Immobilized Lipase Preparation Research Articles

Immobilization of Lipase from Thermomyces Lanuginosus and Its Glycerolysis Ability in Diacylglycerol Preparation.

In the glycerolysis process for diacylglycerol (DAG) preparation, free lipases suffer from poor stability and the inability to be reused. To address this, a cost-effective immobilized lipase preparation was developed by cross-linking macroporous resin with poly (ethylene glycol) diglycidyl ether (PEGDGE) followed by lipase adsorption. The selected immobilization conditions were identified as pH 7.0, 35 °C, cross-linking agent concentration 2.0%, cross-linking time 4 h, lipase amount 5 mg/g of support, and adsorption time 4 h. Enzymatic properties of the immobilized lipase were analyzed, revealing enhanced pH stability, thermal stability, storage stability, and operational stability post-immobilization. The conditions for immobilized enzyme-catalyzed glycerolysis to produce DAG were selected, demonstrating the broad applicability of the immobilized lipase. The immobilized lipase catalyzed glycerolysis reactions using various oils as substrates, with DAG content in the products ranging between 35 and 45%, demonstrating broad applicability. Additionally, the changes during the repeated use of the immobilized lipase were characterized, showing that mechanical damage, lipase leakage, and alterations in the secondary structure of the lipase protein contributed to the decline in catalytic activity over time. These findings provide valuable insights for the industrial application of lipase.

Immobilization and bioimprinting strategies to enhance the performance in organic medium of the metagenomic lipase LipC12

The present work evaluates the immobilization of LipC12 on different supports in tandem with bioimprinting technique, in order to improve its activity and stability in organic medium. Oleic acid was selected as the bioimprinting molecule. The immobilized LipC12 was applied in the synthesis of pentyl oleate by esterification reaction and in the production of fatty acids, mono, and diglycerides via hydrolysis of triacylglycerols, in n-heptane reaction media. For all immobilized lipase preparations, an increase in the conversion of oleic acid to pentyl oleate was observed when immobilization in tandem with bioimprinting treatment was carried out versus immobilization without bioimprinting. The highest conversions were achieved using LipC12 immobilized on hydrophobic supports. The reuse potential of the immobilized preparations was evaluated. The preparations were used in eight successive cycles of esterification reactions and the best results were obtained for LipC12 immobilized on Immobead 150 and chitosan. The activity for the hydrolysis of soybean oil was improved by bioimprinting treatment only for LipC12 immobilized on commercial polypropylene and Accurel MP-1000. LipC12 immobilized on hydrophilic supports or on Immobead150 could be used to hydrolyze tricaprylin to obtain diglycerides with a high proportion of 1,2-diglycerides in reaction times as short as 30 min.

Influence of the Procedure to Immobilize Lipase on SBA-15 for Biodiesel Production from Palm Kernel Oil

Microbial lipase from Burkholderia cepacia was immobilized by covalent bond and physical adsorption on SBA-15 mesoporous support and its catalytic efficiency was measured in the formation of fatty acids ethyl esters from palm kernel oil. The immobilized biocatalyst and its support were characterized by X-ray Diffraction, Fourier Transform Infrared Spectroscopy, N2 Adsorption–desorption, and thermal analysis TG/DTG. The immobilization of the enzyme onto the support maintained the typical structure of the SBA-15. From the TG measurements, it can be deduced that about 25% of lipase was immobilized in SBA-15 via covalent bond and 15% was incorporated via physical adsorption. Both immobilized lipase preparations were used to perform the transesterification reactions using different oil: ethanol molar ratios and time. The biocatalyst prepared by physical adsorption promoted better results, measured by the apparent yield (> 90%) and conversion (98.9%) in all reactions, which were also confirmed by viscosity and density data. The regeneration of SBA-15 support was found to be feasible and demonstrated efficiency for 5 times, reducing the associated costs of the process.

Tailoring a robust nanozyme formulation based on surfactant stabilized lipase immobilized onto newly fabricated magnetic silica anchored graphene nanocomposite: Aggrandized stability and application.

