High-Throughput Screening Method for Lipases/Esterases

Abstract

High-throughput screening (HTS) methods for lipases and esterases are generally performed by using synthetic chromogenic substrates (e.g., p-nitrophenyl, resorufin, and umbelliferyl esters) which may be misleading since they are not their natural substrates (e.g., partially or insoluble triglycerides). In previous works, we have shown that soluble nonchromogenic substrates and p-nitrophenol (as a pH indicator) can be used to quantify the hydrolysis and estimate the substrate selectivity of lipases and esterases from several sources. However, in order to implement a spectrophotometric HTS method using partially or insoluble triglycerides, it is necessary to find particular conditions which allow a quantitative detection of the enzymatic activity. In this work, we used Triton X-100, CHAPS, and N-lauroyl sarcosine as emulsifiers, β-cyclodextrin as a fatty acid captor, and two substrate concentrations, 1 mM of tributyrin (TC4) and 5 mM of trioctanoin (TC8), to improve the test conditions. To demonstrate the utility of this method, we screened 12 enzymes (commercial preparations and culture broth extracts) for the hydrolysis of TC4 and TC8, which are both classical substrates for lipases and esterases (for esterases, only TC4 may be hydrolyzed). Subsequent pH-stat experiments were performed to confirm the preference of substrate hydrolysis with the hydrolases tested. We have shown that this method is very useful for screening a high number of lipases (hydrolysis of TC4 and TC8) or esterases (only hydrolysis of TC4) from wild isolates or variants generated by directed evolution using nonchromogenic triglycerides directly in the test.

Bacillus subtilis Lipase A—Lipase or Esterase?

“…As vinyl acetate was found to negatively affect the Karl Fischer titration, it was freshly distilled and dried with activated molecular sieves for 16 h before use. The activity of the different enzyme preparations was also followed with the tributyrin and p-nitrophenol acetate activity assays [2,5,[53][54][55][56] during equilibration, to establish optimal equilibration times. For BSLA, a small loss of activity over time was observed, while both CALB preparations were stable.…”

“…A tributyrin assay for determining lipase activity was performed according to the literature [54]. The assay is based on pH change by acid formation when tributyrin is hydrolyzed by the enzyme.…”

A broad pH range indicator-based spectrophotometric assay for true lipases using tributyrin and tricaprylin

“…Activities of rHPL, PPL, RGL, rDGL, TLL, YLLIP2, and rAnFaeA were determined according to a HTS method implemented in our laboratory, adapted from previous work ( 32 ), by measuring the apparition of FFAs upon hydrolysis of TG(4:0) or TG(8:0). Each samples.…”

“…Maximum sensitivity is obtained at maximum concentration of substrate was prepared as follows: one volume of the substrate (50 mM dissolved in tert -butanol), also containing the pH indicator, was mixed vigorously on a vortex with nine volumes of buffer solution to reach a fi nal substrate concentration of 5 mM that was found to be optimum for lipase activity measurements ( 32 ). Stock solutions (10×) of pH indicators and buffer solutions were prepared at the concentrations indicated in Table 1 .…”

“…Moreover a great and stable interfacial area is needed to obtain measurable kinetic reads. This can be achieved by adding a detergent ( 32 ). NaTDC, a bile salt that is widely used in standard tests of lipase activity (45)(46)(47), can be used for this purpose at a minimum concentration of 0.5 mM.…”