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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.

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).

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 verruculosum This work was supported by the Ministry of Science and Higher Education of the Russian Federation.

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.

We studied the autopsy material obtained from 7 children who died in the neonatal period in order to evaluate the composition of lymphocytes of the intestinal mucosa against the background of morphological changes in the tissues of the gastrointestinal tract in newborns with sepsis. The main group consisted of 4 children with neonatal sepsis, the control group – of 3 newborns who died from other causes. The research material included the specimen of the small and large intestine.Results. Small intestine: it was found that there were less CD4 + lymphocytes in the small intestinal mucosa in the group of children who died from neonatal sepsis in 75% of cases than in the control group, but this difference was not statistically significant (p=0.1). There were no differences in the number of CD8 + and CD20 + cells in the studied groups. Large intestine: the number of CD4 + lymphocytes of the mucous membrane of the colon was greater in the main group of children than in the control group (p=0.03). An increase in the number of CD4 + cells was registered in 3 of 4 cases of neonatal sepsis. The number of CD8+ and CD20+ lymphocytes in the studied groups was the same (р>0.05).Conclusion. The increase in T-lymphocytes CD4+ in the mucous membrane of the large intestine is probably connected with the antigenic stimulation of opportunistic intestinal bacteria. We found no morphological signs of the suppression of the cells of adaptive immunity associated with the intestinal mucosa.

Non-edible cellulose-containing biomass is a promising and abundant feedstock for simple sugar production. This study presents the results of different cellulose-containing materials (CCM) hydrolysis experiments with P. verruculosum enzyme complexes in laboratory conditions. Among the non-pretreated substrates, only a few had a relatively high convertibility—soy bean husks (31%) and sugar beat pulp (20%)—while wheat straw, oat husks, sunflower peals, and corn stalks had a low convertibility of 3% to 12%. This indicates that a major part of CCM needs pretreatment. Steam-exploded (with Ca(OH)2) soy bean and oat husks (76% and 58%), fine ball-milled aspen wood and nitric acid-pretreated aspen wood (62% and 78%), and steam-exploded (with sulfuric acid) corn stalks (55%) had a high convertibility. Woody biomass pretreated with pulp and paper mills also had a high convertibility (56–78%)—e.g., never dried kraft hardwood and softwood pulp (both bleached and unbleached). These results demonstrate that effective cellulose-containing material processing into simple sugars is possible. Simple sugars derived from CCM using P. verruculosum preparation are a promising feedstock for the microbiological production of biofuels (bioethanol and biobutanol), aminoacids, and organic acids (e.g., lactic acid for polylactic acid production).

The composition and properties of a wide variety of domestic and foreign enzyme preparations (EP) used as additives to feeds of farm animals and poultry are analyzed. Content of the main active enzymes – endoglucanases (beta-glucanases), cellobiohydrolases, and xylanases performing biocatalytic destruction of non-starch polysaccharides, which are anti-nutritional factors in feeds causing their incomplete digestion, is determined. It is shown that based on the component composition data and the level of different types of activity the studied enzyme preparations can be classified into three groups: a) with high xylanase and low cellulase (endoglucanase and cellobiohydrolase) content, b) with high cellulase and low xylanase content, c) containing cellobiohydrolases, endoglucanases and xylanases at a different ratio, but without significant prevalence 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 the groups b) and c) a number of preparations have been identified (Econase XT 25, Agroxyl Plus, Agroxyl Premium, Rovabio Max AP, Sunzyme), which reduced the viscosity of aqueous extract of rye containing xylans most effectively when used at the same dosage with regards to xylanase activity. It was shown that xylanase from precisely these EP was not inhibited by the protein inhibitors of rye. The viscosity of water-soluble beta-glucans of barley was most effectively reduced by the EP Xybeten CELL, Cellulase, Agroxyl, Agrocel, Axtra XB 201, Rovabio Max AP, and Vilzim used at the same dosage with regards to beta-glucanase activity. For all the 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.

Cellular immunity indicators were investigated in infants with localized (и=10) and generalized (и=15) bacterial infections (sepsis). The blood levels of different groups of white blood cells (neutrophils, lymphocytes, monocytes) and lymphocyte subpopulations (CD3, CD4, CD8, CD16/56, and CD19) were estimated. The development of sepsis was accompanied by leukocytosis and leukopenia in 47 and 20% of the cases, respectively. In localized infections, there was leukocytosis in 30% of the infants and leukopenia in 10%. Absolute lymphopenia was present in 40% of the cases of sepsis and in 20% of those of localized infections. The acute period of neonatal sepsis was accompanied by a substantial reduction in the level of CD3+, CD4+, CD8+, and CD16/56+ lymphocyte subpopulations. The highest changes were recorded in the CD4+ and CD16/56+: their counts were decreased by an average of 3,9 and 3,6 times, respectively, compared to the control values; CD8+ lymphocytes dropped by 2,7 times. Localized infections were characterized by a predominant fall in the number of CD4+ and CD16/56+ subpopulations. Moreover, the level of CD4+ lymphocytes was significantly lower than that in the sepsis group. Thus, the acute period of both localized and generalized bacterial infections took place in the presence of cellular immunity suppression. The most marked changes were recorded in sepsis. The fi

