Plant biomass is the main type of organic matter on Earth. Cellulose of plant biomass can be converted enzymatically into glucose, from which further can be obtained different fuels (ethanol, butanol, etc.), ethylene, organic and amino acids, polymers, feed protein and many other useful products. For bioconversion of cellulose-containing raw materials into glucose, hydrolytic enzymes of three types are required: endoglucanase, cellobiohydrolase and β-glucosidase, as well as oxidoreductase (polysaccharide monooxygenase). In this paper, we studied the possibility of improving the hydrolytic ability of the secretory enzyme complex Penicillium verruculosum by adding to it by genetic engineering methods of homologous and heterologous cellulases in various combinations and ratios: polysaccharide monooxygenase (endoglucanase IV) Trichoderma reesei , endoglucanase II and cellobiohydrolase I P. verruculosum , and β-glucosidase Aspergillus niger . The optimal ratio of the components of the cellulase complex to increase the efficiency of the enzymatic destruction of cellulose was determined, an increase in the catalytic activity of enzyme preparations obtained with the help of new recombinant strains-producers was achieved up to two times compared with the basic enzyme preparation of the initial strain of P. verruculosum .
Penicillium verruculosum, cellulases, renewable plant biomass, cellulose enzymatic conversion, genetic engineering, Penicillium verruculosum
1. Bioprocessing of renewable resources to commodity bioproducts. First edition. Wiley and Sons, ed. V.S.Bisaria, A. Kondo, New York, 2014.
2. From the Sugar Platform to biofuels and biochemical. Final report for the European Commission Directorate-General Energy, April 2015, 136 p.
3. Gusakov A.V., Sinitsyn A.P. Depolymerization of natural biopolymers. Enzymatic hydrolysis of cellulose. Chemistry of biomass: biofuels and bioplastics, Moscow, 2017, pp. 65-69.
4. Jung K., Hee J.L., In-Geol C., Kyoung Hean K. Synergistic proteins for the enhanced enzymatic hydrolysis of cellulose by cellulose. Appl.Microbiol.Biotechnol, 17 August, 2014, epub, DOI:https://doi.org/10.1007/s00253-014-6001-3.
5. Harris P. V., Welner D., McFarland, K.C., Re E., Poulsen J.C.N., Brown K., Salbo R., Ding H., Vlasenko E., Merino S., Xu F., Cherry J., Larsen S., Leggio L.L. Stimulation of lignocellulosic biomass hydrolysis by proteins of glycoside hydrolase Family 61: structure and function of large, enzymatic family. Biochemistry, 2010, vol. 49, pp. 3305-3316.
6. Skomarovsky A.A., Gusakov A.V., Okunev O.N., Soloveva I.V., Bubnova T.V., Kondrateva E.G., Synitsyn A.P. Studies of hydrolytic activity of enzyme preparation of Penicillium and Trichoderma fungi. Appl.Biochem.Microbiol, 2005, vol. 41, pp. 182-184.
7. Martins L.F., Kolling D., Camassola M., Dillon A.J., Ramos L.P. Comparison of Penicillium echinulatum and Trichoderma reesei cellulases in relation to their activity against various cellulosic substrates. Bioresource Technol., 2008, vol. 99, pp. 1417-1424.
8. Dotsenko G.S., Gusakov A.V., Rozhkova A.M., Korotkoba O.G., Sinitsyn A.P. Heterologous β-glucosidase in a fungal cellulase system: comparison of different methods for development of multienzyme cocktail. Process Biochemistry, 2015, vol. 50, pp. 1258-1263.
9. Sinitsyn A.P., Osipov D.O., Rozhkova A.M., Bushina E.V., Dotsenko G.S., Sinitsyna O.A., Kondratieva E.G., Zorov I.N., Okunev O.N., Nemashkalov V.A., Matys V.Yu., Koshelev A.V. Production of highly effective enzyme complexes of cellulases and hemicellulases for the hydrolysis of plant materials based on the strain Penicillium verruculosum. Biotechnologia, 2013, no. 5, pp. 40-53.
10. Proskurina O.V., Korotkova O.G., Rozhkova A.M., Matys V.Yu., Koshelev A.V., Okunev O.N., Nemashkalov V.A., Sinitsyna O.A., Sinitsyn A.P. The use of technology "fusion" to create high-performance biocatalysts, based on recombinant strains of the fungus Penicillium verruculosum for the conversion of biomass cellulose containing biomass. Catalysis in Industry, 2013, no. 5, pp. 65-73.
