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.

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Recombinant strains of Penicillium canescens producing homologous pectin lyase A and heterol ogous 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 prep arations (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 pec tin lyase (10%), endo 1,5 arabinase (26%),α L arabinofuranosidase and arabinoxylan arabinofuranohy drolase (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.

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. cane scens 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 optimum of temperature and pH 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.

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