Distinction between esterases and lipases: A kinetic study with vinyl esters and TAG

Chahinian, Henri; Nini, Lylia; Boitard, Elisabeth; Dubès, J.P.; Comeau, Louis-Claude; Sarda, Louis

doi:10.1007/s11745-002-0946-7

Cited by 91 publications

(73 citation statements)

References 35 publications

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“…The maximal TPL activity (430 U/mg) was reached at a vinyl acetate concentration that exceeded the solubility limit. As described by Chahinian et al, 15 it is difficult to estimate the value of K 0.5 from the irregular kinetic curves, as can be seen in Fig. 1; it appears that half-maximal activity is reached at an ester concentration in the range of 280 mM, near the solubility limit of vinyl acetate.…”

Section: Resultsmentioning

confidence: 82%

“…1; it appears that half-maximal activity is reached at an ester concentration in the range of 280 mM, near the solubility limit of vinyl acetate. According to Chahinian et al, 15 esterases express maximal activity against vinyl acetate in the soluble concentration range with a substrate concentration corresponding to half-maximal activity as low as 4 mM. This recently observed catalytic property was used as a criterion to distinguish lipases from esterases.…”

Section: Resultsmentioning

confidence: 99%

“…Assays were performed in 30 ml of 2.5 mM Tris-HCl buffer, pH 7.0, containing 0.1 M NaCl. 15 One lipase unit corresponds to 1 mmol of fatty acid released per minute.…”

Section: Lipase Activity Determinationmentioning

confidence: 99%

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Kinetic properties of turkey pancreatic lipase: A comparative study with emulsified tributyrin and monomolecular dicaprin

Fendri¹,

Sayari²,

Gargouri³

2004

Chirality

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Using the classical emulsified system and the monomolecular film technique, we compared several interfacial properties of turkey pancreatic lipase (TPL) and human pancreatic lipase (HPL). TPL, like HPL, presented the interfacial activation phenomenon when vinyl ester was used as substrate. In the absence of colipase and bile salts, using tributyrin emulsion or monomolecular films of dicaprin at low surface pressure, TPL, unlike HPL, hydrolyzes pure tributyrin emulsion as well as dicaprin films maintained at low surface pressures. TPL was also able to hydrolyze triolein emulsion in the absence of any additive and despite the accumulation of long-chain free fatty acids at the interface. The difference of behaviors between TPL and HPL can be explained by the penetration power of each enzyme. The enzyme that presents the maximal pi(c) (TPL) interacts more efficiently with interfaces, and it is not denaturated at high interfacial energy. Turkey pancreatic lipase is more active on rac-dicaprin than HPL; a maximal ratio of 9 was found between the catalytic activities of the two lipases measured at their surface pressure optima (20 mN m(-1)). A kinetic study on the surface pressure dependency, stereospecificity, and regioselectivity of TPL was performed using enantiopure diglyceride (1,2-sn-dicaprin and 2,3-sn-dicaprin) and a prochiral isomer (1,3-dicaprin) that were spread as monomolecular films at the air-water interface. At low surface pressure (15 mN m(-1)), TPL acts preferentially on primary carboxylic ester groups of the diglyceride isomers (1,3-dicaprin), but at high surface pressure (23 mN m(-1)), this enzyme prefers both adjacent ester groups of the diglyceride isomers (1,2-sn-dicaprin and 2,3-sn-dicaprin). HPL prefers adjacent ester groups of the diglyceride isomers (1,2-sn-dicaprin and 2,3-sn-dicaprin). Furthermore, TPL was found to be markedly stereospecific for the sn-1 position of the 1,2-sn-enantiomer of dicaprin at low surface pressure (15 mN m(-1)), while at high surface pressure (23 mN m(-1)), this lipase presents a stereopreference for the sn-3 position of the 2,3-sn-enantiomer of dicaprin. HPL is stereospecific for the sn-1 position of the 1,2-sn-enantiomer of dicaprin both at 15 and 23 mN m(-1).

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Section: Resultsmentioning

confidence: 82%

Section: Resultsmentioning

confidence: 99%

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Kinetic properties of turkey pancreatic lipase: A comparative study with emulsified tributyrin and monomolecular dicaprin

Fendri¹,

Sayari²,

Gargouri³

2004

Chirality

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“…The results for the dienophiles are in agremeent with earlier experimental and theoretical studies on the binding of small ketones and aldehydes to CALB and other lipases; the binding constants in water are typically in the range of 0.1-1 M, and generally decrease with increasing hydrophobicity of the substrates. [21,85,86] Our lowest binding constant is obtained for cyclopentadiene 4, which has no specific binding to the active site. This supports the interpretation that the main driving force for binding is hydrophobic interactions.…”

