Uncodified intertidal marine habitats of the NWBS and nearby areas were inventory. Because of research, 17 new habitats were identified: “Pontic supralittoral rock with marine Diptera”, “Limestone pavement with supralittoral rockpools”, “Drainage of fresh water on the rocks”, “Diptera and Enchytraeidae on strandline”, “Pontic multispecies communities on strandline”, “Freshwater springs on the sandy beaches and associated pools”, “Long-existing pools and ponds with marine fauna on the sandy beach”, “Pontic mediolittoral granule with Saccocirrus papillocerus”, “Mediolittoral sands with Enchytraeidae”, “Pontic mediolittoral gravel with Donacilla cornea and Saccocirrus papillocerus”, “Pontic mediolittoral fine-medium sands with Pontogammarus maeoticus and bivalves”, “Pontic mediolittoral shelly gravel with crustaceans”, “Pontic soft rock beds with Barnea candida burrows”, “Pontic infralittoral coarse sediments with Rhodophyta”, “Pontic infralittoral sands with crustaceans”, “Pontic scoured sublittoral cobbles and pebbles with Mytilaster”, “Aggregation dead remnants of angiosperms in infralittoral zone”. Some of the selected habitats are covered by the less detailed “Bern” habitats “A2.2: Littoral sand and muddy sand”; “Bern” habitat “A3: Infralittoral rock and other hard substrata”; “Bern” habitat “A5: Sublittoral sediment”. These habitats should be protected for a long time by creating the Emerald Network of Ukraine. Selected habitats relatively well covered by protected areas, with the exception of those associated with soft rocks. The creation of protected areas covering these landscapes between Chornomorsk City and Tiligul Liman is necessary.

A checklist of the northwestern Black Sea supralittoral fauna is presented. It includes 18 species: Ophelia bicornis, Namanereis pontica, Cryptorchestia cf. garbinii, Deshayesorchestia deshayesii, Orchestia montagui, Orchestia gammarellus, Orchestia montagui, Armadilloniscus ellipticus, Halophiloscia cf. couchii, Ligia italica, Tylos ponticus, Chthamalus stellatus, Microeuraphia depressa, Thalassomyia frauenfeldi, Donacilla cornea, Myosotella myosotis, Truncatella subcylindrica, and Melarhaphe neritoides. This paper provides distribution maps for this species in the northwestern Black Sea, based on field studies. The changes that occurred in species composition are discussed. The regional IUCN categories are proposed.

Lytic polysaccharide monooxygenases (PMO) discovered several years ago are enzymes classified as oxidoreductases. In nature, they participate in microbial degradation of cellulose together with cellulases that belong to the hydrolytic type of enzymes (class of hydrolases). Three PMO from ascomycetes–Thielavia terrestris, Trichoderma reesei, and Myceliophthora thermophila–were isolated and purified to homogeneous state using various types of chromatography. The first two enzymes are recombinant proteins heterologously expressed by the Penicillium verruculosum fungus, while the third is a native PMO secreted by M. thermophila. When acting on microcrystalline cellulose, all these PMOs displayed synergism with the cellulase complex of the P. verruculosum fungus. Replacing 10% of cellulases (by protein concentration) with PMO in the presence of 6.25 mM gallic acid or 2.5 μM of cellobiose dehydrogenase from M. thermophila, used as electron donors for PMO, resulted in the 17-31% increase in the yield of reducing sugars after 24-48 h of the enzymatic reaction.

