Development and Optimization of Lipase-Catalyzed Synthesis of Phospholipids Containing 3,4-Dimethoxycinnamic Acid by Response Surface Methodology

The interesterification reaction of egg-yolk phosphatidylcholine (PC) with ethyl ester of 3,4-dimethoxycinnamic acid (E3,4DMCA) catalyzed by Novozym 435 in hexane as a reaction medium was shown to be an effective method for the synthesis of corresponding structured O-methylated phenophospholipids. The effects of substrate molar ratios, time of the reaction and enzyme load on the process of incorporation of 3,4DMCA into PC were evaluated by using the experimental factorial design of three factors and three levels. The results showed that a substrate molar ratio is a crucial variable for the maximization of the synthesis of 3,4-dimethoxycinnamoylated phospholipids. Under optimized parameters of 1/10 substrate molar ratio PC/E3,4DMCA, enzyme load 30% (w/w), hexane as a medium and incubation time of 3 days, the incorporation of aromatic acid into phospholipid fraction reached 21 mol%. The modified phosphatidylcholine (3,4DMCA-PC) and modified lysophosphatidylcholine (3,4DMCA-LPC) were obtained in isolated yields of 3.5% and 27.5% (w/w), respectively. The developed method of phosphatidylcholine interesterification is the first described in the literature dealing with 3,4DMCA and allows us to obtain new O-methylated phenophospholipids with potential applications as food additives or nutraceuticals with pro-health activity.

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Enzymatic Synthesis of O-Methylated Phenophospholipids by Lipase-Catalyzed Acidolysis of Egg-Yolk Phosphatidylcholine with Anisic and Veratric Acids

Catalysts ◽

10.3390/catal10050538 ◽

2020 ◽

Vol 10 (5) ◽

pp. 538 ◽

Cited By ~ 3

Author(s):

Marta Okulus ◽

Anna Gliszczyńska

Keyword(s):

Egg Yolk ◽

Solvent System ◽

Biologically Active ◽

Molar Ratio ◽

Binary Solvent ◽

Molar Ratios ◽

Egg Yolk Phosphatidylcholine ◽

Screening Experiments ◽

Acidolysis Reaction ◽

System 1

Lipase-catalyzed acidolysis reactions of egg-yolk phosphatidylcholine (PC) with anisic (ANISA) and veratric (VERA) acids were investigated to develop a biotechnological method for the production of corresponding biologically active O-methylated phenophospholipids. Screening experiments with four commercially available immobilized lipases indicated that the most effective biocatalyst for the incorporation of ANISA into phospholipids was Novozym 435. None of the tested enzymes were able to catalyze the synthesis of PC structured with VERA. The effects of different solvents, substrate molar ratios, temperature, enzyme loading, and time of the reaction on the process of incorporation of ANISA into the phospholipids were evaluated in the next step of the study. The mixture of toluene/chloroform in the ratio 9:1 (v/v) significantly increased the incorporation of ANISA into PC. The acidolysis reaction was carried out using the selected binary solvent system, 1/15 substrate molar ratio PC/ANISA, 30% (w/w) enzyme load, and temperature of 50 °C afforded after 72 h anisoylated lysophosphatidylcholine (ANISA-LPC) and anisoylated phosphatidylcholine (ANISA-PC) in isolated yields of 28.5% and 2.5% (w/w), respectively. This is the first study reporting the production of ANISA-LPC and ANISA-PC via a one-step enzymatic method, which is an environmentally friendly alternative to the chemical synthesis of these biologically active compounds.

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Enzymatic Production of Biologically Active 3-Methoxycinnamoylated Lysophosphatidylcholine via Regioselctive Lipase-Catalyzed Acidolysis

Foods ◽

10.3390/foods11010007 ◽

2021 ◽

Vol 11 (1) ◽

pp. 7

Author(s):

Marta Okulus ◽

Magdalena Rychlicka ◽

Anna Gliszczyńska

Keyword(s):

