T. Kent Kirk

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.

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 .

Wood Decay by Brown-Rot Fungi: Changes in Pore Structure and Cell Wall Volume

Sweetgum (Liquidambar styraciflua L.) wood blocks were decayed by Postia (= Poria) placenta in soilblock cultures. Decay was terminated at various weight losses, and the pore volumes available to four low molecular weight molecules, (water, 4 Å,; glucose, 8 Å,; maltose, 10 Å; and raffinose, 128,) and three dextrans (Mr 6,000, 38 Å; 11,200, 51 Å; and 17,500, 61 Å) were determined by the solute exclusion technique (Stone and Scallan 1968b). The volume in sound (undecayed) wood that was accessible to the seven probes varied from 1.0 ml g–1 for the three largest to 1.35 ml g–1 for water. Thus, the volume in sound wood attributable to lumens, pits, and other large openings was 1.0 ml g–1 and that accessible to water in the cell wall was 0.35 ml g–1. Of this volume, 80% was inaccessible to molecules > 12 Å in diameter. As the wood was decayed, the volume of pores in the cell wall increased steadily to 0.7 ml g–1 at 35% weight loss. New cell wall volume was accessible to the four low molecular weight probes but not to molecules of Mr ≥ 6,000. The increase in accessible pore volume to the four smallest probes was gradual. Most of the new cell wall volume created by removal of components during decay was in the pore size range of 12 Å, to 38 Å. Within experimental error, no pores of > 38 Å, were observed in sound or decayed wood. Our results are consistent with the hypothesis that the initial depolymerization of cellulose, characteristic of brown rot, is caused by a diffusible agent. The molecular diameter of the agent is apparently in the range 12 Å, to 38 Å, and it causes erosion and thus enlargement of the pores to which it has access.

Relationship of nitrogen to the onset and suppression of ligninolytic activity and secondary metabolism in Phanerochaete chrysosporium

Ligninolytic activity in the white-rot fungus Phanerochaete chrysosporium was previously found not to be induced by lignin, but to develop in cultures in response to nitrogen starvation. Added NH4+suppressed existing activity. The present study examined amino acid profiles and protein concentrations during onset of ligninolytic activity (synthetic 14C-lignin→14CO2) in nitrogen-limited cultures, and defined some characteristics of subsequent suppression by added nutrient nitrogen. During the transition between depletion of medium nitrogen and the onset of ligninolytic activity, total free intracellular amino acids increased, then rapidly decreased; changes in glutamate concentration played a major role. Intracellular protein concentration fluctuated in a manner roughly converse to that of the concentration of free amino acids. Protein turnover was rapid (5–7%/h) during the transition period. Glutamate, glutamine, and histidine were the most effective of 14 nitrogenous compounds in suppressing ligninolytic activity after its onset. The suppressive effect was not mediated through carbon (glucose)-catabolite repression or by alterations in culture pH. Activities responsible for oxidation of lignin and the ligninrelated phenol, 4-hydroxy-3-methoxyacetophenone, responded similarly to added nitrogen. Synthesis of a secondary metabolite, veratryl alcohol, like lignin oxidation, was suppressed quite sharply by glutamate and significantly by NH4+. Results indicate that nitrogen metabolism affects ligninolytic activity as a part of secondary metabolism, and suggest a role for glutamate metabolism in regulating this phase of culture development.

Degradation of gymnosperm (Guaiacyl) vs. angiosperm (syringyl/guaiacyl) lignins by Phanerochaete chrysosporium

This study examined the relative degradabilities of guaiacyl and syringyl/guaiacyl lignins by the white-rot fungus Phanerochaete chrysosporium Burds. Synthetic syringyl/guaiacyl lignin (Cpin syringyl units) was depolymerized much more rapidly than synthetic guaiacyl lignin (Cp), although the two were oxidized to CO2 at the same rate. Milled wood lignin or birch, labeled with H at Ce, was also depolymerized more rapidly than similarly labeled spruce milled wood lignin. During degradation two to three times äs much of both the synthetic and natural guaiacyl lignins became mycelium-bound äs the syringyl/guaiacyl lignins. This result is interpreted to reflect a greater resistance of the guaiacyl lignins. Collectively, our results point to a more facile initial degradation of the syringyl/guaiacyl type of lignins. This finding helps explain the more rapid degradation of angiosperm wood than gymnosperm wood by white-rot fungi.

