The use of microscale processing technologies for quantification of biocatalytic Baeyer-Villiger oxidation kinetics.
Microscale processing techniques would be a useful tool for the rapid and efficient collection of biotransformation kinetic data as a basis for bioprocess design. Automated liquid handling systems can reduce labor intensity while the small scale reduces the demand for scarce materials such as substrate, product, and biocatalyst. Here we illustrate this concept by establishing the use of several microwell formats (96-round, 96-deep square and 24-round well microtiter plates) for quantification of the kinetics of the E. coli TOP10 [pQR239] resting cell catalyzed Baeyer-Villiger oxidation of bicyclo[3.2.0]hept-2en-6-one using glycerol as a source of reducing power. By increasing the biocatalyst concentration until the biotransformation rate was oxygen mass-transfer limited we can ensure that kinetic data collected are in the region away from oxygen limitation. Using a 96-round well plate the effect of substrate (bicyclo[3.2.0]hept-2en-6-one) concentration on the volumetric CHMO activity was examined and compared to data collected from 1.5-L stirred-tank experiments. The phenomenon and magnitude of substrate inhibition, observed at the larger scale, was accurately reproduced in the microwell format. We have used this as an illustrative example to demonstrate that under adequately defined conditions, automated microscale processing technologies can be used for the collection of quantitative kinetic data. Additionally, by using the experimentally determined stoichiometry for product formation and glycerol oxidation, we have estimated the maximum oxygen transfer rates as a function of well geometry and agitation rate. Oxygen-transfer rates with an upper limit of between 33 mmol. L(-1). h(-1) (based solely on product formation) and 390 mmol. L(-1). h(-1) (based on product formation and glycerol oxidation) were achieved using a 96-square well format plate shaken at 1300 rpm operated with a static surface area to volume ratio of 320 m(2). m(-3).
Doig
Steven D
SD
Department of Biochemical Engineering, University College London, Torrington Place, London, WC1E 7JE, United Kingdom.
Pickering
Samuel C R
SC
Lye
Gary J
GJ
Woodley
John M
JM
D019149:Bioreactors; D001643:Bridged Bicyclo Compounds / Q000378:metabolism*; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000378:metabolism; D002384:Catalysis; D002460:Cell Line; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005990:Glycerol / Q000378:metabolism; D007783:Lactones / Q000378:metabolism; D008832:Microchemistry / Q000295:instrumentation* / Q000379:methods*; D008904:Miniaturization; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*; D010105:Oxygenases / Q000235:genetics / Q000378:metabolism*; D010865:Pilot Projects; D015203:Reproducibility of Results; D012371:Robotics / Q000379:methods*; D012680:Sensitivity and Specificity; D013237:Stereoisomerism
D001643:Bridged Bicyclo Compounds; D019086:Bridged Bicyclo Compounds, Heterocyclic; D007783:Lactones; C472066:bicyclo(3,2,0)hept-2-en-6-one; D010105:Oxygenases; C019961:cyclohexanone oxygenase; D005990:Glycerol; D010100:Oxygen