Reaction Time ◽

Design Method ◽

Reaction Medium ◽

Egg Yolk ◽

Major Product ◽

Biologically Active ◽

Molar Ratio ◽

Enzymatic Production ◽

Egg Yolk Phosphatidylcholine ◽

Level 3

Enzymatic acidolysis of egg-yolk phosphatidylcholine (PC) with 3-methoxycinnamic acid (3-OMe-CA) was investigated to produce biologically active 3-methoxycinnamoylated phospholipids. Four commercially available lipases were screened for their ability to incorporate 3-OMe-CA into PC. The results showed that Novozym 435 is the most effective biocatalyst for this process, while during the examination of organic solvents, heptane was found propriate reaction medium. The other reaction parameters including the substrate molar ratio, enzyme load and reaction time were designed using an experimental factorial design method. According to three-level-3-factor Box-Behnken model it was shown that all of studied parameters are crucial variables for the maximization of the synthesis of structured PLs. The optimum conditions derived via response surface methodology (RSM) were: 30% of lipase of the total weight of substrates, 1:15 molar ration of PC/3-OMe-CA and reaction time 4 days. The process of acidolysis performed on the increased scale at optimized parameters afforded two products. The major product, 3-methoxycinnamoylated lysophosphatidylcholine (3-OMe-CA-LPC) was isolated in high 48% yield, while 3-methoxycinnamoylated phosphatidylcholine (3-OMe-CA-PC) was produced in trace amount only in 1.2% yield. Obtained results indicate that presented biotechnological method of synthesis of 3-methoxycinnamoylated lysophosphatidylcholine is competitive to the previously reported chemical one.

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Screening of phospholipase A activity and its production by new actinomycete strains cultivated by solid-state fermentation

PeerJ ◽

10.7717/peerj.3524 ◽

2017 ◽

Vol 5 ◽

pp. e3524 ◽

Cited By ~ 4

Author(s):

Priscila Sutto-Ortiz ◽

María de los Angeles Camacho-Ruiz ◽

Manuel R. Kirchmayr ◽

Rosa María Camacho-Ruiz ◽

Juan Carlos Mateos-Díaz ◽

...

Keyword(s):

Solid State ◽

Solid State Fermentation ◽

High Throughput Screening ◽

Egg Yolk ◽

Specific Substrate ◽

Phospholipase Activity ◽

High Accumulation ◽

Egg Yolk Phosphatidylcholine ◽

Potential Applications ◽

Routine Assay

Novel microbial phospholipases A (PLAs) can be found in actinomycetes which have been poorly explored as producers of this activity. To investigate microbial PLA production, efficient methods are necessary such as high-throughput screening (HTS) assays for direct search of PLAs in microbial cultures and cultivation conditions to promote this activity. About 200 strains isolated with selected media for actinomycetes and mostly belonging toStreptomyces(73%) andMicromonospora(10%) genus were first screened on agar-plates containing the fluorophore rhodamine 6G and egg yolk phosphatidylcholine (PC) to detect strains producing phospholipase activity. Then, a colorimetric HTS assay for general PLA activity detection (cHTS-PLA) using enriched PC (≈60%) as substrate and cresol red as indicator was developed and applied; this cHTS-PLA assay was validated with known PLAs. For the first time, actinomycete strains were cultivated by solid-state fermentation (SSF) using PC as inductor and sugar-cane bagasse as support to produce high PLA activity (from 207 to 2,591 mU/g of support). Phospholipase activity of the enzymatic extracts from SSF was determined using the implemented cHTS-PLA assay and the PC hydrolysis products obtained, were analyzed by TLC showing the presence of lyso-PC. Three actinomycete strains of theStreptomycesgenus that stood out for high accumulation of lyso-PC, were selected and analyzed with the specific substrate 1,2-α-eleostearoyl-sn-glycero-3-phosphocholine (EEPC) in order to confirm the presence of PLA activity in their enzymatic extracts. Overall, the results obtained pave the way toward the HTS of PLA activity in crude microbial enzymatic extracts at a larger scale. The cHTS-PLA assay developed here can be also proposed as a routine assay for PLA activity determination during enzyme purification,directed evolution or mutagenesis approaches. In addition, the production of PLA activity by actinomycetes using SSF allow find and produce novel PLAs with potential applications in biotechnology.

