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Daldinia concentrica degrades the recalcitrant nonphenolic structures in lignin. One of the processes responsible is the [C.sub.alpha]-[C.sub.beta] cleavage of the lignin propyl side chain.

The kinetic parameter level and the inactivation rate of fungal laccases are crucial factors in determining the oxidation-reduction reaction of nonphenolic lignin dimers. Using violuric acid as mediator, it has been found that Trametes villosa possesses the greatest redox rate among nonphenolic laccases, followed by Pycnoporus cinnabarinus and Botrytis cinerea. Differences in the glycosylation level and the carbohydrate content of dimers have also been shown to influence the copper retention, thermal stability and proteolytic degradation of laccases.

The homologous expression of the gene encoding lignin peroxidase isozyme H8, lip2, has been investigated in primary metabolic cultures of Phanerochaete chrysosporium. The expression of lip2 was driven using the glyceraldehyde-3-phosphate dehydrogenase promoter while the expression vector pUGL was employed to transform a Phanerochaete chrysosporium Ura11 auxotroph to prototrophy. Results show that both the steady-state and transient-state kinetic characteristics of the oxidation of veratryl alcohol between wild-type LiPh8 and rLiPh8 were similar, as were the molecular mass, optical absorption spectrum and pI.

Experiments were conducted to clone the ligZ gene encoding a ring cleavage enzyme for 2,2',3-trihydroxy-3'-methoxy-5,5'-dicarboxybiphenyl (OH-DDVA) oxygenase gene of Sphingomonas paucimobilis. An OH-DDVA and DDVA growth-deficient mutant was created by mutagenesis to identify the gene responsible for the ring cleavage. Deletions in the gene fragment were constructed and their oxygen consumption activity was studied. The fragments exhibited two open reading frames with overlapping and opposing transcriptional directions.

The role of simple organic acids on the stimulation of manganese-independent biobleaching of eucalyptus oxygen-delignified kraft pulp (OKP) was investigated. While an inverse relationship was obtained with the glycolate concentration and the kappa number or delignification, pulp brightness showed a direct association with optimum glycolate concentration. Although biobleaching occurred in both the control and experimental cultures in the first period, it stopped in the control during the second stage.

A study was conducted to study the fate of residual lignin during the delignification of kraft pulp by the fungus Trametes versicolor. CO2 trap contents were prepared with scintillation vials and were determined using a Beckman model LS6800 liquid scintillation counter. Kappa values were obtained using the micro kappa method while cellulase treatment was scaled down to a liquid volume of 40 ml. Results showed that the incubation of T. versicolor increased the amounts of 14C and ultraviolet absorbing materials that could be extracted from the pulps.

A research was conducted to study the application of in situ reverse transcription (ISRT) for detecting and quantifying lignin-degrading bacterial species in lignin enrichment communities and Pseudomonas putida F1 which expressed a toluene degradation gene when they were added to seawater exposed to toluene. The non-labeled primer was hybridized to the RNA target site while a general DNA stain was utilized to visualize all sample cells. Results showed that the ISRT protocol was sensitive enough to detect 16s rRNA and mRNA in complex microbial communities.

A research was conducted to examine the reactivities of phenothiazines, 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS), 1-hydroxy-benzotriazole (HBT), and several mediators on kraft pulp and lignin model compounds in combination with various fungal laccases. Crude preparations of laccase from various white-rot fungi were capable of delignifying kraft pulps when coupled to ABTS. Results showed major differences concerning laccase reactivities toward oxidation of veratryl alcohol in the presence of ABTS and HBT.

The identification of a laccase gene family in the new lignin degrading basidiomycete CECT 20197 was investigated using the effluents from sugar cane by-product fermentation industry. The study selected lignin-degrading white rot fungus and demonstrated its ability to produce and secrete several potent isozymes with laccase activity which decolorizes the effluent of a sugar cane by-product. Results reveal that white rot fungus degrade lignin efficiently in the absence of laccase. Furthermore, the production of laccase is repressed by glucose.

