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

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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 hydrolase families 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) media with glucose and cellobiose at various temperatures and pH6 and 8. Relative quantification of the expression levels of all three genes was studied by real-time PCR with the comparative Ct method (2(-ΔΔCt)) using the gyrB housekeeping gene as a normalizer. Results showed that the genes had remarkably different genetic expression levels under the conditions evaluated, with increased expression of all genes obtained on MMB with cellobiose at 30°C. Specific growth rate and specific β-glucosidase activity were also determined for all the culture conditions. Shewanella sp. G5 was able to grow on both media at 4°C, showing the maximum specific growth rate on LB with cellobiose at 37°C. The specific β-glucosidase activity obtained on MMB with cellobiose at 30°C was 25 to 50% higher than for all other conditions. At pH8, relative activity was 34, 60, and 63% higher at 30°C than at 10°C, with three peaks at 10, 25, and 37°C on both media. Enzyme activity increased by 61 and 47% in the presence of Ca(2+) and by 24 and 31% in the presence of Mg(2+) on LB and MMB at 30°C, respectively, but it was totally inhibited by Hg(2+), Cu(2+), and EDTA. Moreover, this activity was slightly decreased by SDS, Zn(2+), and DTT, all at 5mM. Ethanol (14% v/v) and glucose (100mM) also reduced the activity by 63 and 60%, respectively.