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Optimization of Enzymatic Synthesis of Betulinic Acid Amide in Organic Solvent by Response Surface Methodology (RSM)

Indonesian Journal of Chemistry ◽

10.22146/ijc.34903 ◽

2019 ◽

Vol 19 (4) ◽

pp. 849

Author(s):

Nurul Atikah Amin Yusof ◽

Nursyamsyila Mat Hadzir ◽

Siti Efliza Ashari ◽

Nor Suhaila Mohamad Hanapi ◽

Rossuriati Dol Hamid

Keyword(s):

Response Surface Methodology ◽

Reaction Time ◽

Response Surface ◽

Reaction Temperature ◽

Betulinic Acid ◽

Enzymatic Synthesis ◽

Acid Amide ◽

Molar Ratio ◽

Percentage Yield ◽

Substrate Molar Ratio

Optimization of the lipase catalyzed enzymatic synthesis of betulinic acid amide in the presence of immobilized lipase, Novozym 435 from Candida antartica as a biocatalyst was studied. Response surface methodology (RSM) and 5-level-4-factor central-composite rotatable design (CCRD) were employed to evaluate the effects of the synthesis parameters, such as reaction time (20–36 h), reaction temperature (37–45 °C), substrate molar ratio of betulinic acid to butylamine (1:1–1:3), and enzyme amounts (80–120 mg) on the percentage yield of betulinic acid amide by direct amidation reaction. The optimum conditions for synthesis were: reaction time of 28 h 33 min, reaction temperature of 42.92 °C, substrate molar ratio of 1:2.21, and enzyme amount of 97.77 mg. The percentage yield of actual experimental values obtained 65.09% which compared well with the maximum predicted value of 67.23%. The obtained amide was characterized by GC, GCMS and 13C NMR. Betulinic acid amide (BAA) showed a better cytotoxicity compared to betulinic acid as the concentration inhibited 50% of the cell growth (IC50) against MDA-MB-231 cell line (IC50 < 30 µg/mL).

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Interesterification of Egg-Yolk Phosphatidylcholine with p-Methoxycinnamic Acid Catalyzed by Immobilized Lipase B from Candida Antarctica

Catalysts ◽

10.3390/catal10101181 ◽

2020 ◽

Vol 10 (10) ◽

pp. 1181

Author(s):

Magdalena Rychlicka ◽

Anna Gliszczyńska

Keyword(s):

Reaction Time ◽

Design Method ◽

Immobilized Lipase ◽

Reaction Medium ◽

Solvent System ◽

Novozym 435 ◽

Molar Ratio ◽

Binary Solvent ◽

Methoxycinnamic Acid ◽

Substrate Molar Ratio

The p-methoxycinnamic acid (p-MCA) is one of the most popular phenylpropanoids, the beneficial impact of which on the human health is well documented in the literature. This compound has shown many valuable activities including anticancer, antidiabetic, and neuro- and hepatoprotective. However, its practical application is limited by its low bioavailability resulting from rapid metabolism in the human body. The latest strategy, aimed at overcoming these limitations, is based on the production of more stability in systemic circulation bioconjugates with phospholipids. Therefore, the aim of this research was to develop the biotechnological method for the synthesis of phospholipid derivatives of p-methoxycinnamic acid, which can play a role of new nutraceuticals. We developed and optimized enzymatic interesterification of phosphatidylcholine (PC) with ethyl p-methoxycinnamate (Ep-MCA). Novozym 435 and a binary solvent system of toluene/chloroform 9:1 (v/v) were found to be the effective biocatalyst and reaction medium for the synthesis of structured p-MCA phospholipids, respectively. The effects of the other reaction parameters, such as substrate molar ratio, enzyme dosage, and reaction time, on the degree of incorporation of p-MCA into PC were evaluated by use of an experimental factorial design method. The results showed that substrate molar ratio and biocatalyst load have significant effects on the synthesis of p-methoxycinnamoylated phospholipids. The optimum conditions were: Reaction time of three days, 30% (w/w) of Novozym 435, and 1/10 substrate molar ratio PC/Ep-MCA. Under these parameters, p-methoxycinnamoylated lysophosphatidylcholine (p-MCA-LPC) and p-methoxycinnamoylated phosphatidylcholine (p-MCA-PC) were obtained in isolated yields of 32% and 3% (w/w), respectively.