The stabilization of lignin peroxidases (LiP) in white rot fungi by tryptophan was investigated to identify L-tryptophan (TRP) substrates for LiP oxidation. The TRP stimulation considered two possibilities such as direct induction of LiP gene expression and protection against inactivation. Results show that supplementation of various cultures of white rot fungi with tryptophan have a large stimulatory effect on lignin peroxidase activity levels.

The effects of photosynthetic precursors or aromatic acids on the synthesis of aryl metabolites by Bjerkandera sp. strain BOS55 were analyzed in the presence and absence of Mn. The synthesis of veratryl alcohol and veratraldehyde was stimulated by addition of lignin degradation products such as veratrate, p-anisate and 3-chloro-p-anisate in the presence of Mn. However, Mn inhibited the formation of aryl metabolites such as p-anisaldehyde and 3-chloro-p-anisaldehyde by the white rot fungi.

The mechanism by which the fungus Ceriporiopsis subvermispora degrades the nonphenolic structures in lignin was investigated. During cleavage of these structures, C. subvermispora generated metabolites that target the high-molecular-weight polyethylene-glycol-linked lignin model, which is the major nonphenolic beta-O-4 structure of lignin. Other substances such as manganese peroxidase were also generated. The rate of cleavage increased as the enzyme synthesis increased, suggesting that manganese influences cleavage of nonphenolic lignin structures.

A mutant form of the fungus Trametes versicolor that does not produce manganese peroxidase (MnP) appears unable to delignify and bleach kraft pulp and has reduced ability to oxidize dyes. Researchers isolated the M49 mutant strain of the white rot fungus and found that, in comparison to a wild-type strain, it produced less laccase, ehthylene, and methanol when exposed to different substrates. When MnP was added to the culture some of the normal fungal activity was restored.

The fungus Phlebia (P.) radiata 79 may produce large amounts of the enzymes lignin peroxidase (LiP), manganese peroxidase (MnP), laccase, and glyoxal oxidase (GLOX) while breaking down wheat straw. Researchers extracted enzymes produced by the fungus during solid-state fermentation of wheat straw. In comparison to liquid fementation, solid-state cultures produced new forms of MnP and GLOX and higher concentrations of the form of LiP known as LiP2.

A study of the effect of laccase and manganese peroxidase(MnP) on lignin biodegradation by Ceriporiopsis subvermispora shows that an increase in the pH of the cultures resulted in the inverse correlation between lignin mineralization and enzyme titers detected in Ceriporiopsis subvermispora with 10mM ammonium tartrate(AT). Experimental evidence shows that rate of 14CO(sub 2) evolution and level of enzyme and mycelium was higher in solution containing 1% of glucose.

Penicillium chrysogenum, separated from pine forest brown soils in Canary Islands, degenerates and transforms synthetic dehydrogenative polymerisate, organosolv and kraft lignins in a medium containing high concentration of nitrogen and low level of carbon source and glucose. P. chrysogenum partially mineralizes synthetic and industrial lignins to 14CO2. Some lignins are transformed to 4C-intermediates that are soluble in water.

Ruminal anaerobic fungus Neocallimastix patriciarum solubilizes lignin but does not degenerate it. Most solubilized lignin segments are lignin carbohydrate complexes comprising hydroxycinnamic acids that are linked to ether. N. patriciarum solubilizes the lignin carbohydrate complex via xylan dissolution in the lignin-xylan matrix. Soluble lignin does not comprise of only ester-linked phenolics.

Lignin peroxidase biosynthesis of Phanerochaete chrysosporium, reveals a reduction in the intercellular pH and a growth medium pH increases, when Tween 80 is supplemented in cultures. UV-visible diffuse reflectance spectroscopy is used to study lignin peroxidase biosynthesis. Phanerochaete chrysosporium, a white rot fungus, generates several extracellular lignin peroxidases, which are used in various commercial applications such as the paper industry.

The construction of DNA probes specific for genes encoding vital lignin peroxide (LIP) isozymes H2, H8 and H10 of Phanerochaete chrysosporium, was conducted. These probes were employed to examine the temporal expression of the three lip genes in determined low-nitrogen mediums. H2 gene transcripts were synthesized in high levels on days 4, 5 and 7. Low levels of H2 gene transcripts were synthesized on day 6. No relationship was discerned between lip transcript and isozyme levels.

