Lipase immobilization on modified zirconia nanoparticles: Studies on the effects of modifiers

Zirconia nanoparticles modified with carboxylic acid with a long alkyl chain can significantly enhance the activity of immobilized lipases used for asymmetric synthesis in organic media. In this study, various carboxylic acids with different alkyl chain lengths were grafted to zirconia. Lipase from Pseudomonas cepacia (PCL) was immobilized on the modified zirconia nanoparticles and used for the resolution of (R,S)-1-phenylethanol through acylation in isooctane. The results showed that among the straight-chained carboxylic acids that formed a closely packed layer on the support, stearic acid with a long alkyl chain gave the best activity/enantioselectivity. The initial activity obtained was about 10 times higher than that from lipase loaded onto unmodified zirconia. This improvement could be attributed to the interaction between the long hydrophobic chain of the modifier and the lipase, leading to an interfacial activation effect. The stability of the immobilized PCL was investigated, and the lipase was found to be stable for at least ten consecutive runs with a substrate concentration of 5 g/L. At a high substrate concentration of ca. 100 g/L, the activity of the lipase gradually decreased, which suggests that deactivation was induced by substrate toxicity.     

Synthesis of short chain alkyl esters using cutinase from Burkholderia cepacia NRRL B2320

Short chain alkyl esters are well appreciated for fruity flavors they provide. These are mainly applied to the fruit-flavored products like jam, jelly, beverages, wine and dairy. Cutinase from Burkholderia cepacia NRRL B 2320 was found to be active in catalyzing the synthesis of alkyl esters in organic solvent. The optimal temperature range for the enzyme catalyzed synthesis was found to be from 35 °C to 40 °C. The maximum conversion (%) during synthesis of ester was obtained for butyric acid (C4) and valeric acid (C5) with butanol reflecting the specificity of the enzyme for short-chain length fatty acids. In case of alcohol specificity, butanol was found to be most preferred substrate by the enzyme and conversion (%) decreased with increasing carbon chain length of alcohol used in the esterification reaction. The kinetic analysis for the synthesis of butyl butyrate by varying concentration of one substrate at a time (butanol or butyric acid), showed that Ping–Pong Bi Bi model with acid inhibition and influence of initial water is most suitable model for the prediction of the reaction kinetics.     

Lipase immobilisation: an equilibrium study of lipases immobilised on hydrophobic and hydrophilic/hydrophobic supports

This work investigates the enzyme-support equilibrium behaviour in immobilised lipase biocatalysts. Equilibrium data determines the maximum enzyme up-take by unit weight of support. Four lipases were immobilised on two polymeric supports, respectively. They were Lipase PS from Pseudomonas, Lipolase 100L from Humicola, SP871 from Rhizomucor miehel and QL from Alcaligenes. The supports were Accurel EP100 (a polypropylene material) and 45SAA (a polypropylene/silica composite). Experimentally, equilibrium was expressed in terms of lipase loading (LU/g support) versus residual lipase concentration (LU/dm³). Activity, efficiency and operational stability of the immobilised lipases were assayed by solvent-free esterification of oleic acid and octanol.Equilibrium data were modelled by the Langmuir, Freundlich and Redlich–Peterson formulae. It was found that Lipolase 100L/Accurel, PS/45SAA and SP871/45SAA systems conformed to the Langmuir behaviour, while Lipase PS/Accurel and SP871/Accurel systems followed the Freundlich behaviour and Lipolase 100L/45SAA, QL/45SAA and QL/Accurel EP100 resembled Redlich–Peterson behaviour. Whereas immobilisation on Accurel EP100 resulted in classical equilibrium isotherms with all four lipases, immobilisation on support 45SAA resulted in two-plateau equilibrium curves which included a step change in the isotherm for all lipases studied, except for SP871. Quantitatively, for 1 g lipase, Accurel and 45SAA had a maximum capacity of 140 and 260 kLU for PS, 112 and 550 kLU for Lipolase 100L, 320 and 800 kLU for SP871 and 18 and 29 kLU for QL, respectively.     