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Continuous Catalyst-Free Esterification of Oleic Acid in Compressed Ethanol

International Journal of Chemical Engineering ◽

10.1155/2014/803783 ◽

2014 ◽

Vol 2014 ◽

pp. 1-5 ◽

Cited By ~ 8

Author(s):

Ana Carolina de Araujo Abdala ◽

Vitor Augusto dos Santos Garcia ◽

Caroline Portilho Trentini ◽

Lúcio Cardozo Filho ◽

Edson Antonio da Silva ◽

...

Keyword(s):

Oleic Acid ◽

Residence Time ◽

Tubular Reactor ◽

Reaction Medium ◽

Ethyl Esters ◽

Molar Ratio ◽

Molar Ratios ◽

Catalyst Free ◽

Free Process ◽

Positive Effect

The esterification of oleic acid in a continuous catalyst-free process using compressed ethanol was investigated in the present study. Experiments were performed in a tubular reactor and variables investigated were temperature, pressure, and oleic acid to ethanol molar ratio for different residence time. Results demonstrated that temperature, in the range of 473 K to 573 K, and pressure had a positive effect on fatty acid ethyl esters (FAEE) production. In the experimental range investigated, high conversions can be obtained at low ethanol concentrations in the reaction medium and it was observed that oleic acid to ethanol molar ratios greater than 1 : 6 show no significant increase in conversion. Nonnegligible reaction conversions (>90%) were achieved at 573 K, 20 MPa, oleic acid to ethanol molar ratio of 1 : 6, and 20 minutes of residence time.

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Application of Statistical Experimental Design and Surface Plot Technique to Optimize Oxygenated Apatite Synthesis

Advances in Materials Science and Engineering ◽

10.1155/2020/1034959 ◽

2020 ◽

Vol 2020 ◽

pp. 1-6

Author(s):

Soumia Belouafa ◽

Hassan Chaair ◽

Khalid Digua

Keyword(s):

Response Surface Methodology ◽

Experimental Design ◽

Reaction Medium ◽

Atomic Ratio ◽

Precipitation Method ◽

Molar Ratio ◽

Statistical Experimental Design ◽

Anova Analysis ◽

Synthesis Parameters ◽

Surface Plot

This work deals with oxygenated apatite synthesis from calcium chloride and phosphoric acid using a wet precipitation method. An experimental design is applied to precise the influence of the synthesis parameters (pH of the reaction medium, atomic ratio Ca/P of the reagents, concentration of the calcium solution (Ca2+), temperature of the reaction medium (T), and duration of the reaction (D)) on the chemical composition (Ca/P molar ratio, % O2, and % O22−). An empirical model was developed and validated by applying the ANOVA analysis incorporating the interaction effects of all parameters and optimized using the response surface methodology. A reproducible synthesis of more oxygenated apatite with speed of dissolution adaptable to that of the osseous neoformation and allowing a progressive diffusion of oxygenated species (Ca/P = 1.575) is attained.

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FOUR-FACTOR RESPONSE SURFACE OPTIMIZATION OF THE ENZYMATIC SYNTHESIS OF WAX ESTER FROM PALM KERNEL OIL

Indonesian Journal of Chemistry ◽

10.22146/ijc.21653 ◽

2010 ◽

Vol 8 (1) ◽

pp. 83-90 ◽

Cited By ~ 4

Author(s):

Erin Ryantin Gunawan ◽

Dedy Suhendra

Keyword(s):

Response Surface Methodology ◽

Response Surface ◽

Palm Kernel ◽

Molar Ratio ◽

Wax Ester ◽

Central Composite Rotatable Design ◽

Palm Kernel Oil ◽

Kernel Oil ◽

Substrate Molar Ratio ◽

Central Composite

The synthesis of wax ester using refined, bleached and deodorized (RBD) palm kernel oil (PKO) and oley alcohol catalyzed by Lipozyme IM was carried out. Response surface methodology (RSM) based on a five-level, four-factor central composite rotatable design (CCRD) was used to evaluate the interactive effects of synthesis, of reaction time (5-20 h), temperature (20-50 oC), amount of enzyme (0.1-0.2 g) and substrate molar ratio (palm kernel oil to oleyl alcohol, 1:1-1:5) on the percentage yield of wax esters. The optimum condition conditions derived via RSM were reaction time 8.46 h, temperature 44.4 oC, amount of enzyme 0.182 g, substrate molar ratio 1 to 3.7. The actual experimental yield was 92.9 % under optimum condition, which good accordance to the maximum predicted value of 92.4 %. Keywords: response surface methodology, central composite rotatable design, palm kernel oil, lipozyme, alcoholysis, wax ester

