molecules Review Biosensors and Bioassays Based on Lipases, Principles and Applications, a Review Miroslav Pohanka Faculty of Military Health Sciences, University of Defence, Trebesska 1575, CZ-500 01 Hradec Kralove, Czech Republic; [email protected] or [email protected]; Tel.: +420-973253028 Academic Editor: Ondrej Zitka Received: 27 January 2019; Accepted: 8 February 2019; Published: 10 February 2019 Abstract: Lipases are enzymes responsible for the conversion of triglycerides and other esterified substrates, they are involved in the basic metabolism of a wide number of organisms, from a simple microorganism and to mammals. They also have broad applicability in many fields from which industrial biotechnology, the production of cleaning agents, and pharmacy are the most important. The use of lipases in analytical chemistry where it can serve as a part of biosensors or bioassays is an application of growing interest and has become another important use. This review is focused on the description of lipases chemistry, their current applications and the methods for their assay measurement. Examples of bioassays and biosensors, including their physical and chemical principles, performance for specific substrates, and discussion of their relevance, are given in this work. Keywords: amperometry; bioassay; biosensor; biorecognition; catalysis; enzyme; ester; lipase; nanoparticle; nanostructure; potentiometry; voltammetry 1. Introduction While standard analytical methods and devices for various assays are common in laboratories and specialized workplaces, a single device for reliable handheld assays is not able to cover the whole spectrum of analytes that are measured for diagnostic, environmental, technological and other purposes. For the reason, hand-held devices for the most relevant analytes are demanded. Most biosensors belong to the group of handheld assays and there are also significant off-the-shelf devices conforming to the description of a biosensor. Biosensors are a combination of two important parts with unique properties [1,2]. The first part is a sensor, also called a physico-chemical transducer, this part is responsible for providing a physically measurable signal. Biological origin is the second compartment of a biosensor and this part is necessary for providing specificity to an analyte. Lipase can be entitled as one of the parts of biological origin. Lipases are frequently used in biosensors construction because of their wide substrate specificity and good commercial availability. Various source cells can be used as a source of lipases. Bacterial, fungal and mammal lipases such as the one from the porcine pancreas are available [3–5]. Once a lipase becomes a part of the biosensor, it can be used for an assay of either enzymatic substrate or an inhibitor [6–8]. A general principle of biosensors with immobilized lipase is depicted in Figure1. Molecules 2019, 24, 616; doi:10.3390/molecules24030616 www.mdpi.com/journal/molecules Molecules 2019, 24, 616 2 of 14 Molecules 2019, 24, x FOR PEER REVIEW 2 of 15 isolation of a lipase pure lipase source cell lipase immobilization esterified analyte biosensor acid + alcohol sensor Figure 1. A general idea of a lipase biosensor construction. Figure 1. A general idea of a lipase biosensor construction. This review is focused on the survey of actual literature in the field of lipase biosensors, bioassays and similarThis review tools and is methods.focused on Discussion the survey of theirof actu applicabilityal literature and in suitability the field in of the lipase current biosensors, analytical praxisbioassays is also and included similar intools this and work. methods. A search Discussion in a recent of paper their onapplicability the issue with and proper suitability citations in the is presentedcurrent analytical here. praxis is also included in this work. A search in a recent paper on the issue with proper citations is presented here. 2. Lipases and Their Applicability 2. Lipases and Their Applicability Lipases are a group of enzymes belonging to the lipase-esterase superfamily and having basic structuralLipases motives are a commongroup of inenzymes this superfamily belonging such to the as serinelipase-esterase in the active superfamily site and inand the having similar basic way thatstructural they are motives folded common after the in expression this superfamily of genetic such information as serine in the [9,10 active]. Lipases site and are in structurally the similar veryway closethat they to cholinesterases: are folded after acetylcholinesterase the expression of genetic and butyrylcholinesterase information [9,10]. areLipases both widelyare structurally applicable very for biosensorclose to cholinesterases: construction because acetylcholinesterase