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.

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.

[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%.

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.

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.

Purification, biochemical characterization, and structure of recombinant endo-1,4-ß-xylanase XylE.

The gene xylE encoding endo-1,4-ß-xylanase from the 10th family of glycosyl hydrolases produced by the mycelial fungus Penicillium canescens has been expressed under the control of the strong promoter of the bgaS gene encoding ß-galactosidase from P. canescens. As a result, a strain-producer of endoxylanase XylE was developed. The recombinant enzyme was isolated and purified to homogeneity with specific activity of 50 U/mg. The physicochemical and biochemical properties of the endoxylanase were studied. The maximal enzymatic activity was observed at pH 6.0 and 70°C. Endoxylanase XylE was shown to be a highly thermostable enzyme with half-inactivation period τ(1/2) of 7 h at 60°C. The kinetic parameters were 0.52 mg/ml (K(m)) and 75 µmol/min per mg (V(max)) using birch xylan as the substrate. Crystals of endoxylonase XylE were obtained, and the 3D structure was solved at 1.47 Å resolution. The 3D structure of an endo-1,4-ß-xylanase from the 10th family containing carbohydrate and unique cyclic structure located at the C-terminus of the polypeptide chain was obtained for the first time.

[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.

[Production of enzyme preparations on the basis of Penicillum canescens recombinant strains with a high ability for the hydrolysis of plant materials].

An enzyme preparation has been produced on the basis of Penicillium canescens strains with the activity of cellibiohydrolase I, II; endo-1,4-beta-gluconase of Penicillium verruculosum; and beta-glucosidase of Aspergillus niger. It was shown that for the most effective hydrolysis of aspen wood pulp the optimal ratio of cellobiohydrolase and endo- 1,4-3-gluconase in enzyme preparations was 8 : 2 (by protein). It was also established that the homologous xylanase secreted by the Penicillium canescens fungus is a required component for the enzyme complex for hydrolysis of the hemicellulose matrix of aspen wood.

[Creation of a heterologous gene expression system on the basis of Aspergillus awamori recombinant strain].

A heterologous gene expression system was created in a domestic Aspergillus awamori Co-6804 strain, which is a producer of the glucoamylase gene. Vector pGa was prepared using promoter and terminator areas of the glucoamylase gene, and A. niger phytase, Trichoderma reesei endoglucanase, and Penicillium canescens xylanase genes were then cloned into pGa vector. Separation of enzyme samples using FPLC showed the amount of the recombinant proteins to be within the 0.6-14% range of total protein.

Mucoadhesive polyelectrolyte microparticles containing recombinant human insulin and its analogs aspart and lispro.

Microparticles containing recombinant human insulin and its analogs aspart and lispro were prepared using an alternate adsorption of chitosan and dextran sulfate from solutions onto microaggregates of protein-dextran sulfate insoluble complex. The following properties of polyelectrolyte hormone-containing microparticles were studied: pH stability, surface charge, mucoadhesive properties, Ca(2+) binding, degradation under the influence of proteases (trypsin, chymotrypsin). The influence of the self-association ability of encapsulated insulins on the form of protein releasing from microparticles was studied. Insulins aspart and lispro released from the microparticles as monomers were more liable to proteolysis than human insulin released as a hexamer. The combined effect of properties of polyelectrolyte microparticles and of encapsulated recombinant proteins on the bioavailability of insulin under peroral administration is discussed.

[Increase in glucoamylase productivity of Aspergillus awamori strain by combination of radiating mutagenesis and plasmid transformation methods].

Increase in the level of amylolytic genes activator protein encoded by amyR gene was shown to result in enhancement of glucoamylase productivity of A. awamori strain by 30%. However, the same effect equal to 30% increase can be achieved by introduction of extra copies of gla gene encoding glucoamylase. These two effects were not additive, which gave the possibility to suggest an additional limitation in the egulation mechanism of glucoamylase gene expression in Aspergillus family strains while introducing an additional copies of amyR and gla genes.

Enzymological properties of endo-(1-4)-beta-glucanase Eg12p of Penicillium canescens and characteristics of structural gene egl2.

Gene egl2 of secreted endo-(1-4)-beta-glucanase of glycosyl hydrolase family 5 of the mycelial fungus Penicillium canescens was cloned. The gene was expressed in P. canescens under control of a strong promoter of the bgaS gene encoding beta-galactosidase of P. canescens, and endoglucanase producing strains were obtained. Chromatographically purified recombinant 48 kDa protein had pH and temperature optima 3.4 and 60 degrees C, respectively, exhibited specific activity of 33 IU, and had K(m) and V(max) in CM-cellulose hydrolysis of 10.28 g/liter and 0.26 micromol/sec per mg, respectively.

[Stabilization of alkaline proteinase and cellulases via complex formation with chitosan for use as detergent components].

An effective approach to the stabilization of hydrolytic enzymes (alkaline proteinase and cellulases) via the complex formation with chitosan for their further use as detergent components has been developed. Interaction with chitosan results in a 35-50% increase in the level of catalytic activity of the enzymes after incubation for 60 min under the conditions of detergent use (alkaline pH, increased temperature, the presence of anionic surfactants) as compared to the system in the absence of chitosan both due to the enzyme stabilization and the increase of the starting level of catalytic activity. A twofold decrease of the enzyme inactivation constant is observed under the aforementioned conditions in the case of alkaline proteinase. In the case of cellulase preparation, the method for the control of the concentration of the active enzyme in the system modeling synthetic detergents has been suggested. The method is based on the enzymatic destruction of the stabilizing agent, chitosan, by enzymes of the cellulase complex. The destruction of chitosan removed the stabilizing effect, thus resulting in the inactivation of cellulases. The developed approaches allow for the widening of the field of the possible application of enzymes as detergent components.

