Äîêóìåíò âçÿò èç êýøà ïîèñêîâîé ìàøèíû. Àäðåñ îðèãèíàëüíîãî äîêóìåíòà : http://www.mgumus.chem.msu.ru/publication/2013/2013-fedorova-comparative.pdf Äàòà èçìåíåíèÿ: Mon Jan 20 13:28:26 2014 Äàòà èíäåêñèðîâàíèÿ: Thu Feb 27 21:24:38 2014 Êîäèðîâêà: Ïîèñêîâûå ñëîâà: trees

ISSN 0003 6838, Applied Biochemistry and Microbiology, 2013, Vol. 49, No. 6, pp. 570­580. © Pleiades Publishing, Inc., 2013. Original Russian Text © T.V. Fedorova, N.V. Shakhova, O.I. Klein, O.A. Glazunova, L.G. Maloshenok, N.A. Kulikova, N.V. Psurtseva, O.V. Koroleva, 2013, published in Prikladnaya Biokhimiya i Mikrobiologiya, 2013, Vol. 49, No. 6, pp. 570­579.

Comparative Analysis of the Ligninolytic Potential of Basidiomycetes Belonging to Different Taxonomic and Ecological Groups
T. V. Fedorovaa, N. V. Shakhovab, O. I. Kleina, O. A. Glazunovaa, L. G. Maloshenoka, N. A. Kulikovaa, c, N. V. Psurtsevab, and O. V. Korolevaa
b

Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow, 119071 Russia e mail: fedorova_tv@mail.ru Komarov Botanical Institute, Russian Academy of Sciences, St. Petersburg, 197376 Russia c Department of Soil Science, Moscow State University, Moscow, 119991 Russia
Received June 7, 2013

a

Abstract--Screening of the ligninolytic activity of basidiomycetes from the Komarov Botanical Institute Basidio mycetes Culture Collection (LE BIN), Russian Academy of Sciences, belonging to different taxonomic and eco logical groups was performed. The patterns of the position of taxa of active producers of ligninolytic enzymes in the modern system of fungi were identified. Cluster analysis showed that the group of fungi with the greatest ligni nolytic and degradation potential includes representatives of the families Pleurotaceae, Polyporaceae, and Phan erochaetaceae, which perform the first stages of wood decomposition. As a result, species of interest for the further study of their oxidative potential and use in biotechnology were selected. DOI: 10.1134/S0003683813060082

Currently, the interest towards the bioconversion of lignocellulosic waste is steadily growing, which is determined by an increased volume of waste of the pulp and paper industry (PPI) and the woodworking industry and, as a consequence, an increased anthro pogenic impact on the environment [1]. For example, according to the Federal State Statistics Service, the total amount of waste in the Russian pulp and paper industry has increased from 2991.2 million tons in 2005 to 4303.3 million tons in 2011. At the same time, the main methods for disposing of waste in our coun try remain warehousing in landfills and incineration after dewatering and compaction. The use of biotechnological approaches for con verting the pulp and paper industry's waste is limited by its toxicity due to the presence of compounds, such as polynuclear aromatic hydrocarbons, high concen trations of xenobiotics (dioxins, chlorine derivatives, etc.), and lignin, which is one of the most difficult biopolymers to degrade. That is why basidio mycetes--the most active lignin destructors--attract the attention of researchers as the key component of the lignocellulosic waste bioconversion technology. Of all known representatives of Basidiomycota, white rot fungi are the most efficient lignin destructors [2]. The process of degradation of lignocellulosic substrates by these fungi consists of a large number of steps and depends on the presence of an enzymatic system (a unique ligninolytic complex consisting of laccase (EC 1.10.3.2) and various peroxidases, including the man ganese (EC 1.11.1.13) and lignin (EC 1.11.1.14) per oxidases) in them [3­5]. It is known that some species of white rot fungi (wood destructors) have a unique

mechanism of detoxification of both lignin degrada tion products and various xenobiotics [6, 7]. For this reason, researchers constantly work to search for, iso late, and study new strains of basidiomycetes promis ing for use in bioconversion and bioremediation tech nologies [8­10]. This study is the first step in the development of a biotechnology for the processing of solid waste from the pulp and paper industry using basidiomycetes. The goal of this study was to screen and assess the ligninolytic and degradation potential of white rot fungi from different ecological and taxonomic groups and to select the most promising strains for use in the utilization of lignin containing waste of the pulp and paper industry. MATERIALS AND METHODS Screening of cultures. More than 520 cultures of 330 species of higher (basidiomycete) fungi belonging to different taxonomic and ecological groups from the Komarov Botanical Institute Basidiomycetes Culture Collection, Russian Academy of Sciences (St. Peters burg), were used to assess the ligninolytic and degrada tion potential by rapid methods [11]. The most active strains shown in Table 1 were selected for further work. All cultures were stored in tubes on wort agar slants (wort 4%, agar 1.5%) at 4°C. Species identification. The species of fungi were verified morphologically using conventional methods with subsequent genotyping. Strains were grown on an MEA medium with 2% malt extract (Dia M, Russia) and 1.6% agar (Difco,

