Ming Tien

Lignin peroxidase of Phanerochaete chrysosporium

Publisher Summary Several procedures have been described for growing Phanerochaete chrysosporiurn for ligninase production. These procedures differ somewhat in the medium formulation and types of growth vessels: (1) shallow stationary cultures, (2) agitated liquid cultures, and (3) rotating biological contactors (RBCs; disk fermenters). The recently developed use of agitated culture for production of ligninase permits easier “scale up.” Although ligninase can be produced in agitated flask cultures, the reliable use of stirred tank fermenters awaits further development, which is ongoing in several laboratories. This chapter describes the production of ligninase in shallow stationary cultures and in agitated cultures. Shallow stationary cultures (10 ml) are grown in rubber-stoppered, 125-ml Edenmyer flasks at 39 ° under 100% oxygen. The stationary cultures give somewhat more reliable and reproducible results than the agitated cultures.

Lignin-Degrading Enzyme from the Hymenomycete Phanerochaete chrysosporium Burds

The extracellular fluid of ligninolytic cultures of the wood-decomposing basidiomycete Phanerochaete chrysosporium Burds. contains an enzyme that degrades lignin substructure model compounds as well as spruce and birch lignins. It has a molecular size of 42,000 daltons and requires hydrogen peroxide for activity.

Production of multiple ligninases by Phanerochaete chrysosporium: effect of selected growth conditions and use of a mutant strain

Abstract Two methods are described for increasing the production of ligninase by cultures of Phanerochaete chrysosporium grown in a nitrogen-limiting medium. The first method involves addition of veratryl alcohol (0.4 mM) and excess trace metals to stationary flask cultures. (Veratryl alcohol is both a substrate for ligninase and a secondary metabolite of P. chrysosporium. ) The control ligninase activity (20 units l −1 ; as measured by veratryl alcohol oxidation) increases approximately fivefold as a result of these additions. H.p.l.c. analyses of the extracellular proteins produced by these flask cultures revealed at least 13 proteins, ten of which absorb at 409 nm, suggestive of haemproteins; six of these have ligninase (veratryl alcohol oxidizing) activity. The second method entails scale-up using a disc fermenter with a mutant strain which adheres well to the plastic discs, in contrast to the wild type, and which in addition produces high titres of ligninase. The ligninases produced by the mutant and wild-type strains were analysed by native-gel electrophoresis and visualized by silver staining and Western blot analysis. They were also compared by V8 protease digestion analyses. Results indicate a high degree of homology between the ligninases within each strain in addition to homology between the corresponding ligninases of the two stains.

Characterization of an electron conduit between bacteria and the extracellular environment

A number of species of Gram-negative bacteria can use insoluble minerals of Fe(III) and Mn(IV) as extracellular respiratory electron acceptors. In some species of Shewanella, deca-heme electron transfer proteins lie at the extracellular face of the outer membrane (OM), where they can interact with insoluble substrates. To reduce extracellular substrates, these redox proteins must be charged by the inner membrane/periplasmic electron transfer system. Here, we present a spectro-potentiometric characterization of a trans-OM icosa-heme complex, MtrCAB, and demonstrate its capacity to move electrons across a lipid bilayer after incorporation into proteoliposomes. We also show that a stable MtrAB subcomplex can assemble in the absence of MtrC; an MtrBC subcomplex is not assembled in the absence of MtrA; and MtrA is only associated to the membrane in cells when MtrB is present. We propose a model for the modular organization of the MtrCAB complex in which MtrC is an extracellular element that mediates electron transfer to extracellular substrates and MtrB is a trans-OM spanning β-barrel protein that serves as a sheath, within which MtrA and MtrC exchange electrons. We have identified the MtrAB module in a range of bacterial phyla, suggesting that it is widely used in electron exchange with the extracellular environment.

The requirement for ferric in the initiation of lipid peroxidation by chelated ferrous iron.

When certain ferrous chelates are added to lipid, peroxidation of the lipid occurs following a short lag. This suggests that a product of ferrous autoxidation is required to initiate lipid peroxidation. This autoxidation product is apparently ferric iron, rather than the oxygen radicals which also result from ferrous autoxidation. Studies with oxy-radical scavengers and catalase suggest that O2-., H2O2, or the .OH are not involved in the initiation reactions, therefore, we propose that a ferrous-dioxygen-ferric chelate complex may be the initiating species.

Lignin degradation in wood-feeding insects

The aromatic polymer lignin protects plants from most forms of microbial attack. Despite the fact that a significant fraction of all lignocellulose degraded passes through arthropod guts, the fate of lignin in these systems is not known. Using tetramethylammonium hydroxide thermochemolysis, we show lignin degradation by two insect species, the Asian longhorned beetle (Anoplophora glabripennis) and the Pacific dampwood termite (Zootermopsis angusticollis). In both the beetle and termite, significant levels of propyl side-chain oxidation (depolymerization) and demethylation of ring methoxyl groups is detected; for the termite, ring hydroxylation is also observed. In addition, culture-independent fungal gut community analysis of A. glabripennis identified a single species of fungus in the Fusarium solani/Nectria haematococca species complex. This is a soft-rot fungus that may be contributing to wood degradation. These results transform our understanding of lignin degradation by wood-feeding insects.

