The extracellular laccase of white-rot fungus, Cerrena unicolor, was purified from culture by Sephadex G-25 and ion-exchange chromatography on DEAE-Toyopearl column and immobilized on various supports. Purified laccase showed two times higher activity after desalting process and 3.7 to 13.4 times activity after DEAE chromatography. During immobilizing process, 93.8% protein and 100% of laccase activity were coupled to the supports. Following immobilization, the optimal pH (5.5) for immobilized laccase was slightly shifted wide pH values (from 5.0 to 6.0) in the case of some supports, and decreased more gradually in alkaline region. Both free and immobilized laccases showed the highest activity at 60 degrees C, however, the immobilized enzyme was more resistant to the wide range of temperature (50 similar to 80 degrees C). Even at the 90 degrees C, treated glass beads and Supp-I maintained almost 80% activity. After 10 days of storage at 4 degrees C, the immobilized laccases retained 95 similar to 100% of its initial activity. It was also more stable during storage at 4 degrees C. While, after the same storage time only 34% the initial activity was retained by the free enzyme. Immobilized laccase on Glass supports was retained 85 similar to 90% activity over 20 days storage, while free enzyme almost zero activity. The immobilized laccases which are more stable and temperature resistant than free enzyme, seem to be more useful.

This study focuses on the laccase and mediator co-operation in modelling lignin dimer veratrylglycerol-beta-vanillate ether (VVE) oxidation. Earlier we stated that constitutive laccase isolated from Cerrena unicolor can cooperate with acetovanillone (AV) and acetosyringone (AS) in degradation of high molecular fraction of lignosulfonates. The fungus C. unicolor is known as a very good producer of cheap constitutive laccase. Its cultivation for this reason does not require stimulation by the expensive and toxic inducer like 2,5-xylidine. The main purpose of present study is to examine capability of C. unicolor laccase to oxidize VVE, which is a non-phenolic lignin model compound with external mediator HBT, and AV and AS in order to choose one, most efficient system. In the presence of laccase, generation of monomeric aromatic acids from non-phenolic lignin model dimer veratrylglycerol-beta-vanillate ether (VVE) was observed. The addition of acetovanillone (AV) or acetosyringone (AS) intensified this process, i.e. transformation was more extensive than in the experiments omitting mediators. Among the products, isovanillic (IA) and vanillic (VA) acids were identified.

Among 97 fungal strains from the soil collected from the high mountain areas in the Jeju Island, Korea, Penicillium chrysogenum 9 was found to be the best lipase producer. Its lipase productivity reached 42 U/ml in the culture medium. Factors affecting lipase production by Penicillium chrysogenum 9 were studied using fermentation media of different chemical compositions. Under optimal conditions we noted a 1.6-fold increase of lipase activity. The maximum lipase activity was 68 U/ml of culture medium on the 5th day of incubation at pH 6.0 and 20 degrees C; therefore, Penicillium chrysogenum 9 was classified as a psychrotrophic microorganism. The enzyme from Penicillium chrysogenum 9 showed a maximum activity at 30 degrees C and pH 5.0 for synthetic substrates (triacylglycerols) or at pH 7.0 for natural substrates (oils).

Growth and production of lipase by a new isolant Geotrichum-like R59 were studied. Production of extracellular lipase was substantially enhanced when the initial pH of the culture medium, types of carbon and nitrogen sources, those of inducers of lipase biosynthesis, temperature and time of growth were optimized. Sucrose and triolein were the most effective compounds for lipase production, Maximum lipase production was obtained when urea was used as nitrogen source (146 U/ml). Temperature of 30 degrees C, the initial pH of 6.0 and incubation time of 48h were found as optimum conditions for cell growth and production of lipase by Geotrichum-like R59 strain. The enzyme presented a good thermostability and still exhibited very high activity after 1 h incubation at 60 degrees C.

