Properties of recombinant endo-ß-1,6-glucanase from Trichoderma harzianum and its application in the pustulan hydrolysis.

The gene encoding Trichoderma harzianum fungus pustulanase (ThBGL1.6, GH5 family, endo-ß-1,6-glucanase, EC 3.2.1.75) was cloned and heterologously expressed by the highly productive Penicillium verruculosum fungus. The recombinant ThBGL1.6 was purified and its properties were studied. The ThBGL1.6 had an observed molecular mass of 46 kDa (SDS-PAGE data) and displayed maximum of the enzyme activity at pH 5.0 and 50 °C. At 45 °C, the ThBGL1.6 was stable for at least 3 h. The Km was 1.0 g/L with pustulan as the substrate. Reaction product analysis by HPLC clearly indicated that ThBGL1.6 has an endo-hydrolytic mode of action against pustulan as specific substrate. It was also identified that gentiobiose is the main reaction product at studying of long-term pustulan hydrolysis.

Creation of Chitinase Producer and Disruption of Micromycete Cell Wall with the Obtained Enzyme Preparation.

A recombinant strain producing a complex of extracellular enzymes including chitinase from Myceliophtora thermophila was created based on the fungus Penicillium verruculosum. The activity of the enzyme preparations obtained from the cultural fluid of the producer strain was 0.55, 0.53, and 0.66 U/mg protein with chitin and chitosans with the molecular weight of 200 and 1000 kDa, respectively. The temperature optimum for the recombinant chitinase was 52-65°C; the pH optimum was 4.5-6.2, which corresponded to the published data for this class of the enzymes. The content of heterologous chitinase in the obtained enzyme preparations was 47% of total protein content in the cultural fluid. Enzyme preparations produced by the recombinant P. verruculosum XT403 strain and containing heterologous chitinase were able to degrade the mycelium of micromycetes, including phytopathogenic ones, and were very efficient in the bioconversion of microbiological industry waste.

Isolation of Homogeneous Polysaccharide Monooxygenases from Fungal Sources and Investigation of Their Synergism with Cellulases when Acting on Cellulose.

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.

A New Bacillus licheniformis Mutant Strain Producing Serine Protease Efficient for Hvdrolvqis of Sov Meal Proteins.

Induced mutagenesis with y-irradiation of the industrial strain Bacillus licheniformis-60 VKM B-2366,D was used to obtain a new highly active producer of an extracellular serine protease, Bacillus licheni- formis7 145. Samples of dry.concentrated preparations of serine protease produced by the original and mutant strains were obtained, and identity of their protein composition was'established. Alkaline serine protease sub- tilisin DY was the main component of the preparations. The biochemical and physicochemical properties of the Protolkheterm-145 enzyme preparation obtained from the mutant strain were studied. It exhibited pro- teolytic activity (1.5 times higher than the preparation from the initial strain) within broad ranges of pH (5- 11) and temperature (30-70'C).-Efficient hydrolysis of extruded soy meal protein at high concentrations (2 to 50%) in-the reaction mixture was.the main advantage of the Protolikheterm 145 preparation. Compared to,. the preparation obtained using the initial strain, the new preparation with increased proteolytic-activity pro- vided for more complete hydrolysis of the main non-nutritious soy,proteins.(glycinin and 0-conglycinin) with the yield of soluble protein increased by 19-28%, which decreased the cost of bioconversion of the protein- aceous material and indicated promise of the new preparation in resource-saving technologies for processing soy meals and cakes.

[Novel Enzyme Preparations with High Pectinase and Hemicellulase Activity Based on Penicillium canescens Strains].

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.

[Construction of Producers of Cellulolytic and Pectinolytic Enzymes Based on the Fungus Penicillium verruculosum].

Based on the fungus Penicillium verruculosum, we created strains with a complex of extracellular enzymes that contains both cellulolytic enzymes of the fungus and heterologous pectin lyase A from P. canescens and endo- 1,4-α-polygalacturonase from Aspergillus niger. The endopolygalacturonase and pectin lyase activities of enzyme preparations obtained from culture media of the producer strains reached 46-53 U/mg of protein and 1.3-2.3 U/mg of protein, respectively. The optimal temperature and pH values for recombinant pectin lyase and endopolygalacturonase corresponded to those described in the literature for these enzymes. The content of heterologous endopolygalacturonase and pectin lyase in the studied enzyme preparations was 4-5% and 23% of the total protein content, respectively. The yield of reducing sugars upon the hydrolysis of sugar beet and apple processing wastes with the most efficient preparation was 41 and 71 g/L, respectively, which corresponded to a polysaccharide conversion of 49% and 65%. Glucose was the main product of the hydrolysis of sugar beet and apple processing wastes.

