Immobilized Candida Antarctica Lipase Research Articles

Promiscuous lipase catalyzed a new P–C bond formation: Green and efficient protocol for one‐pot synthesis of α‐aminophosphonates

Abstractα‐Aminophosphonates are valuable substructures with important biological and pharmacological properties. Lipase catalytic promiscuity is a new method in the organic synthesis for the preparation of α‐aminophosphonates via multicomponent reaction in one pot. This efficient, simple, and eco‐friendly method proceeds in the presence of immobilized Candida Antarctica lipase as catalyst under solvent‐free conditions at room temperature. The new α‐aminophosphonates are synthesized in high yields (up to 96%). Moreover, enzymatic‐catalyzed P–C bond formation through a Kabachnik‐Fields reaction was achieved for the first time.

Enzymatic synthesis of 1,3-dihydroxyphenylacetoyl-sn-glycerol: Optimization by response surface methodology and evaluation of its antioxidant and antibacterial activities

In this study, the enzymatic synthesis of phenylacetoyl glycerol ester was carried out as a response to the increasing consumer demand for natural compounds. 1,3-dihydroxyphenylacetoyl-sn-Glycerol (1,3-di-HPA-Gly), labeled as “natural” compound with interesting biological properties, has been successfully synthesized for the first time in good yield by a direct esterification of glycerol (Gly) with p-hydroxyphenylacetic acid (p-HPA) using immobilized Candida antarctica lipase as a biocatalyst. Spectroscopic analyses of purified esters showed that the glycerol was mono- or di-esterified on the primary hydroxyl group. These compounds were evaluated for their antioxidant activity using two different tests. The glycerol di-esters (1,3-di-HPA-Gly) showed a higher antiradical capacity than that of the butyl hydroxytoluene. Furthermore, compared to the p-HPA, synthesized ester (1,3-di-HPA-Gly) exhibited the most antibacterial effect mainly against Gram + bacteria. Among synthesized esters the 1,3-di-HPA-Gly was most effective as antioxidant and antibacterial compound. These findings could be the basis for a further exploitation of the new compound, 1,3-di-HPA-Gly, as antioxidant and antibacterial active ingredient in the cosmetic and pharmaceutical fields.

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.

One pot biocatalytic synthesis of a biodegradable electroactive macromonomer based on 3,4-ethylenedioxytiophene and poly(l-lactic acid)

A novel electroactive macromonomer based on poly(l-lactic acid) (PLLA) with (3,4-ethylenedioxythiophene) (EDOT) functional end groups, was prepared by a traditional approach of organometallic polymerization with stannous octanoate [Sn(oct2)] and enzymatic polymerization using immobilized Candida antarctica Lipase B (CAL-B) and Amano lipase Pseudomonas cepacia(PS-IM), as catalysts. In the synthetic strategy, (2,3-dihydrothieno[3,4-b] dioxin-2-yl)methanol (EDOT-OH) was used to initiate the ring opening polymerization of lactide to yield PLLA with EDOT end group. All macromonomers (EDOT-PLLA) were characterized by 1H and 13C RMN, MALDI-TOF, GPC and EDX. Moreover, ICP-OES analysis showed the presence of Sn traces in the material synthesized by the traditional approach, but that pathway led to macromonomers with higher molecular weight while the enzymatic route led to completely metal-free macromonomers with medium and lower molecular weights. Also, electrochemical and chemical polymerization of EDOT-PLLA were tested showing that it is possible to prepare degradable conducting polymers based on poly(3,4-ethylenedioxythiphene) (PEDOT). The biocatalytic synthesis is a very promising and environmental friendly pathway for the preparation of biodegradable materials for short time applications.