Candida rugosa lipase (CRL) was treated with surfactants and immobilized onto the novel formulated magnetic graphene anchored silica nanocomposite (Fe3O4/SiO2/Gr NC). For this purpose, the surface of lipase was initially coated with Triton-X 100 and cetyltrimethylammonium bromide surfactants, to stabilize enzyme in its open form and was then adsorbed onto aminated Fe3O4/SiO2/Gr NC. Glutaraldehyde (GA) was then utilized to cross-link the adsorbed lipase onto the NC. The fabricated NC and conjugated lipase was characterized by various techniques such as FT-IR, XRD, TGA, SEM, TEM, CLSM, CD and Fluorescence spectroscopy. The magnetic character of the as-synthesized NC was verified by AGM investigation. CD and fluorescence spectroscopic analysis demonstrated slight structural rearrangements in lipase upon conjugation. The surfactant stabilized immobilized lipase demonstrated significantly enhanced thermostability, tolerance to various metal ions and inhibitors. The immobilization yield obtained owing to lipase interfacial activation by Triton X 100 and CTAB was remarkably enhanced by 6-folds and 3-folds, respectively which were remarkably higher in comparison to free immobilized lipase. The fabricated nanobiocatalysts were employed to synthesise green apple flavour ester, ethyl valerate via esterification reaction. Triton X 100 stabilized immobilized lipase was a better performer in yielding green apple flavour ester, demonstrating about 90% ester yield as compared to 78% yield obtained by CTAB stabilized immobilized lipase preparation. The obtained outcomes suggested that enzyme structure was stabilized by the GA treatment if executed in the absence or in the presence of detergent, and that, in the company of detergent, a conformation of the lipase with the exposed active center to the medium provided an aggrandized catalytic performance.

Green synthesis of methyl-12-hydroxyoctadec-9-enoate

In the current paper, methyl-12-hydroxyoctadec-9-enoate was synthesized by biocatalytic transesterification of castor oil with methanol in presence of two different indigenously immobilized lipase preparations. Aiming for optimum yield (94% methyl-12-hydroxyoctadec-9-enoate), the batch reaction was conducted to evaluate the influence of reaction time, oil to methanol ratio, and water content. The atom economy (91.67%) and E-factor (0.297) for the studied reaction was uncovered, as well as an analysis of the adherence of the twelve principles of green chemistry was summarized. The green credentials were assessed using an efficient tool in strategic planning, a strengths-weaknesses-opportunities-threats (SWOT) analysis. The operational stability of immobilized lipase reactor column (60 °C) was determined for a prolonged period (>300 h) with the intermittent water addition to maintaining live enzyme. Furthermore, the purification of methyl-12-hydroxyoctadec-9-enoate using liquid-liquid extraction aided purity of 98%, confirmed by HPLC and GC-MS analysis.

Immobilization of Rhizomucor miehei lipase onto montmorillonite K-10 and polyvinyl alcohol gel

Immobilization of enzymes from different sources on various supports in designed systems increases enzymes’ stability by protecting the active site of it from undesired effect of reaction environment. Also, immobilization decreases the cost of separation and facilities the reuse of the enzymes. Therefore, the design of new immobilization enzyme preparations has been an inevitable area of modern biotechnology. Herein, Rhizomucor miehei lipase (RML) was immobilized on montmorillonite K-10 (MMT-RML) by adsorption and in polyvinyl alcohol (PVA-RML) by entrapment to obtain a more stable and active lipase preparation. The free and immobilized lipase preparations were characterized for p-nitrophenyl palmitate hydrolysis. The apparent Michaelis–Menten (Kmapp) constant was almost the same for the free RML and PVA-RML, whereas the corresponding value was 17.7-fold lower for MMT-RML. PVA-RML and MMT-RML have shown a 1.1 and 23.8 folds higher catalytic efficiency, respectively, than that of the free RML. The half-lives of PVA-RML and MMT-RML were found to be 7.4 and 3.4 times longer than the free RML at 35 °C, respectively. PVA-RML and MMT-RML maintained 65% and 87% of their initial activities after four reuses. These results showed that the catalytic performance of RML has improved significantly by immobilization.

