Exhaustive enzymatic hydrolysis is performed for semi-bleached sulfate hardwood cellulose (a semi-finished pulp and paper product) at ultra-high concentrations of it in a reaction mixture (up to 300 g/L per dry compound). Russian commercial enzyme preparations are used for hydrolysis. The best seems to be Agroxil Plus, which has high cellulase and endoxylanase activities. A total of 290 g/L of sugars (including 210 g/L of glucose and 30 g/L of xylose) is obtained using Agroxil Plus (20 mg protein/1 g substrate) in combination with an auxiliary β-glucosidase enzyme preparation (2 mg protein/1 g substrate) at an initial semi-bleached cellulose concentration of 300 g/L. The dosage of Agroxil Plus can be halved (10 mg of protein/1 g of substrate with a total concentration of semi-bleached cellulose of 300 g/L) with a high yield of hydrolysis product (270 g/L of sugars, including 200 g/L of glucose and 30 g/L of xylose), due to the fractional addition of a substrate.

Микроскопические грибы широко используются для промышленного производства технических ферментов; важным условием для этого является создание высокоактивных штаммов – продуцентов ферментов. Показана возможность использования для получения таких штаммов двух подходов: индуцированного мутагенеза и методов генетической инженерии. Разработаны схемы индуцированного мутагенеза, обеспечивающие получение высокоактивных продуцентов технических ферментов. В результате гамма-мутагенеза получены мутантные штаммы грибов рода Trichoderma – продуценты целлюлаз, ксиланаз и пектиназы, с различным компонентным составом карбогидразного комплекса, позволяющие получать в культуральной жидкости (КЖ) до 35–40 г/л внеклеточного белка. С помощью комбинированной обработки УФ и гамма-облучением получен стабильный мутантный штамм Aspergillus awamori с увеличенной продукцией глюкоамилазы. Создана система экспрессии Penicillium verruculosum В1-537 (ΔniaD), позволяющая трансформировать реципиентный штамм экспрессионными конструкциями, содержащими целевые гетерологичные или гомологичные гены, функционально связанные с промотором и терминатором сильного индуцибельного промотора гена мажорного секреторного белка целлобиогидролазы I (cbh1) P. verruculosum. Технология создания и отбора активных рекомбинантных штаммов проста и надежна, состав питательной среды и условий культивирования рекомбинантных штаммов – продуцентов различных целевых ферментов стандартизирован. Затраты времени на получение продуцента целевого фермента составляют от 3 до 6 мес. Реципиентный штамм P. verruculosum характеризуется высокой секреторной способностью (до 60 г/л внеклеточного белка в КЖ), а рекомбинантные штаммы-продуценты – высокой продуктивностью целевых ферментов; полученные с их помощью ферментные препараты (ФП) содержат 30‒70 % целевых рекомбинантных ферментов (в некоторых случаях до 80 %) от общего пула белка. Созданы рекомбинантные штаммы – продуценты целлюлаз, β-глюканаз, ксиланаз, фитазы, кислой протеазы, пектин-лиазы, β-глюкозидазы, экзо- и эндоинулиназ. Мутантные и рекомбинантные штаммы – продуценты различных ферментов – используются для промышленного производства на заводе ООО «Агрофермент» различных технических ФП для пищевой промышленности и кормопроизводства. Microscopic fungi are widely used for the industrial production of technical enzymes; an important condition for this is the creation of highly active strains – producers of enzymes. The possibility of using such approaches to obtain highly active strains as induced mutagenesis and genetic engineering methods is shown. Schemes of induced mutagenesis have been developed that ensure the production of highly active producers of technical enzymes. As a result of gamma-mutagenesis, mutant fungal strains of Trichoderma genus were obtained – producers of cellulases, xylanases and pectinase, with different component composition of the carbohydrase complex. The methods used made it possible to obtain up to 35–40 g/l of extracellular protein in the cultural liquid (CL). Using UV and gamma rays combined treatment, a stable mutant Aspergillus awamori strain with increased glucoamylase production was obtained. An expression system for Penicillium verruculosum B1-537 (ΔniaD) has been developed, which makes it possible to transform a recipient strain with expression constructs containing target heterologous or homologous genes functionally linked to the promoter and terminator of the strong inducible promoter of the P. verruculosum major secretory protein cellobiohydrolase I (cbh1) gene. The technology for creating and selection of active recombinant strains is simple and reliable, the composition of the nutrient medium and cultivating conditions for recombinant strains producing various target enzymes are standardized. The time spent on obtaining the target enzyme producer is from 3 to 6 months. The recipient P. verruculosum strain is characterized by a high secretory capacity (up to 60 g/l of extracellular protein in CL), and the recombinant strains are characterized by a high productivity of target enzymes. Enzyme preparations (EP) derived from the recombinant strains contain 30–70 % of the target recombinant enzymes in the total protein pool (in some cases, up to 80 %). Recombinant strains producing cellulases, β-glucanases, xylanases, phytase, acid protease, pectin-lyase, β-glucosidase, exo- and endo-inulinases have been developed. Mutant and recombinant strains – producers of various enzymes for the food industry and feed production are successfully used for industrial production of various technical EPs at the plant LLC «Agroferment».

