Gallic Acid-Based Alkyl esters Synthesis in a Water-Free System by Celite-Bound Lipase of Bacillus licheniformis SCD11501 Shivika Sharma and Shamsher S. Kanwar Dept. of Biotechnology, Himachal Pradesh University, Summer Hill, Shimla 171 005, India
Priyanka Dogra and Ghanshyam S. Chauhan Dept. of Chemistry, Himachal Pradesh University, Summer Hill, Shimla 171 005, India DOI 10.1002/btpr.2072 Published online March 18, 2015 in Wiley Online Library (wileyonlinelibrary.com)
Gallic acid (3, 4, 5- trihydroxybenzoic acid) is an important antioxidant, anti-inflammatory, and radical scavenging agent. In the present study, a purified thermo-tolerant extra-cellular lipase of Bacillus licheniformis SCD11501 was successfully immobilized by adsorption on Celite 545 gel matrix followed by treatment with a cross-linking agent, glutaraldehyde. The celite-bound lipase treated with glutaraldehyde showed 94.8% binding/retention of enzyme activity (36 U/g; specific activity 16.8 U/g matrix; relative increase in enzyme activity 64.7%) while untreated matrix resulted in 88.1% binding/retention (28.0 U/g matrix; specific activity 8.5 U/g matrix) of lipase. The celite-bound lipase was successfully used to synthesis methyl gallate (58.2%), ethyl gallate (66.9%), n-propyl gallate (72.1%), and n-butyl gallate (63.8%) at 55oC in 10 h under shaking (150 g) in a water-free system by sequentially optimizing various reaction parameters. The low conversion of more polar alcohols such as methanol and ethanol into their respective gallate esters might be due to the ability of these alcohols to severely remove water from the protein hydration shell, leading to enzyme inactivation. Molecular sieves added to the reaction mixture resulted in enhanced yield of the alkyl ester(s). The characterization of synthesised esters was done through fourier transform infrared (FTIR) C 2015 American Institute of Chemical Engispectroscopy and 1H NMR spectrum analysis. V neers Biotechnol. Prog., 31:715–723, 2015 Keywords: gallic acid, lipase, esterification, immobilization, methanol, ethanol, propanol, butanol
Introduction Lipases are a group of enzymes naturally endowed with the property of performing reactions in aqueous as well as organic solvents.1 Lipases are attractive enzymes for preparation of some important intermediates such as chiral alcohols, carboxylic acids, and amines, and have been extensively used in food, textile, detergents, paper, chemical, pharmaceutical, and environmental industries in the last few decades.2 The enzymatic trans-esterification has been performed in solvents and solvent-free media by different immobilized lipases.3,4 From the application viewpoint, enzymatic synthesis in organic solvents is not suitable due to toxicity, flammable nature of solvent and damaging effects on the environment. Thus enzymatic solvent-free systems are being developed to make the enzymatic process competitive.5 The biocatalysts properties are being inclined by selection of different immobilization methods.6 Inorganic supports with the stability in anhydrous and oil/water medium can provide an easily separable and reusable system in continuous fabrication and the modification of inorganic material is in favour of improving its biocompatibility with enzymes, contributing to the improved enzymatic activity and stability.2 Gallic acid Correspondence concerning this article should be addressed to S. Sharma at [email protected]. C 2015 American Institute of Chemical Engineers V
