Increase in thermostability of the 1,4-b-glucanase from Penicillium sp. by site-directed mutagenesisстатьяКраткое сообщение
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Дата последнего поиска статьи во внешних источниках: 20 января 2021 г.
Аннотация:Efficient cellulosic biomass hydrolysis is a critical step in the production of biofuels and high value chemicals from non-food plant ligno-cellulosic biomass. At the most, the most efficient way to convert cellulose to fermentable sugars for the production of bioethanol, for example, or more valuable compounds is the use of cellulolytic enzyme cocktail, but the conversion is highly inefficient at the momentfor the any reasons, including low thermostability of individual enzymes. Thus, the goal of the work was to develop mutated 1,4-b-glucanase, one of the most important component of cellulase cocktail,which has a higher thermostability than the native form of the enzyme.This can be important when using new recombinant enzyme in the bleaching processes of cellulose pulp and also when used in agriculure for granulation of feed.Creation of–S-S- bridges is one of the most popular approaches to increase thermostability, since it makes it possible to strengthen the rigidity of the protein structure. Using site-directed mutagenesis, 3additional disulphide bridges were introduced into the protein globule of 1,4-b-glucanase from micelial fungi Penicillium verruculosum. New recombinant strains have been obtained, mutant homogenous enzymes isolated by chromatographic methods and described by their biochemial (enzymatic activity to soluble polymer substrates, pH-optimum, T-optimum) and kinetic properties.As a result, the introduction of two of the three disulfide bridges led to a stabilization of the protein globule. Especially noticeable effect was at 70 and 80C. For example, the mutant T2 lost 10% of activity to b-glucan in 10 min, 30% in one hour, and 80% after 3 h of incubation. While the native protein lost 30% and 50% for 10 min and anhour of incubation, respectively, and after 2 h native 1,4-b-glucanase was completely inactivated. It is interesting to note that the effects of stabilization were observed to b-glucan, and were not observed when carboxymethylcellulose was used as a substrate