Lipase PS Research Articles

Emulsifying Properties of Lecithin Containing Different Fatty Acids Obtained by Immobilized Lecitase Ultra‐Catalyzed Reaction

AbstractLecitase Ultra and 6 triacylglycerol lipases (lipases PS, M, AH, AY, R, and AK) were immobilized on Amberlite XAD 7HP and used to catalyze the acidolysis reaction between lecithin and capric acid (C10:0) for comparison. The highest molar incorporation value (51.0 mol%) was observed for the immobilized Lecitase Ultra. Further, immobilized Lecitase Ultra was selected for catalyzing acidolysis between lecithin and fatty acids with different chain lengths (C6:0, C8:0, C10:0, C12:0, and C14:0). After reaction, free fatty acids were removed by SPE and the resultant was called modified lecithin fraction 1 (MLF1). The highest molar incorporation value was obtained for C10:0 (51.0 mol%) at 45 °C with a mole ratio of 10/1 (C10:0/lecithin) for 72 h. After removal of lysophosphatidylcholine by solid‐phase extraction from MLF1, the resultant modified lecithin fraction 2 (MLF2) was used to prepare an oil‐in‐water emulsion. All emulsions prepared with MLF2 exhibited significantly higher emulsion stability (ES) values (16.2–17.7) and smaller particle sizes (d32 0.40–0.49 μm, d43 0.75–1.01 μm) than the emulsion prepared with unmodified lecithin (ES 14.1, d32 0.76 μm, d43, 1.26 μm) (P < 0.05). Furthermore, less clarification and droplet aggregation were observed in emulsions prepared with MLF2 than in lecithin‐based emulsions. Overall, the MLF2s showed better emulsifying properties than lecithin.

Towards regioselective enzymatic hydrolysis and glycerolysis of tricaprylin in miniemulsion and the direct preparation of polyurethane from the hydrolysis products

Abstract Miniemulsion droplets are used as the reaction environment for the enzyme catalyzed hydrolysis and glycerolysis of tricaprylin. The reaction was conducted at slightly elevated temperatures in aqueous dispersion without any organic solvent. Lipase PS from Pseudomonas cepacia, known as a lipase without site preference (unspecific), and lipase from Rhizopus arrhizus (RAL), a lipase with sn-1,3 preference, were used for the reactions. The time courses of the conversions were studied in detail with NMR and HPLC. RAL exhibited sn-1,3 preference and Lipase PS clearly showed significant reaction in sn-2 position during hydrolysis of the triglyceride as well as during glycerolysis. From the hydrolysis products, polyurethane-based polymers were synthesized by directly adding isophoronediisocyanate to the miniemulsions after different reaction times. The properties of the products, e.g. the Tg, can be controlled by time-dependent addition of the isocyanate.

Kinetic resolution of propargylic alcohols via stereoselective acylation catalyzed by lipase PS-30

Abstract By using lipase PS-30 as catalyst, the kinetic resolution of a series of racemic propargylic alcohols has been achieved via stereoselective acylation. The value of kinetic enantiomeric ratio (E) reached up to 139. Substituent effect is briefly discussed.

Synergetic Activation of Lipase by an Amino Acid with Alkyl–PEG Sulfate Ionic Liquid

Abstract The synergetic effect of amino acids and an ionic liquid, 1-butyl-2,3-dimethylimidazolium α-cetylpolyoxyethylene(10) ether sulfate (IL1), as coating materials on lipase was discovered: coating on lipase PS using a certain amino acid with IL1 was found to be very effective for accelerating the lipase-catalyzed transesterification of secondary alcohols while maintaining perfect enantioselectivity.

Effect of sub‐ and super‐critical CO2 pretreatment on conformation and catalytic properties evaluation of two commercial enzymes of CALB and Lipase PS

AbstractBACKGROUNDIt is vitally important to understand the effect of sub‐/super‐critical CO2 pretreatment on enzyme conformation and its activity, which should improve the understanding of enzymatic biotransformation applications in nonaqueous media. This study evaluated the effect of sub‐/super‐critical CO2 treatment, including pressure (6 and 10 MPa), exposure time (20, 30 and 150 min) and temperature (35 and 40 °C), on the conformation (e.g. 1D, 2D and 3D structures) and catalytic properties (e.g. residual activity, kinetics constants (Km and Vmax), activation energies (Eα), thermo‐stability and organic solvent tolerance) of two commercial enzymes, CALB and lipase PS in their solution forms.RESULTSCompared with the blank control, the 1D structure of both treated lipases was unchanged, whereas their 2D and 3D structures were altered to some extent. The highest relative activities were 105% and 116% for CALB and lipase PS, respectively. For CALB, Vmax/Km value significantly increased, while for lipase PS, Vmax/Km value was almost constant. Both treated enzymes showed high thermo‐stability with recovery of enzyme activity up to 76% and 80%, respectively. Also, both treated enzymes presented high organic solvent tolerance.CONCLUSIONSIt was speculated that 2D, 3D structure alterations were probably responsible for the satisfactory catalytic properties of enzymes treated with sub‐/super‐critical CO2. © 2013 Society of Chemical Industry