(3, 4, 5-trihydroxybenzoic acid), from gallnut and green tea, is known to be antioxidant, anti-inflammatory, and radical scavenger.7 Gallic acid is an organic substance occurring in many plants either as a free molecule or as part of tannic acid molecule.8 Gallic acid has been known to induce apoptosis in tumour cell lines and renal fibroblasts9 and has proapoptotic and anti-inflammatory effects on fibroblast-like synoviocytes from patients with rheumatoid arthritis.10 Alkyl gallates are used as antibacterial and anti-viral agents11 and found to affect microbial cell viability,12 virus activity,13 and human leukaemia cell proliferation.14 Gallic acid and its esters are hydroxybenzoic derivatives which are used as antioxidant additives in food and pharmaceutical industry. Oxidation of polyunsaturated lipids by reactive oxygen species is a serious and continuing problem for the food industry. Peroxidation produces rancid odours and off-flavors, decreases shelf life, alters food texture, and appearance, and may reduce nutritional quality and food safety.15 E-310 (propyl gallate) and E-311 (octyl gallate), which are known to protect cells against oxidative damage induced by reactive oxygen species, as hydroxyl radicals or hydrogen peroxide and reactive sulphur species.16,17 Synthetic galloyl esters were found to hinder cytokine-induced nuclear translocation of nuclear factor (NF)-kappa B, as well as expression of leukocyte adhesion molecules in vascular endothelium cells.18 Gallic acid and its derivatives have 715
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been extensively evaluated for their anti-tumor activity against a variety of cell lineages.19 Gallic acid inhibits activation of NF-jB and Akt signalling pathways along with the activity of cyclooxygenase, ribonucleotide reductase and glutathione (reduced). Moreover, gallic acid activates ataxia telangiectasia mutated kinase signalling pathways to prevent the processes of carcinogenesis.20 The present study was carried out to exploit the enzymatic potential of a solvent–tolerant thermophilic Bacillus licheniformis isolate SCD1105 originally isolated from a thermal spring produced an extra cellular alkaliphilic lipase under optimal physico-chemical conditions toward the synthesis of a few useful free radical scavenger esters of gallic acid. The purified lipase of B. licheniformis strain was adsorbed on to celite matrix and immobilized enzyme was used for esterification of gallic acid with chain of alcohols. The effects incubation time, concentrations of reactant, immobilized biocatalyst; reaction temperature and presence of molecular sieves on the rate of synthesis of methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate were consecutively evaluated. The synthesized esters were characterized by fourier transform infrared (FTIR) spectroscopy and nuclear magnetic resonance (NMR). Previously we have reported the synthesis of alkyl coumarate esters from the celite-bound lipase of B. licheniformis strain21 albeit at low yields in a water-free system. The present study was conducted to explore wether celite bound-lipase of a thermophilic B. licheniformis bacterium could produce improved yields of alkyl esters of gallic acid in a water-free reaction system by manipulation of vital reaction parameters.
Materials and Methods Materials Celite 545, diethylaminoethyl (DEAE) matrix (S.D. FineChem Ltd., Hyderabad, India); p-nitrophenyl palmitate (pNPP) and glutaraldehyde (Lancaster Synthesis, England); ˚ 3 1.5 mm), methanol, ethanol, n-promolecular sieves (3A panol, and n-butanol (MERCK, Mumbai, India); Bovine serum albumin, deuterated chloroform (CDCl3) solution, Tris buffer, and gallic acid (Himedia Laboratory, Ltd., Mumbai, India) were procured from various commercial suppliers. All chemicals were of analytical grade and were used as received. A chemical reactor with stirring and heating (MiniBlockTM, China) was used to perform ester synthesis using celite-immobilized lipase of B. licheniformis SCD11501. Microorganism B. licheniformis strain SCD11501 (GenBank Accession Number JN998712.1) was isolated from hot springs of Tatapani, District: Mandi, Himachal Pradesh (India). This bacterium was identified by Xcelris Labs Ltd. Ahmedabad-380 054, India on the basis of 16S RNA sequence. Purification and immobilization of lipase of B. licheniformis strain SCD11501 A thermo-tolerant extra cellular lipase produced by B. licheniformis strain SCD11501 was purified (37.0 U/mg protein) by successive techniques of salting out using ammonium sulphate, dialysis, and DEAE anion-exchange chromatography to 10.6-fold with an overall yield of 8.4% (experimental data not provided here).