Optimization of medium chain length fatty acid incorporation into olive oil catalyzed by immobilized Lip2 from Yarrowia lipolytica

Triacylglycerols (TAG) enriched with medium chain fatty acids (M) present specific nutritional, energetic and pharmaceutical properties. Structured lipids (SL) were produced by acidolysis between virgin olive oil and caprylic (C8:0) or capric (C10:0) acids in solvent-free media, catalyzed by the main extracellular lipase from Yarrowia lipolytica lipase 2 (YLL2), immobilized in Accurel MP 1000. Response surface methodology was used for modeling and optimization of the reaction conditions catalyzed by immobilized YLL2. Central composite rotatable designs were performed as a function of the reaction time (2.5–49.5 h) and the molar ratio of medium chain fatty acid/TAG (MR; 0.6–7.4), for both acids, and also of temperature (32–48 ̊C) for C8:0 experiments. As for capric acid, the incorporation of caprylic acid in olive oil showed not to depend of the temperature, within the tested range. The response surfaces, fitted to the experimental data, were described by a first-order polynomial equation, for C8:0 incorporation, and by a second-order polynomial equation for C10:0 incorporation. Under optimized conditions (48 h reaction at 40 ̊C, with a molar ratio of 2:1 M/TAG) the highest incorporation was reached for C8:0 (25.6 mol%) and C10:0 (21.3 mol%).     

Biosynthesis of ethyl caproate and other short ethyl esters catalyzed by cutinase in organic solvent

The main objective of this work was to study the enzymatic synthesis of short chain ethyl esters, a group of relevant aroma molecules, by Fusarium solani pisi cutinase in an organic solvent media (iso-octane), and to assess the influence of different parameters on the reaction yield.Cutinase displayed high initial esterification rates in iso-octane, which amounted to 1.15 μmol min⁻¹ mg⁻¹ for ethyl butyrate (C₄ acid chain) and 1.06 μmol min⁻¹ mg⁻¹ for ethyl valerate (C₅ acid chain). High product yields, 84% for ethyl butyrate and 96% for ethyl valerate, were observed after 6 h of reaction, for an initial equimolar concentration of substrates (0.1 M).The highest product yield (97%) was observed for ethyl caproate (C₆) synthesis, a compound which is a part of natural apple and pineapple flavour, for an alcohol:acid molar ratio of 2 (0.2 M ethanol concentration).Cutinase affinity for short chain length carboxylic acids (C₄–C₆) in ester synthesis in iso-octane confirmed previous observations in reversed micellar system.     

Biocatalytic synthesis,structural elucidation,antioxidant capacity and tyrosinase inhibition activity of long chain fatty acid acylated derivatives of phloridzin and isoquercitrin

Our present investigation describes the regioselective enzymatic acylation of two series of acylated derivatives of phloridzin and isoquercitrin with six different long chain saturated, mono- and poly-unsaturated fatty acids. The biocatalytic synthesis was optimized to achieve 81–98% yields, using immobilized lipase B, from Candida antarctica (Novozym 435®), in acetone at 45 °C. The synthesized esters have been analyzed by ¹H NMR, ¹³C NMR spectroscopy and evaluated for their antioxidant capacity and tyrosinase inhibition, using in vitro assays. Among all the phloridzin and isoquercitrin derivatives, the greatest potential for inhibition of tyrosinase activity (p ?0.05) was exhibited by the α-linolenic acid ester of isoquercitrin.     

Optimization of Candida sp. 99-125 lipase catalyzed esterification for synthesis of monoglyceride and diglyceride in solvent-free system

Esterification of glycerol and oleic acid catalyzed by lipase Candida sp. 99-125 was carried out to synthesize monoglyceride (MAG) and diglyceride (DAG) in solvent-free system. Beta-cyclodextrin as an assistant was mixed with the lipase powder. Six reaction variables, initial water content (0–14 wt% of the substrate mass), the glycerol/oleic acid molar ratio (1:1–6:1), catalyst load (3–15 wt% of the substrate mass), reaction temperature (30–60 °C), agitator speed (130–250 r/min) and beta-cyclodextrin/lipase mass ratio (0–2) were optimized. The optimal conditions to the synthesis of MAG and DAG were different: the optimal glycerol/oleic acid molar ratio, beta-cyclodextrin/lipase mass ratio, catalyst load and reaction temperature were 6:1, 0, 5%, 50 °C for MAG, and 5:1, 1.5, 10%, 40 °C for DAG, respectively. The optimal water content and agitator speed for both MAG and DAG were 10% and 190 r/min, respectively. Under the optimal conditions, 49.6% MAG and 54.3% DAG were obtained after 8 h and 4 h, respectively, and the maximum of 81.4% MAG plus DAG (28.1% MAG and 53.3% DAG) was obtained after 2 h under the DAG optimal condition. Above 90% purity of MAG and DAG can be obtained by silica column separation.     