Enzyme‐catalysed hydrolysis of phosphatidylcholine for the production of lysophosphatidylcholine

AbstractBackgroundProduction of lysophosphatidylcholine (LPC) via enzyme‐catalysed hydrolysis was studied. Three enzymes were employed to conduct the reactions at different temperatures. The starting material, phosphatidylcholine (PC), was dispersed in water (system A) and in ethanol (system B) to define the reaction mixture to carry out the trials.ResultsIt was found that the media employed and the type of biocatalyst (free or immobilized), clearly determine the kinetics of the reactions. PC was well dissolved in ethanol but an opaque emulsion was obtained when it was dissolved in water. Immobilized PLA1 and Novozym 435 were able to convert PC into LPC in ethanol, with yields of 50% and 58.51%, respectively, after 48 h at 50 °C. The highest degree of hydrolysis (70%) was reached with Lipase PS after 48 h at 60 °C in water.ConclusionsBoth reaction media enabled fairly good yields but water was better. The present work proposes a simple reaction scheme compared with other reports in the literature where different substrates, additives and polar solvents have been employed. LPC has interesting properties as a bioemulsifier, which led us to develop new methods for its production. © 2013 Society of Chemical Industry

Recent Lipase-Catalyzed Hydrolytic Approaches to Pharmacologically Important β-and λ-Amino Acids

Kinetic and sequential kinetic enzymatic routes for the synthesis of enantiomeric β- and λ-amino acids through enzymatic ring cleavage of the corresponding lactams in organic solvents or solvent-free systems or a supercritical CO2 medium, and for the enantioselective hydrolysis of the corresponding amino esters in organic solvents are reviewed. In the frame of a practical guide, a simple and rapid GC enantioseparation technique for amino acids, including exact descriptions of selected enzymatic reactions, is additionally presented. Keywords: β-amino acid, λ-amino acid, β-amino ester, burkholderia cepacia lipase PS, candida antarctica lipase B, enantiomer, enzymatic catalysis, GC enantioseparation, kinetic resolution, β-lactam, λ-lactam, lipase, organic solvent, sequential resolution, solvent-free system, supercritical CO2.

Enzymatic production of (S)-3-cyano-5-methylhexanoic acid ethyl ester with high substrate loading by immobilized Pseudomonas cepacia lipase

(S)-3-Cyano-5-methylhexanoic acid ethyl ester is a valuable synthetic intermediate for pregabalin. Immobilized lipase PS from Pseudomonas cepacia was screened and shown to be the best biocatalyst for the enantioselective hydrolysis of 3-cyano-5-methylhexanoic acid ethyl ester, a racemic mixture involving a β-stereocenter. The optimum temperature and pH for the biocatalytic process were 35°C and 6.0, respectively. Lipase PS IM exhibited a strong tolerance toward high substrate concentrations of up to 2.0M (366g/l). In the scaled-up biotransformation, (S)-3-cyano-5-methylhexanoic acid ethyl ester was produced in 0.89M (162.9g/l), 99.2% ee, and 44.5% yield. These results indicated that lipase PS IM catalyzed the preparation of (S)-3-cyano-5-methylhexanoic acid ethyl ester and could be used as an efficient route for the large-scale production of pregabalin.

Lipase-catalyzed interfacial polymerization of ω-pentadecalactone in aqueous biphasic medium: A mechanistic study

The synthetic activity of lipases in biphasic o/w systems was investigated with respect to their use in the synthesis of polyester chains via transesterification reactions. Lipase-catalyzed ring-opening polymerization (ROP) of pentadecalactone (ω-PDL) dispersed in water was used as a model reaction to understand the synthetic activity of lipases in biphasic o/w system. We conducted a systematic investigation of the influence of reaction conditions on the macromolecular characteristics of oligo(ω-PDL) encompassing chemical, thermophysical and colloidal properties of the reaction medium. A model was proposed assuming Michaelis–Menten interfacial kinetics followed by chain extension via lipase-catalyzed linear polycondensation. The solidification of oligo(ω-PDL) chains with a degree of polymerization of approximately three was identified as a major factor limiting the molecular weight of obtained oligomers to ∼870 g mol−1, despite the fast reaction rate and complete conversion of ω-PDL. The addition of toluene into the dispersed phase at a volumetric ratio of 0.3–0.5 of toluene to ω-PDL allowed us to circumvent this problem and increase the molecular weight of obtained oligomers up to 1460 g mol−1. The molecular weight of polymer product according to this model was thus inversely related to the weight ratio percentage of interfacial lipase PS to ω-PDL per droplet and correspondingly correlated with the activity of lipase. Taking into account all these parameters allowed increasing the molar mass of oligo(ω-PDL) from 870 g mol−1 to 3507 g mol−1.

