Title: Lipase-catalyzed Synthesis of Oleoyl-lysophosphatidylcholine by Direct Esterification in Solvent-free Medium without Water Removal Mnasri T, Ergan F, Herault J, Pencreac'h G Ref: J Oleo Sci, 66:1009, 2017 : PubMed
In this work, the synthesis of oleoyl-lysophosphatidylcholine by lipase-catalyzed esterification of glycerophosphocholine (GPC) and free oleic acid in a reaction medium without solvent is presented. The complete solubilisation of GPC, which is a crucial issue in non-polar liquids such as melted free fatty acids, was reached by heating the GPC/oleic acid mixture at high temperature during a short time. The immobilized lipase from Rhizomucor miehei (Lipozyme RM-IM) was shown to catalyze the reaction more efficiently than the immobilized lipases from Thermomyces lanuginosus (Lipozyme TL-IM) and Candida antarctica (Novozym 435). The condition reactions leading to the highest yield were as follows: substrate ratio: 1/20 (GPC/oleic acid); amount of catalyst: 10% (w/w of substrates); temperature: 50 degC. Under these conditions, a yield of 75% of oleoyl-lysophosphatidylcholine was achieved in 24 h under stirring and almost no dioleoyl-lysophosphatidylcholine was produced. Unlike other studies dealing with the esterification of GPC with free fatty acids, the removal of the water produced while the reaction proceeds was not necessary to reach high yields.
Chlorogenic acid (5-caffeoyl quinic acid (CQA)) extracted from Hydrangea macrophylla (44%, w/w) with 98% purity, was acylated with palmitic acid by Novozym 435 to yield mono-acylated CQA. Acylation of CQA was achieved in 2-methyl-2-butanol at 60 degrees C, and yielded two mono-acylated products: a major product acylated at the C-4 of the quinic moiety (4-O-palmitoyl chlorogenic acid) and a minor product acylated at the C-3 (3-O-palmitoyl chlorogenic acid). The bioconversions obtained in 7 days ranged from 14 to 60% and were influenced by the molar ratio of palmitic acid/CQA, which ranged from 10 to 80. The regioselectivity (4-O-palmitoyl/3-O-palmitoyl ratio) of the reaction was also affected by the molar ratio, and ranged from 90 to 70%. The scavenging activities against 1,1-diphenyl-2-picryl-hydrazyl radicals demonstrated that these palmitoyl CQA derivatives are associated with antioxidant activity (70% vs CQA).
Title: An ultraviolet spectrophotometric assay for measuring lipase activity using long-chain triacyglycerols from Aleurites fordii seeds Pencreac'h G, Graille J, Pina M, Verger R Ref: Analytical Biochemistry, 303:17, 2002 : PubMed
In this study, we designed a specific, continuous, and sensitive UV spectrophotometric lipase assay using natural triacylglycerols (TAGs) from the Aleurites fordii seed oil (tung oil). alpha-Eleostearic acid (9,11,13-cis, trans,trans-octadecatrienoic acid) is the main fatty acid component (it accounts for up to 70%) of the TAGs from tung oil. The conjugated triene present in alpha-eleostearic acid constitutes an intrinsic chromophore, which confers strong UV absorption properties on both the free fatty acid and the TAGs from tung oil. The lipase assay is based on the difference between the apparent molar extinction coefficients of the two types of alpha-eleostearic acid present, that which is esterified into TAGs and that which is released into the reaction medium. This difference is responsible for the variations in the UV absorption spectrum of the reaction medium occurring upon enzymatic TAGs hydrolysis. Using the purified lipase from Thermomyces lanuginosa (TLL) and the detergent sodium taurodeoxycholate (NaTDC, 4 mM), it was established that the most suitable method of measuring lipolysis consisted of monitoring the decrease in the OD at 292 nm, which was linear with time and proportional to the amount of lipase added. In order to be able to estimate the specific activity of TLL, we determined an apparent molar extinction coefficient of alpha-eleostearic acid (epsilon = 13,900 M(-1) cm(-1)) under the assay conditions. Amounts of pure TLL as small as 1 ng can be easily detected in the presence of 4 mM NaTDC. Interestingly, the NaTDC concentration can be decreased as far as 0.05 mM. In comparison with other well-known methods of lipase assay, the detection limit of this new method is 100-fold lower than with the pH-stat method and similar to that of a fluorescent assay recently developed at our laboratory.
Title: A novel extracellular esterase from Bacillus subtilis and its conversion to a monoacylglycerol hydrolase Eggert T, Pencreac'h G, Douchet I, Verger R, Jaeger KE Ref: European Journal of Biochemistry, 267:6459, 2000 : PubMed
A novel gene lipB, which encodes an extracellular lipolytic enzyme, was identified in the Bacillus subtilis genomic DNA sequence. We have cloned and overexpressed lipB in B. subtilis and Escherichia coli and have also purified the enzyme from a B. subtilis culture supernatant to electrophoretic homogeneity. Four different lipase assays were used to determine its catalytic activity: pH-stat, spectrophotometry, fluorimetry and the monomolecular film technique. LipB preferentially hydrolysed triacylglycerol-esters and p-nitrophenyl-esters of fatty acids with short chain lengths of <= 10 carbon atoms. Triolein, which is a typical substrate for true lipases, was not hydrolysed at all. These results led us to classify LipB as an esterase rather than a lipase. The catalytic triad of LipB consists of residues Ser78, Asp134, and His157 as demonstrated by amino-acid sequence alignments and site-directed mutagenesis. The nucleophile Ser78 is located in a lipase-specific consensus sequence, which is Ala-X-Ser-X-Gly for most Bacillus lipases. All other bacterial lipases contain a glycine residue instead of the alanine at position-2 with respect to the catalytic serine. We have investigated the role of this alanine residue by constructing LipB variant A76G, thereby restoring the lipase-specific consensus motif. When compared with LipB this variant showed a markedly reduced thermostability but an increased stability at pH 5-7. Determination of the specific activities of wild-type LipB and variant A76G using a monomolecular film of the substrate monoolein revealed an interesting result: the A76G substitution had converted the esterase LipB into a monoacylglycerol hydrolase.
Title: Properties of free and immobilized lipase from Pseudomonas cepacia Pencreac'h G, Leullier M, Baratti JC Ref: Biotechnol Bioeng, 56:181, 1997 : PubMed
The purified lipase from Pseudomonas cepacia (PS, Amano) was immobilized on a commercially available microporous polypropylene support. The enzyme was rapidly and completely adsorbed on the support. Special attention was devoted to the demonstration of the lack of diffusional limitations, either internal or external, when a soluble substrate (p-nitrophenylacetate, pNPA) was used. The activity yield was high (100%) with pNPA and very low (0.4%) with p-nitrophenylpalmitate (pNPP). These values clearly showed that the immobilized enzyme was fully active as soon as activity was assayed on a soluble substrate rather than an insoluble one. With the latter one, the low activity was due mainly to a slow rate of substrate diffusion inside the porous support. The same diffusional phenomenon could explain the complete change of fatty acid specificity of the immobilized lipase. After immobilization, the lipase was mainly specific for short chain fatty acid esters, whereas the free enzyme was mainly specific for long chain esters. The activity-versus-temperature profiles were not greatly affected by immobilization with maximal reaction rates in the range 45 degrees to 50 degrees C for both enzyme preparations. However, immobilization increased enzyme stability mainly by decreasing the sensitivity to temperature of the inactivation reaction. Half-lives at 80 degrees C were 11 and 4 min for the immobilized and free enzymes, respectively. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 181-189, 1997.
