Quantification of the Genetic Expression of bgl-A, bgl, and CspA and Enzymatic Characterization of β-Glucosidases from Shewanella sp. G5

HÉCTOR A. CRISTÓBAL; HUGO R. POMA; CARLOS M. ABATE; VERÓNICA B. RAJAL

MARINE BIOTECHNOLOGY

SPRINGER

Lugar: Berlin; Año: 2016 vol. 18 p. 396 - 408

1436-2228

Shewanella sp. G5, a psychrotolerant marine bacterium,has a cold-shock protein (CspA) and three β-glucosidases,two of which were classified in the glycosyl hydrolasefamilies 1 and 3 and are encoded by bgl-A and bgl genes,respectively. Shewanella sp. G5 was cultured on Luria-Bertani (LB) and Mineral Medium Brunner (MMB) mediawith glucose and cellobiose at various temperatures andpH 6 and 8. Relative quantification of the expression levelsof all three genes was studied by real-time PCR with the comparativeCt method (2-ΔΔCt) using the gyrB housekeepinggene as a normalizer. Results showed that the genes had remarkablydifferent genetic expression levels under the conditionsevaluated, with increased expression of all genes obtainedonMMB with cellobiose at 30 °C. Specific growth rate andspecific β-glucosidase activity were also determined for allthe culture conditions. Shewanella sp. G5 was able to growon both media at 4 °C, showing the maximum specific growthrate on LB with cellobiose at 37 °C. The specific β-glucosidase activity obtained on MMB with cellobiose at30 °C was 25 to 50 % higher than for all other conditions.At pH 8, relative activity was 34, 60, and 63%higher at 30 °Cthan at 10 °C, with three peaks at 10, 25, and 37 °C on bothmedia. Enzyme activity increased by 61 and 47 % in thepresence of Ca2+ and by 24 and 31 % in the presence ofMg2+ on LB and MMB at 30 °C, respectively, but it wastotally inhibited by Hg2+, Cu2+, and EDTA. Moreover, thisactivity was slightly decreased by SDS, Zn2+, and DTT, allat 5 mM. Ethanol (14 %v/v) and glucose (100 mM) alsoreduced the activity by 63 and 60 %, respectively.