Ability of Vasconcellea × heilbornii lipase to catalyse the synthesis of alkyl esters from vegetable oils

Lipase-catalysed synthesis of alkyl esters is regarded as a potential alternative to chemical catalysis. Owing to its availability as a waste material from the babaco fruit production, its strong lipolytic activity and its natural immobilization, the dried latex of Vasconcellea × heilbornii appears as a good candidate to produce alkyl esters. The ability and performance of this lipase to catalyse the alcoholysis of sunflower oil with various primary alcohols was evaluated in a solvent-free system. A linear correlation between the final reaction rate and the alcohol polarity was established. For methanolysis, the influence of substrates ratio on final conversion rate was studied at different temperatures. At 30 °C, the lipase was inactivated by shaking in a mixture containing more than 0.5 molar equivalents of methanol; the minimum methanol concentration for enzyme deactivation increased with temperature. Moreover, for a 0.5:1 methanol/TAG molar ratio, conversion rates of 73, 66 and 55% were obtained at 30, 40 and 55 °C respectively, showing that the increase of temperature diminished the final methanolysis conversion rate. These facts were associated to the miscibility of methanol in oil and to the thermodynamic state of the medium. To overcome the inactivation of the lipase by methanol, alcoholysis was carried out by fractionated addition of methanol. In those conditions, Vasconcellea × heilbornii latex could catalyse the conversion of 70% of sunflower TAGs in methyl esters at 30 °C.     

Optimized synthesis of (Z)-3-hexen-1-yl caproate using germinated rapeseed lipase in organic solvent

(Z)-3-hexen-1-yl esters are important green top-note components of food flavors and fragrances. Effects of various process conditions on (Z)-3-hexen-1-yl caproate synthesis employing germinated rapeseed lipase acetone powder in organic solvent were investigated. Rapeseed lipase catalyzed ester formation more efficiently with non-polar compared to polar solvents despite high enzyme stability in both types of solvents. Maximum ester yield (90%) was obtained when 0.125 M (Z)-3-hexen-1-ol and caproic acid were reacted at 25 °C for 48 h in the presence of 50 g/L enzyme in heptane. Enzyme showed little sensitivity towards a_w with optimum yield at 0.45, while added water did not affect ester yield. Esterification reduced by increasing molecular sieves (>0.0125%, w/v). The highest yields of caproic acid were obtained with isoamyl alcohol (93%) followed by butanol and (Z)-3-hexen-1-o1 (88%) respectively reflecting the enzyme specificity for straight and branched chain alcohols. Secondary alcohols showed low reactivity, while tertiary alcohol had either very low reactivity or not esterified at all. A good relationship has been found between ester synthesis and the solvent polarity (log P value); while no correlation for the effect of solvents on residual enzyme activity was observed. It may be concluded that germinated rapeseed lipase is a promising biocatalyst for the synthesis of valuable green flavor note compound. The enzyme also showed a wide range of temperature stability (5–50 °C).     

Ultrasound assisted lipase catalysed synthesis of isoamyl butyrate

The present work illustrates the incorporation of ultrasound and its improved impact in the lipase catalysed esterification. Synthesis of isoamyl butyrate from isoamyl alcohol and butyric acid using immobilised Novozym 435, has been carried out in the presence of ultrasound. The optimisation of various parameters affecting the synthesis of ester in presence of ultrasound was done. The systematic experimentation involves change of one working parameter at one time while keeping the others constant. For the maximum conversion, optimum parameters such as the ultrasound of 25 kHz frequency with power of 70 W, at the temperature of 60 °C with stirring speed of 80 rpm, mole ratio of alcohol:acid followed as 2:1, use of molecular sieves weighing 2 g, with immobilised enzyme loading of 2% (m/v) and duty cycle of 83%, were obtained. The optimum parameters collectively, gave 96% conversion of the product in 3 h as compared with 10 h in absence of ultrasound. The immobilised biocatalyst, Novozym 435 has an added benefit of reusability till 7 repetitive cycles. Besides, the synthesis is executed in the solvent free system that contributes the production of flavour in greener way.     

