Production of Omega-3 Triacylglycerol concentrates using a new food grade immobilized Candida antarctica Lipase B

Triacylglycerol concentrates of eicosapentaenoic and docosahexaenoic omega-3 fatty acids were synthesized either via transesterification or esterification of glycerol with the corresponding ethyl ester or free fatty acid concentrates, respectively. A newly developed food grade immobilized Candida antarctica lipase Β system using an Amberlite FPX-66 hydrophobic matrix, was compared with a commercially available non-food grade commercial system, for their ability to catalyze these reactions. For either system, the transesterification required higher temperature (90◦C) than esterification (70°C) to achieve maximum triacylglycerol yields. The newly developed immobilized system efficiently catalyzes the esterification of free fatty acids with glycerol and differs from the existing commercial system in that it is food grade and has a more uniform and larger particle distribution. The new system significantly improves flow in a packed bed reactor, enabling multiple reuse of the catalyst for up to 80 repeats.

Effects of lipoprotein lipase gene variations, a high-carbohydrate low-fat diet, and gender on serum lipid profiles in healthy Chinese Han youth

A high-carbohydrate low-fat (HC/LF) diet and lipoprotein lipase gene (LPL) Ser447Stop and Hind III polymorphisms have separately been found to be associated with triacylglycerol (TG) and high density lipoprotein cholesterol (HDL-C). This study sought to test the effects of LPL polymorphisms and an HC/LF diet on the serum lipid profile of Chinese with a lower incidence of coronary artery disease (CAD) consuming a diet with less fat and more carbohydrates. Fifty-six healthy subjects (22.89 ± 1.80 years) were given a control diet of 30.1% fat and 54.1% carbohydrates for 7 days, followed by an HC/LF diet of 13.8% fat and 70.1% carbohydrate for 6 days; there were no changes in the fatty acid composition or restrictions on total energy. Serum lipid profiles at baseline, before and after the HC/LF diet, and LPL polymorphisms were analyzed. After 6 days of the HC/LF diet, TG and the homeostasis model assessment of insulin resistance (HOMAIR) index were found to increase only in females with S447S. No decrease in HDL-C was noted. In subjects with Hind III polymorphism, increased TG was found in all females but not in males. Increased HDL-C, together with apolipoprotein (apo) AI, was found in male H- carriers but not in males with H+/H+ and females. In conclusion, LPL Ser447Stop and Hind III polymorphisms modified the effects of an HC/LF diet on the serum lipid profiles of a young Chinese population in different ways. Effective strategies for dietary interventions targeted at younger populations should take into account the interplay between genetic polymorphisms, diet, and gender.

Enzymatic synthesis of isopropyl myristate using immobilized lipase from Bacillus cereus MTCC 8372

A purified alkaline thermo-tolerant bacterial lipase from Bacillus cereus MTCC 8372 was immobilized on a Poly (MAc- co -DMA- cl -MBAm) hydrogel. The hydrogel showed approximately 94% binding capacity for lipase. The immobilized lipase (2.36 IU) was used to achieve esterification of myristic acid and isopropanol in n -heptane at 65 °C under continuous shaking. The myristic acid and isopropanol when used at a concentration of 100 mM each in n -heptane resulted in formation of isopropyl myristate (66.0 ± 0.3 mM) in 15 h. The reaction temperature below or higher than 65°C markedly reduced the formation of isopropyl myristate. Addition of a molecular sieve (3 Å × 1.5 mm) to the reaction mixture drastically reduced the ester formation. The hydrogel bound lipase when repetitively used to perform esterification under optimized conditions resulted in 38.0 ± 0.2 mM isopropyl myristate after the 3 ^rd cycle of esterification.

Purification and characterization of a low molecular mass alkaliphilic lipase of Bacillus cereus MTCC 8372

A low molecular mass alkaliphilic extra-cellular lipase of Bacillus cereus MTCC 8372 was purified 35-fold by hydrophobic interaction (Octyl-Sepharose) chromatography. The purified enzyme was found to be electrophoretically pure by denaturing gel electrophoresis and possessed a molecular mass of approximately 8 kDa. It is a homopentamer of 40 kDa as revealed by native-PAGE. The lipase was optimally active at 55 °C and retained approximately half of its original activity after 40 min incubation at 55 °C. The enzyme was maximally active at pH 8.5. Mg ²⁺ , Cu ²⁺ , Ca ²⁺ , Hg ²⁺ , Al ³⁺ and Fe ³⁺ at 1 mM enhanced hydrolytic activity of the lipase. Interestingly, Hg ²⁺ ions synergized and Zn ²⁺ and Co ²⁺ ions antagonized the lipase activity. Among surfactants, Tween 80 promoted the lipase activity. Phenyl methyl sulfonyl fluoride (PMSF, 15 mM) decreased 98% of original activity of lipase. The lipase was highly specific towards p -nitrophenyl palmitate and showed a V _max and K _m of 0.70 mmol.mg ⁻¹ .min ⁻¹ and 32 mM for hydrolysis of p NPP.