Isolation and properties of fungal beta-glucosidases.

Using chromatography on different matrixes, three beta-glucosidases (120, 116, and 70 kDa) were isolated from enzymatic complexes of the mycelial fungi Aspergillus japonicus, Penicillium verruculosum, and Trichoderma reesei, respectively. The enzymes were identified by MALDI-TOF mass-spectrometry. Substrate specificity, kinetic parameters for hydrolysis of specific substrates, ability to catalyze the transglucosidation reaction, dependence of the enzymatic activity on pH and temperature, stability of the enzymes at different temperatures, adsorption ability on insoluble cellulose, and the influence of glucose on catalytic properties of the enzymes were investigated. According to the substrate specificity, the enzymes were shown to belong to two groups: i) beta-glucosidase of A. japonicus exhibiting high specific activity to the low molecular weight substrates cellobiose and pNPG (the specific activity towards cellobiose was higher than towards pNPG) and low activity towards polysaccharide substrates (beta-glucan from barley and laminarin); ii) beta-glucosidases from P. verruculosum and T. reesei exhibiting relatively high activity to polysaccharide substrates and lower activity to low molecular weight substrates (activity to cellobiose was lower than to pNPG).

[Cloning of the Penicillium canescens endo-1,4-beta-glucanase gene egl3 and the characterization of the recombinant enzyme].

The gene egl3 of the filamentous fungus Penicillium canescens endo-1,4-beta-glucanase, belonging to family 12 glycosyl hydrolases, was cloned and sequenced. The gene was expressed in P. canescens under the control of the strong promoter of gene bgaS, coding for beta-galactosidase of this fungus, and efficient endoglucanase producer strains were obtained. The recombinant protein was isolated from the culture liquid of the producer strain EGL3-13 and purified to homogeneity; its specific activity was 31.7 IU; molecular weight, 26 kDa; and pH and temperature optimums, 3.2 and 54 degrees C, respectively. The Km and Vm values for CMC hydrolysis were determined; they amounted to 17.1 g/1 and 0.31 microM/(mg s), respectively.

[Hydrolysis of wheat flour with amylase preparations and individual enzymes].

Rheological properties of wheat flour were studied in the course of its processing (cooking and saccharification). The effects of commercial alpha-amylase preparations were compared during flour preparation. Test preparations were equally potent in decreasing the viscosity of an all-grain batch. Homogenous glucoamylases isolated from Aspergillus differed in the presence or absence of the starch-binding domain. The starch-binding domain provided for the high activity of glucoamylase on insoluble starch, but gave no advantages in saccharification of pretreated wheat flour.

[Effectiveness of enzyme preparations of fodder in the degradation of nonstarch polysaccharides from grain substrates].

The effects of commercial and laboratory preparations were compared in the course of treatment of components of compound fodder. The most potent preparations were selected for the treatment of soybean flower, sunflower meal, and wheat and barley flour. Preparation 181-1008, which had a high proteinase activity, provided the highest yield of protein from soybean flour and sunflower meal. Preparations aGA, AG20X, and VR, characterized by high activities of pectinase and alpha-galactosidase, as well as laboratory preparation B2000Mix with a high activity of alpha-galactosidase, provided the highest yield of sugars from soybean flour. Preparations with high alpha-galactosidase activity were the most potent in hydrolyzing soluble carbohydrates from soybean flour. The highest yield of reducing sugars was observed after treatment of wheat and barley flour with preparations B2000Mix and aGa. Xylanase activity of these preparations was lower than that of preparations 3.130.2 and TG20X. Preparations 3.130.2 and TG20X were the most potent in hydrolyzing wheat middlings.

[Isolation and properties of cellobiase from Penicillium verruculosum]. / Vydelenie i svoistva tsellobiazy iz Penicillium verruculosum.

Cellobiase (beta-D-glucosidase) with a molecular weight of 100 kDa and pI 5.2 was isolated from the cellulolytic system of Penicillium verruculosum. Kinetic parameters of enzymatic hydrolysis of cellobiose, gentiobiose, sophorose, and synthetic substrates, i.e. methylumbelliferyl and p-nitrophenyl sugar derivatives were determined. Glucose and D-glucose-delta-lactone competitively inhibited cellobiase (Ki = 0.19 mM and 17 microM, respectively). Glucosyl transfer reactions were studied with cellobiose as a single substrate and in the mixture of cellobiose and methylumbelliferyl cellobioside. The product composition was determined in these systems. The ratio of hydrolysis and transfer reaction rates for cellobiose conversion was calculated.

Electrochemical assay of endo-depolymerase activity of cellulases.

A method for determination of endo-1,4-beta-D-glucanase activity of cellulase samples based on the indirect measurement of decrease in viscosity of a carboxymethylcellulose solution in an electrochemical cell in the presence of an electron carrier was developed. A rotating disk electrode is used as the working electrode. When two reactions (enzymatic and electrochemical) proceeded in the cell simultaneously, the limiting diffusion current at a constant applied potential increases as the viscosity of the solution decreases. Conditions where the initial rate of change of diffusion current (dI/dt) is proportional to the enzyme concentration were found. A good correlation between the new method and a previously known viscometric method for determination of endoglucanase activity was observed.