The possibility of exhaustive enzymatic hydrolysis of semi-bleached sulfate hardwood pulp, a semi-finished product of pulp and paper production, at its super high concentrations in the reaction mixture (up to 300 g/l) is shown. For hydrolysis, Russian commercial enzyme preparations were used, the best of them was Agroxyl Plus, which has a high activity of cellulases and endoxylanase. With the help of Agroxyl Plus (at its dosage of 20 mg protein/g substrate) in the presence of an auxiliary enzyme preparation of β-glucosidase (2 mg protein/g substrate) at an initial concentration of semi-bleached cellulose of 300 g/l, 290 g/l sugars (210 g/l glucose, 30 g/l xylose) were obtained. Due to fed-batch enzymatic hydrolysis, it was possible to halve the dosage of Agroxyl Plus (10 mg protein/g of substrate at a total concentration of semi-bleached cellulose of 300 g/l) while maintaining a high yield of hydrolysis products – 270 g/l sugars (200 g/l glucose, 30 g/l xylose).

Пшеничный глютен является источником незаменимых аминокислот и биоактивных пептидов, а его ферментативные гидролизаты широко используются в качестве пищевых добавок и усилителей вкуса. В то же время высокое содержание пролина препятствует гидролизу глютена пищеварительными протеазами, что приводит к возникновению аллергических реакций и нарушениям пищеварительной системы. Глубокий ферментативный гидролиз способствует снижению аллергенности глютена, повышает его пищевую ценность и расширяет область применения в пищевой промышленности. Некоторые комплексные ферментные препараты (ФП), содержащие эндо- и экзопептидазы, за счет их синергетического действия обеспечивают эффективный гидролиз глютена. На основе штамма Aspergillus oryzae 21-154 LAP – продуцента комплекса эндо- и экзопептидаз с увеличенной активностью лейцинаминопептидазы (LAP) – получен комплексный ФП Протооризин LAP, по активности LAP сравнимый с широко используемым в пищевой промышленности ФП Flavourzyme® 1000L, при высоком уровне общей протеолитической активности, обусловленном действием эндопептидаз. По увеличению содержания растворимого белка и свободных аминокислот при гидролизе пшеничного глютена Протооризин LAP превосходил импортные ФП Flavourzyme® 1000L и Alcalase 2.4L как при индивидуальном, так и при совместном применении. Электрофоретический анализ полученных гидролизатов показал, что Протооризин LAP интенсивно расщеплял все основные белки глютена. Через 24 ч гидролиза оставалась только слабая полоса белка с молекулярной массой ~37 кДа. ФП Alcalase 2.4L расщеплял пшеничный белок с меньшей эффективностью, чем Протооризин LAP, оставляя через 24 ч, помимо полосы на уровне ~37 кДа, некоторое количество пептидов с молекулярной массой менее 14,4 кДа. Наименьшей способностью к гидролизу белков пшеницы обладал Flavourzyme® 1000L, однако при совместном использовании с Alcalase 2.4L препарат эффективно гидролизовал низкомолекулярные белковые фракции. Протооризин LAP успешно заменял совместное действие ФП Flavourzyme 1000L и Alcalase 2.4L, что показывает перспективность его использования при получении гидролизатов пшеничного глютена. Wheat gluten is a good source of essential amino acids and bioactive peptides. Its hydrolysates are widely used as food additives and flavor enhancers. At the same time, the high proline content interferes with the gluten hydrolysis by digestive proteases, which leads to allergic reactions and digestive system disorders. Deep enzymatic hydrolysis contributes to the reduction of gluten allergenicity, increases its nutritional value and expands its scope in the food industry. Some complex enzyme preparations (EP) containing endo- and exopeptidases, due to their synergistic action, provide effective hydrolysis of gluten. Using Aspergillus oryzae 21-154 LAP strain, a producer of endo- and exopeptidases complex with increased leucine aminopeptidase (LAP) activity, the EP Protoorizin LAP was obtained, in terms of LAP activity comparable to Flavourzyme® 1000L widely used in the food industry, and possessing a high level of total proteolytic activity due to endopeptidases presence. During wheat gluten hydrolysis Protoorizin LAP was superior to Flavourzyme® 1000L and Alcalase 2.4L, both when used individually and when combined, by the increase in the soluble protein and free amino acids content. Electrophoretic analysis of the obtained hydrolysates showed that Protoorizin LAP intensively cleaves all major gluten proteins. After 24 h of hydrolysis, the ~14 kDa faint band remained at the level of ~37 kDa. Alcalase 2.4L cleaved wheat protein with less efficiency than Protoorizin LAP, leaving after 24 h, in addition to the ~37 kDa band, some peptides with a molecular weight of less than 14.4 kDa. Flavourzyme® 1000L had the least ability to hydrolyze gluten, however, when used together with Alcalase 2.4L, the EP contributed to a more complete hydrolysis of low molecular weight protein fractions. Protoorizin LAP successfully replaced the combined action of Flavourzyme 1000L and Alcalase 2.4L, which shows the promise of its use in the wheat gluten hydrolysates production.

