Purification and biochemical characterization of the LIP2 lipase from Yarrowia lipolytica

Aloulou, Ahmed; Rodríguez, Jorge A.; Puccinelli, Delphine; Mouz, Nicolas; Leclaire, Julien; Leblond, Y.; Carrière, Frédéric

doi:10.1016/j.bbalip.2006.12.006

Cited by 91 publications

(61 citation statements)

References 36 publications

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“…5C ), similarly to what was reported by Carrière et al ( 46 ), at pH 6.0 for YLLIP2 using both TGs with 4 mM NaTDC ( Fig. 5D ), similarly to what was reported by Aloulou and colleagues ( 37,45 ), at pH 8.2 for PPL using both TGs ( Fig. 5E ), similarly to what was reported by Borgström and Erlanson ( 49 ), at pH 9.2 for TLL ( Fig.…”

Section: Study Of Lipase Inhibition Using Phiblasupporting

confidence: 78%

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

Camacho-Ruiz¹,

Mateos-Díaz²,

Carrière

et al. 2015

Journal of Lipid Research

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subsequent catalysis sensu stricto ( 6, 7 ). Most of the lipases are water-soluble enzymes acting on water insoluble substrates (super-substrates). The two-dimensional nature of enzyme catalysis by lipases does not obey MichaelisMenten kinetics and critically depends on the quality of the interface ( 3,8,9 ). Obtaining accurate, i.e., substratespecifi c, measurements of lipase activity as well as developing reliable lipase assay systems requires taking these unique features into account.Many biotechnological applications for lipases have been described in the food, cosmetic, detergent, and pharmaceutical industries ( 3, 10, 11 ), and an interest exists for new sources of this kind of enzyme in an industrial setting.Novel lipases can be obtained either by isolating them from various natural sources or by using classical protein engineering methods and/or directed evolution procedures (12)(13)(14). All these studies require convenient, sensitive, and specifi c assays for measuring lipase activity ( 15 ). In addition, screening procedures require continuous assays and substrate stability that are compatible with high sample throughput.Considerable progress has been made in the past few years in the development of high-throughput screening (HTS) methods for carboxyl-ester hydrolases. These HTS assays have been developed using chromogenic or fl uorogenic substrates such as butyrate, octanoate, and palmitate of nitrophenol or 4-methylumbelliferone. However, all of these synthetic esters are liable to undergo nonenzymatic alkaline hydrolysis as well as hydrolysis by the nonspecifi c carboxyl-ester hydrolases often present in biological Abstract A continuous assay is proposed for the screening of acidic, neutral, or alkaline lipases using microtiter plates, emulsifi ed short-and medium-chain TGs, and a pH indicator. The lipase activity measurement is based on the decrease of the pH indicator optical density due to protonation which is caused by the release of FFAs during the hydrolysis of TGs and thus acidifi cation. Purifi ed lipases with distinct pH optima and an esterase were used to validate the method. The rate of lipolysis was found to be linear with time and proportional to the amount of enzyme added in each case. Specific activities measured with this microplate assay method were lower than those obtained by the pH-stat technique. Nevertheless, the pH-dependent profi les of enzymatic activity were similar with both assays. In addition, the substrate preference of each enzyme tested was not modifi ed and this allowed discriminating lipase and esterase activities using tributyrin (low water solubility) and tricaprylin (not water soluble) as substrates. This continuous lipase assay is compatible with a high sample throughput and can be applied for the screening of lipases and lipase inhibitors from biological samples. Lipases (TG ester hydrolases, EC 3.1.1.3) are lipolytic carboxylester hydrolases which catalyze the hydrolysis of the ester bonds of TGs to form FFAs and glycerol in some cases . They are widely distr...

