Jerzy Rogalski

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.

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.

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.

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.

Hybrid biobattery based on arylated carbon nanotubes and laccase.

Single-walled carbon nanotubes (SWCNT) were covalently modified with anthracene and anthraquinone and used for the construction of cathodes for biocatalytic reduction of dioxygen. The nanotubes with aromatic groups casted onto the electrode increased the working surface of the electrode and enabled efficient direct electron transfer (DET) between the enzyme and the electrode. The aryl groups enter the hydrophobic pocket of the T1 center of laccase responsible for exchanging electrons with the substrate. Glassy carbon electrode covered with arylated SWCNT and coated with a layer of neutralized Nafion containing laccase was found to be a very efficient cathode in the hybrid battery. Zn wire covered with a Nafion film served as the anode. The cell parameters were determined: power density was 2 mW/cm(2) and the open circuit potential was 1.5 V.

Thermoresponsive poly(N-isopropylacrylamide) gel for immobilization of laccase on indium tin oxide electrodes.

We report on the properties of hydrogel matrix for the immobilization of laccase on conductive supports. The poly(N-isopropylacrylamide) gel is attached firmly to the indium-tin oxide (ITO) electrode, following its silanization with dimethylethoxyvinylsilane. The enzyme entrapped in the gel structure remained active longer than in the solution, and its redox and catalytic properties could be investigated by voltammetric methods. The reduction signals of the active sites, T1 and T2, of the Cerrena unicolor laccase were determined to be 0.79 and 0.38 V, respectively. The laccase catalytic activity toward oxygen in poly(N-isopropylacrylamide) was found to depend strongly on temperature. Reversible swelling/shrinking of the matrix was studied at 30 and 35 degrees C. Shrinking of the gel at higher temperature considerably decreased the efficiency of the catalytic reaction, however, interestingly, did not lead to irreversible changes in the enzyme structure. At temperatures below that corresponding to volume phase transition, the catalytic properties of the film were fully restored. High catalytic efficiency of the gel immobilized enzyme made it possible to employ the gel covered electrode for monitoring oxygen in solutions.

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.

Optimization of glucose oxidase synthesis in submerged cultures of Aspergillus niger G-13 mutant

Both shaken flask cultures and aerated fermenter cultures of the previously selected Aspergillus niger G-13 mutant were optimized for glucose oxidase production (β-d-glucose: oxygen 1-oxidoreductase, EC 1.1.3.4). The enzyme synthesis was strongly influenced by glucose and calcium carbonate. Optimum results were obtained after about 36 h of fermenter culture when the concentrations of both ingredients were respectively 8 and 3.5%. The crude enzyme preparate obtained by gel filtration Sephadex G-25 column and lyophilization was highly specific for glucose and 2-deoxyglucose, but the latter substrate was oxidized about three times more slowly. The heat stability and pH sensitivity of the enzyme were also studied.