Highly selective synthesis of 1,3-oleoyl-2-palmitoylglycerol by lipase catalysis

Schmid, Ulrike; Bornscheuer, Uwe T.; Soumanou, Mohamed M.; McNeill, Gerald P.; Schmid, Rolf D.

doi:10.1002/(sici)1097-0290(19990920)64:6<678::aid-bit6>3.0.co;2-9

Cited by 94 publications

(59 citation statements)

References 20 publications

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“…Schmid et al [15] and Pfeffer et al [16] synthesized the structured TAG oleicpalmitic-oleic by a two-step enzymatic process: alcoholysis of tripalmitin with ethanol to produce 2-monopalmitin and esterification of the latter with oleic acid, catalyzing both reactions with 1,3 specific lipases. Schmid et al [15] obtained the structured TAG OPO with 96% PA at position 2 and 90% oleic acid at positions 1 and 3.…”

Section: Introductionmentioning

confidence: 99%

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Production of triacylglycerols rich in palmitic acid at position 2 as intermediates for the synthesis of human milk fat substitutes by enzymatic acidolysis

Jiménez

Esteban

Robles

et al. 2010

Process Biochemistry

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This paper studies the synthesis of triacylglycerols (TAGs) rich in palmitic acid (PA) at position 2, from palm oil stearin (POS), a vegetable oil highly rich in this acid (60%). The developed process consists of two steps: (1) obtaining PA enriched free fatty acids (FFAs), and (2) enrichment of POS in PA by acidolysis of this oil with PA enriched FFAs, catalyzed by lipase Novozym 435. In step (1) two PA enriched FFA mixtures were obtained: one by saponification of POS, and a PA concentrate (75.1% PA) obtained by crystallization at low temperature in solvents. The latter was obtained carrying out two crystallizations in acetone at -24 and -20 ºC, from which PA was recovered in the solid phases with a total yield of 84%. These PA enriched FFA mixtures were used in step (2) of acidolysis of POS, along with commercial PA (98% PA). In this acidolysis step four factors were studied: temperature, hexane/reaction mixture ratio, FFA/POS molar ratio and the intensity of treatment (IOT = lipase amount × reaction time/POS amount). The best results (TAGs with 79% PA and 75% PA at position 2) were obtained with commercial PA, at 37 ºC, 10 mL hexane/g reaction mixture, a FFA/POS molar ratio 3:1 (1:1 w/w) and an IOT = 9.6 g lipase × h/g POS (for example 48 h, 10 g lipase and 50 g POS). PA enriched TAGs were purified neutralizing the FFAs by KOH hydroethanolic solutions and extracting the TAGs with hexane. In this way 99% pure acylglycerols (TAG+DAG) were obtained; the recovery yield of this purification step was 95%. The experiments carried out with POS demonstrated that it is possible to use only this oil (60% PA, 23% PA at position 2) as a source of PA to obtain a TAG with 70.7% PA and 70.5% PA at position 2. This process consists of four steps:(1) saponification of POS, (2) crystallization of FFAs to obtain PA enriched FFAs (75.1% PA), (3) acidolysis of POS with these FFAs, catalyzed with Novozym 435, to produce PA enriched TAGs at position 2 (70.5% PA) and (4) purification of TAGs to obtain approximately 95% purity and yield. These PA enriched TAGs could be used to obtain structured TAGs rich in PA at position 2 and in oleic acid at positions 1 and 3 (OPO), which is the principal TAG of human milk fat.

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Section: Introductionmentioning

confidence: 99%

“…Schmid et al [15] obtained the structured TAG OPO with 96% PA at position 2 and 90% oleic acid at positions 1 and 3. Pfeffer et al [16] produced 2-monopalmitin with 77% purity and 73% yield in the alcoholysis reaction and, finally, OPO with 95% purity and 90% yield in the esterification reaction.…”

Section: Introductionmentioning

confidence: 99%

Production of triacylglycerols rich in palmitic acid at position 2 as intermediates for the synthesis of human milk fat substitutes by enzymatic acidolysis

Jiménez

Esteban

Robles

et al. 2010

Process Biochemistry

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“…Balcao and Malcata [19] also use the acidolysis reaction to increase the level of unsaturated fatty acid of a butterfat by acidolysis of this one with oleic acid; so decreases the level of saturated fatty acids, such as myristic and palmitic acids. Schmid et al [16] synthesized the structured TAG oleicpalmitic-oleic (OPO) by a two-step enzymatic process: alcoholysis of tripalmitin with ethanol to produce 2-monopalmitin and esterification of the latter with oleic acid, catalyzing both reactions with 1,3 specific lipases. These authors obtained OPO with 96% PA at sn-2 position and 90% oleic acid at sn-1,3 positions.…”

Section: Introductionmentioning

confidence: 99%

Production of triacylglycerols rich in palmitic acid at sn-2 position by lipase-catalyzed acidolysis

