ИСТИНА

The use of renewable raw materials, such as agricultural by-products, is becoming increasingly important as a starting material for new biotech products. Agricultural and forestry materials, including (ligno) cellulose or starch, are first converted to sugars, which are later converted to chemicals, bioplastics, biofuels, and pharmaceuticals by fermentation [1]. Cellulose conversion occurs under the action of a complex of cellulolytic enzymes, which include exo-1,4-β-glucanase (CBH), endo-1,4-β-glucanase (EG) and β-glucosidase(BG). Cellobiohydrolases catalyze the degradation of the crystalline sections of cellulose, sequentially, by the processive mechanism, cleaving cellobiose from the ends of the polysaccharide chain. Endoglucanases catalyze the hydrolysis of amorphous sites of cellulose, splitting internal 1,4- β-glucosidic bonds by a disordered mechanism, thereby reducing the degree of substrate polymerization and creating new sites for the action of cellobiohydrolases. β-glucosidases hydrolyze cellobiose and cellooligosaccharides to the final product - glucose [2, 3]. Lytic polysaccharide monooxygenases (LPMO) catalyze oxidative cleavage of cellulose and cello-oligosaccharides. LPMOs are able to oxidise the C–H bond of the glycoside linkage connecting the sugar units in polysaccharides, which ultimately leads to cleavage of the glycoside link and hence boost generation of fermentable sugars. These enzymes are secreted by various fungal strains and are important components of enzyme cocktails used for industrial biomass conversion [4,5]. Improvement of the properties of new enzyme coctails and fine tuning of biocatalytical processes of cellulose-to-biofuels can lead to ecologically friendly and cost-efficient alternatives to fossil based technologies. LPMO from Penicillium verruculosum was overexpressed in the same fungal strain. The LPMO enzyme preparations with the “basal” cellulolytic enzyme complex, which contains EGs, CBHs, and bG were produced. The yield of fermentable sugars was 45 and 30% higher in microcrystalline cellulose (MCC) and pretreated aspen wood (AW) hydrolysis for some preparations. Results and discussion MCC and AW was efficiently hydrolyzed by Penicillium verruculosum enzyme preparations with 5- 12% of cloned LPMO, while the efficiency decreases dramatically for preparations with higher content of LPMO. The yield of reducing sugars was ~10% higher in case of AW to compare to MCC. The yield of glucose was also higher with LPMO-enriched enzyme preparations, while the xylose yield remains approximately the same. Conclusions New high productive Penicillium fungal strains with overexpression of LPMO were developed. The yield of reducing sugars as well as glucose were up to 45% higher for enzyme preparations with 5-12% of LPMO to compare to Penicillium basal cellulase enzyme complex. The level of total reducing sugars was approx. 10% higher in case of pretreated aspen wood hydrolysis to compare to microcrystalline cellulose. The content of LPMO does not affect much the level of xylose release in case of aspen wood. References [1] R. Kumar, S.Singh, O.V.Singh, J. Industrial Microbiol.Biotechnol. 35 (2008) 377-391. [2] S.T.Merino, J.Cherry, Adv. Biochem Eng/Biotechnology 108 (2007) 95-120. [3] R.E.H. Sims, W.Mabee, J.N.Saddler, M.Taylor, Bioresource Technology 101 (2010) 1570-1580. [4] M.A.S. Kadowaki, A. Várnai, J-K. Jameson, A.E.T. Leite et al., PLoS ONE 13(2018): e0202148. https://doi.org/10.1371/journal.pone.0202148 [5] E.D. Hedegård, U. Rydea, Chem Sci. (2018) 9(15) 3866–3880.