ИСТИНА

The most promising source of raw materials for the production of -C5, -C6 sugars, used as a substrate for further microbiological synthesis, is cellulose-containing feedstocks, such as fast-growing trees (for example, poplar, aspen), lumber, pulp and paper waste, various agricultural waste - straws, stalks and corn cobs, grass, reeds, as well as solid household waste. On average, Russia annually produces 15 billion tons of plant biomass, 800 million tons of timber, 50 million tons of forest waste (mostly unused wood residues), 250 million tons of agricultural waste, and 50-60 million tons of solid municipal waste. Plant biomass and cellulose-containing feedstocks consist mainly of three components: cellulose, hemicellulose and lignin. The cellulose conversion is carried out by hydrolytic enzymes of carbohydrase complex, which includes endo-depolymerases (endoglucanases, EG), exo-depolymerase (cellobiohydrolase, CBH), and β-glucosidase (cellobiase, BG) [1]. Ascomycete fungi Trichoderma and Penicillium are the most popular producers of carbohydrases due to the high secretory ability of microorganisms at relatively low costs of cultivation. Both producers secrete a potent carbohydrase complex, and the enzyme complex produced by the Penicillium verruculosum fungus is more aggressive than the Trichoderma reesei fungal carbohydrase complex, which is the basis of most commercial enzyme preparations for the biorefinery [2,3]. Effective hydrolysis requires the use of highly active, stable and commercially available enzyme preparations. It should be noted that the efficiency of the multi-enzyme complex is ensured both by the catalytic activity of individual cellulases and by the synergistic interaction between the components of the enzyme complex. One of the goal of research of the biotechnology laboratory of enzymes FIT Biotechnology RAS is the protein engineering of individual cellulases of the fungus Penicillium verruculosum. It was shown that removal of one of the sites of N-glycosylation on the surface of the protein globule CBHI, CBHII and EGII P.verruculosum leads to an increase in the specific activity of the enzymes and does not significantly affect the properties such as thermal stability, temperature and pH-optima [4,5]. The introduction of point amino acid substitutions into positions W183F and D213A of the EGII protein globule does not lead to a change in substrate specificity, while kcat increases by 65% and 70% in the hydrolysis of -glucan, respectively. Interestingly, the degree of inhibition of cellobiose for mutated forms of W183F and D213A was lower than for native EGII [6]. Another problem of enzyme preparations of cellulases is insufficient operational stability, i.e. stability in the conditions of large-scale industrial application for bioconversion of different types of plant biomass (a number of technologies existing in this area involve the use of elevated temperatures to intensify the bioconversion process). There are several approaches to solving the problem of insufficient thermostability of cellulases. And the first of these is the rational design of the protein globule, based on standard approaches of protein engineering [7]. In particular, the replacement of amino acids in the unstructured regions of the protein globule with cysteines leads to the creation of additional disulfide bonds, which imparts rigidity to the protein globule and, as a result, leads to an increase in thermal stability. This approach was applied to the protein structures of EGII and CBHI P.verruculosum. It was shown that the introduction of a disulfide bridge in the position of S127C-A165C in EGII leads to a loss of 10% of the activity for -glucan for 10 minutes, 30% for one hour and 80% after 3 hours of incubation. While the native protein lost 30% and 50% activity after 10 minutes and an hour of incubation, respectively