Lipase-catalyzed synthesis of phenolic lipids from fish liver oil and dihydrocaffeic acid

The enzymatic synthesis of phenolic lipids by lipase-catalyzed transesterification of dihydrocaffeic acid (DHCA) with fish liver oil was investigated in a selected organic solvent medium. These synthesized phenolic lipids have potential use as nutraceutical products. Using a molar ratio of 1:8 DHCA to fish liver oil in hexane:2-butanone mixtures of 75:25 and 85:15 (v/v), the lipase-catalyzed reaction resulted in maximum conversion of 55.8 and 65.4%, respectively. The maximum conversion of phenolic monoacylglycerols in hexane:2-butanone mixture of 75:25 and 85:15 (v/v) was 40.3 and 37.7%, respectively; using the same solvent mixtures, the conversions of the phenolic diacylglycerol were 15.8 and 36.8%, respectively. Hexane:2-butanone mixture of 75:25 (v/v) was, therefore, the best organic solvent mixture for the production of phenolic monoacylglycerols, while that of 85:15 (v/v) was best for the production of phenolic diacylglycerols. The phenolic lipids produced from the fish liver oil and DHCA demonstrated antioxidant property as indicated by its free radical scavenging capacity.     

Lipase-catalyzed acidolysis of fish liver oil with dihydroxyphenylacetic acid in organic solvent media

Lipase-catalyzed acidolysis reaction of fish liver oil with dihydroxyphenylacetic acid (DHPA) was investigated in terms of enzyme specificity as well as the effects of enzyme concentration, molar substrate ratio and organic solvent mixture on the bioconversion yield. The highest bioconversion yield of 83% was obtained when Novozym 435 was used as biocatalyst in a hexane:2-butanone mixture of 75:25 (v/v) at a fish liver oil to DHPA substrate molar ratio of 4:1; however, lower bioconversion yield (15%) was obtained when Lipozyme IM 20 was used. The bioconversion yield of phenolic monoacylglycerols (MAGs) increased from 11 to 70% when the ratio of the hexane/2-butanone reaction medium was changed from 85:15 to 75:25 (v/v), whereas that of phenolic diacylglycerols (DAGs) remained relatively unchanged (13–16%). The results also showed that the acidolysis reaction resulted in an increase of C_20:5 ω-3 and C_22:6 ω-3 proportions from 11.5 and 20.2% in the original fish liver oil to 22.6–27.1 and 22.8–23.1% in the phenolic lipids, respectively. The radical scavenging ability of phenolic lipids was determined to be about half-time lower than that of α-tocopherol.     

Lipase-catalyzed transesterification of dihydrocaffeic acid with flaxseed oil for the synthesis of phenolic lipids

Lipase-catalyzed transesterification reaction of dihydrocaffeic acid (DHCA) with flaxseed oil in organic solvent media was investigated. Using equal molar concentration of DHCA and flaxseed oil, only phenolic monoacylglycerols were obtained with a transesterification yield (TY) of 18.9%. A 1:4 DHCA to flaxseed oil ratio resulted in the production of both phenolic mono and diacylglycerols, with TY of 39.6 and 27.8%, respectively. On the other hand, when 1:8 ratio of DHCA to flaxseed oil was used, the TY of phenolic diacylglycerols (46.0%) was higher than that of the phenolic monoacylglycerols (33.3%). The TY of phenolic diacylglycerols increased from 25.1 to 55.8%, when the ratio of the hexane/2-butanone reaction medium was changed from 65:35 to 85:25 (v/v); however, the TY of phenolic monoacylglycerols decreased slightly from 34.0 to 31.8%. The relative proportion of the C(18:3)n-3 was higher in the phenolic mono and diacylglycerols, 64.9 and 59.5%, respectively, as compared to the original flaxseed oil, 53.1%. The radical scavenging ability of phenolic lipids was significant; however, it was about half than that of alpha-tocopherol.     

Enzymatic esterification of dihydrocaffeic acid with linoleyl alcohol in organic solvent media

The enzymatic esterification of dihydrocaffeic acid with linoleyl alcohol, using immobilized lipases (Lipozyme IM 20 and Novozym 435), was investigated in selected organic solvent media. Novozym 435 was found to be more efficient for catalyzing the esterification reaction. The highest enzymatic activity of 0.89 μmol esterified linoleyl alcohol/g solid enzyme/min was obtained in a hexane/2-butanone mixture of 75:25 (v/v), with an esterification yield of 75%; however, an increase in the 2-butanone proportion in the mixture up to 50% (v/v) resulted in a decrease in enzymatic activity and esterification yield to 0.38 μmol esterified linoleyl alcohol/g solid enzyme/min and 40%, respectively. The maximum esterification yield of 99.3% was obtained with a dihydrocaffeic acid to linoleyl alcohol ratio of 1:8. The electrospray ionization-mass spectroscopic structural analysis of the end products confirmed the biosynthesis of dihydrocaffeic acid ester of linoleyl alcohol, which demonstrated an anti-radical activity using 2,2-diphenyl-1-picrylhydrazyl as a radical model.     