New recombinant strains of Penicillium verruculosum with a high level of expression of homologous endo-β-1,4-glucanase II and heterologous phytase A of A. niger, as well as heterologous endo-1,4-β-xylanase E of P. canescens and phytase A of A. niger, are created. This allows us to obtain highly active feed enzyme preparations (EPs) capable of simultaneously significantly reducing the viscosity of nonstarch polysaccharides (NPSs), as well as increasing the bioavailability of phosphorus and minerals of grain-based feed and, thus, increasing the digestibility of nutrients of agricultural animal and poultry feed. Specific EP activities for specific substrates (xylan, β-glucan, and phytate) are studied and the qualitative and quantitative component composition of new EPs is determined. A recipient strain δnia D, into which heterologous endo-1,4-β-xylanase E of P. canescens is successfully cloned, is obtained based on a new enzyme preparation containing homologous endo-β-1,4-glucanase II and heterologous phytase A of A. niger. In this way, a promising producer of three target enzymes is obtained simultaneously.

A new recombinant Aspergillus niger tannase (tannin acyl hydrolase) produced by the Penicillium verruculosum fungus has been studied. A strain with a high level of extracellular tannase (TAN2) secretion (80% of the total extracellular protein) was obtained by cloning the tan2 gene (PDB Acc. No: MT828303) into the recipient strain. The tannase enzyme preparation degraded tannins in black tea extracts. TAN2 was isolated in homogeneous form using chromatographic methods; the enzyme had a high activity against gallotannin (53 U/mg) and less activity against propyl gallate (4.7 U/mg). Homogeneous TAN2 showed temperature and pH optima of 45 °C and 3.5, respectively. At a temperature of 50 °C, TAN2 retained above 80% activity for 3 h; at 60 °C, it retained about 75% of its activity for 90 min; at 70 °C, the enzyme was completely inactivated within 10 min. Tannase was characterized by a high tolerance to NaCl, the activity against gallotannin exceeded 50% of the initial value in solutions with a salt concentration of up to 5 M. The tannase activity was stimulated by Ca2+, Mg2+, Zn2+, Mn2+, Cu2+, Cd2+, Pb2+ by 3--64% and inhibited by 4-65% in the presence of Co2+, Fe3+ and Fe2+ ions.tannase, tannins, Aspergillus niger, Penicillium verruculosumThis work was supported by the Ministry of Science and Higher Education of the Russian Federation.

A method has been developed for the analysis of the induction of the cbh1 gene transcription in the filamentous fungus Penicillium verruculosum after treatment with mono- and oligosaccharides. The method allows obtaining mRNA, which encodes cellobiohydrolase-1 (CBH1) in an amount sufficient for RT-PCR. Citrate, a compound that does not cause carbon catabolic repression, was used as a carbon source. It was shown for the first time that xylose, gentiobiose, and mainly cellobiose induce the expression of the cbh1 gene in P. verruculosum, while sophorose and a mixture of xylooligosaccharides with a degree of polymerization from 3 to 5 are most likely to serve as precursors of inducers. Penicillium verruculosum, qPCR, RT-PCR, monosaccharides, oligosaccharides, inducer The work was supported by the Russian Foundation for Basic Research (RFBR) (grant no.18-29-07070).

Studies were conducted to obtain a complex enzyme preparation that allows efficient hydrolysis of the main plant nonstarch polysaccharides (cellulose, xylans, and pectin). The goal of the studies was to optimize the composition of a fermentation medium for the submerged cultivation of the new mutant strain T. reesei Co-44, a highly active producer of endo-carbohydrases. A concentrate of low molecular soy components was chosen as the inducer of the key carbohydrase biosynthesis by the strain. This concentrate provided the maximum (more than eightfold) increase in polygalacturonase activity and an increased level of endoglucanase and xylanase biosynthesis. After the cultivation of T. reesei Co-44 under optimized conditions, a complex enzyme preparation, Xylorizin K4, was obtained, and its physicochemical properties were studied. The presence of endopolygalacturonase (GH28) T. reesei in Xylorizin K4 was confirmed via electrophoresis and MALDI⎯TOF mass spectrometry. The studies show the potential of Xylorizin K4 application for the production of soy-protein concentrates to eliminate the main soybean nonstarch polysaccharides.

