Application of ESI FT-ICR MS to Study Kraft Lignin Modification by the Exoenzymes of the White Rot Basidiomycete Fungus TrametesHirsutaLE-BIN 072

Trameteshirsuta is a wood rotting fungus that possesses a vast array of lignin degrading enzymes, including7 laccases, 7 ligninolyticmanganese peroxidases, 9 lignin peroxidases and 2 versatile peroxidases. In this study,electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS)was used to examine kraft lignin modification by the enzymatic system of this fungus.The observed pattern of lignin modification suggested that before the 6th day of cultivation,the fungal enzymatic system tended to degrade more oxidized moleculesand, hence, less recalcitrant molecules, with the production of hard-to-modify reduced molecular species. At some point after the 6th day of cultivation,the fungal enzymatic system tended to degrade more oxidized moleculesand, hence, less recalcitrant molecules, with the production of hard-to-modify reduced molecular species. At some point after the 6th day of cultivation,the fungus started to degrade less oxidized, more recalcitrant, compounds, converting them into the more oxidized forms. The altered pattern of lignin modification enabled changes in the fungal enzymatic system. These changes were further attributed to the appearance of the particular ligninolyticmanganese peroxides enzyme(MnP7), which was added by the fungus to the mixture of enzymes that had already been secreted (VP2 and MnP5). Keywords: wood rotting fungi, kraft lignin, mass spectrometry, peroxidases

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.

Characterization of a Dye-Decolorizing Peroxidase from Irpex lacteus Expressed in Escherichia coli: An Enzyme with Wide Substrate Specificity Able to Transform Lignosulfonates

A dye-decolorizing peroxidase (DyP) from Irpex lacteus was cloned and heterologously expressed as inclusion bodies in Escherichia coli. The protein was purified in one chromatographic step after its in vitro activation. It was active on ABTS, 2,6-dimethoxyphenol (DMP), and anthraquinoid and azo dyes as reported for other fungal DyPs, but it was also able to oxidize Mn2+ (as manganese peroxidases and versatile peroxidases) and veratryl alcohol (VA) (as lignin peroxidases and versatile peroxidases). This corroborated that I. lacteus DyPs are the only enzymes able to oxidize high redox potential dyes, VA and Mn+2. Phylogenetic analysis grouped this enzyme with other type D-DyPs from basidiomycetes. In addition to its interest for dye decolorization, the results of the transformation of softwood and hardwood lignosulfonates suggest a putative biological role of this enzyme in the degradation of phenolic lignin.

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% b-O-4’ ether bond cleavage, 27.0% Ca-C1 carbon bond cleavage and 3.6% Ca-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 lignin depolymerization should include targeting stability at low pH.

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, including cleavage of both β-aryl 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-aryl 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 cleavages in a phenolic model lignin dimer we found that degradation of the lignin dimer was greatly enhanced at lower pH, and the acid-stable lignin peroxidase isozyme H8 increased the degradation of the lignin dimer to products 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% b -aryl ether bond cleavage, 27.0% C a -aryl carbon bond cleavage and 3.6% C a -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 hydration of hydronium cation on aliphatic and phenolic hydroxyl groups under, which extremely acidic conditions resulted in lower energetic barriers for bond-cleavages, especially β-ether 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 lignin and further suggest that lignin peroxidase isozyme H8 engineering efforts include targeting stability at low pH.

Mechanisms of Lignin-Degrading Enzymes

Lignin is abundant in nature. It is a potentially valuable bioresource, but, because of its complex structure, it is difficult to degrade. However, enzymatic degradation of lignin is effective. Major lignin-degrading enzymes include laccases, lignin peroxidases, and manganese peroxidases. In this paper, the mechanisms of degradation of lignin by these three enzymes is reviewed, and synergy between them is discussed.

ROS production by localized SCHENGEN receptor module drives lignification at subcellular precision

Production of reactive-oxygen species (ROS) by NADPH oxidases (NOXs) impacts many processes in animals and plants and many plant receptor pathways involve rapid, NOX-dependent increases of ROS. Yet, their general reactivity has made it challenging to pinpoint the precise role and direct cellular targets of ROS. A well-understood ROS target in plants are lignin peroxidases in the cell wall. Lignin can be deposited with exquisite spatial control, but the underlying mechanisms have remained elusive. Here we establish a full kinase signaling relay that exerts direct, spatial control over ROS production and lignification within the cell wall. We show that polar localization of a single kinase component is crucial for pathway function. Our data indicates that an intersection of more broadly localized components allows for micrometer-scale precision of lignification and that this system is triggered through initiation of ROS production as a critical peroxidase co-substrate.

Recent Advances in Applications of Acidophilic Fungi to Produce Chemicals

Processing of fossil fuels is the major environmental issue today. Biomass utilization for the production of chemicals presents an alternative to simple energy generation by burning. Lignocellulosic biomass (cellulose, hemicellulose and lignin) is abundant and has been used for variety of purposes. Among them, lignin polymer having phenyl-propanoid subunits linked together either through C-C bonds or ether linkages can produce chemicals. It can be depolymerized by fungi using their enzyme machinery (laccases and peroxidases). Both acetic acid and formic acid production by certain fungi contribute significantly to lignin depolymerization. Fungal natural organic acids production is thought to have many key roles in nature depending upon the type of fungi producing them. Biological conversion of lignocellulosic biomass is beneficial over physiochemical processes. Laccases, copper containing proteins oxidize a broad spectrum of inorganic as well as organic compounds but most specifically phenolic compounds by radical catalyzed mechanism. Similarly, lignin peroxidases (LiP), heme containing proteins perform a vital part in oxidizing a wide variety of aromatic compounds with H2O2. Lignin depolymerization yields value-added compounds, the important ones are aromatics and phenols as well as certain polymers like polyurethane and carbon fibers. Thus, this review will provide a concept that biological modifications of lignin using acidophilic fungi can generate certain value added and environmentally friendly chemicals.

Biochemical Investigations of Different Mushroom Species for Their Biotechnological Potential

Among bioactive constituents occurring in mushrooms, phenolic compounds focus attention due to their antioxidant activity. Also, a special attention is given to mushrooms secreting extra cellular enzymes such as lignin peroxidases (LiP), manganese peroxidase (MnP) and laccase (Lac), enzymes which can be used in biodegradation processes. The aim of the present study was to investigate ten mushroom species for total phenolic compounds, lignocellulolytic enzymes and for their synthetic dyes decolourisation potential. For this purpose, 70% ethanol extracts of ten dried mushrooms were analysed using spectrophotometric methods. The results revealed that total phenolic compounds in the extracts were the highest in A. campestris, P. ostreatus var. Florida and T. versicolor. Laccase activity showed high values in extracts from P. ostreatus var. Florida, A. campestris, L. edodes, and G. applanatum. Lignin peroxidases (LiP) activity showed high values in extracts from A. campestris, F. velutipes, P. ostreatus var. Florida and T. versicolor, whereas manganese peroxidase (MnP) activity was highest in extracts from P. ostreatus var. Florida, A. campestris and G. applanatum. It was found that some of the fungal extracts showed high activities in decolorizing of synthetic dyes.