Section: Binding Constantsmentioning

confidence: 90%

Computational design of a lipase for catalysis of the Diels-Alder reaction

et al. 2010

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Combined molecular docking, molecular dynamics (MD) and density functional theory (DFT) studies have been employed to study catalysis of the Diels-Alder reaction by a modified lipase. Six variants of the versatile enzyme {\em Candida Antarctica} lipase B (CALB) have been rationally engineered {\em in silico} based on the specific characteristics of the pericyclic addition. A kinetic analysis reveals that hydrogen bond stabilization of the transition state and substrate binding are key components of the catalytic process. In the case of substrate binding, which has the greater potential for optimization, both binding strength and positioning of the substrates are important for catalytic efficiency. The binding strength is determined by hydrophobic interactions and can be tuned by careful selection of solvent and substrates. The MD simulations show that substrate positioning is sensitive to cavity shape and size, and can be controlled by a few rational mutations. The well-documented S105A mutation is essential to enable sufficient space in the vicinity of the oxyanion hole. Moreover, bulky residues on the edge of the active site hinders the formation of a sandwich-like near-attack conformer (NAC), and the I189A mutation is needed to obtain enough space above the face of the $\alpha$,$\beta$-double bond on the dienophile. The double mutant S105A/I189A performs quite well for two of three dienophiles. Based on binding constants and NAC energies obtained from MD simulations combined with activation energies from DFT computations, relative catalytic rates ($v_{cat}/v_{uncat}$) of up to $10^3$ are predicted.Response to Reviewers: We are happy with the favorable comments by the reviewers. We have have corrected the manuscript as suggested by reviewer 1 1. A sentence explaining catalytic promiscuity has been added. 2. A paragraph discussing the relationship between the NAC concept and the Reaction Force has been added.We have shortened the manuscript as suggested by reviewer 2. In particular, we have moved information regarding the structural relaxation of protein structure in MD simulations to the supplementary material. Abstract Combined molecular docking, molecular dynamics (MD) and density functional theory (DFT) studies have been employed to study catalysis of the DielsAlder reaction by a modified lipase. Six variants of the versatile enzyme Candida Antarctica lipase B (CALB) have been rationally engineered in silico based on the specific characteristics of the pericyclic addition. A kinetic analysis reveals that hydrogen bond stabilization of the transition state and substrate binding are key components of the catalytic process. In the case of substrate binding, which has the greater potential for optimization, both binding strength and positioning of the substrates are important for catalytic efficiency. The binding strength is determined by hydrophobic interactions and can be tuned by careful selection of solvent and substrates. The MD simulations show that substrate positioning is sensitive to cavity s...

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“…Cutinases show no interfacial activation and are, therefore, placed as intermediates between lipases and esterases rather than being categorized as classical lipases. [5][6][7] This functional versatility has made them a good alternative to classical lipases for industrial use in detergent products. 8 To function in detergent mixtures, an enzyme needs to retain its catalytic activity under the conditions used for washing, that is, temperatures up to 60 C, and the presence of anionic and nonionic surfactants.…”

Section: Introductionmentioning

confidence: 99%

Thermodynamic and structural investigation of the specific SDS binding of humicola insolens cutinase

et al. 2014

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The interaction of lipolytic enzymes with anionic surfactants is of great interest with respect to industrially produced detergents. Here, we report the interaction of cutinase from the thermophilic fungus Humicola insolens with the anionic surfactant SDS, and show the enzyme specifically binds a single SDS molecule under nondenaturing concentrations. Protein interaction with SDS was investigated by NMR, ITC and molecular dynamics simulations. The NMR resonances of the protein were assigned, with large stretches of the protein molecule not showing any detectable resonances. SDS is shown to specifically interact with the loops surrounding the catalytic triad with medium affinity (K a % 10 5 M 21 ). The mode of binding is closely similar to that seen previously for binding of amphiphilic molecules and substrate analogues to cutinases, and hence SDS acts as a substrate mimic. In addition, the structure of the enzyme has been solved by X-ray crystallography in its apo form and after cocrystallization with diethyl p-nitrophenyl phosphate (DNPP) leading to a complex with monoethylphosphate (MEP) esterified to the catalytically active serine. TheAbbreviations: AA, all-atom; AoC, Aspergillus oryzae cutinase; AOT, sodium bis(2-ethylhexyl) sulfosuccinate; cmc, critical micelle concentration; DEP, diethylphosphate; DNPP, diethyl p-nitrophenyl phosphate; EDTA, ethylene diamine tetraacetate; FsC, Fusarium solani cutinase; GcC, Glomerella cingulata cutinase; HiC, Humicola insolens cutinase; HSQC, heteronuclear singlequantum coherence; IPTG, isopropyl b-D-1-thiogalactopyranoside; ITC, isothermal titration calorimetry; MD, molecular dynamics; MEP, monoethylphosphate; MWCO, molecular weight cut-off; PAGE, polyacrylamide gel electrophoresis; RMSD, root mean square deviation; RMSF, root mean square fluctuation; TES, N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid; SANS, small-angle neutron scattering; SDS, sodium dodecyl sulfate.Additional Supporting Information may be found in the online version of this article.An interactive view is available in the electronic version of the article. enzyme has the same fold as reported for other cutinases but, unexpectedly, esterification of the active site serine is accompanied by the ethylation of the active site histidine which flips out from its usual position in the triad.

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Distinction between esterases and lipases: A kinetic study with vinyl esters and TAG

Cited by 91 publications

References 35 publications

Kinetic properties of turkey pancreatic lipase: A comparative study with emulsified tributyrin and monomolecular dicaprin

Kinetic properties of turkey pancreatic lipase: A comparative study with emulsified tributyrin and monomolecular dicaprin

Computational design of a lipase for catalysis of the Diels-Alder reaction

Thermodynamic and structural investigation of the specific SDS binding of humicola insolens cutinase

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