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COMPARATIVE ANALYSIS OF THE LIGNINOLYTIC POTENTIAL OF BASIDIOMYCETES Table 1. Characteristics of fungal strains studied Strain LE BIN 1998 Species affiliation Antrodiella fagi nea Vampola & Pouzar Taxonomic affiliation Steccheri naceae, Polyporales Geographic origin Russia, the Far East. The substrate is dry branches of deciduo us trees Species ecology

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Secondary xylotroph (typical of heavily decayed wood or remaining on it for 15 years and longer), which causes white rot. Colonizes dead twigs, large branches, and trunks of deciduous trees, more rarely shrubs, of the genera Alnus, Populus, Salix, Padus, Corylus, and Syringa. Xylotroph, which causes white rot. Lives on decom posed wood of angiosperms in serried forest commu nities and tropical mangroves. Wound primary xylotroph performing the first stage of wood decomposition (first seven years), which cau ses white rot. Lives on wood of deciduous trees, mo stly on species of the genera Betula, as well as Acer, Al nus, Carpinus, Corylus, Fagus, Fraxinus, Juglans, Pru nus, Quercus, Salix, Sorbus, Tilia, and Ulmus. Xylotroph, which causes white rot. Lives on dead and dead and down branches, as well as on litter of broad leaved trees of the genera Acer, Alnus, Carpinus, and Quercus. Primary wound xylotroph (performing the first stage of wood decomposition (first seven years), which causes mixed yellow rot. It lives on weakened and dying trees and dead branches and stumps of many deciduous and more rarely coniferous (Picea, Araucaria) tree species.

LE BIN 0677 LE BIN 2047

Polyporaceae, Cuba Coriolopsis caperata (Berk.) Polyporales Murrill Lenzites Polyporaceae, Finland, Lahti district. betulina (L.) Fr. Polyporales The substrate is a birch Betula sp. trunk in a co niferous forest

LE BIN 2009

Phanerocha Byssomerulius etaceae, Poly avellaneus (Bres.) J. Erikss. porales & Hjortstam Pleurotus ostrea tus (Jacq.) P. Kumm.

Russia, the Far East. The substrate is snag branches in an oak grove

LE BIN 0432

Pleurotaceae, Russia, Sochi Agaricales

LE BIN 2142

Peniophora lycii Peniophora (Pers.) HÆhn. & ceae, Russu lales Litsch. Steccherinum murashkinskyi (Burt) Maas Geest.

Russia, Rostov oblast. Xylotroph, which lives on dead branches of various de The substrate is creta ciduous (Acer, Fagus, Rhamnus, and Fraxinus) and oc ceous sediments in the casionally coniferous (Pinus) trees. steppe Secondary xylotroph typical of heavily decayed wood or remaining on it for 15 years and longer. Causes white rot. Lives on dead trunks and branches of va rious deciduous (Acer, Betula, Populus, Salix, and Quercus) and occasionally coniferous (Abies) trees. Xylotroph, which causes actively spreading white rot. Lives on dead (mostly large sized) wood (dead wood, stumps, snags, and treated wood) of decidu ous (usually, on Betula and, more rarely, on Alnus, Carpinus, Cerasus, Fagus, Populus, Salix, Tilia, etc.) and, occasionally, coniferous (Abies, Picea) tree spe cies. A highly competitive species able to slowly dis place many species of wood decaying fungi on the majority of substrates. Xylotroph, which develops on the roots (and possibly on the buried wood) of mostly deciduous trees (Quercus, Fagus). Fruiting bodies are usually found at the base of old heavily damaged tree stumps, which allows this spe cies to be regarded as a secondary xylotroph.

LE BIN 1963

Russia, the Far East, Steccheri naceae, Poly Kamchatka region. Found on a charred porales trunk of the birch Be tula platyphylla

LE BIN 1911

Trametes gibbosa Polyporaceae, Russia, Samara (Pers.) Fr. Polyporales oblast, Stavropol dis trict. Found on lime

LE BIN 1795

Russia, Samara oblast, Xerula radicata Marasmi (Relhan) DÆrfelt aceae, Agari Stavropol district. At the base of a heavily cales damaged stump