Physical and enzymatic properties of lignin peroxidase isoenzymes from Phanerochaete chrysosporium

Abstract Phanerochaete chrysosporium BKM-1767 secretes multiple lignin peroxidase isoenzymes when grown under nitrogen-limited conditions. Here we report the purification of these heme-containing peroxidases, and their physical and catalytic characterization. Ten hemeproteins, designated H1–H10, were separated by anion exchange HPLC. Six of them, H1, H2, H6, H7, H8, and H10, were lignin peroxidases, oxidizing veratryl alcohol in the presence of H 2 O 2 . The other four (three peaks were resolved) exhibited manganese-dependent peroxidase activity, oxidizing vanillylacetone in the presence of H 2 O 2 and Mn +2 . The lignin peroxidases have different isoelectric points, between p14.7 and 3.3, and molecular weights between 38 and 43 kDa, determined by SDS-PAGE. All are N - and probably O -glycosylated. Three organic substrates and H 2 O 2 were used to compare their kinetic properties: the organic substrates were veratryl alcohol, 1,4-dimethoxybenzene, and the lignin model compound 1-(3,4-dimethoxyphenyl)-2-( o -methoxyphenoxy)-propane-1,3-diol. K M and TN values for each of these substrates varied significantly; e.g. for veratryl alcohol K M values were from 86 to 480 μ m and TN values were from 1.3 to 8.3 s -1 . The ranking of the isoenzyme activities differed with the different substrates, suggesting differences in affinities or in active site accessibilities. The K M for H 2 O 2 varied between 13 and 77 μ m . Immunological blot analysis and partial proteolytic digestion patterns showed that the isoenzymes have a high degree of homology. The isoenzyme concentrations in extracellular culture fluid were found to vary relatively and absolutely with culture time. A nomenclature scheme for these 10 hemeproteins has been proposed. This scheme should simplify identification of these proteins in the literature as well as be adaptable to others found in Phanerochaete chrysosporium .

Properties of Ligninase from Phanerochaete Chrysosporium and Their Possible Applications

The wood-degrading fungus Phanerochaete chrysosporium Burds produces a family of enzymes which degrade lignin and lignin-like substrates. These ligninases exhibit a high degree of homology in being hemeprotein peroxidases, in Mr, in cross reactivity to polyclonal antibodies, in being glycosylated, and in catalytic properties. The predominant ligninase is able to generate cation radicals in its aromatic substrates. These radicals can undergo a variety of reactions thus explaining the nonspecific nature of the enzyme. A similar mechanism is suggested for the other isoenzymes. There are numerous potential applications for ligninases. These include: biopulping, waste treatment of byproduct lignins, detoxification of environmental pollutants, and modification of lignins to produce small molecular weight organics.

Iron isotope fractionation during microbial reduction of iron: The importance of adsorption

In experiments investigating the causes of Fe isotope fractionation, the d 56/54 Fe value of Fe(II) remaining in solution (Fe(II)(aq)) after reduction of Fe(III) (goethite) by Shewanella putrefaciens is ;21.2‰ relative to the goethite, in agreement with previous research. The addition of an electron shuttle did not affect fractionation, suggesting that Fe isotope fractionation may not be related to the kinetics of the electron transfer. Furthermore, in abiotic, anaerobic FeCl2(aq) experiments in which approximately one-third of Fe(II)(aq) is lost from solution due to adsorption of Fe(II) onto goethite, the d 56/54 Fe value of Fe(II)(aq) remaining in solution is shifted by 20.8‰ relative to FeCl 2. This finding demonstrates that anaerobic nonbiological interaction between Fe(II) and goethite can generate signif- icant Fe isotope fractionation. Acid extraction of sorbed Fe(II) from goethite in experi- ments reveals that heavy Fe preferentially sorbs to goethite. Simple mass-balance modeling indicates that the isotopic composition of the sorbed Fe(II) pool is ;11.5‰ to 12.5‰ heavier than Fe in the goethite (;2.7‰-3.7‰ heavier than aqueous Fe(II)). Mass balance is also consistent with a pool of heavy Fe that is not released to solution during acid extraction.

Oxidation mechanism of ligninolytic enzymes involved in the degradation of environmental pollutants.

Abstract White rot fungi are the most significant lignin degraders among the wood inhabiting microorganisms. They degrade lignin by extracellular oxidative enzymes. The ligninolytic enzymes also oxidize various environmental pollutants such as polycyclic aromatic hydrocarbons, chlorophenols, and aromatic dyes. The most ubiquitous ligninolytic enzymes produced by these fungi are lignin peroxidases (LP), manganese peroxidases (MnP), and laccases (phenol oxidases). The peroxidases are heme-containing enzymes having typical catalytic cycles, which are characteristic of other peroxidases as well. One molecule of hydrogen peroxide oxidizes the resting (ferric) enzyme withdrawing two electrons. Then the peroxidase is reduced back in two steps of one electron oxidation in the presence of appropriate reducing substrate. The range of the reducing substrates of the two peroxidases is very different due to their altered substrate binding sites. LP is able to oxidize various aromatic compounds, while MnP oxidizes almost exclusively Mn(II) to Mn(III), which then degrades phenolic compounds. Laccases are copper-containing oxidases. They reduce molecular oxygen to water and oxidize phenolic compounds. In this paper, the mechanism of pollutant oxidation by ligninolytic enzymes is discussed giving an overview on the recent results of enzyme kinetics and structure.