Alexey Chernykh

New efficient producers of fungal laccases

Two promising strains of laccase producers—Lentinus strigosus 1566 and Steccherinum ochraceum 1833—were found by screening of basidiomycetes. The cultivation conditions increasing the enzyme yield were selected. The maximal laccase activity was observed in the case of submerged cultivation of the mycelium immobilized on polycaproamide fibers in rich media in the presence of 2 mM CuSO4 in combination with the optimal inducer, namely, 2,6-dimethylphenol for L. strigosus and 2,4-dimethylphenol for S. ochraceum. Under these conditions, the activity of S. ochraceum laccase amounted to 33.1 U/ml and that of L. strigosus, to 186.5 U/ml. Anthracene was transformed with S. ochraceum laccase, and its oxidation to anthraquinone was demonstrated by mass spectrometry.

Reaction intermediates and redox state changes in a blue laccase from Steccherinum ochraceum observed by crystallographic high/low X-ray dose experiments.

The crystal structure of a blue laccase from Steccherinum ochraceum has been solved at 2.0Å of resolution using a classic data acquisition from a single crystal. The overall structural features are typical of this class of enzymes, however, distances inside the trinuclear copper cluster are indicative of a reduction of the metal centers induced by free electrons produced during the X-ray data collection. UV-visible spectra collected during the X-ray exposure support the progressive reduction of the metal centers. In order to better detect the reduction progression steps in the trinuclear copper site, a multicrystal data collection strategy based on a systematic spread of the X-ray dose over many crystals has been employed. This approach is based on collecting multicrystal data sets, then combining the slices of the individual data sets experiencing the same radiation dose to obtain composite complete data sets at progressively higher doses. Applying this technique, we have been able to capture sequential frames of the enzyme during the metal centers and molecular oxygen reduction mechanism obtaining a three-dimensional movie of the X-ray-driven catalytic conversion of the molecular oxygen in the active site of laccase: first, the copper ions reduction, then the molecular oxygen binding and its reductive splitting, thus allowing to reconstruct the entire catalytic cycle for multicopper oxidases.

Selective regulation of laccase isoform production by the Lentinus strigosus 1566 fungus

The effects of a number of culture medium components, such as peptone, yeast extract, mono- and disaccharides, copper ions, 2,6-dimethylphenol, and polycaproamide fiber, on the laccase activity dynamics in the culture liquid and laccase isoform production by the Lentinus strigosus 1566 fungus were studied. It was demonstrated that some saccharides selectively induced or inhibited the synthesis of different laccase isoforms. Similar action was exerted by copper ions, 2,6-dimethylphenol, and polycaproamide fiber, as well as by their combination. Selective in vivo regulation of the production of certain laccase isoforms by basidial fungi by means of altering the culturing medium composition can be utilised for various biotechnological purposes.

Myrothecium verrucaria F-3851, a producer of laccases transforming phenolic compounds at neutral and alkaline conditions

The conditions of submerged cultivation of the ascomycete Myrothecium verrucaria strain F-3851 were optimized in order to increase the yield of laccase in the culture liquid using the natural sources of carbon and energy (fresh rubbed potato tuber or floured grains of buckwheat, barley, oat, wheat, rye, rice, pea, or haricot). The pH-optima of oxidation of a number of laccase substrates (ABTS, 2,6-dimethoxyphenol, syringaldazine, ferulic acid, p-coumaryl alcohol, and coniferyl alcohol) by laccases of the culture liquid as well as substrate selectivity of laccases were investigated. The intermediates of transformation of phenylpropanoids (ferulic acid, p-coumaryl alcohol and coniferyl alcohol) by laccases of the culture liquid at neutral conditions were purified and identified. The ability of laccases of the culture liquid of M. verrucaria strain F-3851 to catalyze polymer compound formation during phenylpropanoid transformation was shown that offers the prospects of application of the laccases of M. verrucaria strain F-3851 for production of pharmacologically valuable polymers in a number of cellular biotechnologies carried out in neutral or alkaline environments.

Novel laccase—producing ascomycetes

Screening of ascomycetes producing laccases during growth on agar medium or submerged cultivation in the presence of various natural sources of carbon and energy (grain crops and potato) was carried out. The conditions of submerged cultivation of the most active strains (Myrothecium roridum VKM F-3565, Stachybotrys cylindrospora VKM F-3049, and Ulocladium atrum VKM F-4302) were optimized for the purpose of increasing laccase activity. The pH-optima and substrate selectivity of laccases in the culture liquid of the strains in relation to ABTS and phenolic compounds (2,6-dimethoxyphenol, syringaldazine, ferulic acid, p-coumaryl alcohol, and coniferyl alcohol) were investigated. High laccase activity at neutral pH was shown for the culture liquids of M. roridum VKM F-3565 and S. cylindrospora VKM F-3049 strains that provides prospects for using laccases of these strains in various cell biotechnologies.

Investigating the Role of Conformational Effects on Laccase Stability and Hyperactivation under Stress Conditions

Fungal laccase from Steccherinum ochraceum 1833 displays remarkable stability under different harsh conditions: organic/buffer mixtures, thermal treatment, and microwave radiation. The behavior is particularly significant in the light of the sharp inactivation observed for two different fungal laccases. Laccase from S. ochraceum 1833 also displays hyperactivation under mild thermal treatment (60 °C). Molecular dynamics simulations at 80 °C explained how this laccase retains the geometry of the electron transfer pathway, thereby assuring electron transfer through the copper ions and thus maintaining its catalytic activity at high temperature. Spectroscopic studies revealed that the thermal activation corresponds to specific conformational changes in the protein. The results indicate that this laccase is potentially applicable under denaturing conditions that might be beneficial for the biotransformation of recalcitrant substrates.

Structural basis for the substrate specificity and the absence of dehalogenation activity in 2-chloromuconate cycloisomerase from Rhodococcus opacus 1CP

2-Chloromuconate cycloisomerase from the Gram-positive bacterium Rhodococcus opacus 1CP (Rho-2-CMCI) is an enzyme of a modified ortho-pathway, in which 2-chlorophenol is degraded using 3-chlorocatechol as the central intermediate. In general, the chloromuconate cycloisomerases catalyze not only the cycloisomerization, but also the process of dehalogenation of the chloromuconate to dienelactone. However Rho-2-CMCI, unlike the homologous enzymes from the Gram-negative bacteria, is very specific for only one position of the chloride on the substrate chloromuconate. Furthermore, Rho-2-CMCI is not able to dehalogenate the 5-chloromuconolactone and therefore it cannot generate the dienelactone. The crystallographic structure of the homooctameric Rho-2-CMCI was solved by molecular replacement using the coordinates of the structure of chloromuconate cycloisomerase from Pseudomonas putida PRS2000. The structure was analyzed and compared to the other already known structures of (chloro)muconate cycloisomerases. In addition to this, molecular docking calculations were carried out, which allowed us to determine the residues responsible for the high substrate specificity and the lack of dehalogenation activity of Rho-2-CMCI. Our studies highlight that a histidine, located in a loop that closes the active site cavity upon the binding of the substrate, could be related to the dehalogenation inability of Rho-2-CMCI and in general of the muconate cycloisomerases.