Improvement of activity and stability of Rhizomucor miehei lipase by immobilization on nanoporous aluminium oxide and potassium sulfate microcrystals and their applications in the synthesis of aroma esters

In this study, Rhizomucor miehei lipase (RML) was immobilized on the hexagonally-ordered nanoporous aluminium oxide membranes (RML-Al2O3-NP) by adsorption and as protein-coated microcrystals (RML-PCMCs) by simultaneously precipitating RML on micron-sized potassium sulfate crystals (K2SO4) in pre-chilled acetone. The hydrolytic activities of immobilized lipase preparations were investigated in terms of p-nitrophenyl palmitate hydrolysis and their esterification activities were examined for the synthesis of some aroma esters such as butyl acetate, isoamyl acetate, hexyl acetate, heptyl acetate, and geranyl acetate. The immobilization yields were 33.8 and 25.1%, respectively for RML immobilized on Al2O3-NP membranes and potassium sulfate crystals. The catalytic efficiency ratios of RML-Al2O3-NP and RML-PCMCs were 2.3- and 3.9-fold higher than that of the free lipase, respectively in terms of hydrolytic activity. The free lipase was stabilized as 4.1- and 10.5-fold, respectively at 40 and 50 °C when immobilized on Al2O3-NP. The corresponding stabilization factors were 4.6- and 12.8-fold higher for RML-PCMCs. RML-Al2O3-NP and RML-PCMCs maintained 84 and 86% of their initial hydrolytic activities, respectively after 10 reuses. Of the synthesized aroma esters, the highest yield was obtained for the geranyl acetate. After 4 h reaction time, no geraniol was detected in the preparative-scale (196 g/L) synthesis of geranyl acetate for both the immobilized lipases when the initial geraniol amount, vinyl acetate amount, RML-PCMCs amount, and reaction temperature values were 1 mmol, 3 mmol, 100 mg (or 300 mg RML-Al2O3-NP), and 50 °C, respectively. These results show that the immobilization of R. miehei lipase by adsorption on nanoporous aluminium oxide and as protein-coated microcrystals leads to the obtention of highly stable, catalytically more active, and reusable lipase preparations.

The Purification and Characterization of Lipases from Lasiodiplodia theobromae, and Their Immobilization and Use for Biodiesel Production from Coconut Oil.

The coconut kernel-associated fungus, Lasiodiplodia theobromae VBE1, was grown on coconut cake with added coconut oil as lipase inducer under solid-state fermentation conditions. The extracellular-produced lipases were purified and resulted in two enzymes: lipase A (68,000Da)-purified 25.41-fold, recovery of 47.1%-and lipase B (32,000Da)-purified 18.47-fold, recovery of 8.2%. Both lipases showed optimal activity at pH 8.0 and 35°C, were activated by Ca2+, exhibited highest specificity towards coconut oil and p-nitrophenyl palmitate, and were stable in iso-octane and hexane. Ethanol supported higher lipase activity than methanol, and n-butanol inactivated both lipases. Crude lipase immobilized by entrapment within 4% (w/v) calcium alginate beads was more stable than the crude-free lipase preparation within the range pH 2.5-10.0 and 20-80°C. The immobilized lipase preparation was used to catalyze the transesterification/methanolysis of coconut oil to biodiesel (fatty acyl methyl esters (FAMEs)) and was quantified by gas chromatography. The principal FAMEs were laurate (46.1%), myristate (22.3%), palmitate (9.9%), and oleate (7.2%), with minor amounts of caprylate, caprate, and stearate also present. The FAME profile was comparatively similar to NaOH-mediated transesterified biodiesel from coconut oil, but distinctly different to petroleum-derived diesel. This study concluded that Lasiodiplodia theobromae VBE1 lipases have potential for biodiesel production from coconut oil.

Production of 6-O-L-Ascorbyl Palmitate by Immobilized Candida antarctica Lipase B.

L-ascorbyl palmitate (ASP) is an oil-soluble derivative of ascorbic acid which is used extensively in food, cosmetics industry, and medical hygiene. Enzymatic synthesis of ascorbyl palmitate in tert-butyl alcohol was carried out using indigenously immobilized lipase preparation PyCal with ascorbic acid and palmitic acid as starting material. The developed batch process under optimized reaction conditions resulted in conversion of 90% with relatively shorter reaction time of 6h. Continuous process in packed bed reactor gave conversion of 50% with space time yield of 15.46g/L/h which was found to be higher than the reported literature on enzymatic synthesis of ascorbyl palmitate. The immobilized lipase used in the present work showed good reusability. Characterization of formed ascorbyl palmitate was carried out by FTIR, MS/MS, H1-NMR, and C13-NMR. The enzymatic process resulted in selective synthesis of 6-O-L-ascorbyl palmitate with purity of 98.6% and no side product formation. The use of underivatized starting materials, high space time yield of 15.46gL-1h-1, high recyclability of catalyst, and no by-product formation make the overall process highly efficient and clean in terms of energy consumption and waste generation, respectively. The optimized reaction parameters for ascorbyl palmitate synthesis in the present study can be used as a useful reference for industrial synthesis of fatty acid esters of ascorbic acid by enzymatic route.

