Improvement of catalytic properties of the endo-1,4-b-glucanase from Penicillium spстатья
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Дата последнего поиска статьи во внешних источниках: 16 января 2019 г.
Аннотация:One of the key challenges of the modern world is a transition from a society based on the increasing use of fossil resources to a more sustainable one, where renewable resources make a growing contribution to energy and material needs. Enzymatic conversion of agricultural and forestry stocks into a wide variety of fine and bulk chemicals, bioplastics, biofuels, pharmaceuticals etc. At present, the use of renewable feed-stock, such as agricultural byproducts are gaining importance as starting material for future biotech products. Agricultural and forestry materials,including (ligno)cellulose or starch are first converted into sugars, which are subsequently transformed into a wide range of products, including ethanol, butanol, and bioplastic building blocks via fermentation. Endo-1,4-b-glucanases (EG) play important role in cellulose biotransformation by converting non-crystalline polymer regions to cellooligosaccharides with low degree of polymerization. Those products can act as the inhibitors of enzymatic reaction catalyzed by endoglucanases. Endoglucanase II (GH5) from Penicillium verruculosum was purified and crystallized in native form and in the presence of cellobiose to determine the amino acids responsible for product binding. Two of them–Trp183 and Asp-213 were selected for substitution by Phe and Ala respectively using site-directed mutagenesis. Recombinant strains EG-W183F and EG-D213A were obtained and endoglucanases were purified by AEX and HIC chromatography. Substrate specificity for native and mutated endoglucanases remains the same, while the Kcat during b-glucan hydrolysis increases 65% and 70% for EG-W183F and EG-D213A respectively. Also 50% increase of Kcat during carboxymethylcellulose hydrolysis were observed for mutated EG-s while Km remains the same for native and mutated forms. The level of inhibition by cellobiose was lower for the mutated forms of EG. The concentration of cellobiose to give the same level of activity loss was 5 and 7 times higher for EG-W183F and EG-D213A in comparison with the native protein.