RESUMO
Thermostability is a fundamental characteristic of enzymes that is of high importance for industrial implementation of enzymatic catalysis. Cellobiohydrolases are enzymes capable to hydrolyze the most abundant natural polysaccharide - cellulose. These enzymes are widely applied in industry for processing of cellulose containing materials. However, structural and functional engineering of cellobiohydrolases for improving their properties is a challenging task. In this study, the thermostability of Penicillium verruculosum Cel7A cellobiohydrolase was increased through rational design of substitutions with proline. The stabilizing substitution G415P resulted in 3.4-fold increase in half-life time at 60 °C compared to wild-type enzyme. Molecular dynamics simulations indicated a clear effect of the stabilizing substitution G415P and the destabilizing substitutions D62P, S191P, and S273P on the stability of the enzyme tertiary structure. The stabilizing substitution G415P decreased flexibility of the lateral sides of the enzyme active site tunnel, while the considered destabilizing substitutions increased their flexibility.
Assuntos
Substituição de Aminoácidos , Celulose 1,4-beta-Celobiosidase , Proteínas Fúngicas , Simulação de Dinâmica Molecular , Mutação de Sentido Incorreto , Talaromyces , Celulose 1,4-beta-Celobiosidase/química , Celulose 1,4-beta-Celobiosidase/genética , Estabilidade Enzimática/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Domínios Proteicos , Talaromyces/enzimologia , Talaromyces/genéticaRESUMO
The pretreatment of softwood and hardwood samples (spruce and hornbeam wood) with 1-butyl-3-methylimidazolium chloride ([Bmim]Cl) was undertaken for further simultaneous enzymatic saccharification of renewable non-food lignocellulosic biomass and microbial fermentation of obtained sugars to ethanol and fumaric acid. A multienzyme cocktail based on cellulases and yeast or fungus cells producing ethanol and fumaric acid were the main objects of [Bmim]Cl influence studies. A complex effect of lignocellulosic biomass pretreatment with [Bmim]Cl on various aspects of the process (both action of cellulases and microbial conversion of hydrolysates to target products) was revealed. Positive effects of the pretreatment with [Bmim]Cl included decreasing the lignin content in the biomass, and increasing the effectiveness of enzymatic hydrolysis and microbial transformation of pretreated biomass. Immobilized cells of both yeasts and fungi possessed improved productive characteristics in the biotransformation of biomass pretreated with [Bmim]Cl to ethanol and fumaric acid.