Synthesis and biological evaluation of new potential inhibitors of N-acylethanolamine hydrolyzing acid amidase

N-Acylethanolamines, including N-palmitoyl-ethanolamine (PEA), are hydrolyzed to the corresponding fatty acids and ethanolamine by fatty acid amide hydrolase (FAAH). Recently, N-acylethanolamine-hydrolyzing acid amidase (NAAA) was identified as being able to specifically hydrolyze PEA. In order to find selective and effective inhibitors of this enzyme, we synthesized and screened several amides, retroamides, esters, retroesters and carbamates of palmitic acid (1–21) and esters with C15 and C17 alkyl chains (22–27). Cyclopentylhexadecanoate (13) exhibited the highest inhibitory activity on NAAA (IC₅₀ = 10.0 μM), without inhibiting FAAH up to 50 μM. Compound 13 may become a useful template to design new NAAA inhibitors.     

Lipase immobilized microstructured fiber based flow-through microreactor for facile lipid transformations

A facile continuous flow-through Candida antarctica lipase B immobilized silica microstructured optical fiber (SMOF) microreactor for application in lipid transformations has been demonstrated herewith. The lipase was immobilized on the amino activated silica fiber using glutaraldehyde as a bifunctional reagent. The immobilized lipase activity in the SMOF was tested calorimetrically by determination of p-nitrophenyl butyrate hydrolysis products. The specific activity of the immobilized lipase was calculated to be 0.91 U/mg. The SMOF microreactor performance was evaluated by using it as a platform for synthesis of butyl laurate from lauric acid and n-butanol in n-hexane and n-heptane at 50 °C, with products identified by gas chromatography–mass spectrometry (GC–MS). Different substrate mole ratios were evaluated, with 1:3, lauric acid:n-butanol showing best performance. Remarkably, percentage yields of up to 99% were realized with less than ∼38 s microreactor residence time. In addition, the SMOF microreactor could be reused many times (at least 7 runs) with minimal reduction in the activity of the enzyme. The enzyme stability did not change even with storage of the microreactor in ambient conditions over one month.     

Optimized enzymatic synthesis of ascorbyl esters from lard using Novozym 435 in co-solvent mixtures

A mild and efficient method for the conversion of fatty acid methyl esters from lard into ascorbyl esters via lipase-catalyzed transesterification in co-solvent mixture is described. A solvent engineering strategy was firstly applied to improve fatty acid ascorbyl esters production. The co-solvent mixture of 30% t-pentanol:70% isooctane (v/v) was optimal. Response surface methodology (RSM) and central composite design (CCD) were employed to estimate the effects of reaction parameters, such as reaction time (12–36 h), temperature (45–65 °C), enzyme amount (10–20%, w/w, of fat acid methyl esters), and substrate molar ratio of fatty acid methyl esters to ascorbic acid (8:1–12:1) for the synthesis of fatty acid ascorbyl esters in co-solvent mixture. Based on the RSM analysis, the optimal reaction conditions were determined as follows: reaction time 34.32 h, temperature 54.6 °C, enzyme amount 12.5%, substrate molar ratio 10.22:1 and the maximum conversion of fatty acid ascorbyl esters was 69.18%. The method proved to be applicable for the synthesis of ascorbyl esters using Novozym 435 in solvent.     

Purification and properties of enantioselective lipase from a newly isolated Bacillus cereus C71

A lipase from Bacillus cereus C71 was purified to homogeneity by ammonium sulfate precipitation, followed by Phenyl-Sepharose chromatography, DEAE ion exchange chromatography and CIM^® QA chromatography. This purification procedure resulted in a 1092-fold purification of lipase with 18% yield. The molecular mass of the purified enzyme was determined to be approximately 42 kDa by SDS-PAGE and mass spectrometer. The lipase was stable in the pH range of 8.5–10.0, with the optimum pH 9.0. The enzyme exhibited maximum activity at 33 °C and retained 92% of original activity after incubation at 35 °C for 3 h. The protein hydrolyzed p-nitrophenyl esters with acyl chain lengths between C4 and C12. Enzyme activity was strongly inhibited in the presence of Cu²⁺ and Zn²⁺ but promoted by non-ionic surfactants. The lipase demonstrated higher enantioselectivity toward R-isomer of ethyl 2-arylpropanoate than the commercial lipases, and can be used potentially as a catalyst to prepare optically pure pharmaceuticals.     

