Enzyme activity deviates due to spatial and temporal temperature profiles in commercial microtiter plate readers

• Published in: Biotechnology journal 2016-04, Vol.11 (4), p.519-529
• Main Authors: Grosch, Jan-Hendrik, Sieben, Michaela, Lattermann, Clemens, Kauffmann, Kira, Büchs, Jochen, Spieß, Antje C.
• Format: Article
• Language: English
• Subjects: Bioreactors - microbiology; Cresol red; Edge effect; Enzyme Activation; Enzymes - metabolism; Industrial Microbiology - methods; Lactobacillus brevis; Lactobacillus brevis ADH (LbADH); Optical thermometer; Phenolsulfonphthalein - analogs & derivatives; Phenolsulfonphthalein - chemistry; Temperature; Temperature measurement
• ISSN: 1860-6768; 1860-7314
• DOI: 10.1002/biot.201500422

Microtiter plates (MTP) and automatized techniques are increasingly applied in the field of biotechnology. However, the susceptibility of MTPs to edge effects such as thermal gradients can lead to high variation of measured enzyme activities. In an effort to enhance experimental reliability, to quantify, and to minimize instrument‐caused deviations in enzyme kinetics between two MTP‐readers, we comprehensively quantified temperature distribution in 96‐well MTPs. We demonstrated the robust application of the absorbance dye cresol red as easily applicable temperature indicator in cuvettes and MTPs and determined its accuracy to ±0.16°C. We then quantified temperature distributions in 96‐well MTPs revealing temperature deviations over single MTP of up to 2.2°C and different patterns in two commercial devices (BioTek Synergy 4 and Synergy Mx). The obtained liquid temperature was shown to be substantially controlled by evaporation. The temperature‐induced enzyme activity variation within MTPs amounted to about 20 %. Activity deviations between MTPs and to those in cuvettes were determined to 40 % due to deviations from the set temperature in MTPs. In conclusion, we propose a better control of experimental conditions in MTPs or alternative experimental systems for reliable determination of kinetic parameters for bioprocess development. In order to enhance experimental reliability and quantify deviations in enzyme kinetics in microtiter plate (MTP), temperature distribution in 96‐well MTPs are comprehensively quantified by using an absorbance dye cresol red as easily applicable temperature indicator in cuvettes and MTPs. To quantify the temperature‐caused variances of enzyme activity in MTP measurements, the authors apply an alcohol dehydrogenase of Lactobacillus brevis (LbADH). The study provides clues for reliable determination of kinetic parameters for bioprocess development.