) at the pH of sea water, especially when the protein is loaded with greater than the requisite four MCO copper atoms, suggesting that the protein has evolved specifically for Mn oxidation.