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.

[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.

[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.

[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%.

[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.

[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.

[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.

[Application of neutral-alkaline pectate lyases to cotton fabric boil off].

Commercial and pilot pectate lyase preparations (EC 4.2.2.2) have been compared. They differ in their effect on pectins with different esterification degrees (ED). The activity of the pilot preparation with respect to a substrate with ED = 70% is tenfold lower than with respect to unesterified polygalacturonic acid. For commercial preparations, this activity ratio ranged within 1.5-2. At equal pectate lyase activities, the commercial preparations better remove pectin from crude cotton fabric during its boil off. The laboratory preparation is more efficient for improving the capillarity (wettability) of the fabric owing to the cooperative effect of the pectate lyase, cellulase, and hemicellulase present in the preparation.

[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 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.