Lipase-catalyzed synthesis of structured phenolic lipids in solvent-free system using flaxseed oil and selected phenolic acids as substrates

Structured phenolic lipids (PLs) were obtained by lipase-catalyzed transesterification of flaxseed oil, in a solvent-free system (SFS), with selected phenolic acids, including hydroxylated and/or methoxylated derivatives of cinnamic, phenyl acetic and benzoic acids. A bioconversion yield of 65% was obtained for the transesterification of flaxseed oil with 3,4-dihydroxyphenyl acetic acid (DHPA). However, the effect of the chemical structure of phenolic acids on the transesterification of flaxseed oil in SFS was of less magnitude as compared to that in organic solvent system (OSS). Using DHPA, the APCI-MS analysis confirmed the synthesis of monolinolenyl, dilinolenyl, linoleyl linolenyl and oleyl linolenyl dihydroxyphenyl acetates as phenolic lipids. A significant increase in the enzymatic activity from 200 to 270 nmol of PLs/g solid enzyme/min was obtained upon the addition of the non-ionic surfactant Span 65. However, upon the addition of the anionic surfactant, sodium bis-2-ethylhexyl sulfosuccinate (AOT), and the cationic one, hexadecyltrimethylammonium bromide (CTAB), the enzymatic activity was decreased slightly from 200 to 192 and 190 nmol of PLs/g solid enzyme/min, respectively. The results also showed that the increase in DHPA concentration from 20 to 60 mM resulted in a significant increase in the volumetric productivity (P(V)) from 1.61 to 4.74 mg PLs per mL reaction mixture per day.     

Application of solvent engineering to optimize lipase-catalyzed 1,3-diglyacylcerols by mixture response surface methodology

1,3-Diacylglycerol has been introduced in Japan as a cooking oil under the trade name of Econa to reduce body fat accumulation. Solvent engineering was applied to determine the optimum solvent mixtures for the lipase-catalyzed synthesis of 1,3-DAG by mixture response surface methodology. n-Hexane was required to maintain the lipase activity and the product selectivity could be adjusted by changing the hydrophobicity of reaction medium. The optimum yield (40%) of 1,3-DAG synthesis was obtained with n-hexane/octane (1:1, v/v).     

Improvement in lipase-catalyzed methanolysis of triacylglycerols for biodiesel production using a solvent engineering method

A solvent engineering strategy was applied to the lipase-catalyzed methanolysis of triacylglycerols for biodiesel production. The effect of different pure organic solvents and co-solvent mixtures on the methanolysis was compared. The substrate conversions in the co-solvent mixtures were all higher than those of the corresponding pure organic solvents. Further study showed that addition of co-solvent decreased the values of |log P_interface − log P_substrate| and thus led to a faster reaction. The more the values of |log P_interface − log P_substrate| decreased, the faster the reaction proceeded and the higher the conversion attained. Different co-solvent ratio was further investigated. The co-solvent mixture of 25% t-pentanol:75% isooctane (v/v) was optimal, with which both the negative effects caused by excessive methanol and by-product glycerol could be eliminated. There was no obvious loss in lipase activity even after being repeatedly used for 60 cycles (720 h) with this co-solvent mixture as reaction medium. Other lipases and lipase combinations can also catalyze methanolysis in this co-solvent mixture. Furthermore, other vegetable oils were also explored for biodiesel production in this co-solvent mixture and it had been found that this co-solvent mixture media has extensive applicability.     