Robust Enzymatic Resolution of 3-Fluoromandelic Acid with Lipase PS Supported on Celite

The resolution of different mandelic acids using the lipase PS “Amano” SD enzyme is described. By supporting the lyophilized enzyme over Celite, both the activity and the stability of lipase PS in organic media were significantly improved, enabling the robust resolution scale-up of 3-fluoromandelic acid. The methodology was extended to produce a range of optically pure (R)-mandelic acids, avoiding tedious extractions or chromatography.

Enzymatic kinetic resolution of hydroxystearic acids: A combined experimental and molecular modelling investigation

Abstract Enantioenriched 7-, 8-, 9-, and 10-hydroxystearic acids (HSA) were obtained, for the first time, by kinetic resolution of their racemates with lipases CALB and PS, in the presence of vinyl acetate. Among them, the best results were obtained for 7-HSA and 9-HSA, whose enantiomeric excess was around 55%. The same resolutions carried out on the hydroxy esters completely failed. For the acid substrates neither the Kazlauskas’ rule nor the 3D-QSAR model could be applied, since both models are focused on the CALB alcohol-pocket evaluation and not on the acyl-pocket one. Therefore, a semiquantitative approach was used, whose results were in accordance with our findings, as far as the absolute configuration of the product is concerned.

ChemInform Abstract: Ionic‐Surfactant‐Coated Burkholderia cepacia Lipase as a Highly Active and Enantioselective Catalyst for the Dynamic Kinetic Resolution of Secondary Alcohols.

AbstractThe commercially available lipase PS is purified and lyophilized in the presence of an ionic surfactant to provide significantly enhanced activity.

Lipase-Catalyzed Incorporation of Different Fatty Acids into Tripalmitin-Enriched Triacylglycerols: Effect of Reaction Parameters

Tripalmitin-enriched triacylglycerols were concentrated from palm stearin by acetone fractionation and as the substrate reacted with a mixture of equimolar quantities of fatty acids (C8:0-C18:3). The incorporation degree and acyl migration level of the fatty acids and acylglycerols composition were investigated, providing helpful information for the production of human milk fat substitutes. Higher incorporation degrees of the fatty acids were obtained with lipase PS IM, Lipozyme TL IM, and Lipozyme RM IM followed by porcine pancreatic lipase and Novozym 435-catalyzed acidolysis. During reactions catalyzed by Lipozyme TL IM, Lipozyme RM IM, and lipase PS IM, incorporation degrees of C12:0, C14:0, C18:1, and C18:2 were higher than those of other fatty acids at operated variables (molar ratio, temperature, and time), and the triacylglycerols content reached the highest (82.09%) via Lipozyme RM IM-catalyzed acidolysis. On the basis of significantly different levels of acyl migration to the sn-2 position, lipases were in the order of lipase PS IM < Lipozyme TL IM < Lipozyme RM IM.

Pheromone synthesis. Part 249: Syntheses of methyl (R,E)-2,4,5-tetradecatrienoate and methyl (2E,4Z)-2,4-decadienoate, the pheromone components of the male dried bean beetle, Acanthoscelides obtectus (Say)

The enantiomers of methyl (E)-2,4,5-tetradecatrienoate (1), a component of the male pheromone of Acanthoscelides obtectus, were synthesized from the enantiomers of 1-undecyn-3-ol (6), which were obtained via asymmetric acetylation of (±)-1-trimethylsilyl-1-undecyn-3-ol (4) with vinyl acetate as catalyzed by lipase PS (Amano). The ortho ester Claisen rearrangement of 6 with triethyl orthoacetate was the key-step to generate the chiral allenic system. A new synthesis of (±)-1 was also executed starting from (±)-6. Three different syntheses of methyl (2E,4Z)-2,4-decadienoate (2), another component of the male pheromone of A. obtectus, were achieved by means of either palladium-catalyzed Heck reaction or a Claisen and an Al2O3 catalyzed thermal rearrangements.

Enantiomeric scaffolding of α-tetralone and related scaffolds by EKR (Enzymatic Kinetic Resolution) and stereoselective ketoreduction with ketoreductases

Stereochemically pure compounds containing an all carbon quaternary stereocenter based on 1-tetralone, 1-indanone and 4-chromanone scaffolds have been synthesized by employing Lipase PS (Burkholderia cepacia) catalyzed kinetic resolution. These scaffolds are further functionalized by microbial ketoreductase enzymes (Geotrichum candidum, Candida parapsilosis and Aspergillus niger) to access stereochemically pure diols which, on further synthetic manipulation, yield novel cyclic compounds.

