Nam-Seok Cho

Fungal laccase: properties and activity on lignin

The sources of ligninocellulose that occur in various forms in nature are so vast that they can only be compared to those of water. The results of several, more recent experiments showed that laccase probably possesses the big ability for “lignin‐barrier” breakdown of ligninocellulose. The degradation of this compound is currently understood as an enzymatic process mediated by small molecules, therefore, this review will focus on the role of these mediators and radicals working in concert with enzymes. The fungi having a versatile machinery of enzymes are able to attack directly the “lignin‐barrier” or can use a multienzyme system including “feed‐back” type enzymes allowing for simultaneous transformation of lignin and carbohydrate compounds.

Activity of free and immobilized extracellular Cerrena unicolor laccase in water miscible organic solvents

The extracellular laccase of white rot fungus Cerrena unicolor was purified from culture by ion-exchange chromatography on DEAE-Toyopearl column and immobilized on silanized porous glass heads. During bonding procedure 94.35% protein and 100 % laccase activity were coupled to the support. Optimum pH for immobilized laccase compared to free enzyme was shifted from 5.5 to 5.7. The immobilized laccase was more resistant for thermal denaturation: at 70°C, the activity of immobilized enzyme was around 20 % higher than that of the free enzyme. It was also more stable during storage at 4°C. After about seven months the immobilized laccase retained 94.81 % of its initial activity, whereas free enzyme only 39.59 %. Nine water-miscible organic solvents tested in an anhydrous form caused an inhibitory effect on both laccase forms, but, in the mixture with water, the enzyme in all cases shows activity. It decreased, however, when the concentration of solvents increased. Among all solvents, the best results were obtained with ethylene glycol and methoxyethanol. When the reaction mixture contained 50 % of ethylene glycol, free and immobilized laccase retained 24 and 31 % respectively of their activity in the buffer. In the case of 50 % methoxyethanol these data were 6 and 36 %. Organic solvents shifted pH optima of both laccase forms to pH 6.0. The calculation from Lineweaver-Burk plot oxidizing capacity of laccase towards syringaldazine was slightly higher for the free enzyme than for immobilized one, 18.5 and 20.0 M/l respectively. These results show that Cerrena unicolor laccase both in free and immobilized form is able to catalyze the oxidation of syringaldazine in organic solvents. It may be usable in transformation of substrates insoluble or sparingly soluble in water. The immobilized laccase, as more stable and temperature resistant than free enzyme, seems to be more useful.

Removal of Hexavalent Chromium from Dilute Aqueous Solution by Coniferous Leaves

Summary The ability of coniferous leaves to remove Cr(VI) from dilute aqueous solutions by physico-chemical adsorption and chemical reduction was investigated. Of 30 coniferous tree species tested, relatively high removal ability was found in Ginkgo, Larix, Pinus, Cryptomeria, Metasequoia, Taxodium, Thuja and Thujopsis spp. To search for optimum operation condition for treatment of wastewater containing Cr(VI), factors affecting Cr(VI) removal were studied using P. densiflora leaves. The extent of the removal was dependent upon the initial solution pH, contact time, temperature and initial concentration of Cr(VI) in solution. The Cr(VI) removal was mainly governed by the physico-chemical adsorption onto the substrate. The adsorption data were well fitted to the Freundlich isotherm.

Adsorption of trivalent chromium from dilute solution by conifer leaves.

Summary Chromium(III), Cr(III) adsorption capacities of the leaves of 34 conifer species were examined. Among these, Ginkgo biloba, Taxus cuspidata, Cephalotaxus harringtonia var. nana, and Taxodiaceae and Cupressaceae spp. showed large capacities to adsorb Cr(III). The adsorption capacities of conifer leaves for Cr(III) (3.12–5.09 mg Cr g−1 adsorbent) compared favorably with those of commercial activated carbons (1.23–2.75 mg g−1). Factors affecting Cr(III) adsorption were studied using G. biloba leaves. The factors included solution pH, contact time, temperature, and the initial concentration of Cr(III). The amount of Cr(III) adsorbed on the adsorbent increased steadily with increasing pH in a pH range from 2 to 5, with increasing contact time, and with increasing temperature ranging 20 to 40 °C. The Cr(III) adsorption was also affected by the initial concentration of Cr(III) in the solution.  A linear relationship was observed between the amount of Cr(III) adsorbed and the equilibrium concentration of Cr(III) in the solution when graphed logarithmically. The maximum capacity of G. biloba leaves was 27.5 mg Cr g−1 adsorbent by column experiments.