Immobilization of bacterial lipase on celite gel matrix Celite 545 (3 g) was incubated with 10 ml 0.05 M Tris HCl buffer of pH 9.5 for 12 h in a glass vial. (2 ml; 60 U of enzyme) enzyme was added to the matrix for 1 h at 37 C. The celite particles were sedimented by centrifugation (5 min at 10,000g at 4 C) followed by addition of glutaraldehyde (1%; v/v) for 1 h at 37 C. The celite was extensively washed with Tris buffer (0.05 M, pH 9.5) to get rid of unbound activating agent. The supernatant was decanted and immobilized biocatalyst was retained. The weight of biocatalyst was recorded and activity was assayed using 40 mg of celite-bound lipase. The immobilized protein in matrix was determined by subtracting unbound protein in the supernatant from the total protein used for immobilization as well as increased/decreased in the total activity was calculated by adding total activity of supernatant and matrix in comparison to total enzyme units of purified lipase incubated earlier. Colorimetric method for lipase assay The lipase activity was assayed using p-NPP as a chromogenic substrate. The stock solution (20 mM) of p-NPP was prepared in HPLC grade iso-propanol. The reaction mixture contained 60 ll of p-NPP stock-solution, 40 mg of enzyme, and Tris buffer (0.05 M, pH 9.0) to make final volume 3 ml. The reaction mixture was incubated at 50oC for 10 min in a waterbath (Bangalore Genei Pvt. Ltd., Bangalore). Further enzymatic reaction was stopped by chilling the reaction mixture at 220oC for 7 min. The absorbance (A410) of heat-inactivated lipase was subtracted from the absorbance of the corresponding test sample. The A410 of p-nitrophenol released was measured (Labindia 30001 UV/vis Spectrophotometer) and expressed in millimoles (mM). Each of the assays were performed in triplicate unless otherwise stated and mean values 6 SD was presented. Stock solutions of various p-nitrophenyl esters para-nitrophenyl formate (p-NPF), para-nitrophenyl acetate (p-NPA), para-nitrophenyl caprylate (p-NPC), para-nitrophenyl palmitate (p-NPP), paranitrophenyl benzoate (p-NPB), and para-nitrophenyl laurate (p-NPL) were also prepared for use in some of the experiments. Unit of lipase activity The unit (U) of enzyme activity was defined as mmole(s) of p-nitrophenol released from p-NPP per min by one ml of free enzyme or one gram of celite-immobilized enzyme (weight of matrix included) under standard assay conditions. Esterification reaction Alkyl gallate esters were synthesized by using 1 M alcohol (methanol, ethanol, propanol or butanol), 1 M gallic acid, and celite-bound purified lipase taken in glass-vial (20 ml capacity). The reaction was performed at 55 C for 10 h in a chemical reactor. The scheme of the synthesis of the methyl gallate, ethyl gallate, propyl gallate, and butyl gallate is presented schematically (Figure 1). The effect(s) of incubation time, reaction temperature, molar concentration of reactants, biocatalyst, and molecular sieves on the rate of synthesis of methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate were separately evaluated. The formed esters, that is methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate were separated on the basis of their
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Figure 1. Scheme of the synthesis of the methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate.
solubility in hot water using a separating funnel. The gallic acid was soluble in hot water while the formed esters (methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate formed in different test tubes) were insoluble in hot water and were separated out using separating funnel. The amount of ester synthesized was determined and represented as % yield. The synthetic reactions in water-free medium were performed in duplicate mean values were presented. Effect of incubation time on synthesis of methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate The reaction mixture (2 g) comprised celite-immobilized lipase (10 mg), gallic acid (1 M): methanol (1 M) or gallic acid (1 M): ethanol (1 M) or gallic acid (1 M): n-propanol (1 M) or gallic acid (1 M): n-butanol (1 M) taken in Teflon-coated capped glass vials (5 ml capacity) in a solvent-free system, respectively. The glass vials were incubated at 55oC in a chemical reactor for 4, 6, 8, 10, and 12 h under shaking (150g). Effect of reaction temperature on the synthesis of methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate The effects of reaction temperature (40, 45, 50, 55, and 60 C) on the synthesis of methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate by the immobilizedlipase (10 mg) were studied at incubation time of 10 h under continuous shaking (150g). Effect of relative molar concentration of reactants on the synthesis of methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate The concentration of one of the reactants (gallic acid) was kept constant at 1 M and varying the concentration of the other reactant (methanol, ethanol, n-propanol, and n-butanol; 1–4 M) in the reaction mixture (2 g) in a solvent-free system. The esterification was carried out at 55 C for optimized time (10 h) under continuous shaking using 10 mg celite-bound lipase. Effect of amount of biocatalyst on synthesis of methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate The synthesis of ethyl gallate, methyl gallate, n-propyl gallate, and n-butyl gallate were studied by taking different
amount of immobilized lipase (0.5–3% of acid weight) in reaction mixture (2 g) containing 1 M of gallic acid: 1 M methanol; 1 M of gallic acid: 1 M ethanol; 1 M of gallic acid: 1 M n-propanol; and 1 M of gallic acid: 1 M n-butanol in a solventfree system at 55 C for 10 h under shaking (150g). Effect of molecular sieves on synthesis of methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate Molecular sieves were added to the reaction system to study their effect on the synthesis of methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate by celiteimmobilized lipase. To the reaction mixture, varying amounts (1–5% of reaction volume of 2 g) of molecular sieves were added. The esterification was carried out at 55oC under shaking (150g) for 10 h. Characterization of methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate Synthesized esters were characterized by Fourier transform infrared spectroscopy and NMR to get evidence of the formation of reaction product. FTIR spectrum was recorded on Perkin Elmer spectrophotometer in transmittance mode in KBr and NMR spectroscopy (INOVA 400 MHz spectrophotometer) using tetramethyl silane (TMS) as internal standard in CHCl3.