The effect of aromatic alcohols, coniferyl, sinapyl, vanillyl and iso-vanillyl alcohols, on the removal of chlorinated phenols from water environment by fungal laccases from Cerrena unicolor and Rhizoctonia praticola was studied. In optimal conditions all tested alcohols removed about 30 to 60% of chlorophenols from the supernatant, compared to that of laccase alone. R. praticola at pH 7.0 significantly removed more chlorophenols from supernatant than in the case of C. unicolor at pH 5.5. The laccase reaction is independent on the enzyme source and favours higher pH condition. Tested chlorophenols were partly coupled with aromatic alcohols and sedimented. It almost linearly depended on amounts of chlorine atoms in chlorophenols; more chlorine atoms resulted in higher yield of coupling reaction.

Manganese dependent peroxidase (MnP) is the most ubiquitous enzyme produced by white-rot fungi, MnP is known to be involved in lignin degradation, biobleaching and oxidation of hazardous organopollutants. Bjerkandera fumosa is a nitrogen-unregulated white-rot fungus, which produces high amounts of MnP in the excess of N-nutrients due to increased biomass yield. The objective of this study was to optimize the MnP production in N-sufficient cultures by varying different physiological factors such as Mn concentration, culture pH, and incubation temperature. The growth of fungus was optimal in pH 4.5 at , -unregulated white-rot fungus produces high amounts of MnP in the excess N-nutrients. The fungus produced the highest level of MnP (up to ) with asparagine and as N source at 1.5 mM concentration, pH value of 4.5 at . Purification of MnP revealed the existence of two isoforms: MnPl and MnP2. The molecular masses of the purified MnPl and MnP2 were in the same range of 42~45 kDa. These isoforms of B. fumosa strictly require Mn to oxidize phenolic substrates. Concerned to kinetic constants of B. fumosa MnPs, B. fumosa has similar Km value and Vmax compared to the other white-rot fungi.

Several wood rotting fungi decolourized Remazol brilliant blue R (RBBR) and carminic acid (CA). Parallel activity of laccase in these fungi was studied. The addition of acetovanillone (AV) or acetosyringone (AS) intensified these processes: decolourization was more extensive than in the experiment omitting these compounds. At the presence of AS the decourization was more extensive than AV. However the level of destaining was relatively low in comparison to laccase activity on syringaldazine. The highly purified constitutive form of Cerrena unicolor and inducible form of Trametes versicolor laccases also decolourized both dyes. The fungus, Lentinus lepideus, which has no laccase activity did not decolourize at all. Nitrogen starvation induced the laccase and decolourization activity in both organisms, irrespective of nitrogen availability. This fact indicates laccase not solely responsible for decolourization, and probably decolourization of dyes involves more than one mechanism.

Selected species of Basidiomycetes (Abortiporus biennis, Cerrena unicolor and Trametes versicolor) were known to produce enhanced extracellular laccase (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) when they exposed to some heavy metal ions, xenobiotics and lignin related compounds. The constitutive and inducible forms of purified laccase showed oxidative and demethoxylating activities, which prove the bifunctionality of the enzyme. The oxidative/dernethoxylating ratios in inducible laccases varied depending on used inducers. The demethoxylating activities of laccases induced by lignin related compounds were Much higher than those induced by various xenobiotic substances.

In order to clarify the diversity of the arbuscular mycorrhizal fungi, 9 individual plant roots and soils were randomly chosen at 27 sites in the general cultivation fields in the Chungbuk- and Chungnam-provinces, middle parts of Korea. In terms of height growth of Sorghum bicolor, the soil in Cheongwon site (host plant Fagopyrum esculentum) resulted in the best growth, and the order of growth was Platycodon grandiflorus, Miscanthus sinensis, Sesamum indicum, and Capsicum annuum. It represents that AM fungi species are having host-specific differences. Concerned to AM fungi colonization of host plant, Sorghum bicolor, AM fungi colonization in the roots was an increasing colonization tendency with the increase of culture periods. At the initial culture stage, colonization upto 10 days was rather slow, and after-wards the rates were increased. This colonization increase has leveled off more than 40 days culture. In addition, both morphology-based and molecular approaches of the spores from various soils of Chungbuk- and Chungnam- provinces in Korea were applied to characterize the AM fungi. By following these approaches, in Chungbuk province, total 9 species of 6 genera, such as 3 species of Glomus, 1 species of Paraglomus, 1 species of Gigaspora, 2 species of Scutellospora, 1 species of Acaulospora, and 1 species of Archaeospora, were identified. In Chungnam province, total 6 species of 5 genera, such as 2 species of Gigaspora, 1 species of Glomus, 1 species of Scutellospora, 1 species of Acaulospora, and I species of Archaeospora, were confirmed.