United States) in Petri dishes (90 mm) at 25° in the dark. The growth of the strains was characterized by the diameter of the colony on days 7 and 14 of growth. The characterization of the macromorphological and micromorphological features of the mycelium was performed for 4 week old colonies by standard meth
APPLIED BIOCHEMISTRY AND MICROBIOLOGY

ods [12, 13]. The macromorphological characteristics included descriptions of the aerial mycelium, color, smell, and reversum of the colonies. The micromor phological description included the characteristic fea tures of the hyphal system and the presence of swell ings, buckles, and anamorphic elements (asexual
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reproductive structures) on the mycelium. The teleo morph stage (sporocarp formation) in the culture was obtained on substrate blocks (birch sawdust with wheat bran, 3 : 1) in a Sanyo MLR 351H climatic chamber at 15° and 85% humidity. For genotyping the fungal strains, we performed partial (for 18S and 28S) and complete (for 5.8S) determinations of the rRNA nucleotide sequences, as well as the DNA sequences located between the genes encoding the 18S, 5.8S, and 28S rRNAs (ITS1 and ITS2 regions), using standard oligonucleotide primers to the conserved regions of the ribosomal genes: ITS1F 5' CTT GGT CAT TTA GAG GAA GTA A 3' and ITS4B 5' CAG GAG ACT TGT ACA CGG TCC AG 3 ' [14]. PCR was performed under the following conditions: 1 cycle of 5 min at 95° and then 25 cycles (1 min at 90°, 1 min at 56°, and 1 min at 72°), followed by 1 cycle of 10 min at 72°. Rapid method for assessing the total ligninolytic and laccase activities. For a qualitative assessment of the ligninolytic and laccase activity by the rapid method [11], the strains were grown in the dark at 25° in Petri dishes (90 mm) in media of the following composition: 4.0% wort and 2.0% agar (S A medium); (g/L): 0.6 KH2PO4, 0.4 K2HPO4, 0.5 MgSO4 7H2O, 0.05 CaCl2, 0.05 MnSO4, 0.001 ZnSO4, 0.0005 FeSO4, 3.0 pep tone, 10.0 g/L glucose, and 2.0 % agar (G P medium); and G P medium supplemented with 0.15 g/L CuSO4 (G P+Cu medium). For inoculation, units of a 7 day old mycelium (diameter, 5 mm) grown on an MEA medium were placed at the edge of a cup with the mycelial side fac ing down. The activity of the ligninolytic enzymes was determined after one and two weeks of growth. Myce lial blocks of 5 mm in diameter were cut near the edge of a growing colony and transferred into the wells of microbiological plates. Then, guaiacol (2 mL/100 mL 2) (Sigma, United States) and 1.0% syringaldazine (Sigma, United State) in C2H5OH were applied drop wise. The activity was evaluated visually by the color reaction intensity on a scale from "­" (no activity) to "+ + +" (the highest activity) at 5 , 15 , 30 , and 60 min and 3 and 24 h intervals. Rapid method for the evaluation of lignin peroxidase activity. A qualitative assessment of the lignin peroxi dase activity was performed by a rapid diffusion method [15]. Strains were grown in the dark at 25° in Petri dishes (90 mm) on S A, G P, and G P+Cu media supplemented with 0.02% Azure B under sterile conditions before aliquoting. For inoculation, blocks of 7 day mycelium (diameter, 5 mm) were placed on an MEA medium at the edge of a dish with the myce lial side facing down. The lignin peroxidase activity was determined after one and two weeks of growth. The activity was assessed visually by the intensity of changes in the dye color on a scale from "­" (no activ ity) to "+ + +" (the highest activity). Cultivation methods. Inocula were grown in 750 mL stationary Erlenmeyer flasks with porcelain beads on an agar free G P medium at 25° for 10­14 days depending on the growth rate of the fungus. Before

