Мицелиальный гриб Penicillium verruculosum (ВКМ F-3972D) является высокопродуктивным продуцентом комплекса ферментов, участвующих в биодеградации полимеров растительной клеточной стенки. Секреторная способность P. verruculosum достигает 60 г/л внеклеточного белка, что позволяет использовать его в качестве базового продуцента ферментов и ферментных комплексов, востребованных промышленными биотехнологиями. На базе гриба P.verruculosum была разработана система экспрессии, основанная на использовании сильного индуцибельного промотора гена cbh1, кодирующего ключевой фермент секретируемого комплекса – целлобиогидролазу 1. Для получения рекомбинантных штаммов методами геномного редактирования был получен ауксотрофный штамм-реципиент Penicillium verruculosum 537, дефектный по гену niaD, кодирующий нитратредуктазу, что позволило осуществить направленную селекцию рекомбинантов на средах с нитратом натрия. Анализ транскрипционного механизма P.verruculosum позволил получить генерацию реципиентных штаммов с нокаутом генов негативных факторов транскрипции (TacA, CreA), что позволило качественно улучшить состав секретируемого комплекса. Частичная дерепрессия штамма по глюкозе позволила оптимизировать схему ферментации рекоминантных штаммов в режиме fed-batch. Используя разработанную систему экспрессии были получены штаммы-продуценты кормовых ферментов (эндоглюканазы, ксиланазы, пептидазы) для гидролиза некрахмальных полисахаридов зерна и соевого белка, штаммы-продуценты комплекса пектиндеградирующих ферментов для различных направлений пищевой промышленности, комплекс инулин-деградирующих ферментов для получения фруктозо-глюкозных сиропов и фруктоолигосхаридов, разрабатывается комплекс ферментов-пептидаз и липаз для моющих средств и т.д.. Важно отметить, что разработки ФИЦ Биотехнологии РАН имеют реальный спрос в секторе биотехнологий РФ. Плотные связи Лаборатории биотехнологии ферментов ФИЦ Биотехнологии РАН с заводом ферментных препаратов ООО «Агрофермент» позволяет внедрять разработки в промышленность и сельское хозяйство. Работа выполнена при поддержке Министерства науки и высшего образования РФ в соответствии с договором № 075-15-2022-318 от 20.04.2022 о предоставлении гранта в виде субсидии из федерального бюджета РФ. Грант предоставлен для государственной поддержки создания и развития НЦМУ «Агротехнологии будущего».

Methods of the pretreatment of reeds (grinding, hydrothermal treatment, treatment with acid or alkali solutions, organosolv, deep eutectic solvents) and their effect on the subsequent enzymatic hydrolysis by cellulase and hemicellulase complexes have been studied. Substrates with the highest reactivity were obtained by exposing the reeds to a weakly alkaline deep eutectic solvent (DES) and an alkali solution. The depth of enzymatic hydrolysis of these pretreated substrates was 63 and 59

A chimeric form of Penicillium verruculosum endo-1,4-β-glucanase II (EGII), the C-terminus of which contains a cellulose-binding module (CBM) of P. verruculosum cellobiohydrolase I, is produced by the genetic engineering method. In the native form, the eglI gene does not have a region encoding CBM. The resulting chimeric enzyme is isolated in a homogeneous form and its properties are studied. The addition of CBM to EGII led to a significant increase in the activity of the chimeric enzyme relative to microcrystalline cellulose (MCC) and the appearance of its adsorption capacity in relation to cellulose. However, the addition of CBM to EGII led to a decrease in activity towards soluble polysaccharide substrates (carboxymethylcellulose and β-glucan). The chimeric form of the enzyme in the composition of the cellulase complex allowed us to hydrolyze cellulose-containing substrates more effectively. The yield of reducing sugars (RS) in the case of MCC hydrolysis after 24 h with a cellulose complex containing cellobiohydrolase I, β-glucosidase, and EGII-CBM in comparison with a complex containing EGII without CBM increased by 22%, while for chopped aspen wood the RS increased by 42%.

The composition and properties of a wide range of domestic and foreign enzyme preparations (EP), used as additives to feeds of farm animals and poultry, are analyzed. The content of the main active enzymes – endoglucanases (beta-glucanases), cellobiohydrolases and xylanases, leading to biocatalytic destruction of non-starch polysaccharides, which are anti-nutritional factors of feeds and causing their incomplete digestion, is determined. It is shown that, based on the data on the component composition and the level of different types of activity, the studied enzyme preparations can be divided into three groups: a) with high xylanase and low cellulase (endoglucanase and cellobiohydrolase) content, b) high cellulase and low xylanase content, c) containing cellobiohydrolases, endoglucanases and xylanases in a different ratio, but without significant predominant of any of these enzymes. The ability of EP to reduce the viscosity of water-soluble non-starch polysaccharides – xylans and beta-glucans- has been studied. Among the enzyme preparations that have xylanase in their composition and belong to groups b) and c), a number of preparations have been determined which, at the same dosage according to xylanase activity, most effectively reduced the viscosity of the aqueous extract of rye containing xylans (Econase XT 25, Agroxyl Plus, Agroxyl Premium, Rovabio Max AP, Sunzyme). It was shown that the xylanase of precisely these EP is not inhibited by protein inhibitors of rye. At the same dosage for beta-glucanase activity, the viscosity of water-soluble beta-glucans of barley was most effectively reduced by the EP Xybeten CELL, Cellulase, Agroxyl, Agrocell, Axtra XB 201, Rovabio Max AP and Vilzyme. For all studied EP, no inhibitory effect of the barley extract on beta-glucanase activity was found.