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Section: Study Of Lipase Inhibition Using Phiblasupporting

confidence: 78%

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

Camacho-Ruiz¹,

Mateos-Díaz²,

Carrière

et al. 2015

Journal of Lipid Research

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“…Increasing attention has been paid to microbial lipases due to their wide range of biochemical properties and relative easiness to produce and isolate in bioreactors [2,4]. The catalytic properties of microbial lipases have rendered many biotechnological applications in the food, cosmetic, detergent, pharmaceutical, and energy industries [3,[5][6][7][8]. This is primarily due to their ability to utilize a broad spectrum of substrates in the synthesis or hydrolysis of lipid compounds, as well as their activity in organic solvents [1,9].…”

Section: Introductionmentioning

confidence: 99%

Purification and characterization of extracellular lipases from Pseudomonas monteilii TKU009 by the use of soybeans as the substrate

Wang

Lin

Liang

et al. 2008

J Ind Microbiol Biotechnol

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A lipase-producing bacterium was isolated and identified as Pseudomonas monteilii TKU009. A lipase (F2) and lipase-like materials (F1) were purified from the culture supernatant of P. monteilii TKU009 with soybean powder as the sole carbon/nitrogen source. The molecular mass of F1 and F2 was estimated to be 44 kDa by SDS-PAGE and gel filtration. The optimum pH, optimum temperature, and pH and thermal stabilities of F2 were 7, 40 degrees C, 8-11, and 50 degrees C; and of F1 were 6, 40 degrees C, 6-7, and 50 degrees C, respectively. F2 was completely inhibited by EDTA and slightly by Mg(2+), Fe(2+), Mn(2+), and SDS. F1 was completely inhibited by EDTA and Fe(2+) and strongly by Zn(2+), Mn(2+), Ca(2+), Mg(2+), and SDS. The activities of both the enzymes were enhanced by the addition of non-ionic surfactants Triton X-100 and Tween 40, especially for F1. F2 preferably acted on substrates with a long chain (C10-C18) of fatty acids, while F1 showed a broad spectrum on those with chain length of C4-C18. The marked activity of F2 in organic solvents makes it an ideal choice for application in a water-restricted medium including organic synthesis.

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“…The purifi ed Thermomyces lanuginosus lipase (TLL), CALA from Candida antarctica , and LIP3 lipase from Candida rugosa (CRLIP3) were a generous gift from Dr. S. Patkar (Novozymes, Denmark). LIP2 lipase from Yarrowia lipolytica (YLLIP2) was produced and purifi ed according to Aloulou et al ( 34 ). Rhizopus homothallicus lipase (RHL) was produced and purifi ed according to Mateos Diaz et al ( 35 ).…”

Section: Uv Spectrophotometric Lipase Assay Using Coated Tagsmentioning

confidence: 99%

An ultraviolet spectrophotometric assay for the screening of sn-2-specific lipases using 1,3-O-dioleoyl-2-O-α-eleostearoyl-sn-glycerol as substrate

Mendoza

Rodríguez

Leclaire

et al. 2012

Journal of Lipid Research

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Lipases (triacylglycerol ester hydrolases, EC 3.1.1.3) are lipolytic carboxylester hydrolases that catalyze the hydrolysis of the ester bonds of long-chain triacylglycerols (TAGs). They are widely distributed in microorganisms, plants, and animals ( 1-3 ), where they play an important role in lipid metabolism ( 4,5 ).Because lipases are water-soluble enzymes that hydrolyze insoluble long-chain TAG substrates, the cleavage reaction has to occur at the lipid-water interface ( 6-8 ). The mechanisms involved in the enzymatic lipolysis depend strongly on the mode of organization of the lipid substrate in interfacial structures such as monolayers, micelles, liposomal dispersions, and oil-in-water emulsions. Lipases interact with these lipid complexes, or "super-substrates," via hydrophobic domains that are exposed upon contact as the result of a substrate-induced conformational change, which sometimes has been called "interfacial activation" ( 3, 9 ). The two-dimensional (2-D) nature of this lipase reaction does not obey Michaelis-Menten kinetics and depends critically on the quality of the interface ( 3,8,9 ). Obtaining accurate, i.e., substrate-specifi c, measurements of lipase activity, as well as developing reliable lipase assay

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Purification and biochemical characterization of the LIP2 lipase from Yarrowia lipolytica

Cited by 91 publications

References 36 publications

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

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

Purification and characterization of extracellular lipases from Pseudomonas monteilii TKU009 by the use of soybeans as the substrate

An ultraviolet spectrophotometric assay for the screening of sn-2-specific lipases using 1,3-O-dioleoyl-2-O-α-eleostearoyl-sn-glycerol as substrate

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