Recombinant strains of Penicillium canescens producing homologous pectin lyase A and heterologous endo-1,5-α-arabinase A and endo-1,4–α-polygalacturonase, as well as enzymes of the host strain (α-L-arabinofuranosidases, xylanases, and others), were obtained by genetic engineering. The enzyme preparations (EPs) obtained from the cultural medium of recombinant P. canescens strains efficiently hydrolyzed raw plant material with a high content of pectin compounds. It was shown that the yield of reducing sugars and arabinose increased 16 and 22% in comparison with the control EP based on the host strain when one of the obtained EPs was used for beet pulp hydrolysis. It was established that the most active EP consisted of pectin lyase (10%), endo-1,5-arabinase (26%),α-L-arabinofuranosidase and arabinoxylan-arabinofuranohydrolase (12%), and xylanase (10%). The activities of pectin lyase, polygalacturonase, and arabinase of the EP in reactions with various substrates were determined. The specificity, pH and T-optima, and thermal stability of the homogenous recombinant endo-1,5-α-arabinase were investigated. The kinetic parameters ( K m , k cat ) of the linear arabinan hydrolysis were determined.

The effect of polysaccharide monooxygenase (endoglucanase IV) from the fungus Trichoderma reesei on the hydrolysis of polysaccharide substrates by cellulases secreted by the fungus Penicillium verruculosum has been investigated. Supplementation of the enzyme complex from P. verruculosum by endoglucanase IV from T. reesei has been shown to elevate the efficiency of cellulose hydrolysis by 45%.

На основе гриба Penicillium verruculosum созданы штаммы, комплекс внеклеточных ферментов которых содержит как целлюлолитические ферменты этого гриба, так и гетерологичные пектинлиазу А из P. canescens и эндо-1,4- -полигалактуроназу из Aspergillus niger. Активность эндо-полигалактуроназы ферментных препаратов, полученных из культуральной жидкости штаммов-продуцентов, достигала 46–53 ед./мг белка, а пектинлиазы – 1.3–2.3 ед./мг. Оптимумы температуры и рН рекомбинантных пектинлиазы и эндо-полигалактуроназы соответствовали описанным в литературе для этих ферментов. Содержание гетерологичной эндо-полигалактуроназы в исследованных ферментных препаратах составило 4–5% от общего белка, пектинлиазы – 23%. Выход восстанавливающих сахаров при гидролизе отходов переработки сахарной свеклы и яблок наиболее эффективным ферментным препаратом составил 41 и 71 г/л, что соответствовало степени конверсии полисахаридов 49 и 65%. Основным продуктом гидролиза отходов переработки сахарной свеклы и яблок была глюкоза.

The possibility of conversion of parchment to glucose by enzymatic hydrolysis using enzyme preparations (EP) based on fungi of the genera Penicillium and Trichoderma was studied. Parchment was hydrolyzed at 50 °C and pH = 5 under constant stirring. The dosage of cellulase EP (P. verruculosum B1-221-131 and T. reesei BioACE) was respectively 10 and 15 enzyme units with respect to filter paper per 1 g of dry substrate. The process was carried out both in the absence and in the presence of excess cellobiase (β-glucosidase) of P. verruculosum F10. It was found that the conversion of parchment to glucose can achieve 70 % without any pretreatment. It is shown that the cellulase EP from P. verruculosum provides a higher yield of glucose than the EP from T. reesei does. Cellobiase (β-glucosidase) plays an important role in the hydrolysis of parchment wastes: in the presence of excess cellobiase, hydrolytic ability of cellulase EP increases. Thus, the possibility of using the parchment as a raw material for production of sugars and, further, of other valuable products is demonstrated.

Методами генетической инженерии были получены рекомбинантные штаммы Penicillium canescens, которые продуцировали гомологичную пектинлиазу А и гетерологичные эндо-1,5- -арабиназу А и эндо-1,4- -полигалактуроназу, а также ферменты исходного штамма ( -L-арабинофуранозидазы, ксиланазы и другие ферменты). Ферментные препараты (ФП), полученные из культуральной жидкости этих штаммов P. canescens, эффективно гидролизовали растительное сырье с высоким содержанием пектиновых веществ. Показано, что при гидролизе свекловичного жома одним из полученных ФП выход восстанавливающих сахаров и арабинозы повышался на 16 и 22% по сравнению с контрольным ФП на основе исходного штамма. Установлено, что в состав наиболее активного ФП входили пектинлиаза (10%), эндо-1,5- -арабиназа (26%), -L-арабинофуранозидаза и арабиноксилан-арабинофураногидролаза (12%) и ксиланаза А (10%). Определены активности пектинлиазы, полигалактуроназы и арабиназы ФП по отношению к различным субстратам. Изучены специфичность, оптимум рН и температуры, термостабильность гомогенной рекомбинантной эндо-1,5- -арабиназы. Определены ее кинетические параметры (Km, kcat) гидролиза линейного арабинана.