Lipid Extraction from <Emphasis Type="Italic">Tetraselmis</Emphasis> sp. Microalgae for Biodiesel Production Using Hexane-based Solvent Mixtures

Lipid extraction is a critical step in the downstream processing of biodiesel production from microalgae. Solvent extraction using mixtures of non-polar and polar solvents is one of the most well-known processes for this purpose. Hexane is the most common solvent of choice for large-scale lipid extractions due to its technical and economic advantages, especially its high selectivity toward lipids and low cost. In this study, extractions using mixtures of hexane and polar solvents were evaluated for their performance in order to develop a more efficient method for large-scale lipid extraction from microalgae. The combination of hexane and methanol resulted in the highest fatty acid methyl ester (FAME) yield for lipids from Tetraselmis sp. The effects of extraction conditions, including proportions of methanol to hexane, ratios of total solvent volume to dry biomass, and extraction time, on extraction yields were evaluated to determine optimum conditions providing higher lipid and FAME yields. The optimal conditions were as follows: proportion of hexane to methanol of 1:1, ratio of total solvent volume to dry biomass of 10 mL/g, and extraction time of 120 min. Finally, the selected solvent mixture and optimal conditions were applied to larger scale extraction experiments with scale-up factors of 10, 50, and 100. FAME yields of large-scale extractions were almost completely consistent with increasing scale-up factors. The results of this study suggest that a hexane and methanol mixture is a promising solvent for large-scale lipid extraction from microalgae.     

Enzymatic desymmetrization of 3-(4-fluorophenyl)glutaric anhydride through enantioselective alcoholysis in organic solvents

The enzymatic desymmetrization of 3-(4-fluorophenyl)glutaric anhydride (3-FGA) was investigated through lipase-catalyzed enantioselective alcoholysis in organic solvents. An immobilized Lipase B from Candida Antarctica (Novozym 435) was found to be an efficient biocatalyst for the enantioselective alcoholysis of 3-FGA. Methyl tert-butyl ether (MTBE) and methanol were chosen as the suitable reaction medium and acyl acceptor, respectively. The optimum reaction temperature, molar ratio of methanol to 3-FGA and 3-FGA concentration were 25°C, 2:1 and 100 mM, respectively. Under these conditions, complete conversion was achieved and methyl (S)-3-(4-fluorophenyl)glutarate ((S)-MFG) was obtained in a moderate ee value of 80%. Furthermore, the reaction was performed on a gram scale and the ee value of (S)-MFG was enriched to 96% after treatment with a toluene/hexane (2/1, v/v) mixture.     

Improvement in lipase-catalyzed methanolysis of triacylglycerols for biodiesel production using a solvent engineering method

A solvent engineering strategy was applied to the lipase-catalyzed methanolysis of triacylglycerols for biodiesel production. The effect of different pure organic solvents and co-solvent mixtures on the methanolysis was compared. The substrate conversions in the co-solvent mixtures were all higher than those of the corresponding pure organic solvents. Further study showed that addition of co-solvent decreased the values of |log P_interface − log P_substrate| and thus led to a faster reaction. The more the values of |log P_interface − log P_substrate| decreased, the faster the reaction proceeded and the higher the conversion attained. Different co-solvent ratio was further investigated. The co-solvent mixture of 25% t-pentanol:75% isooctane (v/v) was optimal, with which both the negative effects caused by excessive methanol and by-product glycerol could be eliminated. There was no obvious loss in lipase activity even after being repeatedly used for 60 cycles (720 h) with this co-solvent mixture as reaction medium. Other lipases and lipase combinations can also catalyze methanolysis in this co-solvent mixture. Furthermore, other vegetable oils were also explored for biodiesel production in this co-solvent mixture and it had been found that this co-solvent mixture media has extensive applicability.     

Effects of crude rapeseed oil on lipid composition in Arctic charr Salvelinus alpinus

This study investigated the effects of crude rapeseed oil (RO) on lipid content and composition in muscle and liver of Arctic charr Salvelinus alpinus . Triplicate groups were fed diets containing fish oil (FO):RO ratio of 100:0, 75:25, 50:50 and 25:75 until two-fold mass increase. Total lipid content increased significantly in the liver with higher proportion of RO in the diet. Profound effects were seen in the fatty acid composition in the analysed tissues with a reduction in 20:5n-3 and 22:6n-3 and an increase in 18:2n-6 with higher RO content in the diets. A drop in cholesterol content was seen at 25% inclusion of RO in both tissues. Wild-caught fish contained a considerably higher amount of 20:4n-6 in both storage and membrane lipids of white muscle compared with the experimental fish.     

