Overview of Mycelial Fungi - Lignin Destructors

In this work, the following genuses of mycelial fungi, capable of producing ligninolytic enzymes of various actions, were considered:Penicillium, Aspergillus, Fusariumand Altermaria. Fungi of the genus Aspergilluswere capable of producing laccase, manganese peroxidase and lignin peroxidase in the medium. Penicillium mostly produced laccase. Fusariumproduced laccase, aryl alcohol oxidase, manganesedependent peroxidase, manganese-independent peroxidase and lignin peroxidase. Alternariaproduced laccase, lignin peroxidase and manganese peroxidase. The results demonstrated the possibility of using specific substrates in the study of enzyme activity, as well as the influence of some factors introduced into the medium on the synthesis of enzymes. The auxiliary influence of these fungi on the synthesis of ligninolytic enzymes in symbiosis with otherswas considered. Keywords: mycelial fungi, ligninolytic enzymes, Penicillium, Aspergillus, Fusarium, Altermaria

Functional Characterization of Melanin Decolorizing Extracellular Peroxidase of Bjerkandera adusta

Melanin pigmentation in the human skin results from complicated cellular mechanisms that remain to be entirely understood. Uneven melanin pigmentation has been counteracted by inhibiting synthesis or transfer of melanin in the skin. Recently, an enzymatic approach has been proposed, wherein the melanin in the skin is decolorized using lignin peroxidase. However, not many enzymes are available for decolorizing melanin; the most studied one is lignin peroxidase derived from a lignin degrading fungus, Phanerochaete chrysosporium. Our current study reveals that versatile peroxidase from Bjerkandera adusta can decolorize synthetic melanin. Melanin decolorization was found to be dependent on veratryl alcohol and hydrogen peroxide, but not on Mn2+. The degree of decolorization reached over 40% in 10 min at 37 °C and a pH of 4.5. Optimized storage conditions were slightly different from those for the reaction; crude enzyme preparation was the most stable at 25 °C at pH 5.5. Since the enzyme rapidly lost its activity at 50 °C, stabilizers were screened. As a result, glycerol, a major component in several cosmetic formulations, was found to be a promising excipient. Our results suggest that B. adusta versatile peroxidase can be considered for future cosmetic applications aimed at melanin decolorization.

The Influence of Inducers on the Coltricia cinnamomea Laccase Activity and its Ability to Degrade POME

Some species of Basidiomycetes, specifically white rot groups, produce three ligninolytic enzymes, namely, Lignin Peroxidase (LiP), Manganese Peroxidase (MnP) and Laccase (Lac), which have low activity in degrading Palm Oil Mill Effluent (POME). The research objective was to obtain the data on the ability of the Coltricia cinnamomea to produce LiP, MnP, and Lac enzymes to degrade POME. This research also studied the effect of sucrose, alcohol, veratryl alcohol, CuSO4 and ZnSO4,as inducers. Isolates of Coltricia cinnamomea, which were stored in a PDA media at -20℃ were obtained from the Microbiology section of the Research Center for Biology (LIPI). Furthermore, the growth media used were DM, Bean sprout Extract (TE) and PDB. The result indicated that PDB is the most suitable growth media for the production of ligninolytic enzymes, because in this medium these enzymes showed the highest activity. It was also observed that sucrose increased the laccase activity by 40.80%. Furthermore, Coltricia cinnamomea was able to reduce the concentration of Poly R-478 by 60.74%, after the addition of ZnSO4. In addition, it degraded and decreased the color and COD of POME, by 72.63% and 91.19% respectively, after the addition of veratryl alcohol, and incubation for 10 days. Therefore, this fungus can be used to degrade POME in order to prevent environmental pollution. Coltricia cinnamomea has not been used for POME degradation. By using Coltricia cinnamomea, we obtained new data regarding the activity of laccase and its ability to degrade POME.

Lignin peroxidase: Optimization of media for higher productivity by single factor optimization method

Lignin peroxidase belongs to ligninolytic enzyme group and is one of the industrial important enzymes as it has wide applications in different sectors. Lignin peroxidase is produced by submerged fermentation process which requires optimization of physical and chemical parameters to achieve higher activity and make the process cost effective. The present study aimed at the optimization of physical as well chemical parameters of production medium. The optimization includes physical parameter such as incubation time, inoculum size, temperature, pH, RPM (Rotation per minute) while chemical parameters include carbon source, nitrogen source and different mineral elements. Form the optimization study, it was observed that highest lignin peroxidase production was achieved after 72 hours of incubation at temperature 300C, pH 6 and RPM 120. Optimization of chemical parameters reveals that incorporation of sodium nitrite (9g/L) in the media gave significant increase in enzyme activity. It was found that the maximum productivity achieved after optimization was 2214 U/ml which was four times higher than process without optimized parameters.