Results Effect of incubation time on synthesis of methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate using celite-bound lipase of B. licheniformis SCD11501 The effect of reaction time on synthesis of alcoholic esters of gallic acid (methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate) by celite-immobilized lipase (10 mg) was studied at 55 C in a chemical reactor under continuous shaking (Figure 2) up to 12 h in a solvent-/water-free system. The yield of methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate were 55.7%, 63.0%, 67.6%, and 60.4%, respectively after 10 h. The maximum yield was recorded for n-propyl gallate (67.6%) while minimum was observed for methyl gallate (55.7%). It seems that the low conversion with more polar alcohols such as methanol and
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Figure 2. Effect of incubation time on the synthesis of methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate.
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Figure 4. Effect of relative molar concentration of reactants on the synthesis of methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate.
Figure 3. Effect of reaction temperature on the synthesis of methyl gallate, ethyl gallate, n-propyl gallate, and nbutyl gallate.
Figure 5. Effect of amount of biocatalyst on synthesis of methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate.
ethanol might be due to their capacity to severely remove water from the protein hydration shell, there by leading to enzyme inactivation. Thus in the subsequent esterification reactions, a reaction time of 10 h for ethyl gallate, methyl gallate, n-propyl gallate, and n-butyl gallate at 55 C was considered optimum using celite-bound lipase. After 10 h, there might be a gradual loss of lipase activity at 55oC on account of heat denaturation and/or aggregation/precipitation of protein/enzyme in water-free solvent system. Such an adverse effect on the biocatalyst might have lead to decrease in yield(s) of esters. Previously, maximum yield of n-propyl gallate was recorded at 12 h incubation time.22 In an another study, immobilization of Pseudomonas aeruginosa lipase onto a synthetic poly (AAc-co-HPMA-cl-EGDMA) hydrogel catalysed the esterification of methanol and acrylic acid into methyl acrylate in a relatively short period of 6 h at 55 C.23
after which the yield of the synthesized ester(s) decreased. This decline in ester yield might be on account of rapid denaturation of the matrix-bound lipase for an extended period of time at 55oC. A more or less a similar yields of methyl gallate (56.3%), ethyl gallate (63.9%), n-propyl gallate (68.1%), and n-butyl gallate (61.0%) were achieved at 55oC in 10 h under shaking. In a previous study, 40 C was the most desirable operating temperature for enzymatic trans-esterification reaction where the process attained 100% fatty acid methyl ester yield at 4 h.24 A reaction temperature of 55 C was also found to be optimum for the synthesis of ethyl propionate using a commercial lipase, Steapsin.25 In a previous study the optimal temperature for the synthesis of ethyl ferulate using celite-bound commercial lipase was found to be 45 C.26
Effect of reaction temperature on the synthesis of methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate by celite-bound lipase The effect of change in the reaction temperature on the synthesis of methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate by celite-immobilized biocatalyst (10 mg) was also studied (Figure 3) in a water-free system. The B. licheniformis SCD11501 possesses a GC content of 55% establishing a thermo-tolerant nature of enzyme. A maximum increase in yield of esters of gallic acid was recorded at a temperature of 55 C in 10 h under shaking