[SUPPRESSION OF TUMOR GROWTH AFTER XENOGRAFTING OF HUMAN COLORECTAL CARCINOMA CELLS].

Previously we've described the obtainment of a subpopulation of cancer stem cells from a human colorec- tal carcinoma cell line MIP101. These cells possess elevated clonogenic and tumorigenic capacities. According to our data, depletion of stem compartment in a cancer cell population blocks its tumorigenicity. The current work is dedicated to the comparison of tumorigenic potential between cell populations with enriched or depleted stem compartment. We show that tumor growth following xenografting of enriched stem cell population can be suppressed by intramuscular injections of ganciclovir. Thus, we report a method to obtain a cell population with high Oct4 promoter expression within the MIP101 colorectal carcinoma cell line and to eliminate these cells from the population in vitro as well as in vivo.

[Cultivation of a novel cellulase/xylanase producer, Trichoderma longibrachiatum mutant TW 1-59-27: production of the enzyme preparation and the study of its properties].

As a result of gamma-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 degrees C; it occurred in xylanase at 60 degrees 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 secretion of endoglucanase-1. An enzyme preparation with increased endoglucanase-1 content is promising for use as a feed additive in agriculture.

Properties of enzyme preparations and homogeneous enzymes - endoglucanases EG2 Penicillium verruculosum and LAM Myceliophthora thermophila.

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 ß-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 ß-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 ß-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 ß-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 New Enzyme Preparation with High Penicillopepsin Activity Based on the Producer Strain Penicillium canescens].

The producer of fungal penicillopepsin, an aspartate protease, has been created by genetic engineering. The biochemical and physicochemical properties of the penicillopepsin enzyme preparation obtained from the culture liquid of the producer were studied. Properties of the new enzyme preparation and the commercially available aspergillopepsin were compared. Their proteolytic activities were found to be 670-680 U/g of the preparation. The soluble protein yield upon the wheat flour hydrolysis with penicillopepsin was 2.7 times higher than with aspergillopepsin. It is probably caused by the presence of the xylanase activity in the penicillopepsin preparation.

[Use of Endoglucanase IV from Trichoderma reesei to Enhance the Hydrolytic Activity of a Cellulase Complex from the Fungus Penicillium verruculosum].

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%.

[Hydrolysis of chitozan with an enzyme complex from Myceliophthora sp].

Samples of low-molecular weight chitosan with molecular masses of 8-24 kDa, identical deacetylation degrees (85%), and polydispersity indexes soluble at pH 5-7 were obtained by enzymatic hydrolysis using an enzyme complex from the micelial fungi Myceliophthora fergusii with yields of 50-80%. The optimal conditions for hydrolysis were found (pH 5.6, 37 degrees C, an enzyme/substrate ratio 1/800, 15-60 min). The obtainment of chitosan sample sets with different characteristics will enable the selection of the most efficient ones for comparison in in vitro/in vivo experiments.

Comparative study of biochemical properties of glucoamylases from the filamentous fungi Penicillium and Aspergillus.

Here we report the first isolation to homogeneous forms of two glucoamylases from the fungus Penicillium verruculosum and their study in comparison with known glucoamylases from Aspergillus awamori and Aspergillus niger. Genes that encode glucoamylases from P. verruculosum were cloned and expressed in the fungus Penicillium canescens, and the recombinant glucoamylases were obtained with subsequent study of their molecular weights, isoelectric points, optimal temperature and pH values, and stability. The catalytic activities of the recombinant glucoamylases were determined in relation to soluble potato starch. Changes in molecular mass distribution and content of low molecular weight products during starch hydrolysis by glucoamylases from P. verruculosum, A. awamori, and A. niger were studied. An exo-depolymerization mechanism was established to be the pathway for destruction of starch by the glucoamylases.

