ABSTRACT
BACKGROUND: In most microbial cultivations D-glucose is the main carbon and energy source. However, quantification of D-glucose especially in small scale is still challenging. Therefore, we developed a FRET-based glucose biosensor, which can be applied in microbioreactor-based cultivations. This sensor consists of a glucose binding protein sandwiched between two fluorescent proteins, constituting a FRET pair. Upon D-glucose binding the sensor undergoes a conformational change which is translated into a FRET-ratio change. RESULTS: The selected sensor shows an apparent Kd below 1.5 mM D-glucose and a very high sensitivity of up to 70% FRET-ratio change between the unbound and the glucose-saturated state. The soluble sensor was successfully applied online to monitor the glucose concentration in an Escherichia coli culture. Additionally, this sensor was utilized in an at-line process for a Corynebacterium glutamicum culture as an example for a process with cell-specific background (e.g. autofluorescence) and medium-induced quenching. Immobilization of the sensor via HaloTag® enabled purification and covalent immobilization in one step and increased the stability during application, significantly. CONCLUSION: A FRET-based glucose sensor was used to quantify D-glucose consumption in microtiter plate based cultivations. To the best of our knowledge, this is the first method reported for online quantification of D-glucose in microtiter plate based cultivations. In comparison to D-glucose analysis via an enzymatic assay and HPLC, the sensor performed equally well, but enabled much faster measurements, which allowed to speed up microbial strain development significantly.
Subject(s)
Biosensing Techniques/methods , Fluorescence Resonance Energy Transfer/methods , Glucose/analysis , Cell Culture Techniques/methods , Corynebacterium glutamicum/metabolism , Escherichia coli/genetics , Escherichia coli/metabolismABSTRACT
The development of process steps catalyzed by immobilized enzymes usually encompasses the screening of enzyme variants, as well as the optimization of immobilization protocols and process parameters. Direct immobilization of biocatalysts by physical entrapment into hydrogels can be applied to reduce the effort required for immobilization, as the enzyme-specific optimization of the immobilization procedure is omitted. Physical entrapment is applicable for purified enzymes as well as crude cell extracts. Therefore, it can be used to quickly assess and compare activities of immobilized enzymes. For the application in flow reactors, we developed 3D-printed hydrogel lattices for enzyme entrapment as well as matching housings, also manufactured by 3D-printing. Testing the resulting enzyme reactors for three different enzymes, namely alcohol dehydrogenase from Lactobacillus brevis, benzoylformate decarboxylase from Pseudomonas putida and ß-galactosidase from Aspergillus oryzae, and four different enzymatic reactions showed the broad applicability of the approach but also its limitations. The activity of the immobilized biocatalysts was measured in batch experiments and compared to the kinetics of the respective free enzymes in solution. This comparison yields an effectiveness factor, which is a key figure to describe the extent the immobilized catalyst is effectively utilized. For the examined systems the effectiveness factor ranged between 6 and 14% and decreased with increasing absolute activity of the entrapped enzymes due to mass transfer limitations. To test the suitability of the hydrogel lattices for continuous operation, they were inserted into 3D-printed reactor housings and operated at constant flow. Stable product formation could be monitored over a period of 72 h for all four enzymatic systems, including two reactions with redox cofactor regeneration. Comparing calculated and experimental conversion in the continuous setup, higher values of the effectiveness factor in batch experiments also hint at good performance in continuous flow. This can be used to optimize complex biocatalytic reactions on a small scale.
ABSTRACT
Easy, fast and gentle immobilization for the efficient reuse of important biocatalysts is highly demanded. We used the commercially available HaloTag™ technology (Promega), so far relatively unknown in the context of biocatalysis, to immobilize the benzaldehyde lyase from P. fluorescence (PfBAL). Immobilization mediated by this fusion tag proceeds rapidly within minutes from crude extracts yielding covalently attached enzymes in high purity, making expensive and laborious previous chromatographic purification steps obsolete, which strongly reduces the costs for biocatalyst immobilization. Further, we introduce a novel design of HaloTag fusions and demonstrate the positive effect of the tag on soluble expression and activity of PfBAL. The immobilized biocatalyst was stable at 4°C for months and was successfully reused in several repetitive batches for the carboligation of aggressive aldehydes.
Subject(s)
Bacterial Proteins/chemistry , Biotechnology/methods , Enzymes, Immobilized/chemistry , Recombinant Proteins/chemistry , Aldehyde-Lyases/chemistry , Aldehyde-Lyases/metabolism , Bacterial Proteins/metabolism , Enzymes, Immobilized/metabolism , Genetic Engineering , Pseudomonas fluorescens/enzymology , Recombinant Proteins/metabolism , RhodococcusABSTRACT
The linear plasmid pDJ12 from Micrococcus D12, isolated from the high-altitude volcanic Diamante Lake in the northwest of Argentina, was completely sequenced and annotated. It is noteworthy that the element is probably conjugative and harbors genes potentially instrumental in coping with stress conditions that prevail in such an extreme environment.