Effect of relative molar concentration of reactants on the synthesis of methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate When esterification using celite-bound biocatalyst (10 mg) was performed by keeping the concentration of one of the reactants (gallic acid) constant at 1 M and varying the concentration of the other reactant (methanol, ethanol, n-propanol, and n-butanol; 1–4 M) in the water-free system (Figure 4). The molar ratio of 1:1 (acid: alcohol) was found to be optimum for the synthesis of methyl gallate (56.3%) while maximum ester yield for ethyl gallate (64%), n-propyl gallate (68.8%), and n-butyl gallate (61.8%) was observed at
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1:2 molar concentrations. Earlier, molar ratio of 1: 1 was recorded to be optimum for the synthesis of isoamyl butyrate using Lipozyme TL IM.27In another study, a molar ratio of 1:3 (ethanol: propionic acid) was optimum for the synthesis of ethyl propionate in hexane.25 Generally it is seen that either an equimolar ratio of acid and alcohol or a molar excess in favour of alcohol results in higher yield of ester in a water free or water-restricted system.26,27
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Effect of amount of biocatalyst on synthesis of methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate The synthesis of methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate was studied by employing different amounts of immobilized lipase (0.5–3%, w/v) at 55oC for 10 h under shaking. The maximum synthesis of methyl gallate (57.4%) and ethyl gallate (65.2%) was obtained with 2.5% (w/v) of immobilized lipase while maximum yield of n-propyl gallate (70.1%) and n-butyl gallate (62.6%) were obtained with 1.5% of celiteimmobilized lipase (Figure 5). As reported in the synthesis of methyl acrylate 12.5 mg/ml immobilized lipase from Pseudomonas aeruginosa was used to get maximum yield of ester.23 Effect of addition of molecular sieves to the reaction system on synthesis of methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate
Figure 6. Effect of molecular sieves on synthesis of methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate.
Molecular sieves were added in the reaction mixture to which the celite-bound lipase was introduced as a biocatalyst and the ester synthesis was performed at 10 h at 55oC. The solvent-free reaction mixture (gallic acid and alcohol viz. methanol, ethanol, n-propanol, or n-butanol) was treated with varying amounts (1–5% of reaction volume of 2 g) of molecular sieves to remove traces of water molecules, if any (Figure 6). The maximum amount of methyl
Figure 7. FTIR spectrum (a) methyl gallate, (b) ethyl gallate, (c) n-propyl gallate, and (d) n-butyl gallate.
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Figure 8.
NMR spectrum of (a) methyl gallate, (b) ethyl gallate, (c) n-propyl gallate, and (d) n-butyl gallate.
gallate (58.2%), ethyl gallate (66.8%), n-propyl gallate (72.1%), and n-butyl gallate (63.8%) was recorded when the molecular sieves were used at a concentration of 3%, (w/v) of reaction mixture 55 C for 10 h under shaking. Any further addition of molecular sieves to the reaction mixture had an adverse affect on the amount of ester synthesized.
An improvement in rate of esterification has been previously reported for esterification of lauric acid and methanol in the presence of molecular sieves.28 The excess of molecular sieves often over-scavenge the water content in the water-free reaction system there by rendering lipase molecules inactive.
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Figure 8.
Characterization of methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate Characterization of the synthesized esters was carried out by FTIR spectroscopy, 1H NMR spectra were recorded on in deuterated chloroform (CDCl3) solution with internal standard TMS (0 ppm), and chemical shifts were reported in parts
(Continued)
per million (d/ppm). NMR is one of the most important techniques for the estimation of product (ester) as it deals with the protons vicinity of the ester/compound. This technique explained the change of environment of proton when the one reactant is converted into desired product. FTIR spectrum of methyl gallate has a sharp peak at 1730 cm21 which was
Table 1. Characteristic Peaks of Various Esters Formed From Gallic Acid 1
FTIR Characteristic Peaks S. No. 1. 2. 3. 4. 5.