Fungal degradation of kraft lignin and lignin sulfonates prepared form synthetic 14C-lignins

Kraft lignins (KL), bleached kraft lignins (BKL), and lignin sulfonates (LS) were prepared from synthetic 14C-lignins labeled in the aromatic nuclei or in the propyl side chains. These and control lignins (CL) were incubated with the lignin-decomposing white-rot fungus, Phanerochaete chrysosporium Burds., in a defined culture medium containing cellulose as growth substrate. Decomposition was monitored by measuring the 14CO2 evolved. Average percentages of the [ring-14C]- and [side chain-14C]-lignins, respectively, recovered as 14CO2 at the cessation of 14CO2 evolution were: KL, 41 and 31; BKL, 42 and 26; LS, 28 and 21; and CL, 26 and 24. Gel permeation chromatography of radiolabeled materials extracted from spent cultures showed that substantial degradation to nonvolatile products had occurred. The polymeric components in the extracts were further degraded in fresh cultures. These results indicate that industrial lignins are significantly bioalterable, and that under favorable conditions industrial lignins are substantially biodegradable.

Characteristics of Cotton Cellulose Depolymerized by a Brown-Rot Fungus, by Acid, or by Chemical Oxidants

Summary Wood decay fungi of the brown-rot type destroy the strength of wood before significant weight loss occurs.This is due to extension depolymerization of the cellulose. Evidence indicates that enzymes cannot gainaccess to the cellulose in wood and that the depolymerizing agent might be oxidative. Our objectiveshere were to gain information about the nature of the agent by characterizing pure cellulose that hadbeen depolymerized by a brown-rot fungus (BR) and to compare its characteristics with those of cellulosedepolymerizcd by acid (A). by Fenton’s reagent (Fe 2+ + H 2 O 2 ) (F), or by periodic acid/bromine/water(P). All four types of depolymerized cellulose exhibited molecular size characteristics indicating that de-polymerization was due to cleavages within the noncrystalline regions. Carbonyl contents of the sampleswere similar, from 1.4 to 1.9 per cellulose molecule. Carboxyl contents were 0.44, 0.00, 0.24, and 7.38per molecule for samples BR, A, F, and P, respectively; uronic acids were absent except for a trace insample P. On complete acid hydrolysis the samples gave the expected amounts of glucose, except forsample P, which contained nonglucosyl moietics that did not contain carbonyl or carboxyl groups detectedby our analytical procedures. With theglucose in the acid hydrolysates we found glyceric, crythronic,ararbonic, and gluconic acids in samples BR and F, and crythronic acid in sample P. Our results indicatethat the brown-rotted cellulose resembles sample F more than samples A or P.

Potential applications of bio-ligninolytic systems

Abstract Members of various fungal taxa and actinomycetes have been shown to degrade lignin at least partially. The white-rot wood-decomposing basidiomycetes completely metabolize the complex polymer, exhibit the highest reported rates, and are the most studied. Evidence indicates that their degradation of lignin involves oxidative, non-specific reactions, but the nature of the catalysts and the reactive species remain undefined; the catalysts have not been separated from living cells. Culture conditions optimal for lignin metabolism by white-rot fungi have been described, and several potential applications of whole ligninolytic cultures have been explored preliminarily: (a) partial delignification for the production of cellulosic products (bio-mechanical pulping, bio-bleaching); (b) conversion of lignocellulosics (improving ruminant digestibility, cultivating edible mushrooms) into feed and food; and (c) treatment of lignin-derived wastes (decolorizing, removing BOD, COD). The possibility to biomodify by-product lignins to yield valuable polymeric or low molecular weight chemicals has not been approached experimentally, but is another area of potential application. Improved waste treatment processes might well be the first intentional application of bioligninolytic systems.

Carboxylic Acids Produced Through Oxidative Cleavage of Aromatic Rings During Degradation of Lignin in Spruce Wood by Phanerochaete Chrysosporium

Abstract Pre-extracted spruce wood chips were decayed by Phanerochaete chrysoaporium to a 20% loss in lignin (8% loss in total weight), and extracted successively with petroleum ether, chloroform, acetone, methanol, and aqueous dioxane. The low molecular weight fraction of the methanol extract was analyzed by gas chromatography/ high resolution mass spectrometry after acetylation and nethylation. Examination of the spectra resulted in structural assignments for 28 compounds, 10 of which were aromatic acids identified and reported previously (Holzforschung 36,3 (1982)). At least 13 of the remaining compounds were formed via aromatic ring cleavage. In addition to ring cleavages, the new structures revealed oxidation of α- and γ-hydroxyl groups, oxidative cleavage of Cα−Cβ and Cβ–Cγ bonds, and 3-O-demethylation. It is postulated that oxidative cleavage of aromatic rings in the lignin units with an ether linkage at C-4 of the guaiacyl group involves 3-O-demethylation, hydroxylation at C-2 and subsequent o-cle...

Partial delignification of unbleached kraft pulp with ligninolytic fungi

SummaryUnbleached kraft pulp is partially delignified on incubation under specified conditions with ligninolytic fungi, thereby decreasing requirements for bleaching chemicals. Studies withPhanerochaete chrysosporium demonstrated important effects of nutrient nitrogen and molecular oxygen concentrations. Possible approaches to rate enhancement are suggested.