A. V. Koshelev

The selection and properties of Penicillium verruculosum mutants with enhanced production of cellulases and xylanases

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 β-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 β-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.

Use of a preparation from fungal pectin lyase in the food industry

A new enzyme preparation of fungal pectin lyase (EC 4.2.2.10) was shown to be useful for the production of cranberry juice and clarification of apple juice in the food industry. A comparative study showed that the preparation of pectin lyase is competitive with commercial pectinase products. The molecular weight of homogeneous pectin lyase was 38 kDa. Properties of the homogeneous enzyme were studied. This enzyme was most efficient in removing highly esterified pectin.

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.

Application of the “fusion” approach for the production of highly efficient biocatalysts based on recombinant strains of the fungus Penicillium verruculosum for the conversion of cellulose-containing biomass

Hydrolysis of cellulose-containing biomass mediated by biocatalysts (enzyme preparations, EP) is one of the most advanced and environmentally friendly methods of obtaining a range of useful substances. A new approach to creating recombinant EPs with predefined properties, which consists in applying fusion constructs for the cloning of genes encoding target enzymes, was used in the present study. A number of EPs with different properties was derived from a strain of the fungus Penicillium verruculosum using fusion constructs; these preparations are of interest primarily as additives enhancing the hydrolytic capacity of the basic cellulolytic complex from P. verruculosum. Use of the new EPs in combination with the basic EP from P. verruculosum resulted in an increase of the biocatalytic (hydrolytic) efficiency of the latter towards cellulose-containing raw materials of plant origin. Addition of 20% of the new EP to the basic EP without changing the total EP dose in the reaction mixture resulted in a significant (up to 70%) increase of the efficiency of hydrolysis of cellulose-containing substrates (ground aspen wood and shredded deresined pine wood).

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-β-gluconase of Penicillium verruculosum; and β-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-β-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.

The production of highly effective enzyme complexes of cellulases and hemicellulases based on the Penicillium verruculosum strain for the hydrolysis of plant raw materials

Methods for the production and analysis of cellulase and hemicellulase enzyme preparations of various compositions based on the Penicillium verruculosum carbohydrase complex and intended for the effective hydrolysis of different types of cellulose-containing materials (CCMs) have been developed. New recombinant strains of P. verruculosum producing multienzyme carbohydrase complexes with increased activities of cellulases (due to the expression of endo-β-1,4-glucanases I and IV and cellobiohydrolase II from Trichoderma reesei) and hemicellulases (due to the expression of endo-β-1,4-xylanases from P. canescens and T. reesei and endo-β-1,4-mannanase from T. reesei) were constructed. The hydrolytic efficiency of the enzyme preparations (EPs) produced by the new recombinant strains during continuous hydrolysis of three CCM types (milled aspen, depitched pine wood, and milled bagasse) was studied. It was shown that new EPs containing recombinant proteins and retaining their own basic cellulase complex are characterized by the highest hydrolytic ability, exceeding that of the EP based on the original P. verruculosum strain. The recombinant enzyme preparations were highly stable; the optimal pH and temperature values for cellulase, xylanase and mannanase activities were in the range of 3.5–5.5 and 50–80°C, respectively.

Production of biocatalysts on the basis of recombinant heterologous xylanase producer strains in the Penicillium verruculosum fungus: Their application in the hydrolysis of timber and wood processing industry wastes

The aim of this work was to create biocatalysts with an increased heterologous expression of endo-β-1,4-xylanase of P. canescens using recombinant P. verruculosum strains, to analyze the properties of new enzyme preparations, and to study the saccharifying activity of these preparations in the hydrolysis of plant raw materials, such as hogged aspen and detarred pine wood wastes of the timber and wood processing industries. The xylanase activity of the existing enzymatic preparations is insufficiently high to hydrolyze a xylan-rich biomass. The creation of increasingly xylanolytically active P. verruculosum-based recombinant strains containing homologous or heterologous genes of xylanase and mannanase is therefore a problem of great interest.Using the methods of genetic engineering, we obtained enzymatic preparations that are biocatalysts for the hydrolysis of plant raw material wastes of the sawmilling and wood processing industries and, according to the data of chromatographic fractionation, have compositions of 45–60% cellulase and 20–50% xylanase (which is optimal for the saccharifying of bagasse, along with aspen and pine wood). The originality of our technique lies in the creation of biocatalysts with predetermined properties, thus reducing appreciably the cost of enzyme preparation by eliminating the need to mix components of the carbohydrase complex for the hydrolysis of plant raw materials, e.g., aspen and pine wood.

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%.

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 (Km, kcat) of the linear arabinan hydrolysis were determined.

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