Compound Gallic acid Methylgallate Ethylgallate Propyllgallate Butylgallate
Functional Groups AC@O, AC@O, AC@O, AC@O, AC@O,
AOH, ACH AOACAO, ACH AOACAO, ACH AOACAO, ACH AOACAO
21
H NMR Peaks Value in
Absorption Values (cm )
Functional Groups
Values in ppm
1693, 3500, 2929.1 1730, 1215.5, 2900 1719.2, 1220, 2965 1730.9, 1245, 2900 1726.3, 1245, 2999
ACOOH, Benzene ring ACH3, Benzene ring ACH2CH3, Benzene ring ACH2CH2CH3, Benzene ring ACH2CH2CH2CH3, Benzene ring
12.74, 7.7, 7.5 3.89, 6.95 3.92, 2.3, 6.95 3.92, 2.2, 2.3, 6.95 1.5, 2.2, 2.3, 3.92, 6.95
ascribed to AC@O stretching of ester group and when compared with the spectrum of gallic acid this peak of ester was not present in it that clearly confirmed that the lipasecatalysed esterification reaction had occurred. The other peak at 2939.5 cm21 was due to the C-H stretching of methyl group present in methyl gallate (Figure 7a). Three spectra of ethyl gallate, n-propyl gallate, and n-butyl gallate also had the same characteristic peak due to the AC@O stretching of the ester group at 1719.2, 1730.9, or 1726.3 cm21, respectively, along with the peaks at 2900–2700 cm21 due to CAH stretching (Figure 7b–d). 1 H NMR spectra of gallate series (methyl, ethyl, n-propyl, and n-butyl) were compiled (Figure 8 and Table 1). Spectrum of methyl gallate had peaks with (d/ppm) 3.95 (singlet, 3H of ester group attached with CH3) and 6.95 (doublet, 1 H of benzene ring; Figure 8a). As the chain length of the ester increased, the corresponding peaks due to the methylene and methyl protons of the alkyl group also appeared with d/ppm values in the range 3.2–1.9 thereby confirming the synthesis of different esters with various alcohols. For ethyl gallate the signals due to AC2H5 and benzene ring attained value at 2.3 (ACH3), 3.92 (ACH2), and 6.95 ppm (Figure 8b). Similarly, for n-propyl and n-butyl gallate the peaks due to the absorption by different protons of AC3H7 and AC4H9 d values at 2.3(ACH3), 2.2(ACH2), 3.92(ACH2), and 6.95 ppm and 1.5(ACH2), 2.2(ACH2), 2.3(ACH2), 3.92(ACH2), 6.95, respectively, were observed (Figure 8c,d).
Conclusion The study explored wether improved yields of alkyl esters of gallic acid (methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate) could be achieved by manipulation of vital reaction parameters by employing a celite-immobilized purified lipase of a thermophilic B. licheniformis SCD11501 strain in a water-free system. Esterification reactions were carried out by reacting gallic acid and methanol, ethanol, n-propanol, or nbutanol in different ratio in the presence of celite-bound lipase at selected temperatures for different time periods in a chemical reactor. Attractive yields of methyl gallate (58.2%), ethyl gallate (66.9%), n-propyl gallate (72.1%), and n-butyl gallate (63.8%) in the presence of celite-bound lipase could be achieved after sequentially optimizing various reaction parameters. Maximum ester synthesis was recorded for n-propyl gallate (72.1%) at 55 C in 10 h at 1.5% biocatalyst concentration in the presence of molecular sieves. The low conversion seen with more polar alcohols such as methanol and ethanol might be because of over-scavenging of water from the protein hydration shell leading to enzyme inactivation.
Acknowledgments The financial support from Department of Biotechnology, Ministry of Science and Technology, Government of India to Department of Biotechnology, Himachal Pradesh Univer-
sity, Shimla (India) is thankfully acknowledged. Moreover, the authors do not have any conflict of interest at their place of work.