of their sensitivity and to butyrylcholinesterase neurotoxic compounds are [1 ,both11–13 widely]. The exampleapplicable of cholinesterasesfor biosensor construction shows where because the development of their sens of lipaseitivity in to bioassays neurotoxic and compounds biosensors can[1,11–13]. go because The theyexample are moreof cholinesterases advanced in theirshows development where the developm due to theirent of use lipase in the in assay bioassays of military and biosensors nerve agents can andgo because pesticides. they are more advanced in their development due to their use in the assay of military nerveIn agents mammals, and pesticides. pancreatic lipase, lipoprotein lipase and hepatic lipase are distinguished [14]. The mostIn mammals, frequently pancreatic applied lipase, lipases lipoprotein are of the proper-namedlipase and hepatic triacylglycerol lipase are distinguished acyl hydrolases [14]. (E.C. The 3.1.1.3)most frequently and can beapplied easily lipases acquired are from of the the proper-named many organisms triacylglycerol from which acyl both hydrolases microorganisms (E.C. 3.1.1.3) and livestockand can be are easily most acquired common. from While the microbialmany organi lipasessms from can be which produced both microorganisms by standard biotechnological and livestock processes.are most common. Mammalian While lipases microbial can belipases isolated can frombe produced livestock by organs standard and biot agricultureechnological is actually processes. the majorMammalian source lipases for this can type be of isolated enzyme. from Vertebrate livestock lipases organs share and largeagriculture sequence is actually identities, the rangingmajor source from 58%for this to 97%type[ 15of ].enzyme. The lipase Vertebrate E.C. 3.1.1.3 lipases is nevertheless share large sequence structurally identities, distinct ranging to lipoprotein from 58% lipase to 97% EC 3.1.1.34[15]. The [15 lipase,16]. E.C. 3.1.1.3 is nevertheless structurally distinct to lipoprotein lipase EC 3.1.1.34 [15,16]. Triacylglycerol isis thethe mainmain substratesubstrate ofof lipaselipase andand glycerolglycerol andand carboxyliccarboxylic acids are thethe mostmost importantimportant productsproducts of of the the catalyzed catalyzed reaction. reaction. The The role ro ofle lipaseof lipase relates relates to basal to basal metabolic metabolic processes processes and itand is involvedit is involved in retinol in retinol biosynthesis, biosynthesis, triacylglycerol triacylglycerol degradation degradation and lipid and metabolism. lipid metabolism. The common The chemicalcommon reactionschemical catalyzedreactions bycatalyzed lipase may by lipase differ may in various differ organisms.in various organism Acetylations. Acetylation [17], the hydrolysis [17], the ofhydrolysis various carboxylicof various esterscarboxylic [18– 20esters] and [18–20] transesterification and transesterification [21,22] are [21,22] the most are typicalthe most reactions typical catalyzedreactions catalyzed by lipases. by lipases. The range of substrates converted byby lipaseslipases cancan bebe learnedlearned fromfrom thethe followingfollowing examples.examples. InIn the workwork byby LianghuaLianghua andand coworkers,coworkers, ibuprofen in R and SS racemateracemate reacts withwith 1-propanol1-propanol in thethe presencepresence ofof lipase from Penicillium expansum [[23]23] while ( S)-ibuprofen n-propylester and ( R)-ibuprofen are thethe products.products. The reaction can be applied for thethe preparationpreparation or manufacturingmanufacturing of an opticallyoptically purepure isomerisomer ofof ibuprofen.ibuprofen. The hydrolysishydrolysis ofof 4-nitrophenyl4-nitrophenyl palmitatepalmitate providingproviding 4-nitrophenol4-nitrophenol andand palmitatepalmitate was described described in in the the same same paper. paper. The The high high activity activity of lipase of lipase can be can found be found in yeast in and yeast it can be used for technological purposes. In a paper devoted to the research on this issue, water hydrolysis of 1,2,3-trihexaicosanoylglycerol to 1,2-dihexaicosanoylglycerol and hexaicosanoate, the Molecules 2019, 24, 616 3 of 14 and it can be used for technological purposes. In a paper devoted to the research on this issue, water hydrolysis of 1,2,3-trihexaicosanoylglycerol to 1,2-dihexaicosanoylglycerol and hexaicosanoate, the hydrolysis of trimyristin to dimyristin and myristate, and the hydrolysis of tripalmitin to dipalmitin and palmitate are described for lipase from yeast Saccharomyces cerevisiae [24]. Tributyrin can be designated as a common substrate for lipases of various origin. In a study, tributyrin hydrolysis to dibutyrin and butyrate was mentioned for lipases from various organisms, including bacterium