inoculation, the inoculum was crushed with porcelain beads at 180 rpm for 20 min to form a homogeneous suspension, which was added to the cultivation flasks to a final volume of 10% under sterile conditions. Submerged cultivation was performed on a shaker at 180 rpm for 20 days in 750 mL conical flasks con taining S A, G P, and G P+Cu media without agar. Sampling of the culture liquid for measuring the lac case activity was performed daily. Determination of laccase activity. The laccase activity was determined spectrophotometrically by the increase in absorbance at = 525 nm after adding 0.018 mL of 0.1% alcoholic solution of syringaldazine to the reaction mixture (1.7 mL of phosphate­citrate buffer (pH 5.0) and 0.2 mL of culture filtrate) [16]. The increase in absorbance in 1 mL of the reaction mixture for 1 min was taken as one unit of enzymatic activity. Isolation of DNA. Fungal chromosomal DNA was isolated from the mycelium on days 8­10 of surface culturing. The mycelium was washed with distilled water and then dried between layers of filter paper. The dried mycelium was ground in a mortar with liquid nitrogen to obtain a homogeneous white­yellow pow der. DNA was isolated from the ground fungal biomass using DNeasy Plant Mini Kit (50) protocols (Qiagen, United States) according to the protocol provided by the manufacturer. The purity of the samples was mon itored by agarose gel electrophoresis. The resulting chromosomal DNA samples were stored at ­20°. Isolation of total RNA. Total RNA was isolated from the ground fungal biomass using a RNeasy Plant Mini Kit (50) (Qiagen, United States) according to the protocol provided by the manufacturer, adapted for plant material and filamentous fungi. Total RNA was used as a template for the synthesis of the first cDNA strand in the reverse transcription reaction. Reverse transcription. A reverse transcription reac tion (RT) was performed using a Mint reagent kit for cDNA synthesis (Evrogen, Russia) according to the manufacturer's instructions. The first cDNA strand was amplified in a PCR reaction to obtain double stranded cDNA (ds cDNA) enriched in full length sequences. The resultant ds cDNA was used as a tem plate for PCR. PCR. The chromosomal DNA and ds cDNA of each test fungus was used as a template for PCR. PCR was performed in a reaction mixture of the following composition: 2 µL of 10 X Encyclo buffer (Evrogen, Russia), 0.4 uµL of 50 X mixture of dNTP (20 mM each) (Evrogen, Russia), 1.0 µL of forward and reverse primers (0.5 µM), 0.4 µL of 50 X Encyclo polymerase (Evrogen, Russia), 1 µL of template DNA, and 14.2 µL of sterile water. The reaction was performed in a C1000 thermocycler (Bio Rad, United States). The amplification products were separated by electro phoresis in a 1.2% agarose gel. The results were visual ized under a UV transilluminator at 260 nm. Laccase gene identification. The presence of the laccase gene was detected by PCR using the following program: 1 cycle of 5 min at 94°, then 35 cycles (15 s
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at 90°, 20 s at 56°, and 30 s at 72°), followed by 1 cycle of 10 min at 72°. The PCR fragments were amplified using the following degenerate primers: LacA1 For 5' CAYTGGCAYGGWTTYTTYCAR 3' LacA1 Rev 5' RTGRAARTCDATRTGRCARTG 3' LacA2 For 5' GGNACNTTCTGGTAYCAYAGYCAY 3' LacA2 Rev5' RTANCGNAGDATAGCVGAGTT 3'. Purification and cloning of PCR fragments. PCR products were purified using a QIAquick Gel Extrac tion Kit (50) (Qiagen, United States) according to the protocol suggested by the manufacturer. The isolated PCR fragments were cloned using the pAL TA vector for the rapid cloning of PCR products (Evrogen, Rus sia) in Escherichia coli XL1 Blue competent cells. Determination of nucleotide sequences. The nucle otide sequences of the cloned DNA fragments were determined using an ABI PRISM® BigDyeTM Termi nator v. 3.1 reagent kit with the subsequent analysis of the reaction products in an ABI PRISM 3730 auto matic DNA sequencer (Applied Biosystems, United States) at the Genome Collective Use Center (Engel hardt Institute of Molecular Biology, Russian Acad emy of Sciences). Sequence analysis. A homology search in GenBank was performed using the BLAST server (http:// www.ncbi.nlm.nih.gov/BLAST) [17]. The amino acid and nucleotide sequences were edited using the Bio Edit Sequence Alignment Editor (http://www. mbio.ncsu.edu/bioedit/bioedit.htmL). Alignment of the amino acid sequences of proteins was performed using the ClustalW program (http://www.ebi.ac.uk/clustalW). Statistical data processing. Three biological and analytical replicates were performed for all experi ments. A statistical analysis of the results was per formed using the STATISTICA 8.0 program. To com bine the fungal strains into groups, the experimental data were subjected to tree clustering. For this pur pose, the preliminary screening results were scored. To form clusters, the Euclidean distance was used as the distance between objects. The clusters were combined using the neighbor joining method (single bond). RESULTS AND DISCUSSION Screening and identification of the species affiliation of cultures. We studied the cultural and morphological characteristics of the studied strains to determine their taxonomic affiliation (Table 1). It was found that the cultural traits of strains Antrodiella faginea 1998, Coriol opsis caperata 0677, Lenzites betulina 2047, Pleurotus ostreatus 0432, Peniophora lycii 2142, Trametes gibbosa 1911, and Xerula radicata 1795 were typical of these species [12, 18]. The morphology of the vegetative mycelium of strains Byssomerulius avellaneus 2009 and Steccherinum murashkinskyi 1963 (Figs. 1a, 1b) was studied for the first time and allowed us to perform their identification, which was confirmed using the basidiomes obtained from the culture (Fig. 3a, 3b). To confirm the correctness of strain identification, we
APPLIED BIOCHEMISTRY AND MICROBIOLOGY