It was shown that the presence of cellobiohydrolase, β-glucosidase, endoglucanase, arabinoxylan-arabinofuranhydrolase, pectin lyase, and polygalacturonase is necessary for efficient hydrolysis of sugar beet pulp (SBP). Optimal multienzyme complexes consisting of the same enzymes and additional endoarabinase or exoarabinase, endogalactanase, β-xylosidase, endoxylanase, and/or α-arabinofuranosidase were determined. These components enabled the conversion of SBP based on a yield of total reducing sugars (RSs) of 61–68%, an arabinose yield of 94%, and a glucose yield of 63–79%. The optimal complex from dry multienzyme preparations (EP) of cellulases, hemicellulases, and pectinases, which are produced by the fungal strains Penicillium canescens, P. verruculosum, and Aspergillus foetidus, enabled SBP hydrolysis based on arabinose and glucose yields close to 100% at an initial SBP concentration of 100–250 g/L and an EP concentration of 5–10 mg protein/g SBP after 24–48 h of hydrolysis.

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.

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.

The genes of endoglucanases EG2 (36.2 kDa) Penicillium verruculosum and LAM (30.8 kDa) Myceliophthora thermophila were cloned in P. verruculosum recombinant strain. New enzyme preparations with highly stable activity against β-glucan and laminarin were obtained and investigated, homogeneous enzymes EG2 (EC 3.2.1.4) and LAM (EC 3.2.1.6) being purified and characterized. For β-glucan, the EG2 K m value was found to be 10 times higher than that for LAM; however, EG2 demonstrated greater processivity due to its higher k cat. The pH and temperature optima of EG2 and LAM activity against barley β-glucan overlapped and were 4.3–4.9 and 61–67°C, respectively, and EG2 appeared to be more stable than LAM. Oligosaccharides with degree of polymerization 2–10 were formed by hydrolysis of β-glucan and laminarin by the studied enzymes. The recombinant enzyme preparations were faster and more effective in decreasing the reduced viscosity of wholegrain barley extract than some commercial enzyme preparations. Thus, the new enzyme preparations seem to be rather perspective as feed additives for degradation of non-starch polysaccharides in grain animal feed.

The genes inuA and inu1, encoding two inulinases (32nd glycosyl hydrolase family) from filamentous fungi Aspergillus niger and A. awamori, were cloned into Penicillium canescens recombinant strain. Using chromatographic techniques, endoinulinase InuA (56 kDa, pI 3) and exoinulinase Inu1 (60 kDa, pI 4.3) were purified to homogeneity from the enzymatic complexes of P. canescens new transformants. The properties, such as substrate specificity, pH- and T-optima of activity, stability at different temperatures, influence of cations and anions on the catalytic activity, etc., of both recombinant inulinases were studied.

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.

The gene encoding the xlnR xylanolytic activator of the heterologous fungus Aspergillus niger was incorporated into the Penicillium canescens genome. Integration of the xlnR gene resulted in the increase in a number of activities, i.e. endoxylanase, β-xylosidase, α-L-arabinofuranosidase, α-galactosidase, and feruloyl esterase, compared to the host P. canescens PCA 10 strain, while β-galactosidase, β-glucosidase, endoglucanase, and CMCase activities remained constant. Two different expression constructs were developed. The first consisted of the nucleotide sequence containing the mature P. canescens phytase gene under control of the axhA promoter region gene encoding A. niger (1,4)-β-D-arabinoxylan-arabinofuranohydrolase. The second construct combined the P. canescens phytase gene and the bgaS promoter region encoding homologous β-galactosidase. Both expression cassettes were transformed into P. canescens host strain containing xlnR. Phytase synthesis was observed only for strains with the bgaS promoter on arabinose-containing culture media. In conclusion, the bgaS and axhA promoters were regulated by different inducers and activators in the P. canescens strain containing a structural tandem of the axhA promoter and the gene of the xlnR xylanolytic activator.

Для получения гидролизатов с ценными биологическими характеристиками проведена оптимизация процесса ферментативного гидролиза кератинсодержащего сырья. Увеличение весового соотношения сырья и воды, количества вносимого ферментного препарата щелочной протеазы С из Acremonium chrysogenum и повышение температуры реакционной смеси приводили к росту выхода продуктов гидролиза и их антиоксиоксидантной емкости. Молекулярные массы основных растворимых продуктов, полученных в оптимальных условиях гидролиза, составляли от 3.55 до 3.60 кДа. Продукты характеризовались высокой антиоксидантной емкостью, 100%-ной перевариваемостью и сбалансированным составом аминокислот.