Jiménez

Esteban

Robles

et al. 2010

Biochemical Engineering Journal

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This paper studies the synthesis of triacylglycerols (TAGs) rich in palmitic acid (PA) at sn-2 position from palm stearin (PS), a vegetable oil highly rich in PA (60%, but only 12.8% of this is located at sn-2 position). These PA rich TAGs were obtained by lipasecatalyzed acidolysis of this oil with free fatty acids (FFAs) highly rich in PA, such as commercial PA (98% PA) and a FFA extract obtained by saponification of PS (60% PA). PA has a melting point of 63 ºC and during the acidolysis reaction the substrates, highly rich in this acid, must remain liquid; therefore high temperatures or solvents must be used. An important objective of this work was to operate without solvent and at the lowest possible temperature. In this acidolysis reaction four factors were firstly studied: type of lipase, temperature, solvent amount and the intensity of treatment (IOT = lipase amount × reaction time/PS amount). The influence of these variables was studied in a stirred tank reactor (STR). The lipases tested were Novozym 435 from Candida antarctica (immobilized on a macroporous acrylic resin), and lipases QLC (immobilized on diatomaceous earth), and QLM (non immobilized), both from Alcaligenes sp., and the one selected was lipase QLC. According to the manufacturer the optimum temperature for this lipase is 65-70 ºC, which allows it to operate without solvent. The best results with lipase QLC (TAGs with 80% PA, both total and at sn-2 position) were obtained with commercial PA, at 65 ºC, a 3:1 FFA/PS molar ratio (1:1 w/w), without solvent and an IOT = 7 g lipase × h/g PS (for example 2.5 g PS, 2.5 g commercial PA, 0.75 g lipase and 24 h). These results were the basis for establishing the operational conditions to obtain PA rich TAGs with the lipase immobilized in a packed bed reactor (PBR), operating by recirculation of the reaction mixture through the lipase bed. In this system TAGs with 75% PA were obtained at an IOT = 8 g lipase × h/g PS. This result and the apparent kinetic constants obtained in both reactors show that the reaction rate is lower in the PBR than in the STR. Subsequently, PA enriched TAGs were separated from FFAs by two procedures: the first one at room temperature and in presence of hexane and the second one at 65 ºC and without hexane. Using the first procedure, 95% of TAGs in the acidolysis reaction mixture were recovered with a purity of 99%. Using the second one, 98% pure TAGs were obtained with a recovery yield of 80%. Therefore, these highly rich PA TAGs can be obtained by acidolysis of PS and PA rich FFAs in solvent-free media, and then these TAGs also can be purified to 98% in absence of hexane, using only a hydroethanolic KOH solution.3

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“…Though crude triglycerides can be directly added to commercial products, a growing industrial interest is devoted to eicosapentaenoic acid/DHA-enriched triglycerides. Fish oils are the best-exploited natural source of -3 fatty acids and are often used as starting material to produce fats and oils with high nutritional value (19,32,33).…”

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confidence: 99%

Enhancement of the Stability of a Prolipase from Rhizopus oryzae toward Aldehydes by Saturation Mutagenesis

Lorenzo

Hidalgo

Molina

et al. 2007

Appl Environ Microbiol

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A prolipase from Rhizopus oryzae (proROL) was engineered in order to increase its stability toward lipid oxidation products such as aldehydes with the aim of improving its performance in oleochemical industries. Out of 22 amino acid residues (15 Lys and 7 His) prone to react with aldehydes, 6 Lys and all His residues (except for the catalytic histidine) were chosen and subjected to saturation mutagenesis. In order to quickly and reliably identify stability mutants within the resulting libraries, active variants were prescreened by an activity staining method on agar plates. Active mutants were expressed in Escherichia coli Origami in a 96-well microtiterplate format, and a stability test using octanal as a model deactivating agent was performed. The most stable histidine mutant (H201S) conferred a stability increase of 60%, which was further enhanced to 100% by combination with a lysine mutant (H201S/K168I). This increase in stability was also confirmed for other aldehydes. Interestingly, the mutations did not affect specific activity, as this was still similar to the wild-type enzyme.In the last several years, a growing number of industrial applications have required the use of lipases in the presence of partly oxidized triglycerides. Biodiesel (fatty acid monoalkyl esters) has attracted considerable attention during the past decade as a renewable, biodegradable, and nontoxic fuel (36). It is produced from various vegetable oils and has a viscosity close to that of diesel, but it is derived from triglycerides by transesterification with alcohol. Recently, enzymatic transesterification using lipases has become more attractive for biodiesel production, since the glycerol produced as a by-product can easily be recovered, and the purification of fatty methyl esters is simple to accomplish. Owing to the higher price of biodiesel relative to petroleum fuel, waste triglycerides (e.g., frying oils) are often used as cheaper feedstock for the biodiesel synthesis (6,8,26), thus helping also to reduce the cost of wastewater treatment in sewage systems and generally assisting in the recycling of resources (12,19,23,24).With enzymatic technology it is also possible to produce new fats according to specific requirements. Special triglycerides of the ABA-type containing medium-chain fatty acids (e.g., C 8 ) in the sn-1,3 positions and long-chain unsaturated fatty acids (e.g., C 16 to C 22 ) in the sn-2 position are effective energy sources for patients with malabsorption, e.g., pancreatic insufficiency. Polyunsaturated fatty acids like eicosapentaenoic acid

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Highly selective synthesis of 1,3-oleoyl-2-palmitoylglycerol by lipase catalysis

Cited by 94 publications

References 20 publications

Production of triacylglycerols rich in palmitic acid at position 2 as intermediates for the synthesis of human milk fat substitutes by enzymatic acidolysis

Production of triacylglycerols rich in palmitic acid at position 2 as intermediates for the synthesis of human milk fat substitutes by enzymatic acidolysis

Production of triacylglycerols rich in palmitic acid at sn-2 position by lipase-catalyzed acidolysis

Enhancement of the Stability of a Prolipase from Rhizopus oryzae toward Aldehydes by Saturation Mutagenesis

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