Discoloration of dyes by Trametes spp

The dyes used in the textile industry contribute significantly to pollution of water sources as they are disposed, most of the time, without proper treatment. The objective of this work was to test three strains of two species of the genus Trametes collected in Brazil against the ability to discolor the indigo carmine dye and to detect the activity of the enzymes laccase, lignin peroxidase and manganese peroxidase. The experiment was carried out in Kirk medium under static, non-sterile condition, at ±28 °C for 120 h. Trametes lactinea (URM8350) discolored 81.40% of the indigo carmine dye, T. lactinea (URM8354) 85.09% and T. villosa (URM8022) 96.11%. Laccase was detected in all specimens. Manganese peroxidase was detected in T. villosa (URM8022) and T. lactinea (URM8354), while lignin peroxidase was not detected in any of the isolates under the conditions of the experiment. The discoloration rates observed demonstrate the ability of the strains to discolor carmine indigo and the promising use in the discoloration processes in wastewater from the textile segment.

Experimental and theoretical insights into the effects of pH on catalysis of bond-cleavage by the lignin peroxidase isozyme H8 from Phanerochaete chrysosporium

Background Lignin peroxidases catalyze a variety of reactions, resulting in cleavage of both β-O-4′ ether bonds and C–C bonds in lignin, both of which are essential for depolymerizing lignin into fragments amendable to biological or chemical upgrading to valuable products. Studies of the specificity of lignin peroxidases to catalyze these various reactions and the role reaction conditions such as pH play have been limited by the lack of assays that allow quantification of specific bond-breaking events. The subsequent theoretical understanding of the underlying mechanisms by which pH modulates the activity of lignin peroxidases remains nascent. Here, we report on combined experimental and theoretical studies of the effect of pH on the enzyme-catalyzed cleavage of β-O-4′ ether bonds and of C–C bonds by a lignin peroxidase isozyme H8 from Phanerochaete chrysosporium and an acid stabilized variant of the same enzyme. Results Using a nanostructure initiator mass spectrometry assay that provides quantification of bond breaking in a phenolic model lignin dimer we found that catalysis of degradation of the dimer to products by an acid-stabilized variant of lignin peroxidase isozyme H8 increased from 38.4% at pH 5 to 92.5% at pH 2.6. At pH 2.6, the observed product distribution resulted from 65.5% β-O-4′ ether bond cleavage, 27.0% Cα-C1 carbon bond cleavage, and 3.6% Cα-oxidation as by-product. Using ab initio molecular dynamic simulations and climbing-image Nudge Elastic Band based transition state searches, we suggest the effect of lower pH is via protonation of aliphatic hydroxyl groups under which extremely acidic conditions resulted in lower energetic barriers for bond-cleavages, particularly β-O-4′ bonds. Conclusion These coupled experimental results and theoretical explanations suggest pH is a key driving force for selective and efficient lignin peroxidase isozyme H8 catalyzed depolymerization of the phenolic lignin dimer and further suggest that engineering of lignin peroxidase isozyme H8 and other enzymes involved in lignin depolymerization should include targeting stability at low pH.

Lignin-Degrading Microorganisms from Organic Soils

The most prevalent aromatic polymer in nature is lignin, produced by higher plants and thought to make up 30-35 percent of the non-fossil organic carbon on the planet. Lignin hydrolyzing enzymes such as lignin peroxidase, laccase, manganese peroxidase, and others produce a variety of aromatic monomers, including ferulic and vanillic acids. However, very little research has been done on the role of microbes in lignin degradation. In the present work, we have isolated 25 ligninolytic bacteria and 25 ligninolytic fungi from organic soils of Koppal, Raichur districts of Karnataka. The bacterial isolates were identified as Pseudomonas putida, Bacillus subtilis, based on biochemical tests, and fungi were identified as Aspergillus niger, Trichoderma viridae, Phanerochaete chrysosporium and Pleurotus ostreatus based on morphological characters. The ligninolytic activity of bacterial isolates was high when compared to fungal isolates. All the isolates produced detectable amounts of lignin peroxidase, manganese peroxidase, and laccase under in vitro conditions. In dye decolorization test, fungal isolates KGST-1, KGST-2, and KKSP could decolorize Ramazol Brilliant Blue R and Congo red.