Expression, purification and characterization of an endoglucanase from Serratia proteamaculans CDBB-1961, isolated from the gut of Dendroctonus adjunctus (Coleoptera: Scolytinae)

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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 {\textdegree}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 {\textdegree}C. The K m and V max values for Spr Cel8A were 6.87 mg/ml and 3.5 $μ$mol/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.

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Cano-Ramírez, C., Santiago-Hernández, A., Rivera-Orduña, F. N., García-Huante, Y., Zúñiga, G., & Hidalgo-Lara, M. E. (2016). Expression, purification and characterization of an endoglucanase from Serratia proteamaculans CDBB-1961, isolated from the gut of Dendroctonus adjunctus (Coleoptera: Scolytinae). AMB Express, 6(1). https://doi.org/10.1186/s13568-016-0233-9

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