Two alternative methods to study the expression of specific lignin peroxidase (LPO) genes were devised. The techniques were employed to determine LPO transcripts from LPO genes. A nucleotide sequencing of a new LPO cDNA of Phanerochaete chrysosporium BKM-F-1767 was also undertaken. It was revealed that the L18-related gene of the P. chrysosporium BKM-1767 was secreted under limited carbon and nitrogen conditions, despite transcript levels being higher in carbon limited cultures.

The synthesis and isoenzyme composition of the ligninolytic system under nonlimiting nutrient conditions in Phanerochaete chrysosporium BKM-F-1767 were characterized using a nonimmersed liquid culture system and conditions close to those occurring in nature. The response of P. chrysosporium to any kind of starvation conditions may be controlled by a broad regulatory mechanism, but the factors that trigger overproduction of the individual components of the ligninolytic system may differ.

The relationships between the rates of synthetic lignin mineralization and the levels of manganese peroxidase (MnP) and laccase produced by the white rot fungus ceriporiopsis subvermispora is examined. Different levels of the extracellular enzymes are produced under different culture conditions and the mineralization rates are determined. A marginal relationship exists between mineralization rate and MnP and laccase production.

The xylosylation of phenolic hydroxyl group of lignin moel compounds in cultures of Coriolus versicolor was demonstrated. Experiments involved the incubation of a mixture of 4-methylguaiacol (MeG) and commercial UDP-xylose with mycelia cell extracts. UDP-xylose was transformed into MeG-xyloside in the process. Results indicate that UDP-xylotransferase is connected to the bioconversion of phenolic hydroxyl groups of lignin model compounds.

Research was conducted to examine the expression of Phanerochaete chrysosporium genes encoding lignin peroxidases, manganese peproxidases, and glyoxal oxidase in wood. Extraction of poly(A) RNA was done from colonized wood chips using magnetic capture while specific transcripts were quantified through competitive reverse transcriptase PCR. Results suggest that genes previously shown to be highly expressed under a wide range of cultural conditions are unlikely to play a significant role in biopulping performance on aspen.

Microbiological research shows that the soil-inhabiting microorganism Fusarium proliferatum can degrade lignin. Synthetic, natural and carbon-14 labelled lignin was used in comparative studies to ascertain degrading activity. Mineralization production occurs during primary metabolism. Residual products are also produced. The production process involves both depolymerization and polymerization activity.

A study was conducted on the ligninolytic capabilities of Pleurotus floridanus MUCL 28518, Pleurotus ostreatus CBS 411.71, Pleurotus pulmonarius CBS 507.85 and Pleurotus sajor-caju MUCL 29757 during solid-state fermentation of 14C lignin-labeled wheat straw. The kinetics of extracellular enzymes related to lignin degradation were monitored. Results indicated that Mn2+-oxidizing enzymes have an important function in lignin degradation.

Lignin peroxidase and Mn peroxidase production in Polyporus ostreiformis contribute to its ability to enhance delignification and decolorization in rice straw. This property of the fungi makes it useful for control of water pollution and generation of gaseous and liquid fuels from lignocellulosic materials.

Oxidative stress in lignin peroxidase (LIP)-producing cultures of Phanerochaete chrysosporium is studied by comparing levels of reactive oxygen species (ROS), cumulative oxidative damage, and antioxidant enzymes with those found in non-LIP producing cultures exchange of atmospheric air. Results suggest that high levels of ROS, preferentially hydroxyl radicals, are required to trigger LIP synthesis.

Results reveal that 3-hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase are important for the detoxification and assimilation of formaldehyde as well as vanillic acid in nonmethylotrophic Burkholderia cepacia TM1. Data indicate that incorporaion of labeled formaldehyde into cell components and degradation of vanillic acid increase by overexpression of the genes encoding the two enzymes.

Manganese peroxidase can be used to increase the bleachability of kraft pulps. It is hypothesized that the bleaching action is brought about by residual lignin chelation by the magnesium ion. However, studies have shown that manganese peroxidase partially oxidize lignin enhancing the extraction of residual lignin by alkali. Manganese peroxidase does not degrade lignin into soluble fragments.