Batch and semicontinuous production of l-ascorbyl oleate catalyzed by CALB immobilized onto Purolite® MN102

l-Ascorbyl oleate is recognized as efficient liposoluble antioxidant and biosurfactant which can be synthesized by using immobilized lipases as catalysts in organic media. However, development of economical production process is still a challenge, particularly in (semi)continuous systems. Hereby, we present application of novel immobilized lipase preparation, lipase B from Candida antarctica hydrophobically adsorbed onto styrene divinylbenzene based support, for the synthesis of l-ascorbyl oleate in batch bioreactor (BB) and fluidized bed bioreactor (FBB) with recirculation of reaction medium. Reaction kinetics was described by Ping Pong bi bi mechanism with low acyl donor inhibition. Considerably higher reaction rates (∼40%) were accomplished in FBB. Hydrodynamic study revealed that reaction is kinetically controlled and unhindered external and internal mass transfer was enabled in both systems. However, much higher shear stress occurred in BB (10.46Pa) comparing to FBB (5×10−2Pa). After 12 consecutive reaction cycles lasting 24h, 23.13g of product per gram of biocatalyst was synthesized in a FBB. Within this study, usage of new immobilized enzyme in semicontinuous system enabled high reaction rates and satisfying operational stability in the synthesis of l-ascorbyl oleate, offering good prospect for further process development.

Improving enantioselectivity of lipase from Candida rugosa by carrier-bound and carrier-free immobilization

The enantioselectivity of carrier-bound and carrier-free immobilized lipase from Candida rugosa (CRL) was studied. CRL was immobilized in six agarose-based carriers functionalized with different reactive groups and in two different CRL cross-linked aggregates. Both, activity and enantioselectivity of all the immobilized lipase preparations were evaluated with different racemic esters under different reaction conditions (temperature, pH and solvent polarity). A strong effect of reaction media and immobilization protocol on enzyme activity and selectivity was found. Enzyme immobilization and reaction engineering allowed us obtaining the best immobilization protocol and reaction conditions to achieve high activity and enantioselectivty of CRL as heterogeneous catalyst. CRL immobilized on an agarose-based carrier activated with primary amino groups preferentially hydrolyzed (S)-phenylethyl acetate with E>200 under pH 7, 4°C and 30% of acetonitrile. On the other hand, CRL aggregated and cross-linked through their carboxylic groups preferentially hydrolyzed the (S)-isomer of ethyl 2-hydroxy-4-phenylbutyrate with an E=39 under pH 5, 4°C and 30% of acetonitrile. This work demonstrates the success of the combinatorial enzyme engineering for the production of highly enantioselective heterogeneous biocatalysts by screening different immobilization protocols and reaction media conditions.

Acyl migration in enzymatic interesterification of triacylglycerols: Effects of lipases from Thermomyces lanuginosus and Rhizopus oryzae, support material, and water activity

