Monitoring Lipase/Esterase Activity by Stopped Flow in a Sequential Injection Analysis System Using p-Nitrophenyl Butyrate

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.

Beyond esterase‐like activity of serum albumin. Histidine‐(nitro)phenol radical formation in conversion cascade of p‐nitrophenyl acetate and the role of infrared light

“…As a result of esterase‐like activity of BSA p ‐NPA is converted to p ‐NP (Scheme .). The transformation is usually evidenced by monitoring evolution of the UV–Vis band centered around 400 nm, which corresponds to the absorption of nitrophenolate ion (yellow at pH values above its pKa = 7.08 at 22°C).…”

Microbial lipases and their industrial applications: a comprehensive review

“…which caused a change in the impedance of the medium 10 nmol/l (fungal lipase) [ 306 ] Candida rugosa (Fungi) Chlorfenvinphos, Malathion Lipase converted p- nitrophenyl acetate to p- nitrophenol and acetic acid, p- nitrophenol was oxidized and a current at 0.024 V was recorded, analyzed inhibited lipase and stopped the reaction. 84.5 µmol/l for chlorfenvinphos and 282 µmol/l for malathion [ 716 ] Optical assays-based on lipase biosensor Candida antarctica, Yarrowia lipolytica and fungus Lipase itself p-nitrophenyl butyrate hydrolysis to butyric acid and p-nitrophenol, coloration caused by p-nitrophenol was measured 0.05 U/ml [ 717 ] Candida antarctica, Mucor miehei, Thermomyces lanuginosus (Fungus) and bacteria Pseudomonas cepacia and P. fluorescens Lipase itself Butyryl 4-methyl umbelliferone (Bu-4-Mu) and methanol in tert-butanol were trans-esterified in the presence of lipase, production of 4-methylumbelliferone was measured fluorometre Not available [ 718 ] …”

Biosensors and Bioassays Based on Lipases, Principles and Applications, a Review

“…P -nitrophenyl butyrate as a substrate was selected by Pliego and coworkers for the determination of lipase activity in a stop flow sequential injection analysis system [63]. The principle of the assay was the same as described in the chapter devoted to the standard methods: p -nitrophenyl butyrate was hydrolyzed to p -nitrophenol and butyric acid, coloration caused by p -nitrophenol was measured photometrically at 415 nm.…”