Stimulation of n-alkane conversion to dicarboxylic acid by organic-solvent- and detergent-treated microbes

Summary A wild-type strain of Cryptococcus neoformans and Pseudomonas aeruginosa were used to convert n-pentadecane to the corresponding dioic acid, tridecane 1,13-dicarboxylic acid (DC-15). Altering the cell permeability by treating C. neoformans with 1% (v/v) toluene or 7% (v/v) Triton X-100 stimulated production of DC-15 by 1.5-fold and fourfold, respectively. Furthermore, DC-15 productivity was increased from 2.5 mg/l per hour to 18 or 30 mg/l per hour, respectively. If 10% (v/v) hexane was used to treat the yeast culture, stimulation of DC-15 production could reach 200% and more viable cells remained compared to the toluene-treated culture. Data from the organic solvent treatment experiment indicated that the solvent with a higher polarity showed a more adverse effect on DC-15 production. P. aeruginosa was vulnerable to most organic solvents; however, Tween 80 could greatly stimulate the conversion of n-pentadecane to DC-15. Although organic solvents and non-ionic detergents could enhance DC-15 formation by microbial conversion, it was inhibited by elevated levels of DC-15.Offprint requests to: E.-C. Chan     

Markedly improving Novozym 435-mediated regioselective acylation of 1-beta-D-arabinofuranosylcytosine by using co-solvent mixtures as the reaction media

A comparative study was made of Novozym 435-catalyzed regioselective acylation of 1-beta-D-arabinofuranosylcytosine with vinyl propionate for the preparation of the 5'-O-monoester in eleven co-solvent mixtures and three pure polar solvents. Novozym 435 displayed low or no acylation activity toward 1-beta-D-arabinofuranosylcytosine in pure polar solvents, although those solvents can dissolve the nucleosides well. When a hexane-pyridine co-solvent system was adopted, both the initial rate and the substrate conversion were enhanced markedly. The polarity of co-solvent mixtures had significant effect on the reaction. Among the solvent mixtures investigated, the higher the polarity of the solvent mixture, the lower the initial reaction rate and the substrate conversion. It was also found that the acylation was dependent on the hydrophobic solvent content, the water activity and the reaction temperature. The most suitable co-solvent, initial water activity, and reaction temperature were hexane-pyridine (28:72, v/v), 0.07, and 50 degrees C, respectively. Under these conditions, the initial rate, the substrate conversion and the regioselectivity were as high as 91.1 mM h(-1), >97% and >98%, respectively, after a reaction time of 6 h. Among the reaction mediums examined, the lowest apparent activation energy was achieved with hexane-pyridine (28:72, v/v), in which Novozym 435 also exhibited good thermal stability.     

Effective recovery of poly‐β‐hydroxybutyrate (PHB) biopolymer from Cupriavidus necator using a novel and environmentally friendly solvent system

This work demonstrates a significant advance in bioprocessing for a high‐melting lipid polymer. A novel and environmental friendly solvent mixture, acetone/ethanol/propylene carbonate (A/E/P, 1:1:1 v/v/v) was identified for extracting poly‐hydroxybutyrate (PHB), a high‐value biopolymer, from Cupriavidus necator. A set of solubility curves of PHB in various solvents was established. PHB recovery of 85% and purity of 92% were obtained from defatted dry biomass (DDB) using A/E/P. This solvent mixture is compatible with water, and from non‐defatted wet biomass, PHB recovery of 83% and purity of 90% were achieved. Water and hexane were evaluated as anti‐solvents to assist PHB precipitation, and hexane improved recovery of PHB from biomass to 92% and the purity to 93%. A scale‐up extraction and separation reactor was designed, built and successfully tested. Properties of PHB recovered were not significantly affected by the extraction solvent and conditions, as shown by average molecular weight (1.4 × 10⁶) and melting point (175.2°C) not being different from PHB extracted using chloroform. Therefore, this biorenewable solvent system was effective and versatile for extracting PHB biopolymers. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:678–685, 2016     

Lipase-catalyzed biosynthesis of cinnamoylated lipids in a selected organic solvent medium

Biosynthesis of cinnamoylated lipids through the lipase-catalyzed transesterification reaction of cinnamic acid with triolein was investigated in organic solvent media. Electrospray ionization-mass spectroscopy (ESI-MS) structural analysis of the reaction mixture revealed the formation of two major end products, monoleyl-1(3)-cinnamate and dioleyl-2-cinnamate. Decreasing the molar ratio of cinnamic acid to triolein from 1:1 to 1:4.5 resulted in an increase in the maximum bioconversion yield of cinnamoylated lipids from 19 to 42%, which remained constant at a lower ratio of 1:6. However, an excess of triolein appeared to have a more beneficial effect on the formation of dioleyl-2-cinnamate than monoleyl-1(3)-cinnamate, leading to different end product compositions at ratios of substrates. With cinnamic acid to triolein ratios of 1:4.5 and 1:6.0, an increase in the bioconversion yield of cinnamoylated lipids to 55% was achieved by adding 2.2 mgmL(-1) silica gel to the reaction mixture. Radical scavenging activity of cinnamoylated lipids, with 50% of radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging, was found to be higher than that of its corresponding phenolic acid.     