The enzymatic interesterification of a solvent‐free, equimolar mixture of trilaurin and 1,3‐palmitin‐2‐olein was studied using three immobilized lipase preparations as catalysts. Analysis of triacylglycerol (TAG) content and fatty acid (FA) distribution monitored the lipase‐catalyzed interesterification in sn‐1,3 positions and FA exchange in the sn‐2 position caused by acyl migration. Lipase from Rhizopus oryzae immobilized on polypropylene showed high sn‐1,3 regioselectivity, and minimal exchange in the sn‐2 position. With lipase from Thermomyces lanuginosus on silica (Lipozyme® TL IM), completely randomized FA distribution was obtained in 24 h. T. lanuginosus lipase on polypropylene caused a moderate rate of FA exchange in the sn‐2 position. Thus, the T. lanuginosus lipase and silica promoted randomization of FA distribution, whereas the R. oryzae lipase and polypropylene did not. Higher water activity promoted hydrolysis and thereby increased concentrations of partial acylglycerols, but at the same time a decrease in the acyl migration rate of these intermediates was also observed. The net result was that at a certain degree of interesterification, there was no significant effect of water activity on the degree of exchange in the sn‐2 position. On the other hand, a low water activity had the major advantage of giving a high yield of TAG.Practical applications: Both the type and the position of FA affect the properties of TAG. In TAG interesterification, there are thus different requirements for regiospecificity in FA exchange, depending on which product is desired. According to our results, lipase‐based catalysts can be used for TAG interesterification either to achieve unchanged FA composition in sn‐2 position or fast FA randomization in all positions. This helps to broaden the application of lipases in interesterification. The production of TAG can be tailor‐made for lipid mixtures with particular TAG composition and FA distribution using proper lipases, support materials, water activity, and reaction time.The enzymatic LLL–POP model reaction was used to investigate effects of lipase, support material, and water activity on acyl migration. (Fig. A): effect of Lipozyme® TL IM, TLPP and ROPP at aw 0.26, (Fig. B): effect of aw between 0.15 and 0.80 using Lipozyme® TL IM as the example.

Bacillus sp. PS35 Lipase-Immobilization on Styrene-Divinyl Benzene Resin and Application in Fatty Acid Methyl Ester Synthesis.

Lipase is an enzyme with immense application potential. Ester synthesis by lipase catalysis in organic media is an area of key industrial relevance. Enzymatic preparations with traits that cater to the needs of this function are hence being intensely researched. The objectives of the study were to immobilize the lipase from Bacillus sp. PS35 by cross-linking and adsorption onto styrene-divinyl benzene (Sty-Dvb) hydrophobic resin and to comparatively characterize the free and immobilized lipase preparations. The work also aimed to apply the immobilized lipase for catalysing the fatty acid methyl ester (FAME) synthesis from palm oil and optimize the process parameters for maximizing the yield. In this study, the purified lipase from Bacillus sp. PS35 was immobilized by adsorption onto styrene-divinyl benzene hydrophobic resin with gluteraldehyde cross-linking. The immobilized enzyme showed better pH and temperature stabilities than the free lipase. Organic solvent stability was also enhanced, with the relative activity in the presence of methanol being shifted from 53% to 81%, thereby facilitating the enzyme's application in fatty acid methyl ester synthesis. It exhibited remarkable storage stability over a 30-day period and after 20 repetitive uses. Cross-linking also reduced enzyme leakage by 49%. The immobilized lipase was then applied for biodiesel production from palm oil. Methanol and oil molar ratio of 5:1, three step methanol additions, and an incubation temperature of 50°C were established to be the ideal conditions favoring the transesterification reaction, resulting in 97% methyl ester yield. These promising results offer scope for further investigation and process scale up, permitting the enzyme's commercial application in a practically feasible and economically agreeable manner.

Immobilized lipase on micro-porous biosilica for enzymatic transesterification of algal oil

Enzymatic transesterification reactions for biodiesel production require harsh conditions, which require methods of enzyme stability enhancements. In this study, we present covalently immobilized lipase on the biosilica-polymer composite as a viable method to obtain enzymes with enhanced stability in such harsh conditions. The fresh water microalgae Scenedesmus quadricauda was cultivated in a batch photo-bioreactor with CO2 aeration, and urea was supplied as nitrogen source (0.075gL−1). Under optimized conditions, the amount of extracted oil was around 29.6%. Finally, the algal oil was utilized for production of biodiesel via enzymatic transesterification reaction which were performed in n-hexane using the free and immobilized lipase preparations. Fatty acid methyl ester (FAME) components were determined using gas chromatography–mass spectrophotometry (GC–MS). The conversion of algal oil to biodiesel was found to be 85.7% and 96.4%, with the free and immobilized enzyme, respectively. The immobilized lipase was highly stable and only 17% of activity was lost after 6 cycles repeated uses.