Investigation on the chemoenzymatic synthesis of threo- and erythro-β-hydroxy-l-glutamic acid derivatives

► Transformation of a malonic semialdehyde derivative in β-hydroxy glutamate precursors by aldolase. ► Separation of the two epimers by lipase PS. ► Assignment of configurations by Kazslauskas’ model and H-2 and H-3 chemical shifts. ► Possibility of transforming the products into β-hydroxy glutamic acid derivatives. A derivative of the malonic semialdehyde was transformed by means of a bioconversion catalyzed by the enzyme l -threonine aldolase from E. coli into a 6:4 epimeric mixture of two precursors of β-hydroxy glutamic acid. The enzyme was selective for the formation of the ( S )-configuration at C-2, whereas the configuration at C-3 was not controlled. The two epimers were separated exploiting a diastereoselective acylation in organic solvent catalyzed by lipase PS. The relative and absolute configurations of the products were preliminarily assigned on the base of the model proposed by Kazslauskas for the stereopreference of lipase PS and by comparison of the chemical shifts of the H-2 and H-3 protons of the two homologues. The possibility of transforming the obtained products into β-hydroxy glutamic acid derivatives by conventional chemical reactions was demonstrated.

Ionic‐Surfactant‐Coated Burkholderia cepacia Lipase as a Highly Active and Enantioselective Catalyst for the Dynamic Kinetic Resolution of Secondary Alcohols

The widerapplications of these DKR procedures in organic synthesis,however, can be hampered by the narrow specificity, moder-ate enantioselectivity, and low activity of the enzymeemployed. Therefore, it is of great importance to develop ahighly active enzyme having high enantioselectivity toward awide range of substrates for DKR. We herein report thationic-surfactant-coated Burkholderia cepacia lipase(ISCBCL) has great potential as such an excellent enzyme.Commercially available Burkholderia cepacia lipase(BCL; brand names: Lipase PS and Lipase PS-C) displayssignificantly lower activity than Candida antarctica lipase B(CALB; brand name: Novozym 435) (Table 1, entries 1–3);the latter has been most frequently employed in the metallo-enzymatic DKR. Fortunately, we have developed a practicalapproach for enhancing its activity dramatically. In a typicalprocedure, buffer-free soluble BCL (24% protein),

Novel Sol‐Gel Lipases by Designed Bioimprinting for Continuous‐Flow Kinetic Resolutions

AbstractThe bioimprinting effect in sol‐gel immobilization of lipases was studied to develop efficient novel immobilized biocatalysts with significantly improved properties for biotransformations in continuous‐flow systems. The bioimprinting candidates were selected systematically among the substrate mimics already found in the active site of experimental lipase structures. Four lipases (Lipase AK, Lipase PS, CaLB and CrL) were immobilized by a sol‐gel process with nine bioimprinting candidates using various combinations of tetraethoxysilane (TEOS), phenyltriethoxysilane (PhTEOS), octyltriethoxysilane (OcTEOS) and dimethyldiethylsilane (DMDEOS) as silica precursors. The biocatalytic properties of the immobilized lipases were characterized by enantiomer selective acylation of various racemic secondary alcohols in two different multisubstrate systems (mixture A: a series of alkan‐2‐ols rac‐1a–e and mixture B: heptan‐2‐ol rac‐1f and 1‐phenylethanol rac‐1g). Except with Lipase AK, the most significant activity enhancement was found with the imprinting molecules already found as substrate mimics in X‐ray structures of various lipases. The synthetic usefulness of the best biocatalysts was demonstrated by the kinetic resolution of racemic 1‐(thiophen‐2‐yl)ethanol (rac‐1h) in batch and continuous‐flow systems.

Kinetic resolution of allylic alcohols via stereoselective acylation catalyzed by lipase PS-30

By using lipase PS-30 as catalyst, the kinetic resolution of a series of racemic allylic alcohols has been achieved via stereoselective acylation. The value of kinetic enantiomeric ratio ( E) reached up to 968. Substituent effect is briefly discussed.

ChemInform Abstract: Enantioselective Transesterification of (.+‐.)‐6‐Benzyloxy‐2,5,7,8‐tetramethyl‐3,4‐dihydro‐2H‐1‐benzopyran‐2‐ylm ethanol Catalyzed by the Amano PS Lipase in the Ionic Liquid [bmim] PF6.

AbstractThe chiral benzopyran ((S)‐(—)‐I) represents a key intermediate for the synthesis of α‐tocopherol and α‐tocotrienol.