Casimir C. Akoh

Lipase-catalyzed acidolysis of olive oil and caprylic acid in a bench-scale packed bed bioreactor

Abstract Lipase-catalyzed acidolysis of olive oil and caprylic acid was performed in a bench-scale packed bed bioreactor to produce structured lipids (SL). A 1,3-specific lipase, IM 60 from Rhizomucor miehei was used as the biocatalyst. Reaction products were analyzed by reversed phase high-performance liquid chromatography, with an evaporative light scattering detector. Olive oil is characterized by four major clusters of triacylglycerol species with equivalent carbon number (ECN), C44, C46, C48, and C50. Three monosubstituted products and two disubstituted products were detected after the reaction. Monosubstituted products had ECN of C36, C38, and C40, and disubstituted products had ECN of C30 and C32. The effect of solvent, temperature, substrate mol ratio, and flow rate/residence time were studied. Optimal solvent-free production of SL was obtained at a substrate flow rate of 1 ml/min, residence time 2.7 h, temperature 60°C, and mol ratio 1:5 (olive oil/caprylic acid). Fatty acid distribution at the sn-2 position of olive oil was determined by pancreatic lipase hydrolysis as 74.8% oleic acid and 25.2% linoleic acid. SL produced at optimal conditions had 7.2% caprylic acid, 69.6% oleic acid, 21.7% linoleic acid and 1.5% palmitic acid at the sn-2 position.

Structured lipids: Synthesis and applications

Abstract Structured lipids (SL) are defined as triacylglycerols (TAG) restructured or modified to change the fatty acid composition and/or their positional distribution in glycerol molecules by chemical or enzymatic processes. SL may provide the most effective means of delivering desired fatty acids for nutritive or therapeutic purposes. In this review, the synthesis and applications of structured lipids, nutritional/clinical aspects of fatty acids, immobilization, and characteristics of lipases are discussed.

Effect of reaction parameters on SP435 lipase-catalyzed synthesis of citronellyl acetate in organic solvent

The reaction parameters affecting the enzymatic synthesis of citronellyl acetate by direct esterification were studied. Reaction mixtures were prepared using 2 ml dry n-hexane, 0.1 m citronellol, 0.1 m acetic acid, 10% (w/w) Candida antarctica lipase, SP435, incubated at 30°C and 200 rev min−1 for 24 h. A 98% molar conversion yield was achieved after 14 h incubation, the highest yield ever reported. Acetic acid concentrations > 0.30 m inhibited the esterification activity of SP435. Optimum temperature was 20°C, giving a yield of 98%. Addition of water to the reaction mixtures decreased the esterification activity of SP435. The highest yields were observed for solvents having log P values ≥ 0.85. SP435 was able to give 95.6% conversion even after the tenth reuse, demonstrating its stability and efficiency under the conditions of this study.

Lipase.Catalyzed Incorporation of n-3 Polyunsaturated Fatty Acids into Vegetable Oils

The ability of immobilized lipases IM60 fromMucor miehei and SP435 fromCandida antarctica to modify the fatty acid composition of selected vegetable oils by incorporation of n−3 polyunsaturated fatty acids into the vegetable oils was studied. The transesterification was carried out in organic solvent with free acid and ethyl esters of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) as acyl donors. With free EPA as acyl donor, IM60 gave higher incorporation of EPA than SP435. However, when ethyl esters of EPA and DHA were the acyl donors, SP435 gave higher incorporation of EPA and DHA than IM60. When IM60 and free acid were used, the addition of 5 μL water increased EPA incorporation into soybean oil by 4.9%. With ethyl ester of EPA as acyl donor, addition of 2 μL water increased EPA incorporation by 3.9%. For SP435, addition of water up to 2μL resulted in increased EPA incorporation, but the incorporation declined when the added water exceeded this amount. The addition of water increased the EPA incorporation into Trisun 90 after 24 h reaction but not the reaction rate at early stages of the reaction.

Protein engineering and applications of Candida rugosa lipase isoforms

Commercial preparations of Candida rugosa lipase (CRL) are mixtures of lipase isoforms used for the hydrolysis and synthesis of various esters. The presence of variable isoforms and the amount of lipolytic protein in the crude lipase preparations lead to a lack of reproducibility of biocatalytic reactions. Purification of crude CRL improve their substrate specificity, enantioselectivity, stability, and specific activities. The expression of the isoforms is governed by culture or fermentation conditions. Unfortunately, the nonsporogenic yeast C. rugosa does not utilize the universal codon CTG for leucine; therefore, most of the CTG codons were converted to universal serine triplets by site-directed mutagenesis to gain expression of functional lipase in heterologous hosts. Recombinant expressions by multiple-site mutagenesis or complete synthesis of the lipase gene are other possible ways of obtaining pure and different CRL isoforms, in addition to culture engineering. Protein engineering of purified CRL isoforms allows the tailoring of enzyme function. This involves computer modeling based on available 3-D structures of lipase isoforms. Lid swapping and DNA shuffling techniques can be used to improve the enantioselectivity, thermostability, and substrate specificity of CRL isoforms and increase their biotechnological applications. Lid swapping can result in chimera proteins with new functions. The sequence of the lid can affect the activity and specificity of recombinant CRL isoforms. Candida rugosa lipase is toxicologically safe for food applications. Protein engineering through lid swapping and rationally designed site-directed mutagenesis will continue to lead to the production of CRL isoforms with improved catalytic power, thermostability, enantioselectivity, and substrate specificity, while providing evidence for the mechanisms of actions of the various isoforms.

