Andrzej Leonowicz

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.

Fungi and their ability to decolourize azo and anthraquinonic dyes

Abstract One hundred and fifteen fungi from different physio-ecological groups were compared for their capacity to decolourize two structurally different dyes in agar plates. We found that the azo dye, Acid Red 183, was much more resistant to decolourization by the examined strains in both solid and liquid cultures. Among the tested fungi, 69 strains showed decolourization of the anthraquinonic dye, Basic Blue 22, within 5–14 days, and only 16 strains were able to decolourize the azo dye, Acid Red 183, within 21 days. Furthermore, the potential of selected strains for decolourization of dyes was examined with regard to their extracellular oxidative factors both enzymatic and non-enzymatic. In static aqueous culture, the three selected fungi ( Bjerkandera fumosa , Kuehneromyces mutabilis , and Stropharia rugoso-annulata ) formed fungal mats, which did not decolourize any dye beyond some mycelial sorption. In comparison to the static cultures, the agitated cultures (180 rpm) removed 75 to 100% of the colour of Basic Blue 22 and 20 to 100% of Acid Red 183 colour.

Immobilization of laccase from Cerrena unicolor on controlled porosity glass

Abstract White-rot basidiomycete Cerrena unicolor grown in non-induced and induced conditions was tested for production of laccase, lignin peroxidase (LiP) and manganese-dependent peroxidase (MnP). A typical correlation between the concentration of phenolic compounds in the culture fluid and the extracellular laccase activity was observed. The heterogeneous crude laccase preparation obtained after the non-induced fermentor cultivation was immobilized both on controlled porosity glass (CPG) activated by γ-aminopropyltriethoxysilane (APTES) and on CPG with its surface covered by dextran layers. The laccase activities were tested in the aqueous solution for the native and immobilized preparations using different pH and temperature conditions. Laccase activities were additionally examined for native and immobilized forms of laccase preparations in the aqueous solution containing organic solvents. The greatest activity toward the substrate used in the presence of organic solvents was shown by the laccase preparation coupled with the CPG covered by a dextran layer. Potential inhibitors such as thioglycolic acid, thiourea and EDTA used in 1-mM concentration did not show inhibiting properties towards the laccase preparations.

The effect of fungal laccase on fractionated lignosulphonates (peritan Na)

Abstract Two fractions obtained after chromatography of lignosulphonates on Sephadex G-50, varying in M r s, were treated with extracellular Trametes versicolor laccase. After incubation of the low M r fraction, polymerization was observed, while in the case of the high M r fraction the reverse process occurred. As a result of depolymerization, five new lower M r fractions appeared. The reaction reached peak level after 2 hr of incubation and then the quantities of the products diminished, possibly due to their repolymerization. These studies indicate that laccase possesses both polymerization and depolymerization activity though the former was predominant.

Screening and mutagenesis of moulds for the improvement of glucose oxidase production

Abstract The strain of Aspergillus niger G most effective for producing glucose oxidase (see β- d -glucose:oxygen 1-oxidoreductase, EC 1.1.3.4) was selected out of 110 moulds belonging to 15 different species by the method of test-tube microculture. Conidia of the selected strain were further subjected to mutagenesis with both u.v. and N -methyl- N ′-nitro- N -nitrosoguanidine (NTG) and the products were analysed for glucose oxidase activity with our own diffusion plate method. Among 960 strains isolated after mutagenesis only 12 showed higher activity (from 1.5 to 18%) than the starting strain A. niger G.

Immobilization of laccase from Phlebia radiata on controlled porosity glass

Abstract Laccase from the white-rot fungus Phlebia radiata was immobilized on glass beads which were activated by γ-aminopropyl-triethoxysilane. 98% of the protein and 96% of the laccase activity were coupled to the support. The final preparation contained ca. 1 mg of protein per gram of glass beads. The activity of the immobilized enzyme retained after two weeks preservation at +4°C was 100% and at +25°C over 90%. The activity in the presence of organic solvents was rather similar irrespective of the form of the enzyme, free or bound. However, the catalytic activity of the immobilized laccase was less vulnerable against inhibitors such as Cu-chelators and 2,6-dimethoxy-1,4-benzoquinone.

Cooperation between fungal laccase and glucose oxidase in the degradation of lignin derivatives

Abstract In the presence of Trametes versicolor laccase, generation of quinonoid intermediates formed from a high-molecular-weight fraction of lignosulphonates (Peritan Na) was observed. The addition of glucose oxidase caused a diminution of the quinone level; thioglycolic acid intensified this process. When both laccase and glucose oxidase were incubated with the high-molecular-weight fraction, depolymerization was more extensive than in the experiment omitting glucose oxidase. In the case of the low-molecular-weight fraction, these two enzymes operated in concert and the polymerization process was disturbed due to glucose oxidase activity. Therefore the action of glucose oxidase in reducing quinones improved the efficiency of lignin depolymerization.

Influence of aromatic compounds and lignin on production of ligninolytic enzymes by Phlebia radiata

Abstract Toxic aromatic compounds and lignin preparations stimulated the production of laccase, lignin peroxidase and manganese-dependent peroxidase when supplemented in non-agitated cultures of the white-rot fungus Phlebia radiata . Low amounts (0.02 mM) of xylidine stimulated the production of laccase, whereas a higher amount (1 mM) increased lignin peroxidase production. Low amounts of benzyl alcohol increased the production of lignin peroxidase. Lignosulphonate and synthetic lignin (DHP) preferably stimulated laccase production. In all cases lignin peroxidase activities were only one-tenth to one-twentieth of those which usually were obtained by adding monomeric ligninrelated aromatic compounds, such as veratryl alcohol, into the cultivation medium of the fungus.

Immobilization of cellulase and d-xylanase complexes from Aspergillus terreus F-413 on controlled porosity glasses

The major components of cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] and d-xylanase (see 1,4-β-d-xylan xylanohydrolase, EC 3.2.1.8) complexes have been immobilized on glass beads activated by 3-aminopropyltriethoxysilane or 3-glycidoxypropyltrimethoxysilane. The final preparations contained over 20 mg protein g−1 glass beads. The activity retained was 71.6–98.1% for cellulase complexes and 81–100% for d-xylanase complexes. The immobilization of the enzymes spread their optimum pH range. Cellulose and d-xylan were quantitatively hydrolysed by the immobilized enzymes. The major reaction products were identified as a d-glucose and d-xylose respectively.

Uptake of cadmium ions in white-rot fungus Trametes versicolor: effect of cd (II) ions on the activity of laccase.

The effects of cadmium Cd (II) ions on the physiology and biological activity of Trametes versicolor, a strain belonging to white‐rotting Basidiomycetes, were examined. Cd (II) ions were added to 10‐day‐old cultures grown on a liquid medium, or at the time of inoculation. Our experiments showed that T. versicolor is a good cadmium biosorbent from aqueous solution, this strain removing almost all the Cd (ll) ions over the first 2h of incubation by what appears to be a rapid, energy‐independent surface binding phenomenon, at the rate of ∼2mg Cd per g mycelial dry weight. An additional slower and energy‐dependent transport mechanism was also present, taking in ∼0.3mg Cd (II) perg dry weight. It is also shown that these Cd (II) ions significantly stimulate the activity of extracellular laccase when added to 10‐day‐old cultures.