Integral Stereoselectivity of Lipase Based on the Chromatographic Resolution of Enantiomeric/Regioisomeric Diacylglycerols

Abstract

Stereoselectivity, a distinctive characteristic of lipase (EC 3.1.1.3), refers to the ability to differentiate between enantiomeric positions (sn-1 and sn-3) in triacylglycerol (TAG). This property has been determined based on the time course of enantiomeric excess of diacylglycerol (DAG) considering several consecutive steps of lipase-catalyzed hydrolysis of TAG; however, this concept is insufficient to represent the true nature of lipases which are capable of hydrolyzing the sn-2 position of TAG under the condition acyl migration occurs. Here, we suggest “integral stereoselectivity” to capture the preference of lipases for all ester groups of both TAG and DAG, as a novel index of the stereochemistry of lipase. To determine integral stereoselectivity, we established an analytical system based on the chromatographic resolution of dioleoylglycerol (DO) enantiomers and regioisomers. DO enantiomers were derivatized with 4-nitrophenyl isocyanate, and subsequently, resolved by chiral-phase high-performance liquid chromatography–ultraviolet. Regioisomers of monooleoylglycerol and DO were analyzed by HPLC with an evaporative light-scattering detector. Time-course analysis of three model lipases involved in the hydrolysis of trioleoylglycerol validated the analytical system designed to determine the integral stereoselectivity. As an accurate indicator of lipase stereochemistry reflecting all hydrolysis steps, integral stereoselectivity can expedite the development of lipases with unique stereochemistry from agricultural sources and their application to the food industry.

Kinetic modeling of lipase-catalysed hydrolysis of triacylglycerol in a reverse micelle system for the determination of integral stereoselectivity

“…6C). 19,20 In all cases, the best fit of the models are in good agreement with the experimental results with satisfactory values of determination coefficient (R 2 ), which were calculated to be 0.96, 0.98, and 0.99 for PPL, CVL, and PFL, respectively.…”

“…1, we previously proposed the concept of 'integral stereoselectivity' and developed a chromatographic method for analyses. 19,20 Based on a time-course of TAG, FFA, regioisomers, and enantiomers of DAG (i.e., 1,2-sn-, 1,3-sn-, and 2,3-sn-DAG) and MAG (i.e., 1-sn-, 2-sn-, and 3-sn-MAG) obtained with this analytical method, in this study, the integral stereoselectivity of lipase was quantified as a kinetic constant for each step of the reaction. This is the first presentation of a kinetic model describing integral stereoselectivities of lipases.…”

“…1, we previously proposed the concept of ‘integral stereoselectivity’ and developed a chromatographic method for analyses. 19,20 Based on a time-course of TAG, FFA, regioisomers, and enantiomers of DAG ( i.e. , 1,2- sn -, 1,3- sn -, and 2,3- sn -DAG) and MAG ( i.e.…”

Lipase and Its Unique Selectivity: A Mini-Review

“…e regioselectivity of lipases for triacylglycerols is fundamentally determined by the communication between the enzyme structure and the substrate structure; nevertheless, other extrinsic factors such as temperature, water content, organic solvent hydrophobicity, reaction medium, and immobilization, can induce conformational changes of the protein structure followed by affecting the regioselectivity [41]. e lipase from Pseudomonas fluorescens showed an sn-1,3 regiospecificity for triacylglycerols in oil-in-water emulsion medium [27], as compared to showing nonregiospecific properties in aprotic organic solvent medium with low water contents such as reversed micellar systems [64]. e free enzyme form of Candida antarctica lipase B is reported to be sn-1,3 regiospecific [57], whereas its immobilized form (Lipozyme ® CALB manufactured by Novozymes A/S) is nonregiospecific.…”

Optimization of Spectrophotometric and Fluorometric Assays Using Alternative Substrates for the High-Throughput Screening of Lipase Activity

“…Most lipases from eukaryotes precisely control the accessibility of their active site to their substrates by opening and closing their lid structure at oil/water interface [3,4]. Furthermore, lipases typically exhibit unique selectivities on their substrates, such as typoselectivity, regioselectivity, and stereoselectivity [5,6], though a few lipases show promiscuous behavior toward their substrates [7]. Because of those attractive properties, lipases are being studied in the fields of selective hydrolysis for flavor/texture improvement [8,9], esterification for the synthesis of structured lipids or functional compounds [10,11], and other catalytic reactions.…”