Two novel GH11 endo-xylanases from Myceliophthora thermophila C1 act differently toward soluble and insoluble xylans.

Two novel GH11 endo-xylanases from Myceliophthora thermophila C1 (C1), Xyl7 and Xyl8, were purified and the influence of solubility and molecular structure of various xylans on their efficiency was investigated. Both endo-xylanases were hindered by a high degree of substitution of a xylan. The two GH11 xylanases released different products from the xylans, in which Xyl7 displayed a degradation product composition closer to GH10 xylanases. A correlation of the degradation product composition with a specific residue at position 163 in the amino acid sequence of Xyl8 is suggested: tyrosine in Xyl8; valine in Xyl7. This is confirmed with examples of various endo-xylanases reported in literature. The C1 GH11 xylanases were more efficient on self-associated xylan compared to C1 GH10 endo-xylanases and they released more small xylooligomers from these xylans. This is contrary to the general assumption that GH10 xylanases degrade xylans to a higher degree than GH11 xylanases.

[A comparative analysis of colorectal carcinoma cell lines that differ in metastatic potential].

In the current work we make an attempt to compare cancer cells of one origin, but differing in the expression of CEA protein, a clinical marker of metastatic carcinomas, presumably one of the key factors in metastatic activity. We have explored the morphology of cell colonies in vitro, expression patterns of epithelial markers, the ability of these cells to form tumors and metastases in vivo, and evaluated their stem compartment with the aid of a suicidal genetic construct sensitive to the embryonic stem cell marker, Oct4.

[Investigation of transcriptional regulation of Oct4 (POU5F1) gene with distal enhancer].

Investigations of transcriptional regulation of Oct4 gene in mouse embryonic stem cells have revealed an important cis-element--the distal enhancer (DE). DE consists of two functionally significant elements--DEa and DEb. Both elements are necessary to complete the DE-mediated expression of Oct4 gene in pluripotent cells. The most likely candidates for the binding site DEb are Oct4 itself in complex with Sox2 protein. It remains unclear which transcriptional proteins bind to the DEa site and what is the mechanism of the co-operation between the DEa and the DEb. Through the use of using the EMSA and chromatographic fractionation of proteins from extracts of mouse embryonic stem cells and mouse tissues, were isolated proteins specifically interacting with the sequence DEa Oct4 gene.

[In vitro derivation and characterization of a colorectal cancer stem cell subpopulation].

In present publication we describe for the first time the obtainment of cancer stem cells from a weakly metastatic human colorectal carcinoma cell line MIP101 via selecting from the native population the cells that express intensively an embryonic stem cell marker, POU5F1 (Oct4). We provide the evidence that these cells possess an elevated clonogenic and tumorigenic potential when compared to the native population, and this correlates to the hypothesis of cancer stem cells' primary role in the development of malignant neoplasms.

Cloning, purification, and characterization of galactomannan-degrading enzymes from Myceliophthora thermophila.

Genes of ß-mannosidase 97 kDa, GH family 2 (bMann9), ß-mannanase 48 kDa, GH family 5 (bMan2), and α-galactosidase 60 kDa, GH family 27 (aGal1) encoding galactomannan-degrading glycoside hydrolases of Myceliophthora thermophila C1 were successfully cloned, and the recombinant enzymes were purified to homogeneity and characterized. bMann9 displays only exo-mannosidase activity, the K(m) and k(cat) values are 0.4 mM and 15 sec(-1) for p-nitrophenyl-ß-D-mannopyranoside, and the optimal pH and temperature are 5.3 and 40°C, respectively. bMann2 is active towards galactomannans (GM) of various structures. The K(m) and k(cat) values are 1.3 mg/ml and 67 sec(-1) for GM carob, and the optimal pH and temperature are 5.2 and 69°C, respectively. aGal1 is active towards p-nitrophenyl-α-D-galactopyranoside (PNPG) as well as GM of various structures. The K(m) and k(cat) values are 0.08 mM and 35 sec(-1) for PNPG, and the optimal pH and temperature are 5.0 and 60°C, respectively.

[Development of complex enzymatic preparations of pactinases and celulases for sugar beet marc digestion].

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.

Isolation and properties of recombinant inulinases from Aspergillus sp.

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.