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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).

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.

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.

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.

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.

Degradation of EDTA (ethylenediaminetetraacetic acid) or metal-EDTA complexes by cell suspensions of the bacterial strain DSM 9103 was studied. The activity of EDTA degradation was the highest in the phase of active cell growth and decreased considerably in the stationary phase, after substrate depletion in the medium. Exponential-phase cells were incubated in HEPES buffer (pH 7.0) with 1 mM of uncomplexed EDTA or EDTA complexes with Mg2+, Ca2+, Mn2+, Pb2+, Co2+, Cd2+, Zn2+, Cu2+, or Fe3+. The metal-EDTA complexes (Me-EDTA) studied could be divided into three groups according to their degradability. EDTA complexes with stability constants K below 10(16) (lg K16), such as Mg-EDTA, Ca-EDTA, and Mn-EDTA, as well as uncomplexed EDTA, were degraded by the cell suspensions at a constant rate to completion within 5-10 h of incubation. Me-EDTA complexes with lg K above 16 (Zn-EDTA, Co-EDTA, Pb-EDTA, and Cu-EDTA) were not completely degraded during a 24-hour incubation, which was possibly due to the toxic effect of the metal ions released. No degradation of Cd-EDTA or Fe(III)-EDTA by cell suspensions of strain DSM 9103 was observed under the conditions studied.

Mortierella alpina LPM 301, a producer of arachidonic acid (ARA), was found to possess a unique property of intense lipid synthesis in the period of active mycelium growth. Under batch cultivation of this strain in glucose-containing media with potassium nitrate or urea, the bulk of lipids (28-35% of dry biomass) was produced at the end of the exponential growth phase and remained almost unaltered in the stationary phase. The ARA content of lipids comprised 42-50% at the beginning of the stationary phase and increased continuously after glucose depletion in the medium due to the turnover of intracellular fatty acids; by the end of fermentation (189-210 h), the amount of ARA reached 46-60% of the total fatty acids (16-19% of dry mycelium). Plausible regulatory mechanisms of the growth-coupled lipid synthesis in microorganisms are discussed.

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.