Efficient water removal in lipase-catalyzed esterifications using a low-boiling-point azeotrope

High conversions in lipase-catalyzed syntheses of esters from free acyl donors and an alcohol requires efficient removal of water preferentially at temperatures compatible to enzyme activity. Using a lipase B from Candida antarctica (CAL-B)-mediated synthesis of sugar fatty-acid esters, we show that a mixture of ethyl methylketone (EMK) and hexane (best ratio: 4:1, vo/vo) allows efficient removal of water generated during esterification. Azeotropic distillation of the solvent mixture (composition: 26% EMK, 55% hexane, 19% water) takes place at 59 degrees C, which closely matches the optimum temperature reported for CAL-B. Water is then removed from the azeotrope by membrane vapor permeation. In case of glucose stearate, 93% yield was achieved after 48 h using an equimolar ratio of glucose and stearic acid. CAL-B could be reused for seven reaction cycles, with 86% residual activity after 14 d total reaction time at 59 degrees C. A decrease in fatty-acid chain length as well as increasing temperatures (75 degrees C) resulted in lower conversions. In addition, immobilization of CAL-B on a magnetic polypropylene carrier (EP 100) facilitated separation of the biocatalyst.     

Lipase-catalyzed synthesis of xylitol monoesters: solvent engineering approach

A solvent engineering strategy was applied to the lipase-catalyzed synthesis of xylitol-oleic acid monoesters. The different esterification degrees for this polyhydroxylated molecule were examined in different organic solvent mixtures. In this context, conditions for high selectivity towards monooleoyl xylitol synthesis were enhanced from 6 mol% in pure n-hexane to 73 mol% in 2-methyl-2-propanol/dimethylsulfoxide (DMSO) 80:20 (v/v). On the contrary, the highest production of di- and trioleoyl xylitol, corresponding to 94 mol%, was achieved in n-hexane. Changes in polarity of the reaction medium and in the molecular interactions between solvents and reactants were correlated with the activity coefficients of products. Based on experimental results and calculated thermodynamic activities, the effect of different binary mixtures of solvents on the selective production of xylitol esters is reported. From this analysis, it is concluded that in the more polar conditions (100% dimethylsulfoxide (DMSO)), the synthesis of xylitol monoesters is favored. However, these conditions are unfavorable in terms of enzyme stability. As an alternative, binary mixtures of solvents were proposed. Each mixture of solvents was characterized in terms of the quantitative polarity parameter E(T)(30) and related with the activity coefficients of xylitol esters. To our knowledge, the characterization of solvent mixtures in terms of this polarity parameter and its relationship with the selectivity of the process has not been previously reported.     

Interesterification selectivity in lipase catalyzed reactions of low molecular weight triglycerides

Summary In the absence of an organic solvent, a buffer to enzyme weight ratio of 1 gives maximum selectivity to interesterification over hydrolysis in a lipase-catalyzed mixture of triacetin and tributyrin. Addition of hexane enhances interesterification as does a reversed micelle configuration.     

Radioactive assay for aryl hydrocarbon hydroxylase. Improved method and biological importance

By addition of two volumes of a 1M aqueous KOH/dimethylsulfoxide ($\text{15}\text{85}$; v/v) mixture to the enzymatic incubation medium, it is possible to selectively extract the unmetabolized benzo(a)pyrene in hexane. Therefore, the radio-activity remaining in the water phase corresponds to all the in vitro synthesized metabolites. This isotopic method is very sensitive (2 × 10^?11 moles) and is almost insensitive to the room lighting. The aryl hydrocarbon hydroxylase activities found with this method are 2,3 and 10 times higher in the liver, lung and kidney respectively compared to those obtained with the fluorimetric method.     

Removal of water produced from lipase-catalyzed esterification in organic solvent by pervaporation

Esterification of oleic acid with n-butanol in the presence of Lipozyme(R) was carried out at 25 degrees C in isooctane with various initial water activities. Initial reaction rate as well as equilibrium conversion decreased at high initial water activity. Therefore, removal of water present in the reaction mixtures was essential. A pervaporation process was applied to the lipase-catalyzed synthesis of n-butyloleate to remove water. Pervaporation selectively separated water from the reaction mixture using a nonporous polymeric membrane, cellulose acetate. Therefore, pervaporation is potentially applicable to remove the water produced from various enzymatic processes, such as synthesis of various esters, peptides, and glycosides in a solvent system as well as in a solvent-free system. (c) 1995 John Wiley & Sons, Inc.