Influence of the operating conditions on lipase-catalysed synthesis of ricinoleic acid estolides in solvent-free systems

In this work, the synthesis of ricinoleic acid estolides, also named polyricinoleic acid (PR), in two different solvent-free reaction systems, catalysed by immobilised Candida rugosa lipase is described. First, the esterification was performed in an open air jacketed batch reactor and the influence of amount of enzyme, temperature and initial water content was tested. The best results were obtained when 5 g of soaked immobilised derivative was used as biocatalyst, and the reaction was carried out at 40 °C. It was observed that environmental relative humidity plays an important role in the enzymatic synthesis of ricinoleic acid estolides and, given that this parameter takes on a wide range of values depending on the season, it is considered the main cause of the poor reproducibility achieved in the open air reactor. To solve this problem, the ricinoleic acid estolides were synthesised under controlled atmosphere in a vacuum reactor with dry air intake. The optimum drying time of 7 h was selected. In these conditions, PR with an acid value of 57.5 mg KOH/g was synthesised in 48 h of reaction and the results were entirely reproducible.     

Scaling-up the synthesis of myristate glucose ester catalyzed by a CALB-displaying Pichia pastoris whole-cell biocatalyst

The novel whole-cell biocatalyst Candida antarctica lipase B displaying-Pichia pastoris (Pp-CALB) is characterized by its low preparation cost and could be an alternative to the commercial immobilized Candida antarctica lipase B (CALB). This study addresses the feasibility of using Pp-CALB in large scale glucose fatty acid esters production. 1,2-O-Isopropylidene-α-d-glucofuranose (IpGlc) was used as the acyl acceptor to overcome the low solubility of glucose in an organic solvent and to avoid the addition of toxic co-solvents. IpGlc significantly improved the Pp-CALB catalyzing esterification efficiency when using long chain fatty acids as the acyl donor. Under the preferred operating conditions (50 °C, 40 g/L molecular sieve dosage and 200 rpm mixing intensity), 60.5% of IpGlc converted to 6-O-myristate-1, 2-O-isopropylidene-α-d-glucofuranose (C14-IpGlc) after a 96-h reaction in a 2-L stirred reactor. In a 5-L pilot scale test, Pp-CALB also showed a similar substrate conversion rate of 55.4% and excellent operational stability. After C14-IpGlc was collected, 70% trifluoroacetic acid was adopted to hydrolyze C14-IpGlc to myristate glucose ester (C14-Glc) with a high yield of 95.3%. In conclusion, Pp-CALB is a powerful biocatalyst available for industrial synthesis, and this study describes an applicable and economical process for the large scale production of myristate glucose ester.     

Sustainable preparation of a novel glycerol-free biofuel by using pig pancreatic lipase: Partial 1,3-regiospecific alcoholysis of sunflower oil

The preparation of a novel biofuel denoted as Ecodiesel-100 from the partial 1,3-regiospecific alcoholysis of sunflower oil is reported. Pig pancreatic lipase (PPL) was employed in the reaction as both free and immobilised enzyme on sepiolite. The resulting biofuel is composed of fatty acid ethyl esters and monoglycerides (FAEE/MG) blended in a molar relation 2/1. The novel biofuel has similar physico-chemical properties compared to those of conventional biodiesel and/or petrodiesel, avoiding the production of glycerine as by-product.The biocatalyst was found to be strongly fixed to the inorganic support (87.5%). Nevertheless, the efficiency of the immobilised enzyme was reduced to less than half (42%) compared to that of the free PPL. Quantitative conversions of triglycerides and high yields to FAEE were obtained under mild reaction conditions (20–80 °C, oil/alcohol 2/1 v:v ratio and PPL 0.01–0.1% w/w of total substrate). The immobilised enzyme showed a remarkable stability as well as a great reusability (more than 11 successive reuses) without a significant loss of its initial catalytic activity. Both immobilised and free enzyme exhibited the same reaction mechanism, according to the coincidental results in the Arrhenius parameters (Ln A and E_a). The immobilised PPL was found to be very suitable for the continuous production of biofuel due to its facile recyclability from the reaction mixture.     