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Application of Methylated N-(4-N,N-Dimethylaminocinnamyl) Chitosan for Oral Protein Drug Delivery

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.506.465 ◽

2012 ◽

Vol 506 ◽

pp. 465-468

Author(s):

J. Kowapradit ◽

Theerasak Rojanarata ◽

Tanasait Ngawhirunpat ◽

A. Apirakaramwong ◽

Warayuth Sajomsang ◽

...

Keyword(s):

Drug Delivery ◽

Entrapment Efficiency ◽

Egg Yolk ◽

Molar Ratio ◽

Protein Drug ◽

Egg Yolk Phosphatidylcholine ◽

Model Protein ◽

Protein Drug Delivery ◽

Low Cytotoxicity

In the present study, methylated N-(4-N,N-dimethylaminocinnamyl) chitosan (TM65CM50CS) was synthesized and investigated for oral protein drug delivery by combining with liposomes entrapped bovine serum albumin (FITC-BSA), a model protein. FITC-BSA liposomes composed of egg yolk phosphatidylcholine and sodium oleate in molar ratio of 10:2 were prepared by thin film hydration method. The TM65CM50CS coated liposomal FITC-BSA was evaluated for transport of protein and its cytotoxicity in Caco-2 cells. Moreover, the in vitro stability of BSA in TM65CM50CS coated liposomes was also examined by the degradation of protein from pancreatin. The mean particle size and zeta-potential of liposomes were 101+0.02 nm and -27.44+2.02 mV, respectively. Initial FITC-BSA (2.5% w/w) to lipid showed the highest percentage entrapment efficiency (50.13%) and FITC-BSA content (8.08 mg/g of lipid). The results of FITC-BSA transport showed that TM65CM50CS coated FITC-BSA liposomes enhanced protein permeability across Caco-2 cell monolayers with low cytotoxicity. In addition, these liposomes could protect the degradation of protein from pancreatin. Our studies demonstrated that TM65CM50CS coated liposomes have the potential to be used as an oral protein drug delivery.

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Enhanced Ricinoleic Acid Preparation Using Lipozyme TLIM as a Novel Biocatalyst: Optimized by Response Surface Methodology

Catalysts ◽

10.3390/catal8110486 ◽

2018 ◽

Vol 8 (11) ◽

pp. 486 ◽

Cited By ~ 1

Author(s):

Shangde Sun ◽

Jingjing Guo

Keyword(s):

Response Surface Methodology ◽

Response Surface ◽

Ricinoleic Acid ◽

Efficient Method ◽

Good Alternative ◽

Raw Material ◽

Molar Ratio ◽

Hydrolysis Time ◽

Hydrolysis Of ◽

Enzyme Load

Ricinoleic acid (RA) is an important raw material for plasticizers, emulsifiers, and nanomaterials. In this work, a green and efficient method was developed for RA production. Results showed that Lipozyme TLIM can be used as a novel biocatalyst to catalyze the hydrolysis of castor oil (CO) for RA preparation. Response surface methodology (RSM) was used to evaluate and optimize the effects of reaction variables on the hydrolysis of CO. Reaction conditions were optimized as follows: 41.3 °C, enzyme load 8.9%, 39.2 h, and 40:1 molar ratio of water to oil. Under these optimized reaction variables, the maximum hydrolysis ratio of CO (96.2 ± 1.5%) was obtained. The effect of hydrolysis variables on the reaction was as follows: enzyme load > hydrolysis time > temperature. In conclusion, this is a green, simple, and efficient method for RA preparation and can provide a good alternative method for RA industrial production.

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