Jolanta Luterek

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.

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.

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%.

Immobilization of protocatechuate 3,4-dioxygenase from Pleurotus ostreatus on activated porous glass beads

Abstract Protocatechuate 3,4-dioxygenase (protocatechuate: oxygen 3,4-oxidoreductase, EC 1.13.11.3) purified from the wood-degrading fungus Pleurotus ostreatus was immobilized on glass beads. The per cent attached protein was 78 with a retained enzymatic activity of 90%. The basic properties of the immobilized enzyme were tested and compared with those of a soluble form. Immobilized dioxygenase was stable over wide ranges of pH and temperature. Moreover, the immobilization brought about a slight increase of its specific activity.

Higher fungi as a potential feed and food source from lignocellulosic wastes.

Publisher Summary This chapter describes higher fungi as a potential feed and food source from lignocellulosic wastes. The idea of feed or food biomass production by the submerged culture in the agitated and aerated baffle tanks results from the experiments carried out on penicillin and other antibiotic fermentation processes. In this case, low-cost materials might be used as substrates for fungal mycelium production, and at the same time, the reduction of the biological oxygen requirement by waste by products to an acceptable level could be achieved. It results from the fact that higher fungi are equipped with efficient enzymatic apparatus, which can attack the substrates not acceptable for yeast and bacteria. Solid-stationary or submerged and agitated cultures have been carried to obtain either biomass or some nutritive substances, such as vitamins, amino acids or monosaccharides. The efficiency and quality of biomass depends on the fungal species and methods of culture growth. There are two known kinds of cultures: shallow stationary and submerged, mixed and aerated in the liquid phase.

The effect of fungal protocatechuate 3,4-dioxygenase on sodium lignosulphonate fractions

Abstract Protocatechuate 3,4-dioxygenase (protocatechuate: oxygen 3,4-oxidoreductase, EC 1.13.11.3) from the white-rot fungus Pleurotus ostreatus was incubated with two fractions of sodium lignosulphonate (NaLS) obtained from sulphite waste liquor. By means of gel-permeation chromatography on Sephadex G-50 and UV sepctral analysis, it was shown that there was a decrease in the aromatic structures of the NaLS fractions. Oxygen uptake studies confirmed that the observed transformations of the NaLS frasctions was an enzyme catalysed process. The same method revealed differences in the affinity of the dioxygenase for the NaLS fractions.