Utilization of immobilized lipases as catalysts in the transesterification of non-edible vegetable oils with ethanol

This work reports the use of commercially available immobilized lipase preparations (Novozym® 435 and Lipozyme TL IM, both from Novozymes, and Lipase PS IM from Amano) as catalysts in the transesterification reaction of different alkyl-chain triglycerides with ethanol. The ethanolysis of native oils from Brazilian Amazon plants andiroba (Carapa guianensis), babassu (Orbignya sp.), jatropa (Jatropha curcas), and palm (Elaeis sp.) was studied in a solvent-free system. In a typical reaction, the immobilized preparations were added to the mixture of vegetable oil-to-ethanol in a molar ratio of 1:9. The reactions were performed at 50 ºC for a maximum period of 48 h. Under the conditions used, all the immobilized lipase preparations were able to generate the main esters of fatty acids present in the tested feedstocks, and both the reaction rate and ester yield were dependent on the source of lipase and vegetable oil. The viscosity values for the samples obtained in each reaction displayed a consistent reduction in relation to their original feedstocks, which also confirms the high conversion of triglycerides to ethyl esters (99.8-74.0%). The best performances were obtained with Amano PS IM and Novozym® 435, with the biodiesel samples from the babassu and jatropha oils exhibiting viscosity values in accordance with those predicted by the technical standards of ASTM D6751 (1.9-6.0 mm²/s). Lipozyme TL IM displayed an unsatisfactory performance, indicating that the conditions of the transesterification reaction should be improved. This comparative study using different catalysts and several vegetable oil sources with varying fatty acid compositions is particularly important for all tropical countries with a diversity of native vegetable oil sources.

Asymmetric ammonolysis of (R/S)-mandelic acid by immobilized lipases via direct amidation of mandelic acid in biphasic media

We have investigated the direct enantioselective amidation of mandelic acid with ammonia, catalyzed by a variety of commercial lipases including those from Candida rugosa, Mucor miehei, Pseudomonas sp., Rhizomucor miehei, and Thermomyces lanuginosus covalently immobilized onto Florisil® support via glutaraldehyde and polysuccinimide spacer arms. All the immobilized lipase preparations tested preferentially amidated the R isomer of mandelic acid. The highest amide yields were obtained for immobilized Pseudomonas sp. lipase preparations under the optimized reaction conditions. After 24 h of amidation, the reaction had proceeded with an excellent yield (50%) and enantiopurity (> 99%). The immobilized Pseudomonas sp. lipase preparations catalyzed the amidation reaction with the same yield and enantioselectivity. The enzyme immobilized via a glutaraldehyde spacer arm showed better reusability than that immobilized via a polysuccinimide spacer arm.In view of these results, it is revealed that the direct amidation of mandelic acid catalyzed by the immobilized Pseudomonas sp. lipases is a facile and effective methodology for obtaining (S)-mandelic acid and (R)-mandelamide.

Optimization of immobilization conditions of Mucor miehei lipase onto Florisil via polysuccinimide spacer arm using response surface methodology and application of immobilized lipase in asymmetric acylation of 2-amino-1-phenylethanols

In this study, the immobilization of Mucor miehei lipase onto Florisil® support via polysuccinimide spacer arm was scrutinized by using a 3-factor and 3-level Box–Behnken design. The independent parameters were immobilization pH, immobilization time and initial lipase concentration and the response was the specific activity of immobilized lipase. A quadratic equation was used to explain the relationship between the response and independent parameters. After analysis of variance test, coefficient of determination and adjusted coefficient of determination values were estimated as 0.98 and 0.94, respectively. The optimal immobilization pH, immobilization time and initial lipase concentration were determined as 6.0, 7 h and 1.1 mg mL−1, respectively, after desirability analysis. The specific activity values for three individual experiments were observed as 25.88 ± 0.73, 26.06 ± 0.47 and 25.96 ± 0.52 U mg protein−1 under the optimized conditions. The hydrolytic activities of free and immobilized lipase preparations were characterized using p-nitrophenyl palmitate as substrate. The esterification activity of immobilized lipase preparation was evaluated by asymmetric acylation of 2-(methylamino)-1-phenylethanol, 2-(ethylamino)-1-phenylethanol, 2-(butylamino)-1-phenylethanol, and 2-(hexylamino)-1-phenylethanol with vinyl acetate. The acylation protocol was optimized in terms of the effects of initial water amount, reaction temperature, molar ratio of amino alcohol to vinyl acetate, biocatalyst loading, organic medium and kind of lipases used. The developed protocol provided a facile methodology for the preparation of enantiopure 2-amino-1-phenylethanols which may be used as potential new β-adrenergic receptor antagonists.