Effect of supercritical carbon dioxide on the enzymatic production of biodiesel from waste animal fat using immobilized Candida antarctica lipase B variant

BackgroundWaste animal fat is a promising feedstock to replace vegetable oil that widely used in commercial biodiesel process, however the high content of free fatty acid in waste fat makes it unfeasible to be processed with commercial base-catalytic process. Enzymatic process is preferable to convert waste fat into biodiesel since enzyme can catalyze both esterification of free fatty acid and transesterification of triglyceride. However, enzymatic reaction still has some drawbacks such as lower reaction rates than base-catalyzed transesterification and the limitation of reactant concentration due to the enzyme inhibition of methanol. Supercritical CO2 is a promising reaction media for enzyme-catalyzed transesterification to overcome those drawbacks.ResultThe transesterification of waste animal fat was carried out in supercritical CO2 with varied concentration of feedstock and methanol in CO2. The CO2 to feedstock mass ratio of 10:1 showed the highest yield compared to other ratios, and the highest FAME yield obtained from waste animal fat was 78%. The methanol concentration effect was also observed with variation 12%, 14%, and 16% of methanol to feedstock ratio. The best yield was 87% obtained at the CO2 to feedstock ratio of 10: 1 and at the methanol to feedstock ratio of 14% after 6 h of reaction.ConclusionEnzymatic transesterification to produce biodiesel from waste animal fat in supercritical fluid media is a potential method for commercialization since it could enhance enzyme activity due to supercritical fluid properties to remove mass transfer limitation. The high yield of FAME when using high mass ratio of CO2 to oil showed that supercritical CO2 could increase the reaction and mass transfer rate while reducing methanol toxicity to enzyme activity. The increase of methanol concentration also increased the FAME yield because it might shift the reaction equilibrium to FAME production. This finding describes that the application of supercritical CO2 in the enzymatic reaction enables the application of simple process such as a packed-bed reactor.

Synthesis of Geraniol Esters in a Continuous-Flow Packed-Bed Reactor of Immobilized Lipase: Optimization of Process Parameters and Kinetic Modeling.

With increasing demand for perfumes, flavors, beverages, and pharmaceuticals, the various associated industries are resorting to different approaches to enhance yields of desired compounds. The use of fixed-bed biocatalytic reactors in some of the processes for making fine chemicals will be of great value because the reaction times could be reduced substantially as well as high conversion and yields obtained. In the current study, a continuous-flow packed-bed reactor of immobilized Candida antarctica lipase B (Novozym 435) was employed for synthesis of various geraniol esters. Optimization of process parameters such as biocatalyst screening, effect of solvent, mole ratio, temperature and acyl donors was studied in a continuous-flow packed-bed reactor. Maximum conversion of ~87% of geranyl propionate was achieved in 15min residence time at 70°C using geraniol and propionic acid with a 1:1mol ratio. Novozym 435 was found to be the most active and stable biocatalyst among all tested. Ternary complex mechanism with propionic acid inhibition was found to fit the data.

Lipids from the marine world: Perspectives of an organic chemist

This report describes synthesis of novel structured triacylglycerols (TAGs) and diacylglyceryl ethers (DAGEs) of the 1‐O‐alkyl‐sn‐glycerol type constituting EPA and DHA as the sole fatty acids. One of two TAGs was comprised of a pure EPA located at the 1,3‐positions with pure DHA at the 2‐position and the other had it reversed with a pure DHA at the 1,3‐positions and pure EPA at the 2‐positions. The similarly structured DAGEs were derived from chimyl‐, batyl‐, and selachyl alcohols constituting pure EPA at their sn‐3 position with pure DHA at the sn‐2 position as well as the opposite composition with pure DHA at the sn‐3 position and EPA at the sn‐2 position, the total of six such DAGE products. The syntheses of these compounds were brought about by a two‐step chemoenzymatic process involving a highly regioselective immobilized Candida antarctica lipase to incorporate EPA or DHA activated as acetoxime esters exclusively into the 1,3‐positions of glycerol and the sn‐3 position of the 1‐O‐alkyl‐sn‐glycerols. The second PUFA acyl groups were subsequently introduced to the remaining 2‐positions by EDCI coupling agent to accomplish the title compounds highly efficiently in very high to excellent yields (86–92%).Practical applications: The work described is based on a previously reported methodology to introduce n‐3 PUFAs activated as oxime esters exclusively into the terminal 1,3‐positions of glycerol and 1‐O‐alkyl‐sn‐glycerols to prepare reversed structured TAGs and DAGEs by use of immobilized Candida antarctica lipase. The methodology is ideal for introducing isotopically labeled fatty acids into predetermined positions of TAGs and DAGEs.Novel structured triacylglycerols (TAGs) and diacylglyceryl ethers (DAGEs), constituting EPA and DHA as the sole fatty acids, were obtained by a two‐step chemoenzymatic synthesis comprised of an exclusive incorporation of EPA or DHA, activated as acetoxime esters, into the terminal positions of glycerol and 1‐O‐alkyl‐sn‐glycerols by use of immobilized Candida antarctica lipase, and a subsequent introduction of a second PUFA acyl group to the remaining 2‐positions by EDCI coupling agent.

