Quantitative turbidity assay for lipolytic enzymes in microtiter plates

Abstract

A clearing assay for lipolytic enzymes has been realized in 96-well microtiter plates. A thin layer containing emulsified tributyrin as turbidity-generating substrate was placed on a thicker supporting aqueous layer. Both layers were stabilized by a gel-forming agent. Enzyme addition leads to clearing of the emulsion detected with a standard microtiter plate reader as a decrease of extinction. Dependencies of the signal kinetics on the substrate and enzyme concentrations were studied. For 0.5-1% tributyrin content the reaction rate is not substrate-limited. An initial slope of the signal kinetics is proportional to the lipase activity. A detailed characterization of the assay was performed. Lipolysis of tributyrin was confirmed by glycerol detection. Various gel-forming agents were compared and diffusion conditions in these gels were analyzed. Agar and agarose were found to be the most suitable gel-forming agents, which do not affect enzyme diffusion whereas polyacrylamide gels block lipase diffusion and therefore are not suitable for the assay. The optimized assay prepared from 1% tributyrin emulsion in 2% agar gel was tested with six microbial lipases and porcine pancreatic lipase. The detection limit is 20-60 ng/well which is equivalent to 30 μU/well for T. lanuginosus lipase.

Marinobacter lipolyticusfrom Red Sea for lipase production and modulation of silver nanomaterials for anti‐candidal activities

“…Furthermore, the supernatant lipolytic activity was stable at 6–8 pH and 70°C. In another study, the lipolytic enzymes of bacterial strain in 96‐well plates were determined by turbidity assay [53]. In our investigation, the lipolytic activity in the supernatant was the same in both liquid and solid agar media.…”

“…The biosynthesis of AgNPs were a result of the Ag+ ion reduction to Ag 0 in AgNO 3 solution at room temperature by using an aqueous bacterial extract. After an overnight incubation, the colour changed from light to dark brown, which indicates the biogenic reduction [53]. The reduction was further confirmed by observing the optimal absorption peak around 430 nm (Fig.…”

High Throughput Analysis of Photocatalytic Water Purification

“…The 96-well microplate base is widely used in many fields of sample preparation and analysis, such as drug efficiency screening in pharmacology, 14 enzymatic studies in biochemistry, 15 and high content biological cell bioassays. 16 Utilizing these well plates allows reduction of the reagent and catalyst amount, easier handling, and higher sampling throughput.…”

Design and Synthesis of Cross‐Linked Micellar Particles to Assist Microalgae Lipid Recovery from Aqueous Extract

“…Turbidity test was used for evaluation of lipid binding by CLMP, as it has been used to examine emulsion stability [27,28]. It measures light scattering of an aqueous sample with suspended particles.…”

“…When the lipid quantity in the emulsion exceeded the binding capacity of CLMP, the supernatant after centrifugation was cloudy and the green color became darker with more lipids added. The immiscible minute oil droplets in the emulsion caused the cloudiness, which can be quantified by the turbidity test [27,28]. The binding capacity was determined as the point where the supernatant started to become The tube on the left contained Nannochloropsis lipids in aqueous isopropanol before adding CLMP, and the right one was after adding CLMP (sample corresponding to entry 2 in Table 2) Table 2) binding cloudy, i.e., the absorbance of the supernatant started to increase.…”