Preparation of core–shell polymer supports to immobilize lipase B from Candida antarctica

Abstract Core–shell supports have been prepared and utilized to immobilize lipase B from Candida antarctica . The hydrophobic nature of the supports permitted to immobilize the enzyme via interfacial activation at low ionic strength. Different supports were prepared having different hydrophobicity and crosslinking degree, and compared to the commercially available. Accurel MP 1000 (hydrophobic macroporous polymer of propylene) is a commercial support described as advantageous in different circumstances and it was used as comparative control in the process of immobilization. The immobilized lipase preparations were evaluated in the hydrolysis of p -nitro-phenyl laurate and the esterification of oleic acid with ethanol. On the kinetic resolution of (±)-1,2- O -isopropylidene-3,6-di- O -benzyl- myo -inositol, vinyl acetate was used as activated acyl donor. Results were very diverse, as the lipase properties may be easily tuned via immobilization, and some of the supports permitted to obtain activities even a two fold factor higher than the same amount of lipase immobilized in Accurel MP 1000. Moreover, in many instances, the loading of the support with enzyme produced reduced total activity in some reactions while not in other. This was explained by changes in the physical properties of the support surface that may alter the entry of substrates. Supports PS- co -DVB/PS- co -DVB 25% and PMMA- co -DVB/PMMA- co -DVB 25% presented very good features to immobilize CALB.

Optimization of the Lipase Catalyzed Production of Structured Acylglycerols With Polyunsaturated Fatty Acids Isolated From Sardine Oil

<p>In the present work, direct enzyme-catalyzed esterification of n-3 polyunsaturated fatty acids (n-3 PUFA) isolated from sardine oil was optimized to obtain structured acyglycerols. A n-3 PUFA concentrate was prepared by urea crystallization of refined sardine oil and esterification was carried out mixing free fatty acids and glycerol at different molar ratios (<em>M</em> = 0.48, 1.5, 3.0, 4.5 and 5.52 mol/mol), using an immobilized lipase preparation from <em>Candida antarctica</em> (NV-435) at different temperatures (<em>T</em> = 38, 45, 55, 65 and 72 °C) and reaction times (<em>t</em> = 0.7, 2.75, 5.75, 8.75 and 10.8 h) in a rotatable central composition design. The degree of esterification was determined by analysis of the acylglycerides produced, using liquid chromatography (HPLC-ELSD). Optimization by response surface methodology (RSM) showed that in order to obtain higher esterification levels of n-3 PUFA to glycerol (99.5%), a molar ratio of 1.3 mol n-3 PUFA/mol glycerol, time 8.3 h and temperature 38 °C, are required. However, results of this work show that it is possible to drive the reaction to any determined product (MAG, DAG or TAG) by modifying the reaction conditions.</p>

Optimization of synthesis of fatty acid methyl esters catalyzed by lipase B from Candida antarctica immobilized on hydrophobic supports

Abstract In this work two immobilized preparations of lipase (EC 3.1.1.3) B from Candida antarctica (CALB) were compared for the synthesis of fatty acid methyl esters (FAME) using soybean oil. Commercial Novozym 435 (CALB-435) and CALB immobilized on styrene-divinylbenzene beads (CALB–MCI) were tested for the transesterification reactions. Central composite design (CCD) and response surface methodology (RSM) were used to optimize the reaction parameters, substrate molar ratio, enzyme content, and the added amount of water, on the initial reaction rate as response. The biocatalysts showed different optimal conditions for the production of FAME. For CALB-435, optima conditions were 5.6:1 molar ratio methanol:oil, 25% enzyme, and 5.44% of added water, while for MCI–CALB, these optima were 3:1 molar ratio methanol:oil, 25% enzyme, and 1.18% of added water, resulting in initial reaction rates of 51.47 mmol L −1 h −1 , and 57 mmol L −1 h −1 of FAME, respectively. Conversions of 93.38% using CALB-435, and 99.03% using CALB–MCI were obtained after 72 h of reaction under the optimized conditions. Repeated batches of reaction were carried out to test the operational stability of biocatalysts, with both preparations keeping around 70% of their initial activity after eight batches.