title: Expression, purification and characterization of an endoglucanase from Serratia proteamaculans CDBB-1961, isolated from the gut of Dendroctonus adjunctus (Coleoptera: Scolytinae).
abstract: Serratia proteamaculans CDBB-1961, a gut symbiont from the roundheaded pine beetle Dendroctonus adjunctus, displayed strong cellulolytic activity on agar-plates with carboxymethyl cellulose (CMC) as carbon source. Automatic genome annotation of S. proteamaculans made possible the identification of a single endoglucanase encoding gene, designated spr cel8A. The predicted protein, named Spr Cel8A shows high similarity (59-94 %) to endo-1,4-β-D-glucanases (EC 3.2.1.4) from the glycoside hydrolase family 8 (GH8). The gene spr cel8A has an ORF of 1113 bp, encoding a 371 amino acid residue protein (41.2 kDa) with a signal peptide of 23 amino acid residues. Expression of the gene spr cel8A in Escherichia coli yields a mature recombinant endoglucanase 39 kDa. Cel8A displayed optimal activity at pH 7.0 and 40 °C, with a specific activity of 0.85 U/mg. The enzyme was stable at pH from 4 to 8.5, retaining nearly 40-80 % of its original activity, and exhibited a half-life of 8 days at 40 °C. The K m and V max values for Spr Cel8A were 6.87 mg/ml and 3.5 μmol/min/mg of protein, respectively, using CMC as substrate. The final principle products of Spr Cel8A-mediated hydrolysis of CMC were cellobiose, cello oligosaccharides and a small amount of glucose, suggesting that Spr Cel8A is an endo-β-1,4-glucanase manifesting exo-activity. This is the first report regarding the functional biochemical and molecular characterization of an endoglucanase from S. proteamaculans, found in the gut-associated bacteria community of Dendroctonus bark beetles. These results contribute to improved understanding of the functional role played by this bacterium as a symbiont of bark beetles.
authors: Departamento de Biotecnología y Bioingeniería, CINVESTAV-IPN, México City, México.
authors: Claudia
authors: C
authors: Cano-Ramírez
authors: Departamento de Biotecnología y Bioingeniería, CINVESTAV-IPN, México City, México.
authors: Alejandro
authors: A
authors: Santiago-Hernández
authors: Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, México City, México.
authors: Flor Nohemí
authors: FN
authors: Rivera-Orduña
authors: Departamento de Biotecnología y Bioingeniería, CINVESTAV-IPN, México City, México.
authors: Yolanda
authors: Y
authors: García-Huante
authors: Departamento de Zoología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, México City, México.
authors: Gerardo
authors: G
authors: Zúñiga
authors: Departamento de Biotecnología y Bioingeniería, CINVESTAV-IPN, México City, México. ehidalgo@cinvestav.mx.
authors: María Eugenia
authors: ME
authors: Hidalgo-Lara
keywords: Bark beetles; Cellulase; Gut; Serratia proteamaculans; Symbiosis