Enzyme- and Metal-Catalyzed Synthesis of a New Biobased Polyester

Linear aliphatic polyesters were prepared from Pripol 1012, a diacidic C18 fatty acid dimer, and 1,3-propanediol employing a lipase or a titanium tetrabutanoate. Metal-based catalysis (route M) was carried out with a precondensation at 180 °C and 600 mbar followed by a final condensation at 220 °C and 0.3–0.6 mbar. Enzyme catalysis was carried out with an immobilized Candida antarctica lipase B after either a precondensation step at 180 °C and 600 mbar (route E1) or 80 °C and 100 mbar (route E2) and a final condensation at 80 °C and 0.3–0.6 mbar. Polyesters were obtained along routes M, E1, and E2 with weight-average molecular weights, Mw, at final conversion of 84.6, 26.7, and 15.6 kg mol–1, respectively. The final molecular weight via route E2 was most probably constrained by depletion of 1,3-propanediol during precondensation. Rheological measurements of the polyesters in melt revealed a Newtonian-like behavior at 80 °C. The dynamic viscosities fulfill the Cox–Merz rule. The power law for the viscosity...

Neat lipase-catalysed kinetic resolution of racemic 1-phenylethanol and a straightforward modelling of the reaction

Enzymatic kinetic resolution of racemic 1-phenylethanol catalysed by immobilized Candida antarctica lipase B was investigated in a neat system at the temperature range of 30–70 °C. Synthetic triglycerides, namely glycerol triacetate and glycerol tributyrate, were applied as the esterification agents. Both esterification agents were efficient regarding to the enantioselectivity (>1000). Initial rate of reaction and the kinetic constants were influenced by the applied esterification agent significantly. A detailed modelling approach is presented and verified in the temperature range on 30–60 °C for the tributyrin system.

Ultrasound-assisted lipase catalyzed hydrolysis of aspirin methyl ester

The ultrasound-assisted hydrolysis of aspirin methyl ester (AME) was investigated using immobilized Candida antarctica lipase B (CALB) (1%) in the presence of solvents like triolein, chloroform (CHCl3) and dichloromethane (DCM). The effect of ultrasound and the role of water on the conversion rates have also been investigated. Proton nuclear magnetic resonance spectroscopic (1H NMR) was chosen to calculate hydrolysis convertion rates.We observed that lipase-ultrasound assisted hydrolysis of AME in the presence of triolein and water showed the highest hydrolysis conversion rate (65.3%). Herein low water amount played an important role as a nucleophile being crucial for the hydrolysis yields obtained. Lipase activity was affected by the conjugated action of ultrasound and solvents (35.75% of decrease), however not disturbing its hydrolytic efficiency. It was demonstrated that lipase is able to hydrolyse AME to methyl 2-hydroxy benzoate (methyl salicylate), which applications include fragrance agents in food, beverages and cosmetics, or analgesic agent in liniments.