Immobilized lipase-catalyzed production of structured lipids with eicosapentaenoic acid at specific positions

Structured lipids (SL) were synthesized by the interesterification reaction between medium-chain triacylglycerols and eicosapentaenoic acid (EPA) ethyl ester. The products were partially purified, and the fatty acid at thesn-2 position was determined after pancreatic lipase-catalyzed hydrolysis. The effect of additives (water and glycerol) on the rate of reaction was also investigated. Mol% EPA incorporated into the triacylglycerols was increased by adding water when trilaurin and tricaprylin were the substrates and IM 60 was the biocatalyst. With SP 435, EPA incorporation was always less with additional water than without water. The addition of glycerol (0.005 g or 0.01 g) improved interesterification catalyzed by IM 60 to some degree, but an excess amount (0.02 g) inhibited the reaction. The reaction with glycerol showed no significant difference with SP 435. After scale-up and fractionation by column chromatography, we could recover approximately 0.3–0.4 g of product/g of reaction products. After hydrolysis by pancreatic lipase, we can conclude that IM 60 has a high specificity forsn-1,3 positions. With SP 435 lipase, 34.8–39.3 mol% of EPA was found at thesn-2 position of the recovered SL.

Synthesis of alkyl glycoside fatty acid esters in non-aqueous media byCandida sp. lipase

Alkyl glycoside fatty acid esters were successfully synthesized by lipase-catalyzed transesterification of methyl glucoside, methyl glucoside, methyl galactoside and octyl glucoside with methyl oleate. The experiments were carried out in organic media with lipase enzymes fromCandida sp. as biocatalysts. Time course and the effects of temperature, solvent type, substrate concentration, added water and of immobilizedvs. nonimmobilized enzyme were studied. The optimal conditions for the enzymatic synthesis of alkyl glycoside fatty acid esters were: a molar ratio of alkyl glycoside and methyl oleate of 1:4, an immobilized lipase, SP382 fromCandida sp.; benzene/pyridine (2:1, vol/vol) with no added water; temperature, 55°C; reaction time, 48 h; and shaking at 200 rpm. Acceptable levels of oleic acid incorporation (58.6–100 mol%) onto the alkyl glycosides were achieved.

Four-factor response surface optimization of the enzymatic modification of triolein to structured lipids

The ability of an immobilized lipase to modify the fatty acid composition of (88.8% C18:1, 4.3% C16:0, 3.1% C18:0, and 3.8% C18:2 as determined by gas chromatography, and approximately 90% triolein) in hexane by incorporation of a medium-chain fatty acid, capric acid (C10), to form structured triacylglycerol was studied. Response surface methodology was used to evaluate the effect of synthesis variables, such as reaction time (12–36 h), temperature (25–65°C), molar substrate ratio of capric acid to triolein (2:1–6:1), and enzyme amount (10–30% wt% of triacylglycerol), on the yield of structured lipid. Optimization of the transesterification was attempted to obtain maximum yield of structured lipid while using the minimum molar substrate ratio and enzyme amount as much as possible. Computer-generated contour plot interpretation revealed that a relatively high molar substrate ratio (6:1) combined with low enzyme amount (10%) after 30 h of reaction at 25°C gave optimum incorporation of capric acid. A total yield for combined monoand dicaproolein of up to 100% was obtained.

Enzymatic synthesis of geraniol and citronellol esters by direct esterification in n-hexane

SummaryShort chain fatty acid esters of geraniol and citronellol were synthesized by lipase-catalyzed direct esterification with yields ranging from 90 to 100% molar conversion. The effect of the order of addition of substrates, solvent and enzyme was investigated. Yields were highest when the solvent was added first followed by the substrates and then the lipase.

Antioxidant capacity and lipid characterization of six Georgia-grown pomegranate cultivars.

Six pomegranate (Punica granatum) cultivars were investigated for their antioxidant capacity and lipid profile. Total polyphenols were determined according to the Folin-Ciocalteau method. Major organic acids and phenolic compounds were analyzed by RP-HPLC. Two in vitro antioxidant assays, ferric reducing antioxidant power and Trolox equivalent antioxidant capacity, were used to assess antioxidant capacity. Total lipid was extracted according to the Folch method, and fatty acid methyl esters were determined by GC. Tocopherols and phospholipids were identified and quantified by NP-HPLC using a fluorescence detector for tocopherols and an evaporative light scattering detector for phospholipid analysis. Phytosterols were analyzed by GC. The predominant organic acid was citric acid followed by malic acid. The peel fraction had the highest total hydrolyzable tannins content (4792.3-6894.8 mg/100 g of FW). Overall, the highest antioxidant capacity was found in leaves followed by peel, pulp, and seed. Pomegranate seed had an average lipid content of 19.2% with punicic acid as the predominant fatty acid. Pomegranate seed had high contents of alpha-tocopherol (161.2-170.1 mg/100 g) and gamma-tocopherol (80.2-92.8 mg/100 g).