Fig. 5

Whole cell activity assays of P. putida displaying BglA (a), CelK (b) and CelA (c). The cells were cultivated to an OD of 0.5, protein expression was induced by the addition of 0.2 % l-arabinose, and after 4 h of further cultivation the cells were harvested and resuspended in 50 mM sodium citrate buffer, pH 6, to a final OD of 1 (CelK) and 40 (BglA and CelA). The cells were then mixed 1:1 with 10 mM 4-nitrophenyl-β-d-glucopyranoside (BglA), 10 mM 4-nitrophenyl-β-d-cellobioside (CelK) or 2 % carboxymethylcellulose (CelA) and incubated at 55 °C. For BglA and CelK, the generated 4-nitrophenol was quantified by measuring the absorption of the cell supernatant at 405 nm. For CelA, the amount of reducing sugars in the cell supernatant was determined by means of a DNS assay [33]. P. putida without plasmid served as control, no hydrolytic activity could be detected with these cells (not shown)