performed partial (for 18S and 28S) and complete (for 5.8S) sequencing of their rRNA, as well as the ITS1 and ITS2 regions (Table 2). The species affiliation of strains 1998, 0677, 2047, 0432, 2142, 1911, and 1795, determined by the morphological traits, coincided with the results of DNA typing (100% homology). In the case of strains 1963 (Steccherinum murashkinskyi) and 2009 (Byssomerulius avellaneus), the resulting sequences did not coincide completely with any of the sequences in the database. The nucleotide sequence of basidiomycete Byssomerulius avellaneus 2009 had 98% homology with that of Phlebia chrysocreas (gi no. 37622321); distinctions were observed in the ITS1 and ITS2 variable regions and in the 28S ribosome region. In the case of Steccherinum murashkinskyi 1963, the highest homology (95 and 94%, respectively) was found for Steccherinum ochraceum (accession 157057321) and Steccherinum litschaueri (gi no. 56181683), which allowed us to classify the studied strain with Steccherinaceae sp. (Taxonomy ID: 81060). The sequences of 18S rRNA, 5.8S rRNA, and 28S rRNA of the fungi Phanerochaete avellanea (syn onym Byssomerulius avellaneus) and Steccherinum murashkinskyi were added in the NCBI under the accession numbers GU062467 and FJ798705, respec tively. Thus, the group of strains studied included repre sentatives of aphyllophoroid xylotrophic fungi of the families Phanerochaetaceae (P. avellanea), Steccher inaceae (A. faginea and S. murashkinskyi), and Poly poraceae (C. caperata, L. betulina and T. gibbosa), belonging to the order Polyporales; agaricoid fungi of the families Marasmiaceae (Xerula radicata) and Pleurotaceae (P. ostreatus), belonging to the order Agaricales; and the family Peniophoraceae (P. lycii), belonging to the order Russulales. To select the most promising strains, we used a tra ditional technique of screening for ligninolytic activ ity. The total ligninolytic, laccase, and lignin peroxi dase activities were determined using guaiacol [19] syringaldazine [20] and azure B [15], respectively, as model substrates. The bleaching of polyphenol pig ments, including azure B, by basidiomycetes may also indicate the presence of peroxidase and H2O2 produc ing oxidases [8]. As was shown earlier, the ability of some members of aphyllophoroid xylotrophic fungi to bleach polymeric dyes correlates with their ability to degrade various xenobiotics, which allows the Azure B bleaching test to be used in search for fungi with a high detoxification potential [8]. The results of a qualitative assessment showed that the studied strains exhibited a high ligninolytic and laccase activity after one or two weeks of growth in all media (Table 3). The composition of the culture medium had a significant effect both on the growth of the fungi studied and on the production of extracellu lar ligninolytic enzymes by them. Natural wort (S A medium), used in the experiments, is a rich organic medium and is best suited for the cultivation of basid iomycetes. In addition, it also contains many natural organic compounds that can function as inducers of
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FEDOROVA et al. 2 3

(a) c

d

10 µm

10 µm

a a (b) a

g

a 10 µm

Fig. 1. White rot fungi (a) Byssomerulius avellaneus 2009 and (b) Steccherinum murashkinskyi 1963: (1) surface growth of the fungi on an S A medium; (2) micromorphology of fungal mycelium, 1000; (3) fruiting on sawdust blocks. Arrows and Latin letters des ignate (a) clamp connections, (c) anastomoses, (d) gloeocystidia, and (g) hypha incrustation.

ligninolytic enzymes. Semisynthetic medium G P, containing glucose as a carbon source and peptone as a nitrogen source, on the one hand, does not contain natural inductors. On the other hand, when grown on glucose containing media, white rot fungi are able to synthesize glucose oxidase (EC 1.1. 3.4), which is a major source of H2O2 in some basidiomycetes during growth on wood substrates [21]. To stimulate the bio

synthesis of the ligninolytic enzyme laccase, the G P medium was supplemented with CuSO4 (medium G P+Cu). The S A medium was more advantageous for strains A. faginea 1998, L. betulina 2047, P. avellanea 2009, P. ostreatus 0432, and X. radicata 1795. On the G P medium, the fungi C. caperata, P. avellanea, P. ostreatus, S. murashkinskyi, and T. gibbosa grew more slowly: the Petri dish was completely covered