New recombinant strains of Penicillium verruculosum are created using a new plasmid construct based on an inducible promoter of glucoamylase gene (gla1) that secretes heterologous xylanase E (XylE) from P. canescens . New biocatalysts are produced that contain cellulolytic enzyme preparations (EPs) enriched with XylE. The amount of XylE in the recombinant EPs varies in the optimum range of 11–24% of the total protein while generally preserving the P. verruculosum cellulose complex. The hydrolytic activity of the new EPs with respect to polymeric plant-derived substrates exceeds that of EPs produced using other expression systems and commercial preparations. The new EP glaX-17 surpasses in particular a control EP based on the recipient strain by 13% in the efficiency of hydrolyzing aspen wood and is 20% more efficient than the commercial EP Accelerase Duet. The new EP glaX-17 displays 25% greater efficiency (35–43%) during the hydrolysis of wheat bran than the commercial EP Accelease Duet. The effectiveness is demonstrated of using the new gla1 promoter for the production of EPs (biocatalysts) while preserving the balanced cellulose complex of the strain and optimum yield of heterologous XylE required for the deep hydrolysis of xylan-containing plant biomass.

Recombinant strains of Penicillium verruculosum are developed that produce the homologous endoglucanase 2 (Eg2) and the P. canescens heterologous xylanase E (XylE). The recombinant strains are used to obtain new biocatalysts, i.e., enzyme preparations (EPs) that are substantially enriched with Eg2 and XylE. These preparations are highly active with respect to nonstarch plant polysaccharides (NPSes): cellulose, β-glucan, and xylan. The qualitative and quantitative compositions of the new EPs are studied by protein chromatography. It was shown that the EPs contained (in terms of total protein content) ~16–17% Eg2, 48–63% XylE, and 17–30% cellobiohydrolases, while the EP obtained using the recipient strain contained 1.4% Eg2, ~60% cellobiohydrolase and no XylE. The optimum pH values for cellulase (with respect to carboxymethylcellulose, CMC) and the xylanase activity of the EPs are 4.0 and 5.5, respectively. The EPs exhibit the abovementioned activities within a wide range of pH (3 to 7). The EPs exhibit CMC-ase and xylanase activities in the temperature range of 20–80°С with maxima at 60 and 70°C, respectively. The xylanase activity of the new EPs is virtually uninhibited by protein inhibitors of rye.

Recombinant strains Penicillium verruculosum were created which produce homologous endoglucanase 2 (Er2) and heterologous xylanase E (XylE) P. canescens. The recombinant strains were used for preparing new biocatalysts which are enzyme preparations (EP) enriched considerably with Er2 and XylE; the biocatalysts are highly active to plant non-starch polysaccharides (NPS) such as cellulose, β-glucan, xylan. Proteic chromatography was used for determining the qualitative and quantitative composition of the new EP to show that they contain (expressed as proportions of the total protein content) ca. 16–17 % Er2, 48–63 % XylE, 17–30 % cellobiohydrolase, while EP prepared with the recipient strain contained 1.4 % Er2, ca. 60 % cellobiohydrolase and no XylE. pH equal to 4.0 and 5.5 were optimal for the activity to cellulose (with respect to carboxymethylcellulose, CMC) and to xylanase, respectively, EP being active over a wide range of pH from 3 to 7. EP were active to CMC and xylanase at the temperature range from 20 to 80 °C with maxima at 60 and 70 °C. The activity of new EP to xylanase was practically not inhibited by protein inhibitors of rye.

Modern technologies for the enzyme hydrolysis of cellulose-containing raw materials allow the production of sugars from which alcohols (biofuel), organic and amino acids, biopolymers, feed additives, and other value-added products can be obtained via microbiological conversion. Three types of cellulolytic enzymes are required for the bioconversion of cellulose containing materials: endoglucanase, cellobiohydrolase, and ß-glucosidase. The prospects for improving the hydrolytic capabilities of the enzyme complex secreted from Penicillium verruculosum are investigated in this work by means of genetic engineering to add different combinations and ratios of homologous and heterologous cellulases: endoglucanase IV (EGIV) of Trichoderma reesei , endoglucanase II (EGII), and cellobiohydrolase I (CBHI) of P. verruculosum , along with ß-glucosidase (ß-GLU) of Aspergillus niger . The optimum ratio of components is determined and the catalytic activity of enzymatic complexes is increased by as much as 100%.

Application of modern technologies of enzymatic hydrolysis of cellulose-containing raw materials makes it possible to obtain sugars that undergo microbiological conversion to synthesize alcohols (biofuels), organic and amino acids, biopolymers, food additives and other value-added products. Cellulolyticus enzymes of three types – endoglucanase, cellobiohydrolase, beta-glucosidase – are required for the bioconversion of cellulose-containing raw material. The present work is aimed at studying a possibility of application of genetic engineering for improvement of the hydrolytic ability of secretory enzyme complex Penicillium verruculosum by adding homologous and the heterologous cellulases in different combinations and ratios – endoglucanase IV (EGIV) from Trichoderma reesei, endoglucanase II (EGII) and cellobiohydrolase I (CBHI) from P. verruculosum, and also beta-glucosidase (b-GLU) from Aspergillus niger. The optimal ratio of components is determined to double the catalytic activity of the new enzyme complexes.

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%.

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.

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.

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.