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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.

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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%.

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Hydrolytic ability of laboratory enzyme preparations from fungus of the Penicillium genus was investigated using kraft pulp from nonbleached softwood and bleached hardwood cellulose as substrates. The enzyme preparations were shown to efficiently hydrolyze both softwood and hardwood cellulose. The yields of glucose and reducing sugars were 24-36 g/l and 27-37 g/l from 100 g/l of dry substrate in 48 h, respectively, and depended on the number of substrate grinding cycles.

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Commercial and pilot enzyme preparations from fungi of the genera Penicillium and Trichoderma have been compared with regard to their action on conifer wood pretreated with acidified aqueous ethanol (organosolve). In most experiments, enzymes from the genus Penicillium allowed higher yields of reducing sugars and glucose than those from Trichoderma. High beta-glucosidase activity is essential for deep pulp hydrolysis.

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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.

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The paper describes three Penicillium verruculosum 28K mutants with about threefold enhanced production of five industrially important carbohydrases. The two-stage fermentation process that we developed provided a further two- to threefold increase in the production of carbohydrases. Physiological and biochemical studies showed that the synthesis of all five carbohydrases is inducible. Carboxymethylcellulase, xylanase, and beta-glucanase are synthesized under a common regulatory control, as is evident from the concurrent increase in the synthesis of these enzymes in the presence of microcrystalline cellulose. The synthesis of avicelase and beta-glucosidase is evidently induced by other cellulose- and hemicellulose-containing compounds present in the fermentation medium and, hence, is regulated independently of the three aforementioned enzymes.

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Enzyme preparations were isolated from the culture liquid of five mutant strains of the cellulase producer Penicillium verruculosum. The hydrolytic activities of these preparations against unbleached eucalypt cellulose was compared to that of commercial preparations of Trichoderma reesei (T. longibrachiatum). In the majority of cases, P. verruculosum enzymes provided higher yields of reducing sugars (RSs) and glucose. A correlation was found between the yield of RSs and the avicelase activity of the preparations in the reaction mixture.

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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.

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Light-addressable potentiometric sensors were prepared by immobilizing glucose oxidase or alpha-chymotrypsin on the surface of a pH-sensitive electrode. Two methods of immobilization were used: incorporation into a hydrophilic matrix of bovine serum albumin and incorporation into a hydrophobic matrix of modified polyethylenimine. The glucose oxidase and alpha-chymotrypsin preparations immobilized in the polyethylenimine matrix were characterized by the Michaelis constant values of 6.0 and 3.1. mM, respectively, and by pH optimums at 6.0-7.2 and 7.0-8.0, respectively. These values are consistent with results obtained by other methods. Therefore, light-addressable potentiometric sensors can be used in combination with both hydrophilic and hydrophobic matrices. The ability to combine light-addressable potentiometric sensors with hydrophobic matrices provides an opportunity for the development of potentiometric biosensors for detecting substances poorly soluble in water.

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