This study was performed to examine the role of laccase from white-rot fungi, Cerrena unicolor and Trametes versicolor, with low molecular weight mediators, acetovanillone (AV) and acetosyringone (AS), in decolorizing of the textile dye, Chicago sky blue 6B (Direct Blue 1, DB1). The tested fungi were shown the ability to remove colour from DB1. The decolorizing activity of the dye was closely related to the laccase from fungi. In the presence of AV or AS as co-substrate, decolorization was more extensive than that of the control. Moreover, at the presence of AS, the decolorization process was more effective than AV. The highly purified laccase of C. unicolor and T versicolor also decolorized the dye. The addition of AV or AS enhanced this process.

The water soluble fractions(WSF) from Japanese larch wood were isolated, purified by anion exchange resin and Sephadex gel filtration and identified its chemical structure by means of periodate oxidation and methylation reactions. Its major components are arabinose and galactose (1:3.4). Based on the results of periodate oxidation, methylation and gas chromatographic analysis of purified WSF, main chain is composed of - 1,3-glycosidic linkage among D-galactopyranoses, and two different side chains;- l,6-glycosidic linkage among 2-3 units of D-galactopyranoses and - 1,6-glycosidic linkage between 1-2 units of D-galactopyranose and L-arabinopyranose. Addition of WSF to culture media of oak mushroom (Lentinula edodes) accelerated the mycelial growth. In the case of PDA cultures, 2 percent addition of WSF in Sanlim No. 6 strain and 4 percent of WSF in Mok-H strain mostly enhanced the mycelial growth of the mushroom. In the case of sawdust cultures, 4 percent addition of WSF in two strains showed the best mycelial growth. High percentages addition of WSF inhibited mycelial growth of the mushroom. Mushroom production was increased with addition of WSF. By the addition of WSF, ergosterol contents in the media were quite high at the colonized stage and rapidly increased at the fruiting stage. Therefore the ergosterol content could be utilized as an indicator to evaluate the culture maturity for the mushroom fruiting.

Laccases are multicopper-containing enzymes which catalyze the oxidation of phenolic and nonphenolic compounds with the concomitant reduction of molecular oxygen. They often occur as isoenzymes, either constitutive or inducible, that oligomerize to multilateral complexes, what allow for penetration to the woody cell wall structure. White rot basidiomycete fungi may produce a number of laccase isoenzymes, some constitutively and others after induction. Fungal laccase is commonly induced by many ions, such as Cu2+, Cd2+ Ca2+, Li+, Mn2+, Ag+, Hg2+, Mn and Fe3+, phenolic compounds, some organic compounds, such as ethanol, isopropanol, cAMP, caffeine, p-anisidine, viscosinamide and paraquat, and nitrogens and even heat shock. A combination of Cu and pHB (p-hydroxybenzoic acid) made it possible to extend the inducible laccase activities over 30-fold. But the most effective inducer of laccase in the basidiomycete and other higher fungi is 2,5-xylidine, over 160-fold stimulation of laccase activity. The laccases are frequently encoded by gene families, as e.g. in Pycnoporus cinnabarinus, from which the lcc3-1 or the allelic form lac1 and lac3-2 have been cloned and sequenced. In the case of inducible forms the post-inductional laccase formation depends upon the synthesis of mRNA and the induction is due to the synthesis of a new protein.

