Directed evolution of the bacterial endo-β-1,4-glucanase from Streptomyces sp. G12 towards improved catalysts for lignocellulose conversion

• Main Authors: Davide Agostino Cecchini, Olimpia Pepe, Anna Pennacchio, Massimo Fagnano, Vincenza Faraco
• Format: Article
• Language: English
• Published: SpringerOpen 2018-05-01
• Series: AMB Express
• Subjects: Directed evolution; High-throughput screening; Cellulase; Arundo donax; Saccharification
• Online Access: http://link.springer.com/article/10.1186/s13568-018-0602-7

Abstract 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.