A. V. Lisov

Two laccase isoforms of the basidiomycete Cerrena unicolor VKMF-3196. Induction, isolation and properties.

The laccase induction in submerged culture of basidiomycete Cerrena unicolor VKM F‐3196 was investigated. Cu2+ at concentration 0.1 mM was an optimum inducer of C. unicolor laccase. Two isoforms of laccase, namely LacC1 and LacC2, were isolated and characterized. The isoforms were shown to have different physical‐chemical and catalytic properties. On the basis of the MALDI TOF MS analysis of tryptic cleavage products of both the proteins and N‐terminal amino‐acid sequences analysis two isoforms of laccase (LacC1 and LacC2) were classified as products of two different genes. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Hybrid Mn-peroxidases from basidiomycetes

The Mn-peroxidase from the fungus Panus tigrinus 8/18 is a hybrid enzyme. It catalyzes both Mn2+-dependent and Mn2+-independent oxidation of organic substrates. The spectral properties of intermediates and the pathway of the catalytic cycle are typical of hybrid Mn-peroxidases. The enzyme catalyzes the “oxidase” reaction (NADH oxidation) without peroxide and with the presence of Mn2+, which takes part in hydrogen peroxide production via Mn3+ and preserves the enzyme from inactivation. With the presence of organic mediators, the hybrid Mn-peroxidase oxidizes nonphenolic compounds: aromatic alcohols and a nonphenolic lignin model compound. The degree of conversion of 2,4,6-trichlorophenol is higher with the presence of 1-hydroxybenzotriazole.

Xylanase and cellulase of fungus Cerrena unicolor VKM F-3196: Production, properties, and applications for the saccharification of plant material

Under the conditions of submerged cultivation in a medium containing microcrystalline cellulose, the Cerrena unicolor VKM F-3196 basidiomycete is capable of producing xylanase and cellulase. Electrophoretically homogeneous cellulase and xylanase were obtained using ion exchange and hydrophobic chromatography. The molecular weight of both cellulase and xylanase was ∼44 kDa. It was shown that xylanase catalyzed the hydrolysis of xylan with the production of xylose, xylobiose, and xylotetrose and it exhibited properties of endoxylanases. Cellulase hydrolyzed carboxymethylcellulose, xylan, and microcrystalline cellulose with the formation of cellotriose and cellotetraose. For both enzymes, the pH optimum was ∼4.0. The enzymes exhibited moderate thermostability: xylanase retained 35% of the initial activity for 1 h at 60°C; cellulase, 10% under the same conditions. Xylanase, cellulose, and a mixture of these enzymes saccharified plant material (wheat, rye, wheat middling, and oat), indicating the possible use of these enzymes in biotechnology.

[Conversion of polychlorophenols by laccases with 1-hydroxybenzotriazole as a mediator].

The action of purified laccase from the basidial fungi Cerrena unicolor and Trametes sp. on 2,4,6-trichlorophenol (2,4,6-TCP) and pentachlorophenol (PCP) was studied, including reactions involving 1-hydroxybenzotriazole as a mediator. Oxidation of 2,4,6-TCP by laccase without the mediator yielded 2,6-dichlorobenzoquinone as a primary conversion product, whereas PCP was not oxidized. Products of further conversion of 2,4,6-TCP and PCP formed with the presence of the mediator.

A 2,5-Dihydroxybenzoic Acid-Gelatin Conjugate: The Synthesis, Antiviral Activity and Mechanism of Antiviral Action Against Two Alphaherpesviruses.

Various natural and synthetic polyanionic polymers with different chemical structures are known to exhibit potent antiviral activity in vitro toward a variety of enveloped viruses and may be considered as promising therapeutic agents. A water-soluble conjugate of 2,5-dihydroxybezoic acid (2,5-DHBA) with gelatin was synthesized by laccase-catalyzed oxidation of 2,5-DHBA in the presence of gelatin, and its antiviral activity against pseudorabies virus (PRV) and bovine herpesvirus type 1 (BoHV-1), two members of the Alphaherpesvirinae subfamily, was studied. The conjugate produced no direct cytotoxic effect on cells, and did not inhibit cell growth at concentrations up to 1000 µg/mL. It exhibited potent antiviral activity against PRV (IC50, 1.5–15 µg/mL for different virus strains) and BoHV-1 (IC50, 0.5–0.7 µg/mL). When present during virus adsorption, the conjugate strongly inhibited the attachment of PRV and BoHV-1 to cells. The 2,5-DHBA–gelatin conjugate had no direct virucidal effect on the viruses and did not influence their penetration into cells, cell-to-cell spread, production of infectious virus particles in cells, and expression of PRV glycoproteins E and B. The results indicated that the 2,5-DHBA–gelatin conjugate strongly inhibits the adsorption of alphaherpesviruses to cells and can be a promising synthetic polymer for the development of antiviral formulations against alphaherpesvirus infections.