Table 2. Verification of the species affiliation of fungal strains by DNA typing Strain Antrodiella faginea 1998 Coriolopsis caperata 0677 Lenzites betulina 2047 Byssomerulius avellaneus 2009 Pleurotus ostreatus 0432 Peniophora lycii 2142 Steccherinum murashkinskyi 1963 Trametes gibbosa 1911 Xerula radicata 1795 The closest strain from the NCBI database (Taxonomy ID) Antrodiella faginea (92699) Coriolopsis caperata (195176) Lenzites betulinus (5632) Phanerochaete avellanea, synonym Byssomerulius avellaneus, (194676) GU062467 Pleurotus sp. 'Florida' (188765) Peniophora lycii (154539) Steccherinum murashkinskyi (627145)FJ798705 Trametes gibbosa (160864) Hymenopellis radicata, synonym Xerula radicata (937743)
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Identity, % 99 100 99 100 99 100 100 100 100
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COMPARATIVE ANALYSIS OF THE LIGNINOLYTIC POTENTIAL OF BASIDIOMYCETES Table 3. Activity of the ligninolytic enzymes of studied and control fungal strains determined by a rapid method Strain Medium SA LE BIN 1998 GP G P+Cu SA LE BIN 0677 GP G P+Cu SA LE BIN 2047 GP G P+Cu SA LE BIN 2009 GP G P+Cu SA LE BIN 0432 GP G P+Cu SA LE BIN 2142 GP G P+Cu SA LE BIN 1963 GP G P+Cu SA LE BIN 1911 GP G P+Cu Duration of culti vation, days 7 14 7 14 7 14 7 14 7 14 7 14 7 14 7 14 7 14 7 14 7 14 7 14 7 14 7 14 7 14 7 14 7 14 7 14 7 14 7 14 7 14 7 14 7 14 7 14 Growth, mm 33.3 ± 1.1 78.7 ± 0.9 36.3 ± 1.1 89.3 ± 0.9 23.3 ± 4.4 77.3 ± 1.8 71.0 ± 2.7 >90.0 37.0 ± 2.0 75.3 ± 1.8 46.3 ± 2.9 70.6 ± 1.8 78.7 ± 0.9 >90.0 73.7 ± 2.2 >90.0 56.7 ± 3.1 >90.0 56.3 ± 2.4 88.3 ± 2.2 26.7 ± 2.4 69.3 ± 3.8 37.3 ± 0.9 78.7 ± 0.9 75.0 ± 1.3 >90.0 42.3 ± 1.1 63.0 ± 2.0 60.7 ± 1.8 >90.0 25.0 ± 0.1 64.3 ± 3.8 51.0 ± 0.3 87.3 ± 0.4 40.3 ± 1.8 80.0 ± 0.1 34.7 ± 1.8 68.3 ± 4.4 23.7 ± 1.1 47.0 ± 1.3 17.7 ± 0.4 34.7 ± 0.4 73.0 ± 1.3 >90.0 34.7 ± 0.4 72.0 ± 2.0 35.0 ± 2.0 81.7 ± 2.4
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Activity guaiacol + ++ ± ± ± + +++ ++ ++ + +++ ++ ++ +++ ± ± + + + ++ ++ ± ± + ++ ++ ± ± + ± ++ + ± ± + ++ ++ +++ ++ ++ +++ +++ ++ +++ ++ ± +++ ++
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syringaldazine ++ ++ + ± ++ + +++ ++ ++ + +++ ++ ++ +++ ± ± ++ ++ ++ ++ ++ ± ± ­ +++ ++ ± ± + + + + ± ± ++ + ++ +++ +++ ++ +++ +++ ++ ++ +++ + +++ +++

azure B ­ + ­ ± ­ ­ ­ + ­ ­ ­ ­ + +++ ­ ± ­ ­ ­ ± ­ ­ ­ ­ ± +++ ­ ­ ­ ± ­ ± ­ ­ ­ ­ ­ ± ­ ­ ­ ­ ­ +++ ± + ­ ±

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576 Table 3. Contd. Strain Medium SA LE BIN 1795 GP G P+Cu SA Trametes hirsuta 072* G P G P+Cu SA T. maxima 0275* GP G P+Cu SA T. ochracea 093* GP G P+Cu
* Control strains.

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Duration of culti vation, days 7 14 7 14 7 14 7 14 7 14 7 14 7 14 7 14 7 14 7 14 7 14 7 14

