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. 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). 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. Rational design of the protein globule, based on standard approaches of protein engineering, was applied to cellobiohydrolase I (CBHI) from Penicillium verruculosum to improve enzyme thermostability [1]. 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 (DSB), which imparts rigidity to the protein globule and, as a result, leads to an increase in thermal stability. This approach was applied to design CBHI protein globule, because CBHI is a key enzyme of hydrolytical complex to destroy cellulose- most abundant biolpolymer in plant cell wall. As a result the introduction of DSB into positions 4 and 72 leads to stabilization of the CBHI at 70°C and also to a noticeable stabilization at 80 ° C. It should be noted that the change in the thermoinactivation curve in the case of the disulfide bridge introduction has a changed form and corresponds to the type of thermal stabilization. Molecular dynamics calculation proved positive effect of CBHI thermostabilization.