Functional characterization of GH7 endo-1,4- β-glucanase from Aspergillus fumigatus and its potential industrial application

Publication date: October 2018 Source:Protein Expression and Purification, Volume 150 Author(s): Aline Vianna Bernardi, Paula Fagundes de Gouvêa, Luis Eduardo Gerolamo, Deborah Kimie Yonamine, Laís de Lourdes de Lima Balico, Sergio Akira Uyemura, Taisa Magnani Dinamarco A gene encoding an endo-1,4-β-glucanase (Afu6g01800) from A. fumigatus was cloned into the vector pET-28a(+) and expressed in the E. coli strain RosettaTM (DE3) pLysS. Sequence analysis indicated that the enzyme Af-EGL7 belonged to the GH7 family. The gene Af-egl7 encoded a protein comprising 460 amino acids, with a CBM1 domain at residues 424–460 and molecular mass of 52 kDa, as estimated by SDS-PAGE. This enzyme was optimally active at pH and temperatures ranging from 4.5 to 5.5 and from 40 to 60 °C, respectively. Mn2+ addition significantly enhanced the Af-EGL7 cellulase activity by 233%, whereas SDS addition fully inhibited this activity. Higher activity was observed toward β-glucan than toward xyloglucan and CM-Cellulose, suggesting that the enzyme corresponds to a β-1,3-1,4-glucanase. qRT-PCR in different culture media helped to establish the time-course expression profile. Different polysaccharides induced the gene Af-egl7 in a time-dependent manner; in the particular case of the substrate sugarcane exploded bagasse (SEB), Af-egl7 was induced 2500-fold. Upon addition to a commercial cellulase cocktail, Af-EGL7 significantly improved SEB saccharification, which suggested that the enz...
Source: Protein Expression and Purification - Category: Biochemistry Source Type: research