Oxidase reaction of the hybrid Mn-peroxidase of the fungus Panus tigrinus 8/18

The hybrid Mn-peroxidase of the fungus Panus tigrinus 8/18 oxidized NADH in the absence of hydrogen peroxide, this being accompanied by the consumption of oxygen. The reaction of NADH oxidation started after a period of induction and completely depended on the presence of Mn(II). The reaction was inhibited in the presence of catalase and super-oxide dismutase. Oxidation of NADH by the enzyme or by manganese(III)acetate was accompanied by the production of hydrogen peroxide and superoxide radicals. In the presence of NADH, the enzyme was transformed into a catalytically inactive oxidized form (compound III), and the latter was inactivated with bleaching of the heme. The substrate of the hybrid Mn-peroxidase (Mn(II)) reduced compound III to yield the native form of the enzyme and prevented its inactivation. It is assumed that the hybrid Mn-peroxidase used the formed hydrogen peroxide in the usual peroxidase reaction to produce Mn(III), which was involved in the formation of hydrogen peroxide and thus accelerated the peroxidase reaction. The reaction of NADH oxidation is a peroxidase reaction and the consumption of oxygen is due to its interaction with the products of NADH oxidation. The role of Mn(II) in the oxidation of NADH consisted in the production of hydrogen peroxide and the protection of the enzyme from inactivation.

Xylanases of Cellulomonas flavigena : expression, biochemical characterization, and biotechnological potential

Four xylanases of Cellulomonas flavigena were cloned, expressed in Escherichia coli and purified. Three enzymes (CFXyl1, CFXyl2, and CFXyl4) were from the GH10 family, while CFXyl3 was from the GH11 family. The enzymes possessed moderate temperature stability and a neutral pH optimum. The enzymes were more stable at alkaline pH values. CFXyl1 and CFXyl2 hydrolyzed xylan to form xylobiose, xylotriose, xylohexaose, xylopentaose, and xylose, which is typical for GH10. CFXyl3 (GH11) and CFXyl4 (GH10) formed the same xylooligosaccharides, but xylose was formed in small amounts. The xylanases made efficient saccharification of rye, wheat and oat, common components of animal feed, which indicates their high biotechnological potential.

Crystallization and X-ray diffraction studies of a two-domain laccase from Streptomyces griseoflavus.

Laccase (EC 1.10.3.2) is one of the most common copper-containing oxidases; it is found in many organisms and catalyzes the oxidation of primarily phenolic compounds by oxygen. Two-domain laccases have unusual thermostability, resistance to inhibitors and an alkaline optimum of activity. The causes of these properties in two-domain laccases are poorly understood. A recombinant two-domain laccase (SgfSL) was cloned from the genome of Streptomyces griseoflavus Ac-993, expressed in Escherichia coli and purified to homogeneity. The crystals of SgfSL belonged to the monoclinic space group P21, with unit-cell parameters a = 74.64, b = 94.72, c = 117.40 Å, β = 90.672°, and diffraction data were collected to 2.0 Å resolution using a synchrotron-radiation source. Two functional trimers per asymmetric unit correspond to a Matthews coefficient of 1.99 Å(3) Da(-1) according to the monomer molecular weight of 35.6 kDa.

Transformation of cellobiose during the interaction of cellobiose dehydrogenase and β-glucosidase of Cerrena unicolor

This work investigated the regulatory role of the interaction between cellobiose dehydrogenase (CDH) and β‐glucosidase (β‐GLU) in the conversion of cellobiose into cellobionolactone or glucose in vitro. To study the regulation, the two enzymes were isolated from the culture medium of the fungus Cerrena unicolor grown on a medium with microcrystalline cellulose. The enzymes were obtained in an electrophoretically homogeneous state. Their properties were studied. Both enzymes had acidic pH optima and were more stable in the acidic pH range. CDH was moderately thermostable, while β‐GLU had a low thermostability. Both enzymes efficiently catalyzed the transformation of cellobiose. A mixture of CDH and β‐GLU transformed cellobiose to glucose or cellobionolactone in the presence of various concentrations of laccase and hydroquinone. Formation of glucose and cellobionolactone in vitro during the competition between CDH and β‐GLU for cellobiose depended on the availability of quinones, formed as a result of the interaction of laccase and hydroquinone, for CDH. At low laccase and hydroquinone concentrations, the formation of glucose was found to predominate over that of cellobionolactone. The possible physiological role of the enzymes’ interaction is discussed.