Prediction of solvent effect on enzyme enantioselectivity

Enantiomeric ratios for the esterification reaction of 2-phenylpropionic acid with 1-octanol catalyzed by immobilized Candida antarctica lipase B in sixteen organic solvents have been measured. Correlation equations that relate the enzyme enantioselectivity with the van der Waals volume (Vw), the van der Waals area (Aw), and the Hildebrand solubility parameters (δ2) of the solvents were obtained, and they were compared with linear solvation energy relationship (LSER) correlation equations, which relates the enzyme enantioselectivity with polarity, acidity, basicity, and Hildebrand solubility parameters of the solvents. Statistical analysis of the experimental data for the esterification reaction investigated here as well as for seven other reactions taken from the literature showed that the pure solvent Vw, Aw and δ2 parameters, sufficiently describe the dependence of enzyme enantioselectivity on the properties of the solvent, yielding better results than the LSER equations that use one more parameter. Furthermore, the enzyme enantioselectivity has been related to the infinite dilution activity coefficients of substrates in the solvent, which are predicted by the COSMO-RS model. It is shown that the predicted by COSMO-RS ratios of infinite dilution activity coefficients of the two enantiomers can be used to correlate satisfactorily the enantiomeric ratios.

Insight into microwave assisted enzyme catalysis in process intensification of reaction and selectivity: Kinetic resolution of (R,S)-flurbiprofen with alcohols

Biocatalysis has potential applications in pharmaceuticals, fine chemicals and drug industries for the synthesis and separation of chiral compounds. The current work deals with the kinetic resolution of (R,S)-flurbiprofen using immobilized Candida antarctica lipase B under the influence of microwave irradiation. Flurbiprofen (R,S)-[2-(3-fluro-4-phenyl) phenyl] propionic acid is one of the most widely used non-steroidal anti-inflammatory drugs (NSAID). It exists in two enantiomeric forms as (R,S)-flurbiprofen because it has a single stereogenic center. Pharmacological action of flurbiprofen resides with the S form by inhibiting Cox-2 activity and results in anti-inflammatory action and hence (R)-flurbiprofen causes gastrointestinal infection. Novozym 435 was found to be most active among others for the stereoselective esterification of the R-enantiomer in organic reaction media. Microwave irradiation shows a synergistic effect on enzyme catalysis by enhancing both rate of the reaction and conversion of R-enantiomer into an ester. Effects of various reaction parameters such as solvent, speed of agitation, mole ratio, type of alcohol, temperature, catalyst loading, and reusability were studied. Maximum conversion of R-flurbiprofen ester under microwave irradiation was found to be ∼44.4% with enantioselectivity (E) of 478, eeP=98.9% and eeS=79.07% in 6h, whereas in conventional heating the maximum conversion was 17% with enantioselectivity (E) of 126, and eeS=20.12% in 6h. The ping-pong bi–bi mechanism with inhibition by methanol was proposed for the kinetic resolution of (R,S)-flurbiprofen and validated against experimental data.

Effects of methanol and enzyme pretreatment to Ceiba pentandra biodiesel production

ABSTRACTBiodiesel production from Ceiba pentandra oil using immobilized Candida antarctica lipase (Novozym 435) was studied. A drastic change from 20.6% to 78.0% yield for 9:1 methanol to oil molar ratio indicated the significance of methanol stepwise addition method. Tests were done on the effect of enzyme pretreatments to biodiesel yield productivity which resulted in increase of yield when using t-butanol but decreased yield when using sodium chloride solution. The reusability of enzyme was measured at 63.7% relative yield after three batches. Biodiesel production from C. pentandra oil using biocatalyst is feasible and can be improved for industrialization.