Ethyl isovalerate synthesis using Candida rugosa lipase immobilized on silica nanoparticles prepared in nonionic reverse micelles

Uniform and monodispersed silica nanoparticles were synthesized with a mean diameter of 100 ± 20 nm as analyzed by Transmission Electron Microscopy (TEM). Glutaraldehyde was used as a coupling agent for efficient binding of the lipase onto the silica nanoparticles. For the hydrolysis of pNPP at pH 7.2, the activation energy within 25–40 °C for free and immobilized lipase was 7.8 and 1.25 KJ/mol, respectively. The V_max and K_m of immobilized lipase at 25 °C for pNPP hydrolysis were found to be 212 μmol/min/mg and 0.3 mM, whereas those for free lipase were 26.17 μmol/min and 1.427 mM, respectively. The lower activation energy of immobilized lipase in comparison to free lipase suggests a change in conformation of the enzyme leading to a requirement for lower energy on the surface of the nanoparticles. A better yield (7 fold higher) of ethyl isovalerate was observed using lipase immobilized onto silica nanoparticles in comparison to free lipase.     

Enzymatic synthesis and surface properties of novel rhamnolipids

New rhamnolipids were obtained via the development of a synthesis procedure consisting of two biocatalyzed steps. In the first step, naringinase was used to introduce a primary alcohol function onto rhamnose by glycosylation of 1,3-propanediol. In the second step, immobilized lipase B from Candida antarctica catalyzed the esterification of the primary hydroxyl group with mono- and di-carboxylic fatty acids of increasing chain length (from C8 to C14). For the monoic acids, the initial rate and 24 h yield decreased with increasing chain length. For the dioic acid, the number of carbon atoms of the acid did not influence these parameters. The new rhamnolipid obtained with tetradecanoic acid showed very good surface properties. At pH 5, it had a very low critical aggregation concentration of 1.70 μM and it diminished water's surface tension to 27.6 mN/m. It was also able to form stable insoluble monolayers. On the other hand, the rhamnolipid formed with tetradecanedioic acid showed far less interesting surface properties.     

Immobilisation of Pseudomonas gessardii acidic lipase derived from beef tallow onto mesoporous activated carbon and its application on hydrolysis of olive oil

The study was carried out to immobilise the acidic lipase derived from Pseudomonas gessardii onto mesoporous activated carbon (MAC₄₀₀) for the application of hydrolysis of olive oil. MAC₄₀₀ was prepared from rice husk by the two stages process. P. gessardii was isolated from the beef tallow acclimatised soil. The acidic lipase (ALIP) was produced from a slaughterhouse waste, namely beef tallow as a substrate and immobilised onto MAC₄₀₀. The maximum immobilisation capacity of the MAC₄₀₀ was 3570 U/g at optimum immobilisation conditions; time (180 min), pH (5.0) and temperature (30 °C). The immobilised lipase had better thermal stability and reusability than the free lipase. The immobilisation of ALIP onto MAC₄₀₀ (MAC₄₀₀–ALIP) followed pseudo-first-order rate kinetics with rate constant 0.012/min. The Michaelis–Menten constant of MAC₄₀₀–ALIP was lower than that of the ALIP, which confirmed the higher affinity between enzyme and substrate. The immobilization of acidic lipase onto MAC₄₀₀ was confirmed by Fourier transform-infrared spectroscopy (FT-IR), scanning electron microscopy (SEM) and X-ray diffraction (XRD) pattern analysis. Reusability study of MAC₄₀₀–ALIP on olive oil hydrolysis showed 82% of hydrolysis up to 13 runs and 50% of hydrolysis up to 35 cycles of reuse. This work concludes that the acidic lipase immobilised mesoporous activated carbon matrix can be considered as a potential biocatalyst for the hydrolysis of olive oil. Thus, the enzyme immobilised matrix has potential industrial applications.”