Determination of enzyme activity differs from other analytic
Determination of enzyme activity differs from other analytical procedures. In this Erlotinib Hydrochloride sale case, instead of enzyme concentration, a substrate depletion or product accumulation should be monitored. Such approach allows calculating activity basis of calibration curves performed for enzymatic reaction product (Pochart, Dewit, Desjeux, & Bourlioux, 1989) or enzyme standards (Bzura & Fiedoruk-pogrebniak, 2018; Tymecki, Strzelak, & Koncki, 2013) The most popular enzymatic reaction substrates that can be applied for lactase and β–galactosidase activity assays are p–nitrophenyl (Cameron, Manthey, Baker, & Grohmann, 2001; Hansen, DelOlmo, & Burri, 1998) or o–nitrophenyl (Citti, Sandine, & Elliker, 1965) derivatives for photometric methods or 4-methyl-umbelliferyl derivatives for fluorescence measurements (Kwapiszewski et al., 2015, 2014). p–Aminophenyl derivatives are used for amperometry (Laczkaet al., 2010, Mittelmannet al., 2002, Neufeldet al., 2003). Derivatives of more volatile compounds like 2,2,2–trichloroethanol can be applied within gas chromatography (Køppen & Dalgaard, 1985). There are also some other techniques, like liquid chromatography (Gonzalez Andrada, Romero, Morales, & JimenezPerez, 1996) and high-resolution ultrasonic spectroscopy (Altas, Kudryashov, & Buckin, 2016), allowing enzyme activity determination using underivatized disaccharides.
Due to the catalytic nature of enzymatic activity assays the measurements should be performed at the fixed time, fixed concentration change or in a kinetic mode. Ensuring the best reproducibility of such measurement procedure is essential. Such conditions like the reaction time, the moment of reagents dosing, the dynamics of reagent transportation and mixing, should be strictly defined and controlled. Some additional difficulties arise from the fact that the optimal conditions for enzymatic reaction often differ from those for subsequent detection of the product of this biocatalytic process. Thus, in many cases, enzyme activity assays are multistep protocols, where each step is conducted under different conditions. The illustration of such problem is enzyme activity assays based on spectrophotometric detection of nitrophenol. Optimal operating conditions for lactase and their enzyme analogs are at 4-7 pH range, whereas nitrophenol is significantly better detectable in an alkaline environment (see Figure S1-A in the Supplementary Material). Finally, a complete determination routine is often laborious and time-consuming process. An excellent solution to those problems is the mechanization of multistep bioanalytical procedure offered by modern flow analysis systems. Moreover, flow techniques also allow to perform the measurements under the non-equilibrium conditions, and processing consecutive sample before the end of the previous sample analysis, what significantly shortens the time of analysis. Principles, applications and advances of modern flow analysis have been reviewed recently (Gonzálezet al., 2015, Horstkotteet al., 2018, Sasakiet al., 2017, Trojanowicz and Kołacińska, 2016).
In this paper, a fully mechanized multi-pumping flow analysis (MPFA) system for β–galactosidase activity examination is presented. This modern, highly cost-effective system is composed of solenoid micropumps and microvalve powered and actuated by Arduino microcontroller. Compact and extremely economic optoelectronic flow-through detector coupled with this MPFA system for photometric detection of nitrophenol generated in the course of the enzyme reaction is operating according to paired-emitter-detector-diode (PEDD) principle (Tymecki, Brodacka, Rozum, & Koncki, 2009). To the best of the author's knowledge, this is the first example of β–galactosidase activity assay performed in the flow analysis format.
Results and discussion
Acknowledgments This research was supported by the Polish National Science Centre under Project NCN Opus (no.2014/13/B/ST4/04528).