Cooperation of fungal laccase and glucose 1-oxidase in transformation of Björkman lignin and some phenolic compounds

Summary A screening of wood-rotting basidiomycete fungi was conducted for glucose 1-oxidase (GOD) and laccase (LAC) production as well as for ligninolytic activity measured by a Rhemazol reaction. The results showed that genera rich in GOD are lignin degraders as well as effective producers of extracellular LAC. The fungi poor in GOD neither showed LAC, nor ligninolytic activity. The Björkman lignin and 3 phenolic compounds, hydroquinone and syringic and vanillic acids, were tested on the sequential activity of LAC and GOD. In the presence of LAC, quinoid intermediates formed from Björkman lignin and phenolic compounds were observed. The addition of GOD caused a diminution of the quinone level. During incubation of Björkman lignin with LAC and GOD depolymerization occurred, and in the experiments omitting GOD the quantities of low molecular products were markedly lower. Consequently, the consecutive ping-pong activity of LAC and GOD reduced the polymerization and improved the efficiency of depolymerization processes.

Catalyzed steaming as pre-treatment for the enzymatic hydrolysis of bamboo grass culms

Culms of bamboo grass (Sasa senanensis Rehd.) were treated with saturated steam at 197°C for 10 min in the presence of various acid catalysts in order to improve enzymatic susceptibility. Catalyst levels were varied at 5, 10, 20 and 30 mmol kg−1 original material. The catalysts were Lewis acids, NH4Cl, (NH4)2SO4, acetic acid and organic acid anhydrides. The extent of enzymatic hydrolysis of steamed substrate was improved by the addition of catalyst. The acid catalysts strongly modified the steam treatment of bamboo grass culms. Overall recovery of the steamed solids decreased with increase in concentration of the catalyst. Conversion yields from 60 to 70%, based on polysaccharides present in the original material, were achieved by catalyzed steaming followed by enzymatic hydrolysis.

Transcriptional regulation of laccase and cellulase in relation to fruit body formation in the mycelium of Lentinula edodes on a sawdust-based substrate.

Extracellular enzyme activities of laccase and cellulase and their transcriptional regulation were investigated at various growth stages in a sawdust-based substrate forLentinula edodes. Changes of laccase and cellulase activities revealed a clear relationship with fruit body development stages. Laccase and cellulase activities were regulated at the level of gene transcription. The level of laccase mRNA was maximal at the fully colonized stage and declined during fruit body development. Cellulase mRNA began to accumulate at the pin (miniature fruit bodies) formation stage. Cellulase mRNA transcripts were maximally expressed at the veil-break stage of fruit body development. This tendency was clearer in the fruiting cultures with the wide-range-weather strains than in non-fruiting cold-weather strains. Transcription of laccase and cellulase genes was also affected by the water conditions of the sawdust-based substrate. Primordia initiation occurred when the water potential of the medium was high for rapid mRNA transcription by the mycelium.

Effect of coniferyl alcohol addition on removal of chlorophenols from water effluent by fungal laccase

The effect of coniferyl alcohol on removal of chlorinated phenols from a water environment byRhizoctonia praticola andCerrena unicolor laccases was studied. At optimal conditions in which 7 mM coniferyl alcohol and laccase were added to chlorinated phenols over 20h, about 34% of the radioactivity of 4-chlorophenol, 57% of 2,4-dichlorophenol, 66% of 2,4,5-trichlorophenol, and 85% of pentachlorophenol were removed from the supernatants, compared to the level without laccase activity. After 12-h incubation periods at the optimal concentrations of coniferyl alcohol and laccase (added simultaneously), the fast first phase of chlorophenol removal was complete in 1 h, and eventually coniferyl alcohol enhanced the removal of 4-chlorophenol by 40%, 2,4-dichlorophenol by 54%, 2,4,5-trichlorophenol by 60%, and pentachlorophenol by 76%.

Adsorption by coniferous leaves of chromium ions from effluent

The chromium adsorption ability of leaves from 34 conifer species were examined. Among them, deciduous conifer species, such asLarix, Ginkgo, Metasequia, andTaxodium, showed high ability to adsorb chromium ions. Factors affecting chromium adsorption were studied using larch (Larix leptolepis) leaves to determine the optimum adsorption conditions. The factors included solution pH, contact time, temperature, and the initial concentration of chromium ions. Maximum adsorption for Cr3+ was observed at pH 5, and maximum Cr6+ adsorption occurred at pH 3. The amount of Cr6+ adsorbed on the adsorbent increased rapidly during the first 4 h, then gradually increased, and finally reached equilibrium in 16 h. The adsorption rate of Cr3+ was somewhat slower than that of Cr6+. The adsorption isotherm for Cr6+ adsorption was composed of two straight lines, suggesting that the adsorbent could not practically reduce the concentration of Cr6+ in solution below 1.6 mg Cr/L Column experiments using larch leaf packing suggested that the practical operation could be controlled by monitoring the effluent pH.

Changes in phenol oxidases and superoxide dismutase during fruit-body formation of Pleurotus on sawdust culture

Abstract Peroxidase and laccase activities increased rapidly up to the formation of primordia and then declined throughout the entire stage of fruiting. In the case of Pleurotus ostreatus, the level of Mn-dependent peroxidase was very low in primordia and fruiting stages but gradually increased with the growth of the fruit-body, whereas no activity was detected in Pleurotus sajor-caju during all growth stages. Superoxide dismutase activity was observed mainly at the fruiting stages. These results show that changes in concentration of lignin-related enzymes are associated with the fruiting process.