Growth, mm 43.0 ± 0.5 >90.0 45.0 ± 1.34 87.3 ± 1.8 40.3 ± 1.8 81.7 ± 2.4 >90.0 65.7 ± 0.9 >90.0 78.3 ± 1.0 >90.0 78.0 ± 1.3 >90.0 55.3 ± 3.1 84.3 ± 0.9 65.5 ± 2.56 85.5 ± 1.5 67.0 ± 2.7 >90.0 50.7 ± 0.9 84.3 ± 1.6 54.7 ± 2.9 84.7 ± 1.6

guaiacol ± +++ + ++ + +++ ++ ++ +++ ++ +++ +++ +++ + + ± ++ ++ ++ ++ ± ± + +++

Activity syringaldazine ­ +++ + + ++ ++ +++ +++ +++ ++ +++ ++ +++ + ++ ± ++ ++ ++ ++ ± ± + +++

azure B ­ ­ ­ ± ­ ­ ­ ++ ­ ± ­ ­ + +++ ­ ± ­ ­ ­ +++ ­ ­ ­ ­

with mycelium one week later compared to the growth on wort, and the color reaction of the substrates was less intense. Conversely, the strains A. faginea and P. lycii on the G P medium grew faster than on the S A medium; however, the oxidation of guaiacol and syringaldazine in this case was less intense. The addi tion of copper as an inducer (G P+Cu medium) pro moted the production of ligninolytic enzymes, includ ing laccases, in C. caperata, L. betulina, P. lycii and T. gibbosa but had no effect on the production of lign inolytic enzymes in the fungi A. faginea, P. avellanea, and P. ostreatus. The reaction with the guaiacol and syringaldazine of fungi C. caperata 0677, L. betulina 2047, P. ostreatus 0432, and T. gibbosa 1911, as well as three control strains grown on the S A medium, cor related with the Azure B bleaching. Note that the complete bleaching of the dye was only observed for the fungi L. betulina, P. ostreatus, and T. gibbosa, as well as two control strains--T. maxima 0275 and T. ochra cea 093. When white rot fungi are grown on natural substrates, including wort, they synthesize both lignin and peroxidases and various oxidases capable of gener ating hydrogen peroxide, which is required for mani festing the catalytic activity of peroxidase [22]. The presence of the genes encoding these enzymes was shown for Phanerochaete chrysosporium, Peniophora gigantean, Trametes ochracea [22, 23], Trametes versicolor [21], and various species of the genus Pleurotus [24].

Submerged cultivation and detection of the laccase gene in the genome of the studied fungi. The produc tion of laccases in submerged cultures was observed in all tested strains, except P. avellanea 2009 and X. rad icata 1795. It was shown that the composition of the medium had a significant effect on the laccase activity (Fig. 2). For example, for a number of strains, the S A medium was optimal for laccases biosynthesis: the highest activity was exhibited by strains A. faginea 1998 and S. murashkinskyi 1963. Significant induction of laccase activity by cupric salts was observed during the cultivation of C. caperata 0677, L. betulina 2047, and T. gibbosa 1911 (white rot fungi of the family Polypo raceae, which are active producers of laccases) [25]. However, the enzyme activity in T. gibbosa 1911 cul tured on S A and G P+Cu media differed insignifi cantly (3.8 and 2.3 U/mL, respectively). Taking into account the fact that all test strains, according to the rapid screening data, were able to oxi dize syringaldazine and that laccase activity was not detected during the submerged cultivation of the P. avellanea 2009 and X. radicata 1795 strains in any of the three media, we performed experiments for detect ing the laccase genes in the genomes of the studied fungi. It is known that the amino acid sequences of all lac cases described to date have four conserved regions [26]: 61 TSIHWHXFQ 69, 106 FWYHSHLSTQY 116, 394 PHPFHLHGH 402, and 449 WFLH
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COMPARATIVE ANALYSIS OF THE LIGNINOLYTIC POTENTIAL OF BASIDIOMYCETES U/mL 20 18 16 I 14 12 10 8 6 III 4 II 2 0 1

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Fig. 2. Daily average laccase activity measured during sub merged cultivation of fungi (1) A. faginea 1998, (2) C. caperata 0677, (3) L. betulina 2047, (4) P. lycii 2142, (5) P. ostreatus 0432, (6) S. murashkinskyi 1963, and (7) T. gibbosa 1911 on media (I) S A, (II) G P, and (III) G P+Cu.

CHIDXHL 459 (the amino acid residues are enumer ated according to the amino acid sequence of the lac case from Trametes hirsuta, PDB code 3fpx), where X designates the variable amino acid residues that are present in conserved regions. Histidine residues in these regions are responsible for the coordination of copper ions in the T1 copper site and T2/T3 copper
() M 1 2 3 4 5