Semi-pilot scale-up of a continuous packed-bed bioreactor system developed for the lipase-catalyzed production of pseudo-ceramides

Ceramides are sphingolipid compounds that are very attractive as active components in both the pharmaceutical and the cosmetic industries. In this study, the synthesis of 1- O ,3- N -diacyl 3-amino-1,2-propanediol-type pseudo-ceramides was developed at the semi-pilot scale, starting from a two-step continuous enzymatic process with immobilized Candida antarctica lipase B (Novozym ® 435) in a packed-bed bioreactor, previously optimized at the laboratory scale. This process involved the selective N -acylation of 3-amino-1,2-propanediol (step 1), followed by the selective O -acylation of the N -acyl 3-amino-1,2-propanediol synthesized in the first step, with various fatty acids as acyl donors, to produce N , O -diacyl 3-amino-1,2-propanediol-type pseudo-ceramides (step 2). Under partially optimized operating conditions, high synthesis yields and production rates were obtained, within the ranges 76–92% and 3.7–4.6 g h −1 (step 1), or 23–36% and 1–1.4 g h −1 (step 2), respectively, depending on the fatty acids used as acyl donors. The overall synthesis yields varied from 20 to 33%: the best yield was obtained using palmitic acid and lauric acid as first and second acyl donors, respectively. Together with the high production rates also obtained with these acyl donors, this confirms that this two-step process has great potential for the production of differently functionalized 1- O ,3- N -diacyl 3-amino-1,2-propanediol-type pseudo-ceramides on an industrial scale.

Enzymatic synthesis of biobased poly(1,4-butylene succinate-ran-2,3-butylene succinate) copolyesters and characterization. Influence of 1,4- and 2,3-butanediol contents

Different (co)polyesters, poly(1,4-butylene succinate-ran-2,3-butylene succinate) (PBB’S), were enzymatically copolymerized at different 1,4-butanediol (1,4-BDO)/2,3-butanediol (2,3-BDO) molar ratios, with immobilized Candida antarctica lipase B (CALB). The synthesized macromolecular architectures were characterized by NMR, SEC, FTIR, WAXS, MALDI-TOF MS, DSC, TGA and specific optical rotation. Based on an optimized process, PBB’S copolyesters were synthesized in high yield. A random distribution between both BDOs was obtained along the chain. Mw‾ decreased with increasing 2,3-BDO content, from 26 to 6kg/mol. The specific optical rotation decreased linearly with decreasing 2,3-BDO content, and can be used as a characterization technique to determine PBB’S composition. DSC results showed that an increase in 2,3-BDO content raised the Tg and reduced the degree of crystallinity, leading to amorphous materials for 2,3-BDO content higher than 47mol.%. Synthesized copolyesters exhibited a good thermal stability, higher than 250°C, with a degradation profile depending on the 1,4-BDO/2,3-BDO ratio.

In Situ Separation of the Chiral Target Compound (S)-2-Pentanol in Biocatalytic Reactive Distillation

In situ separation of a chiral target compound is realized for the first time as distillate on top of an integrated biocatalytic batch reactive distillation column. The applied reaction system is the racemic resolution of (R/S)-2-pentanol in a transesterification with propyl butyrate, which is catalyzed by immobilized Candida antarctica lipase B (Novozym435). Biocatalyst integration is realized in baskets of wire gauze catalytic packings as column internals. The equilibrium limited reaction is shifted to the product side by fractional distillation of low boiling propanol and the target compound (S)-2-pentanol. Increased molar fractions up to x(S)-2-PeOH = 65 ± 4% with an enantiomeric excess of 90 ± 4% and 51 ± 3% overall conversion are reached for (S)-2-pentanol. Feasibility of the reaction system is investigated in our preselection tool. This tool is developed to evaluate predefined operation conditions in the column setup for transesterification reactions based on criteria regarding biocatalyst stabilit...