Neonatal sepsis is a significant health issue associated with high mortality. Immune responses associated with neonatal sepsis, such as proinflammatory cytokine production, are believed to play a central role in the pathogenesis of this disease. In the present study, serum levels of the proinflammatory cytokines TNF-α, IL1-β, and IL-6 and the anti-inflammatory cytokines IL-4 and IL-10 were evaluated for 25 subjects with neonatal sepsis. We observed that subjects with late onset of sepsis (LOS), as well as those with early onset of sepsis (EOS), had a substantial increase in serum TNF-α. In contrast to EOS, subjects with LOS demonstrated a significant increase in serum levels IL-6 and IL-10. Additionally, we observed a significant difference in cytokine profiles between acute and postacute cases of neonatal sepsis. For instance, the level of proinflammatory cytokines, such as TNF-α and IL-6, was elevated in the acute phase, whereas the production of anti-inflammatory cytokines, such as IL-10, became substantially upregulated during the postacute phase. Additionally, no correlation was observed between cytokine levels and CRP levels or lymphocyte counts. Thus, in contrast to CRP levels and lymphocyte counts, examination of the cytokine profile can provide valuable information when determining the most effective therapy for treating neonatal sepsis. This information may be useful to physicians when determining if anti-inflammatory or immune stimulatory therapy is warranted.

© 2017 Kh. S. Khaertynov et al.Neonatal sepsis is a significant health issue associated with high mortality. Immune responses associated with neonatal sepsis, such as proinflammatory cytokine production, are believed to play a central role in the pathogenesis of this disease. In the present study, serum levels of the proinflammatory cytokines TNF-α, IL1-β, and IL-6 and the anti-inflammatory cytokines IL-4 and IL-10 were evaluated for 25 subjects with neonatal sepsis. We observed that subjects with late onset of sepsis (LOS), as well as those with early onset of sepsis (EOS), had a substantial increase in serum TNF-α. In contrast to EOS, subjects with LOS demonstrated a significant increase in serum levels IL-6 and IL-10. Additionally, we observed a significant difference in cytokine profiles between acute and postacute cases of neonatal sepsis. For instance, the level of proinflammatory cytokines, such as TNF-α and IL-6, was elevated in the acute phase, whereas the production of anti-inflammatory cytokines, such as IL-10, became substantially upregulated during the postacute phase. Additionally, no correlation was observed between cytokine levels and CRP levels or lymphocyte counts. Thus, in contrast to CRP levels and lymphocyte counts, examination of the cytokine profile can provide valuable information when determining the most effective therapy for treating neonatal sepsis. This information may be useful to physicians when determining if anti-inflammatory or immune stimulatory therapy is warranted.

Aim. To assess the lymphocyte apoptosis intensity in children with neonatal sepsis. Methods. Lymphocyte apoptosis was assessed in 20 children [of them 16 (80%) prematurely born] with late neonatal sepsis. Bacteriology tests allowed to identify the causetive agent for sepsis in 9 (45%) cases - Staphylococcus haemolyticus in 3 cases, Klebsiella - in 4 cases, Candida - in 2 cases. The control group consisted of 10 healthy newborns. Lymphocyte apoptosis was assessed by determining the amount of mitochondrial transmembrane potential (ΔΨm). Lymphocytes were stained with fluorochrome DiOC 6. Results were obtained by flow cytometry. A decrease in mitochondrial potential value was defined as the reduction of fluorochrome DiOC 6 fluorescence . This parameter is one of the earliest signs of apoptosis. Results. An increased apoptosis was revealed, which was manifested by an increase in the number of cells with reduced membrane potential (DiOC-negative cells) in all children with neonatal sepsis. In 50% of sepsis cases (10 newborns) the number of lymphocytes in which apoptosis was triggered was higher by 3,4 times compared to control, in 25% of cases (5 newborns) - by 6 times, in another 25% (5 newborns) - by 13.4 times. Meanwhile, an absolute lymphopenia was observed only in 65% of cases. The most extensive apoptosis was observed in patients with minimal intensity of severe acute inflammatory reaction. Recovery period was characterized by a decrease in apoptosis intensity, seen as the reduction of the number of cells with reduced membrane potential by mean of 1.9 times. Reduced intensity of lymphocyte apoptosis at recovery was associated with lymphocyte count increase in peripheral blood. Conclusion. Acute phase of neonatal sepsis occurs on the background of the increased intensity of lymphocyte apoptosis.

