Fig. 8
Primary screening of an E. coli random mutagenesis clone library expressing variants of endoglucanase celA2 applying the RoboLector system. a On-line biomass signal (monitored via scattered light) for a two-step cultivation of 46 E. coli clones consisting of one not induced pre-cultivation and an induced main cultivation. Additionally, a not induced wildtype and plain media served as references. The main cultivation was automatically inoculated from the previous cultivation step. b Volumetric cellulase activity (via 4-MUC assay) of the supernatant after cellulase extraction from E. coli cells including standard deviations of three independent main cultivations. Reference activity of the clone expressing the original enzyme indicated by dashed line and white bar. Not induced wildtype and media are depicted as clone no. 47 and 48. c) Parity plot comparing volumetric and biomass-specific cellulase activities of the supernatants after cellulase extraction from E. coli cells of the main cultivation. For calculation of biomass specific activities, the final scattered light values of the main cultivation were used. Wildtype clone expressing the original enzyme indicated by circle. Cultivation conditions: 96well MTP, VL = 150 μL, n = 995 rpm, d0 = 3 mm, pre-cultivation and main cultivation in Wilms-MOPS medium containing 15 g L−1 glucose, cellulase expression automatically induced by adding 0.1 mM IPTG after reaching the scattered light threshold of 10 a.u.. Activity assay conditions: incubation in 96well MTP, 0.05 mM 4-MUC solution in potassium phosphate buffer (pH = 6.5), VL = 150 μL, 30 °C, fluorescence measurement at 365/455 nm