CO2-expanded bio-based liquids as novel solvents for enantioselective biocatalysis

For the first time, CO2-expanded bio-based liquids were reported as novel and sustainable solvents for biocatalysis. Herein, it was found that by expansion with CO2, 2-methyltetrahydrofuran (MeTHF), and other bio-based liquids, which were not favorable solvents for immobilized Candida antarctica lipase B (Novozym 435) catalyzed transesterification, were tuned into excellent reaction media. Especially, for the kinetic resolution of challenging bulky secondary substrates such as rac-1-adamantylethanol, the lipase displayed very high activity with excellent enantioselectivity (E value > 200) in CO2-expanded MeTHF (MeTHF concentration 10% v/v, 6 MPa), whereas there was almost no activity observed in conventional organic solvents.

Lipase-ultrasound assisted synthesis of polyesters

Poly (ethylene glutarate), poly (ethylene malonate) and poly (ethylene phthalate), were enzymatically synthesized by immobilized Candida antarctica lipase B in solvent free conditions. The synthesis of these polyesters was based on the ester-ester exchange reaction between ethylene glycol diacetate and di-ethyl glutarate, di-benzyl malonate, di-n-octyl phthalate to produce poly (ethylene glutarate), poly (ethylene malonate) and poly (ethylene phthalate), respectively. The effect of ultrasound and PET polymeric beads was evaluated and showed to improve the synthesis of all polyesters. Ultrasound, as a green solvent-free technology, showed high potentiality for the polyester synthesis intensification.

Optimization of batch Novozym435-catalyzed transesterification of waste cooking oil with methanol for biodiesel production in a solvent-free medium

ABSTRACT In this study, response surface methodology based on face center composite – 1/2 factorial fraction design (FCCD) of experiments was employed to optimize the batch transesterification process of waste cooking oil as one of the most abundant and readily available domestic wastes with methanol using immobilized Candida antarctica lipase (Novozym435) to maximize the production of high-purity biodiesel. Statistically significant second-order quadratic model equations (p < 0.0001) were elucidated through multiple regression analysis to describe the interrelationships between response of interest (biodiesel yield wt% and % conversion, i.e. the transesterification efficiency) and five independent variables (methanol:oil molar ratio, enzyme concentration wt%, reaction temperature °C, reaction time h, and mixing rate rpm). The statistical significance of the effect of these variables (factors) and their interactions on the transesterification efficiency was evaluated and the validity of the predicted models was confirmed. The optimum operating conditions were found to be: 3.63:1 M:O, 8.94 wt% Novozym435, 45.23°C, 2.76 h, and 535.84 rpm. This produced 97 wt% biodiesel yield with % conversion of ≈ 91.79%. An overall acceptable agreement was achieved between the produced biodiesel fuel properties at the aforementioned optimal operating conditions and the Egyptian petrodiesel and international biodiesel standards.

Kinetic Resolution of (R,S)-α-Tetralol by Immobilized Candida antarctica Lipase B: Comparison of Packed-Bed over Stirred-Tank Batch Bioreactor

Here, we have demonstrated the flow chemistry approach for kinetic resolution of (R,S)-α-tetralol using the packed-bed reactor of immobilized Candida antarctica lipase B (Novozym 435). We have investigated the performance of different enzymes including Lipozyme RM IM, Lipozyme TL IM, and Novozym 435 and compared their activity and enantioselectivity in nonaqueous media. The kinetics of transesterification of (R,S)-α-tetralol using vinyl acetate as the acylating agent was studied in a packed bed of immobilized enzyme. A continuous-flow packed bioreactor shows better behavior in terms of enantioselectivity and conversion vis-a-vis a stirred-tank reactor. A conversion of ∼50% of the (R,S) mixture with ∼100% selectivity (E) for (R)-α tetralol (eep ≥ 99.99%) was obtained at 65 °C with a 3 min residence time using Novozym 435 as the catalyst in the continuous-flow packed-bed reactor, whereas a 43.6% conversion of racemic mixture (eep ≥99.99%) was obtained in a stirred-tank batch reactor in 8 h under identical c...