Изучено влияние полисахаридмонооксигеназы (эндоглюканазы IV) гриба Trichoderma reesei на гидролиз полисахаридных субстратов целлюлазами, секретируемыми грибом Penicillium verruculosum. Показано, что внесение эндоглюканазы IV из T. reesei в комплекс ферментов гриба P. verruculosum позволяет повысить эффективность гидролиза целлюлозы на 45%.

As a result of γ-mutagenesis of Trichoderma longibrachiatum TW1 and the subsequent selection of improved producers, a novel mutant strain, TW1-59-27, capable of efficiently secreting cellulase and xylanase was obtained. In a fed-batch cultivation, the new TW1-59-27 mutant was significantly more active compared with the original TW1 strain. For instance, the activities of cellulase (towards carboxymethylcellulose) and xylanase in the culture broth (CB) increased by 1.8 and two times, respectively, and the protein content increased by 1.47 times. The activity of these enzymes in the dry enzyme preparation derived from the CB of the TW1-59-27 mutant was 1.3–1.8 times higher than that in the preparation derived from the original TW1 strain. It was established that the cellulase from the enzyme preparation of the mutant strain demonstrated the maximum activity at 55–65°C; it occurred in xylanase at 60°C. The pH optima of these enzymes were pH 4.5–5.0 and pH 5.0–6.0, respectively. It was shown that the content of endoglucanases in the enzyme preparation increased from 7% to 13.5%; the effect is largely driven by the elevated secretion of endoglucanase-1. An enzyme preparation with increased endoglucanase-1 content is promising for use as a feed additive in agriculture.

В результате -мутагенеза Trichoderma longibrachiatum TW1 и последующей селекции улучшенных продуцентов получен новый мутантный штамм TW1-59-27, способный эффективно секретировать целлюлазы и ксиланазы. При fed-batch культивировании нового мутанта TW1-59-27 активность ферментов значительно возрастала по сравнению с исходным штаммом TW1. Так, активность целлюлазы (по отношению к карбоксиметилцеллюлозе) и ксиланазы в культуральной жидкости мутантного штамма увеличивалась в 1.8 и 2 раза соответственно, а содержание белка – в 1.47 раз. Активность этих ферментов в сухом ферментном препарате, полученном из КЖ мутанта TW1-59-27, возрастала в 1.3–1.8 раз по сравнению с препаратом из исходного штамма TW1. Условлено что целлюлаза ферментного препарата из мутантного штамма проявляла максимальную активность при 55–65°С, а ксиланаза при 60°С. Оптимум рН для действия этих ферментов – 4.5–5.0 и 5.0–6.0 соответственно. Показано что содержание эндоглюканаз в ферментном препарате увеличивалось с 7 до 13.5% в значительной степени вследствие секреции эндоглюканазы 1. Ферментный препарат с повышенным содержанием эндоглюканазы 1 может быть перспективным для применения в качестве кормовой добавки в сельском хозяйстве.

The problem of raising the efficiency of enzyme preparations catalyzing cellulose conversion is among the present-day technological challenges. Here, we report the enhancement of the hydrolytic capacity of cellulase preparations by introducing nonhydrolytic enzymes (polysaccharide monooxygenases) into the cellulolytic complex. An enzyme preparation with an increased hydrolytic capacity has been obtained from the recombinant strain of the fungus Penicillium verruculosum that carries the Trichoderma reesei endoglucanase IV gene. This method allows the efficiency of the cellulase complex to be increased by 20%.