Synthesis of ethyl acetate employing celite-immobilized lipase of Bacillus cereus MTCC 8372

A wide range of fatty acid esters can be synthesized by esterification and transesterification reactions catalyzed by lipases in non-aqueous systems. In the present study, immobilization of a purified alkaline extra-cellular lipase of Bacillus cereus MTCC 8372 by adsorption on diatomaceous earth (celite) for synthesis of ethyl acetate via transesterification route was investigated. B. cereus lipase was deposited on celite (77% protein binding efficiency) by direct binding from aqueous solution. Immobilized lipase was used to synthesis of ethyl acetate from vinyl acetate and ethanol in n -nonane. Various reaction conditions, such as biocatalyst concentration, substrates concentration, choices of solvents ( n -alkanes), incubation time, temperature, molecular sieves (3Å × 1.5 mm), and water activity(a _w ), were optimized. The immobilized lipase (25 mg/ml) was used to perform transesterification in n -alkane(s) that resulted in approximately 73.7 mM of ethyl acetate at 55 °C in n -nonane under shaking (160 rpm) after 15 h, when vinyl acetate and ethanol were used in a equimolar ratio (100 mM each). Addition of molecular sieves (3Å × 1.5 mm) as well as effect of water activity of saturated salt solutions (KI, KCl and KNO ₃ ) to the transesterification efficiency has inhibitory effect. Batch operational stability tests indicated that immobilized lipase had retained 50% of its original catalytic activity after four consecutive batches of 15 h each.

Synthesis of ethyl oleate employing synthetic hydrogel-immobilized lipase of Bacillus coagulans MTCC-6375

Ten polymeric hydrogels were chemically synthesized by varying the concentrations of copolymer (DMA) and cross-linker (MBAm) molecules. An alkaline lipase of Bacillus coagulans MTCC-6375 was immobilized onto a poly (MAc-co-DMA-cl-MBAm)-hydrogel support at pH 8.5 and temperature 55ºC in 16 h. The bound lipase possessed 7.6 U.g⁻¹ (matrix) lipase activity with a specific activity of 18 U.mg⁻¹ protein. Hydrogel bound-lipase catalyzed esterification of oleic acid and ethanol to synthesize ethyl oleate in n-nonane. Various kinetic parameters were optimized to produce ethyl oleate using immobilized lipase. The optimal parameters were bound enzyme/substrate (E/S) ratio 0.62 mg/mM, ethanol/oleic acid 100 mM:75 mM or 100 mM:100 mM, incubation time 18 h and reaction temperature 55ºC that resulted in approximately 53% conversion of reactants into ethyl oleate in n-nonane. However, addition of a molecular sieve to the reaction mixture promoted the conversion to 58% in 18 h in n-nonane, which was equivalent to 55 mM of ethyl oleate produced.

Synthesis of ethyl laurate by hydrogel immobilized lipase of Bacillus coagulans MTCC-6375

An alkaline thermo-tolerant lipase from Bacillus coagulans MTCC-6375 was purified and efficiently immobilized onto a synthetic hydrophobic poly (MAc-co-DMA-cl-MBAm)-hydrogel at pH 8.5 and temperature 55°C in 16 h. The hydrogel bound matrix possessed 7.6 IU g -1 matrix lipase activity with a specific activity of 18 IU mg -1 protein. Immobilized lipase was used to catalyze the esterification of lauric acid and ethanol to produce ethyl laurate in n-nonane. The reaction conditions that were optimized to produce ethyl laurate in n-nonane included enzyme/substrate (E/S) ratio, substrate concentration, reaction time and reaction temperature. The optimized parameters were E/S ratio of 0.5 mg mM -1, ethanol:lauric acid in ratio of 100 mM:100 mM and reaction time of 15 h at 65°C under continuous shaking (200 rpm). Optimized conditions resulted in 66% conversion of reactants into ethyl laurate in n-nonane in the presence of 300 mg molecular sieve mL -1 reaction mixture.

Increased hydrolysis by Thermomyces lanuginosus lipase for omega-3 fatty acids in the presence of a protic ionic liquid

We report that the hydrolytic performance of Thermomyces lanuginosus lipase, TLL, and its selectivity towards concentrating clinically important omega 3 fatty acids was increased by the addition of a protic ionic liquid, pIL, Triethylammonium mesylate, TeaMs. We show that TeaMs has a structure altering effect on TLL, changing both the secondary and tertiary structure of TLL. The thermal activity of TLL was also significantly enhanced by the addition of TeaMs.

Enzymatic synthesis of isopropyl acetate catalysed by immobilized bacillus cereus lipase in organic medium

Selective production of fragrance fatty acid ester from isopropanol and acetic acid has been achieved using silica-immobilizedlipase of Bacillus cereus MTCC 8372. A purified thermoalkalophilic extracellular lipase was immobilized by adsorption onto the silica. The effects of various parameters like molar ratio of substrates (isopropanol and acetic acid; 25 to 100 mM), concentration ofbiocatalyst (25–125 mg/mL), reaction time, reaction temperature, organic solvents,molecular sieves, and initial water activity werestudied for optimal ester synthesis. Under optimized conditions, 66.0mM of isopropyl acetate was produced when isopropanol and acetic acid were used at 100mM: 75mM in 9h at 55◦C in n-heptane under continuous shaking (160 rpm) using bound lipase(25mg). Addition of molecular sieves (3 °A ×1.5mm) resulted in a marked increase in ester synthesis (73.0mM). Ester synthesiswas enhanced by water activity associated with pre-equilibrated saturated salt solution of LiCl. The immobilized lipase retained more than 50% of its activity after the 6th cycle of reuse.