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 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.

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 Km value was found to be 10 times higher than that for LAM; however, EG2 demonstrated greater processivity due to its higher kcat. 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.

A novel strain of bacteria (LPM-4) characterized by a unique EDTA requirement for cell growth was isolated. Suspensions of washed cells of strain LPM-4 degraded EDTA complexes with Ba2+, Mg2+, Ca2+, and Mn2+ at constant rates ( 0.310 ± 0.486 mmol EDTA/(g h)) and Zn-EDTA at an initial rate of 0.137 ± 0.016 mmol EDTA/(g h). The temperature optima for cell growth and EDTA degradation were determined under pH-auxostat cultivation. As compared with the known EDTA-degrading bacteria, strain LPM-4 exhibited a higher specific growth rate (0.095− 1) and lower mass cell yield (0.219 g cells/g EDTA), which is promising for its practical applications for EDTA removal in wastewater treatment plants.

Background Penicillium verruculosum is an efficient producer of highly active cellulase multienzyme system. One of the approaches for enhancing cellulase performance in hydrolysis of cellulosic substrates is to enrich the reaction system with β -glucosidase and/or accessory enzymes, such as lytic polysaccharide monooxygenases (LPMO) displaying a synergism with cellulases. Results Genes bglI, encoding β-glucosidase from Aspergillus niger (AnBGL), and eglIV, encoding LPMO (formerly endoglucanase IV) from Trichoderma reesei (TrLPMO), were cloned and expressed by P. verruculosum B1-537 strain under the control of the inducible gla1 gene promoter. Content of the heterologous AnBGL in the secreted multienzyme cocktails (hBGL1, hBGL2 and hBGL3) varied from 4 to 10% of the total protein, while the content of TrLPMO in the hLPMO sample was ~3%. The glucose yields in 48-h hydrolysis of Avicel and milled aspen wood by the hBGL1, hBGL2 and hBGL3 preparations increased by up to 99 and 80%, respectively, relative to control enzyme preparations without the heterologous AnBGL (at protein loading 5 mg/g substrate for all enzyme samples). The heterologous TrLPMO in the hLPMO preparation boosted the conversion of the lignocellulosic substrate by 10–43%; however, in hydrolysis of Avicel the hLPMO sample was less effective than the control preparations. The highest product yield in hydrolysis of aspen wood was obtained when the hBGL2 and hLPMO preparations were used at the ratio 1:1. Conclusions The enzyme preparations produced by recombinant P. verruculosum strains, expressing the heterologous AnBGL or TrLPMO under the control of the gla1 gene promoter in a starch-containing medium, proved to be more effective in hydrolysis of a lignocellulosic substrate than control enzyme preparations without the heterologous enzymes. The enzyme composition containing both AnBGL and TrLPMO demonstrated the highest performance in lignocellulose hydrolysis, providing a background for developing a fungal strain capable to express both heterologous enzymes simultaneously.

Complex enzymatic preparations demonstrating activities homologous to pectinlyase A and heterologous to endo-1,4-beta-glucanase from Penicilliumverruculosum and beta-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.

Optimization of the process of enzymatic hydrolysis of keratin-containing stock aimed at obtaining hydrolysates of high biological value has been performed. The increasing of the stock/water weight ratio, the amount of the alkaline protease preparation from Acremonium chrysogenium added and the temperature of the reaction mixture resulted in an increase in the yield and antioxidant capacity of hydrolysis products. The molecular masses of soluble products obtained under optimal hydrolysis conditions ranged from 3.55 to 3.60 kDa. High antioxidant capacity, 100% bioavailability and a well-balanced amino acid composition was characteristic of the hydrolysis products.

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, beta-xylosidase, alpha-L-arabinofuranosidase, alpha-galactosidase, and feruloyl esterase, compared to the host P. canescens PCA 10 strain, while beta-galactosidase, beta-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)-beta-D-arabinoxylan-arabinofuranohydrolase. The second construct combined the P. canescens phytase gene and the bgaS promoter region encoding homologous beta-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.

A novel strain of bacteria (LPM-4) was isolated that is characterized by a unique EDTA requirement for cell growth. Suspensions of washed cells of strain LPM-4 degrated EDTA complexes with Ba2+, Mg 2+, Ca2+, and Mn2+ at constant rates (0.310-0.486 mmol EDTA/(g h)) and Zn-EDTA at an initial rate of 0.137 +/- 0.016 mmol EDTA/(g h). The temperature optima for cell growth and EDTA degradation were determined under pH-auxostat cultivation. As compared with the known EDTA-degrating bacteria, strain LPM-4 exhibited a higher specific growth rate (0.095 h(-1)) and lower mass cell yield (0.219 g cells/g EDTA) that is promising for its practical applications for EDTA removal in wastewater treatment plants.