Date Published: May 5, 2018
Publisher: Springer Berlin Heidelberg
Author(s): Davide Agostino Cecchini, Olimpia Pepe, Anna Pennacchio, Massimo Fagnano, Vincenza Faraco.
With the aim to develop biocatalysts for enhanced hydrolysis of (hemi)cellulose into monosaccharides, random diversity by directed evolution was introduced in the gene coding for the endo-β-1,4-glucanase from Streptomyces sp. G12 which had been recombinantly expressed in Escherichia coli and named rCelStrep. The main objectives were therefore to set up a complete strategy for creation and automated screening of rCelStrep evolved direct mutants and to apply it to generate and screen a library of 10,000 random mutants to select the most active variants. The diversity was introduced in the gene by error-prone polymerase chain reaction. A primary qualitative screening on solid plates containing carboxymethylcellulose as the substrate allowed selecting 2200 active clones that were then subjected to a secondary quantitative screening towards AZO-CMC for the selection of 76 improved variants that were cultured in flasks and characterized. Five rCelStrep mutants exhibiting the highest hydrolytic activities than the wild-type enzyme were further characterized and applied to the bioconversion of the pretreated Arundo donax lignocellulosic biomass. It is worth of noting that one of the five tested mutants exhibited a 30% improvement in bioconversion yields compared to the wild-type enzyme, despite the absence of the carbohydrate binding module domain in this variant. Homology models of the three-dimensional structures of the catalytic and binding modules of rCelStrep were obtained and localization of mutations on these models allowed us to speculate on the structure–function relationships of the mutants.
In recent decades, clean renewable resources, used as an alternative to fossil sources, have been becoming a main topic of research due to need to counteract the concerns regarding the shortage, the green-house gas emissions and global warming related to the latter ones (Liguori et al. 2013).
To improve the enzymatic activity of the β-1,4-endo-glucanase from the strain Streptomyces sp. G12 recombinantly expressed in E. coli BL21(DE3) and named rCelStrep (Amore et al. 2012), this enzyme was subjected to random biodiversity generation applying an error-prone PCR (epPCR) mutagenesis approach on the corresponding gene.