The possibility to apply of higher fungi, mushroom, as a potential food and folk medicine sources is reviewed . The total world production of the cultivated edible mushroom has been increased dramatically since the end of the World War II. Some scientist has predicted that worldwide mushroom production will continue to increase at an average annual rate of 8% to the early of 21 century. Much interest has been aroused recently in the possibility of growing microorganism on a large scale to provide a cheap source of protein, food and folk medicine for antitumors and adult diseases. Large-scale culture for mushroom mycelium in submerged liq uid culture has been realized . Despite the small contribution in terms of food and protein resources, mushrooms will remain a valuable food for many people. Not only does the mush room have a unique flavor, its protein value lies somewhere between that of meat and vegeta bles. In addition mushrooms rank highly for their vitamin content and make an important con tribution to the supply of some minerals in the diet. Lately it could not be negligible to have research the effect of the mushroom and its culture filtrates on cancer, antitumor and the other adult diseases and the possibility as very important folk medicines. In order to produce high value-added mushrooms, its fungal mycelia, and its use of food processing, certain technical difficulties in their productions could be overcome.

Laccase enzymes from Cerrena unicolor and Trametes versicolor were immobilized on the activated glass beads (CPG), silica gel (SG) and soil (SL). The heterogeneous matrices were activated by -aminopropyltriethoxysilane (APTES) and glutaraldehyde (GA), and their surfaces were coated by keratin (KER) on activated or non-activated CPG, SG and SL. The laccase activities were tested in the aqueous solution for the native and immobilized preparations using different pH and temperature conditions. By keratin coating on supports, in the cases of CPG-KER and SL-KER, the immobilization yield was increased from about 80% to 90%. Moreover, much less protein was immobilized in keratin coated matrices than in inorganic ones alone (e.g. on CPG-KER 57.6%, whereas on CPG alone 80.6%). Laccase immobilization on keratin coated inorganic matrices was generally more effective than that of non-coated matrices. Concerned to pH dependency, the optima pH for immobilized laccases generally shifted towards to higher values, 5.5-5.8 and even 5.9 in the case of keratin for C. unicolor and from 5.3 to 5.7 for T. versicolor, respectively, and decreased less gradually both in acidic and alkaline regions. The immobilized laccase was more stable against thermal denaturation. This seems particularly true at in the case of C. unicolor, where the activity of immobilized enzyme is > 50% higher than that of the free enzyme. For T. versicolor the respective values were , and 50%.

There are some evidence that active enzymatic proteins, e.g. fungal laccase, exist in the naturally occured soil humus. This study was performed to investigate the covalent binding of fungal laccase to the humic acid-iron complex, and to measure laccase activity of immobilized ones. Seven methods were adopted to form the covalent binding of fungal laccase with soil humic acids complexed with iron. Using these seven methods it was possible to change the dimension of spacer arm between laccase and support, and also to regulate the mode of covalent binding of this enzyme. The spacer arm was regulated from 2C to 11C. There was not observed any straight relationship between the spacer arm longitude and the laccase activity after immobilization, but the binding mode more effective than the former. Three out of the seven methods gave the high activity of immobilized laccase, and which active products of laccase immobilization was stable up to 10 days after the process. It is indicated that natural soil condition might be prevented the laccase activation by the toxic influence of some phenolic humic compounds. It was shown, for the first time, the possibilities to obtain the high activity of fungal laccase by binding to humic acids, and especially in complex with iron.

Highly purified Cerrena unicolor laccase (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) caused the demethoxylation of milled wood lignin and several lignin related substances. The constitutive form of the enzyme produced extracellularly by C. unicolor fermenter culture was isolated and purified by ion-exchange chromatography on the DEAE-Toyopearl column and by affinity chromatography on a ConA-Sepharose and Syringyl-AH-Sepharose 4B columns. The enzyme was further immobilized on functionalized porous glass (CPG) and keratin coated CPG. The demethylating activity was monitored both by estimation of released methanol and by detection of the level of methoxyl groups (also in some water miscible solvents) after incubation of lignin materials with laccase preparations (free and immobilized). The effects of the incubation time and temperature on the demethoxylating activity of immobilized laccase preparations were also studied.