Molecular characterization of SCO0765 as a cellotriose releasing endo-β-1,4-cellulase from Streptomyces coelicolor A(3)

Abstract

The sco0765 gene was annotated as a glycosyl hydrolase family 5 endoglucanase from the genomic sequence of Streptomyces coelicolor A3(2) and consisted of 2,241 bp encoding a polypeptide of 747 amino acids (molecular weight of 80.5 kDa) with a 29-amino acid signal peptide for secretion. The SCO0765 recombinant protein was heterogeneously over-expressed in Streptomyces lividans TK24 under the control of a strong ermE* promoter. The purified SCO0765 protein showed the expected molecular weight of the mature form (718 aa, 77.6 kDa) on sodium dodecyl sulfate-polyacryl amide gel electrophoresis. SCO0765 showed high activity toward β-glucan and carboxymethyl cellulose (CMC) and negligible activity to Avicel, xylan, and xyloglucan. The SCO0765 cellulase had a maximum activity at pH 6.0 and 40°C toward CMC and at pH 9.0 and 50-60°C toward β-glucan. Thin layer chromatography of the hydrolyzed products of CMC and β-glucan by SCO0765 gave cellotriose as the major product and cellotetraose, cellopentaose, and longer oligosaccharides as the minor products. These results clearly demonstrate that SCO0765 is an endo-β-1,4-cellulase, hydrolyzing the β-1,4 glycosidic bond of cellulose into cellotriose.

Cellulase Promotes Mycobacterial Biofilm Dispersal in Response to a Decrease in the Bacterial Metabolite Gamma-Aminobutyric Acid

“…It showed the highest enzyme activity toward CMC at 30 • C (Figure 1g) and pH 6.0 (Figure 1h). This optimum pH of Rv0062 was similar to that of the cellulases from other bacteria, but the optimum temperature of Rv0062 was relatively low [24,25]. In addition, Mg 2+ contributes to the cellulase activity of Rv0062 with an optimum concentration of 10 mM (Figure 1i).…”

“…It showed the highest enzyme activity toward CMC at 30 °C (Figure 1g) and pH 6.0 (Figure 1h). This optimum pH of Rv0062 was similar to that of the cellulases from other bacteria, but the optimum temperature of Rv0062 was relatively low [24,25]. In With the purified Rv0062 protein, the Rv0062 catalytic reactions were performed, and the products were analyzed using thin-layer chromatography (TLC).…”

Haloferax sulfurifontis GUMFAZ2 producing xylanase‐free cellulase retrieved from Haliclona sp. inhabiting rocky shore of Anjuna, Goa‐India

“…Cell‐free supernatant of culture aliquote was obtained by centrifuging the culture broth at 10,000 g at 20°C for 10 min, which was monitored for its cellulase activity , residual sugar and extracellular protein . The degradation products of CMC‐Na were resolved on silica gel 60 F254 thin layer chromatgraphy (TLC) by developing in butanol:acetic acid:water (60:20:20 vol/vol) and visualized with 5% sulphuric acid in ethanol .…”

“…Cell-free supernatant of culture aliquote was obtained by centrifuging the culture broth at 10,000g at 20°C for 10 min, which was monitored for its cellulase activity [12], residual sugar [13] and extracellular protein [14]. The degradation products of CMC-Na were resolved on silica gel 60 F254 thin layer chromatgraphy (TLC) by developing in butanol:acetic acid:water (60:20:20 vol/vol) and visualized with 5% sulphuric acid in ethanol [15]. 2.7 | Effect of NaCl, temperature, and pH on the cellulase activity of the crude enzyme Effect of NaCl on cellulase activity was determined at 0-30% NaCl concentrations with increment of 5% NaCl.…”