cluster. The authors of [27] described primers for iden tifying laccase genes, developed for the first two regions (amino acid residues nos. 61­70 and 106­ 116), using which it is possible to obtain nucleotide fragments approximately 200 bp long. However, as was shown in [27], these primers cannot be used in all cases. Since laccase genes were not found in the P. avellanea and X. radicata genomes using these primers, we designed new primers. Forward primer LacA2 was selected for the second conserved region of laccase gene sequences (106 FWYHSHLSTQY 116). To construct reverse primer LacA2 Rev, we performed alignment of more than 100 basidial laccase sequences from the NCBI database, as a result of which the con served 297 NSAILRY 303 sequence was identified. Test PCR was performed with control strains of basid iomycetes, from the culture liquids of which classic "blue" laccases were isolated and characterized: Tra metes maxima 0275 (synonym Cerrena maxima), Tra metes ochracea 093 (synonym Coriolus zonatus), and Trametes hirsuta 072. The complete nucleotide sequences of the laccase genes of these basidiomycetes were also determined [28]. PCR with the use of LacA2 For­LacA2 Rev primers yielded 750 bp amplicons for all test and control fungal genomes (Fig. 3a). The amplification specificity was verified by sequencing PCR amplification products. Since the presence of a PCR fragment, the nucleotide sequence of which cor responds to laccase, cannot guarantee that this sequence was not amplified from a pseudogene, we performed a transcriptome analysis for the presence of
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Fig. 3. Electrophoresis of amplification products obtained by PCR with primers LacA2 For and LacA2 Rev of (a) chromosomal DNA and (b) cDNA of test and control fungal strains in a 1.2% agarose gel: (1) A. faginea 1998, (2) C. caperata 0677, (3) L. betulina 2047, (4) P. avellanea 2009, (5) P. ostreatus 0432, (6) P. lycii 2142, (7) S. murashkinskyi 1963, (8) T. gibbosa 1911, (9) T. hirsuta* 072, (10) T. max ima* 0275, (11) T. ochracea* 093, and (12) X. radicata 1795. M--DNA markers (1 kB). * Control strains. APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 49 No. 6 2013


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Fig. 4. Comparative analysis of the amino acid sequences of partial laccase sequences obtained using the cDNA of the studied fungi (NCBI accessions: AEQ38861, AEQ38863, AFQ90522, AFQ90521, AEQ38864, AFQ90523, AEQ38865, AEQ38867, and AFQ90524) and the amino acid sequences of the laccases of the control strains. Asterisks indicate the conserved regions. APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 49 No. 6 2013


COMPARATIVE ANALYSIS OF THE LIGNINOLYTIC POTENTIAL OF BASIDIOMYCETES A. faginea S. murashkinskyi P. lycii X. radicata C. caperata T. gibbosa P. ostreatus T. ochracea T. maxima P. avellanea L. betulina T. hirsuta 10 12 14 16 18 Joining distance 20 22 24

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Fig. 5. Tree clustering of test and control fungal strains constructed on the basis of the ligninolytic activity determined by rapid screening, growth rate, and daily average laccase activity in a submerged culture.

using the LacA2 For and LacA2 Rev primers. PCR amplification products approximately 550 bp long were obtained for all test and control fungal genomes (Fig. 3b). Thus, expressed laccase genes were found in the genomes of all studied basidiomycetes (Fig. 4). However, to ensure the synthesis of laccases in sub merged cultures of P. avellanea 2009 and X. radicata 1795, the cultivation conditions for these strains should be optimized, because the biosynthesis of lac case in different strains of white rot fungi may vary depending on many factors, including the type and content of carbon and nitrogen sources, the presence of inducers in the medium, the phase of the biological cycle of the fungus, etc. [29]. Cluster analysis. The results of tree clustering (Fig. 5) showed that all the fungal strains studied can be divided into two major groups that live in nature on the wood of deciduous trees of varying degrees of decomposition (Table 1). An analysis of the ecology of the fungi comprising these groups showed that the first group included fungi of the families Pleurotaceae, Polyporaceae, and Phanerochaetaceae, which are the primary wound xylotrophs or inhabitants of dead, although not heavily decayed, wood. The second group was formed by members of the families Maras miaceae, Peniophoraceae, and Steccherinaceae, which are secondary xylotrophs preferring heavily decomposed wood. A comparison of the ligninolytic activity of the studied strains and the clustering analy sis results showed that the most active ligninolytic fungi belong to the first group, which allows for the primary screening of effective producers of ligni nolytic enzymes among fungi belonging to different taxonomic and ecological groups.
APPLIED BIOCHEMISTRY AND MICROBIOLOGY

Thus, the screening performed in this study revealed basidiomycete strains with a high ligninolytic activity and degradation potential-- Coriolopsis caper ata 0677, Lenzites betulina 2047, Pleurotus ostreatus 0432, and Trametes gibbosa 1911--which are promising for further use in biotechnology. ACKNOWLEDGMENTS This study was supported by the Russian Founda tion for Basic Research (projects nos. 11 04 01539, 11 04 01704) and the special purpose federal pro gram of the Ministry of Education and Science of the Russian Federation "Research and Development on Priority Directions of the Scientific­Technological Complex of Russia for 2007­2013" (state contract no. 14.512.11.0067) and the Program of the Division of Biological Sciences of the Russian Academy of Sci ences "Biological Resources of Russia". REFERENCES
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