Abstract
Lipases and esterases are biocatalysts used at the laboratory and industrial level. To obtain the maximum yield in a bioprocess, it is important to measure key variables, such as enzymatic activity. The conventional method for monitoring hydrolytic activity is to take out a sample from the bioreactor to be analyzed off-line at the laboratory. The disadvantage of this approach is the long time required to recover the information from the process, hindering the possibility to develop control systems. New strategies to monitor lipase/esterase activity are necessary. In this context and in the first approach, we proposed a lab-made sequential injection analysis system to analyze off-line samples from shake flasks. Lipase/esterase activity was determined using p-nitrophenyl butyrate as the substrate. The sequential injection analysis allowed us to measure the hydrolytic activity from a sample without dilution in a linear range from 0.05–1.60 U/mL, with the capability to reach sample dilutions up to 1000 times, a sampling frequency of five samples/h, with a kinetic reaction of 5 min and a relative standard deviation of 8.75%. The results are promising to monitor lipase/esterase activity in real time, in which optimization and control strategies can be designed.
Highlights
During the last few years, the interest in enzymes has increased considerably, since they have important advantages with respect to conventional chemical processes [1]
The most important parameter to be measured in enzyme production is the lipase/esterase activity, defined as: the amount of enzyme that catalyzes the conversion of one μmol of substrate per minute
Adapting the spectrophotometric microplate method using p-nitrophenyl butyrate as the substrate in the Sequential injection analysis (SIA) system required characterizing the performance of the equipment, i.e., determining the flows, operational times, injected volumes, valves sequence, etc
Summary
During the last few years, the interest in enzymes has increased considerably, since they have important advantages with respect to conventional chemical processes [1]. The most important parameter to be measured in enzyme production is the lipase/esterase activity, defined as: the amount of enzyme that catalyzes the conversion of one μmol of substrate per minute. The hydrolytic activity of lipases and esterases can be determined by different methods, such as: titrimetry, spectroscopy, chromatography, turbidimetry, conductometry, immunochemistry, microscopy and biosensors, among others [8,9]. The standard assay to measure the lipase/esterase hydrolytic activity is the pH-stat technique; this approach is time consuming and generates many wastes. To increase the efficiency in measuring the enzymatic activity, spectrophotometric methods employing chromogenic substrates, such as p-nitrophenyl and resorufin esters, have been used [10]. The hydrolysis of p-nitrophenyl butyrate releases a chromophore (p-nitrophenolate), which can be measured spectrophotometrically at 415 nm and at different time intervals [11,12,13]
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