Improving the efficiency of enzyme preparations which performing conversion of cellulose is one of the most pressing technological challenges today. In this paper, the latest method is considered to increase the hydrolytic ability of cellulase preparations comprising administering a non-hydrolytic enzymes of the type of action (polysaccharidemonooxygenase) in cellulolytic complex. Enzyme preparation with increased hydrolytic capacity was obtained by a recombinant strain of the fungus Penicillium verruculosum, carrying gene endoglucanase IV Trichoderma reesei. Using this method it is possible to increase the effectiveness of cellulase complex up to 20 %.

Methods for the production and analysis of cellulase and hemicellulase enzyme preparations of various compositions based on the Penicillium verruculosum carbohydrase complex and intended for the effective hydrolysis of different types of cellulose-containing materials (CCMs) have been developed. New recombinant strains of P. verruculosum producing multienzyme carbohydrase complexes with increased activities of cellulases (due to the expression of endo-β-1,4-glucanases I and IV and cellobiohydrolase II from Trichoderma reesei ) and hemicellulases (due to the expression of endo-β-1,4-xylanases from P. canescens and T. reesei and endo-β-1,4-mannanase from T. reesei ) were constructed. The hydrolytic efficiency of the enzyme preparations (EPs) produced by the new recombinant strains during continuous hydrolysis of three CCM types (milled aspen, depitched pine wood, and milled bagasse) was studied. It was shown that new EPs containing recombinant proteins and retaining their own basic cellulase complex are characterized by the highest hydrolytic ability, exceeding that of the EP based on the original P. verruculosum strain. The recombinant enzyme preparations were highly stable; the optimal pH and temperature values for cellulase, xylanase and mannanase activities were in the range of 3.5–5.5 and 50–80°C, respectively.

Hydrolysis of cellulose biomass under the action of biocatalysts (enzyme preparations) is one of the most advanced and environmentally friendly methods of obtaining a number of useful products. In this paper, a new approach is used to create a recombinant enzyme preparations with the desired properties by using fusion-constructs for gene cloning of target enzymes. A number of enzyme preparations is based on the strain of the fungus Penicillium verruculosum using fusionconstruct. The properties of these enzyme preparations are interest primarily as an additive to increase the capacity of the base hydrolytic cellulolytic complex P.verruculosum. Sharing new enzyme preparations and basic P.verruculosum allowed to increase its biocatalytic (hydrolytic) efficiency for the vegetable cellulose feed materials. By adding 20 % of the new enzyme preparation to the base without altering the total dosage of enzyme preparations in a reaction mixture the increasing of efficiency of hydrolysis of cellulose substrates (ground aspen wood and shredded pine wood deresined ) up to 70 %.

Hydrolysis of cellulose-containing biomass mediated by biocatalysts (enzyme preparations, EP) is one of the most advanced and environmentally friendly methods of obtaining a range of useful substances. A new approach to creating recombinant EPs with predefined properties, which consists in applying fusion constructs for the cloning of genes encoding target enzymes, was used in the present study. A number of EPs with different properties was derived from a strain of the fungus Penicillium verruculosum using fusion constructs; these preparations are of interest primarily as additives enhancing the hydrolytic capacity of the basic cellulolytic complex from P. verruculosum. Use of the new EPs in combination with the basic EP from P. verruculosum resulted in an increase of the biocatalytic (hydrolytic) efficiency of the latter towards cellulose-containing raw materials of plant origin. Addition of 20% of the new EP to the basic EP without changing the total EP dose in the reaction mixture resulted in a significant (up to 70%) increase of the efficiency of hydrolysis of cellulose-containing substrates (ground aspen wood and shredded deresined pine wood).

Complex enzymatic preparations demonstrating activities homologous to pectinlyase A and heterologous to endo-1,4-β-glucanase from Penicilliumverruculosum and β-glycosidase from Aspergillusniger have been obtained on the basis of recombinant strains of the fungus Penicilliumcanescens. Two approaches were utilized: development of an enzymatic preparation on the basis of a new strain, which produced all three enzymes, and development of an enzymatic preparation via combined cultivation of three strains, each of which produced one of the enzymes.