Indoxyl Acetate as a Substrate for Analysis of Lipase Activity

Abstract

Lipases play a crucial role in metabolism of microbes, fungi, plants, and animals, and in analytical chemistry, they are often used in detection of fats and triglycerides. Determination of lipase activity is also important in toxicology, when lipase activity can be both increased and decreased by organophosphates and other pesticides and in medicine for diagnosis of heart diseases. The standard method for lipase activity determination is based on cleaving ester bonds in lipase buffer containing Tween. Our aim was to find a method with faster and more sensitive response. It is known that acetylcholinesterase belongs to the same group of hydrolases enzymes as lipases and it cleaves indoxyl acetate, so we assume indoxyl acetate could report a similar reaction with lipase. Our method is based on indoxyl acetate as a substrate for lipase, where indoxyl acetate is cleaved by lipase to indoxyl and acetate moiety and blue indigo is created. The method was optimized for different times and amount of enzyme and compared with the standard Tween assay. The calibration curve measured in reaction time 20 minutes with 10 μl of lipase exhibited the best analytical parameters, and it showed Michaelis–Menten response with the Michaelis–Menten constant equal to 8.72 mmol/l. The indoxyl acetate-based method showed faster and more sensitive response than the standard method for lipase activity determination, so it has great potential in biosensor construction and it could be used in industry, medicine, toxicology, and common practice where the activity of lipases is need to be measured.

Urine Test Strip Quantitative Assay with a Smartphone Camera

“…Esterase activity can be easily measured by spectrophotometry using indoxyl esters [ 17 ]. In this article, the use of indoxyl acetate in the way previously described for cholinesterase and lipase assays was chosen [ 18 – 20 ].…”

Methods of Immobilization of Microbial Enzymes on Solid Surfaces and Their Use

“…Látková přeměna je základní vlastností všech živých organismů od nejjednodušších virů a bakterií až po savce (1,2). Enzymy jsou katalyzátory biochemických reakcí a mají nezastupitelnou roli v buněčném metabolismu (3).…”

“…Enzymy jsou součástí výrobních procesů již tisíce let. Lidé, byť nevědomky, enzymatickou aktivitu využívali při výrobě mléčných výrobků, alkoholických nápojů, pečiva, octa, masných výrobků, zpracování kůže a v dalších výrobních procesech (16). V posledních padesáti letech, vzhledem k rostoucímu tlaku na snížení výrobních nákladů, na minimalizaci dopadu na životní prostředí a velké produkci za krátkou časovou jednotku, jsou nejhojněji využívány enzymy produkované mikroorganismy (17).…”

Structure and Function of Pancreatic Lipase-Related Protein 2 and Its Relationship With Pathological States

“…Therefore, the active site of HPNLIPRP2 might be directly accessible to a substrate. In contrast, the lid of rat PNLIPRP2 (RPNLIPRP2) lipase is in the closed conformation ( Mancheño et al, 2004 ; Valek et al, 2019 ). Many researchers, including our research group, illustrate the essential role of the lid in determining the substrate specificity and the mechanism of action of lipases ( Roussel et al, 1998b ; Yang et al, 2000 ; Berton et al, 2007 ; Eydoux et al, 2008 ), and the theory is that closed lid means interfacial activation.…”