References 1. Sharma S, Kanwar SS. Organic solvent tolerant lipases and applications. Sci World J. 2014; DOI:10.1155/625258. 2. Dong H, Li Y, Li J, Sheng G, Chen H. Comparative study on lipases immobilized onto bentonite and modified bentonites and their catalytic properties. Ind Eng Chem Res. 2013; DOI: 10.1021/ie4001986. 3. Royon D, Daz M, Ellenrieder G, Locatelli S. Enzymatic production of biodiesel from cotton seed oil using t-butanol as a solvent. Bioresour Technol. 2007;98:648–653. 4. Shah S, Gupta MN. Lipase catalyzed preparation of biodiesel from Jatropha oil in a solvent free system. Process Biochem. 2007;42:409–414. 5. Soumanou MM, Bornscheuer UT. Improvement in lipasecatalyzed synthesis of fatty acid methyl esters from sunflower oil. Enzyme Microb Technol. 2003;33:97–103. 6. Sharma S, Kanwar SS. Properties and stability of celite immobilized lipase of a thermophilic Bacillus sp. STL-7. Int J Biochem Photon. 2013;108:173–182. 7. Farbood Y, Sarkaki A, Hashemi S, Mansouri MT, Dianat M. The effects of gallic acid on pain and memory following transient global ischemia/reperfusion in Wistar rats. Avicenna J Phytomed. 2013;23:543–567. 8. Lokeswari N. Gallic acid production by immobilization of Aspergillus orzae. Int J Sci Eng Res. 2013;4:472–475. 9. Chen CY, Chen KC, Yang TY, Liu HC, Hsu S. Gallic acid induces a reactive oxygen species-provoked c-Jun NH2-Terminal kinase-dependent apoptosis in lung fibroblasts. Evidence-Based Complementary Alt Med. 2013; DOI: 10.1155/613950. 10. Yoon CH, Chung SJ, Lee SW, Park YB, Lee SK. Gallic acid, a natural polyphenolic acid, induces apoptosis and inhibits pro-inflammatory gene expressions in rheumatoid arthritis fibroblast-like synoviocytes. Joint Bone Spine. 2013;80: 274–279. 11. Takai E, Hirano A, Shiraki K. Effects of alkyl chain length of gallate on self-association and membrane binding. J Biochem. 2011;150:165–171. 12. Fujita K, Kubo I. Antifungal activity of octyl gallate. Int J Food Microbiol. 2002;79:193–201. 13. Uozaki M, Yamasaki H, Katsuyama Y, Higuchi M, Higuti T, Koyama AH. Antiviral effect of octyl gallate against DNA and RNA viruses. Antiviral Res. 2007;73:85–91. 14. Dodo K, Minato T, Noguchi-Yachide T, Suganuma M, Hashimoto Y. Anti-proliferative and apoptosis-inducing activities of alkyl gallate and gallamide derivatives related to (-)-epigallocatechin gallate. Bioorg Med Chem. 2008;16:7975–7982. 15. Barreiro SL, Bravo-Dıaz C, Paiva-Martins F, Romsted LS. Maxima in antioxidant distributions and efficiencies with increasing hydrophobicity of gallic acid and its alkyl esters. The pseudophase model interpretation of the “Cutoff Effect. J Agric Food Chem. 2013;61:6533–6543. 16. Masak H, Okamoto N, Sakaki S, Sakurai H. Protective effects of hydroxybenzoic acids and their esters on cell damage induced by hydroxyl radicals and hydrogen peroxides. Biol Pharm Bull. 1997;20:304–312. 17. Klein E, Weber N. In vitro test for the effectiveness of antioxidants as inhibitors of thiyl-radical induced reactions with unsaturated fatty acids. J Agric Food Chem. 2001;49:1224–1230.
Biotechnol. Prog., 2015, Vol. 31, No. 3 18. Murase T, Kume N, Hase T, Shibuya Y, Nishizawa Y, Tokimitsu I, Kita T. Gallates inhibit cytokine-induced nuclear translocation of NF-kappa B and expression of leukocyte adhesion molecules in vascular endothelial cells. Arterioscler Thromb Vasc Biol. 1999;19:1412–1418. 19. Locatelli C, Filippin-Monteiro FB, Creczynski-Pasa TB. Alkyl esters of gallic acid as anticancer agents: A review. Eur J Med Chem. 2013;60:233–239. 20. Verma S, Singh A, Mishra A. Gallic acid: Molecular rival of cancer. Environ Toxicol Pharmacol. 2013;35:473–485. 21. Sharma S, Dogra P, Chauhan GS, Kanwar SS. Synthesis of alkyl coumarate esters by lipase of Bacillus licheniformis SCD11501. J Mol Catal B: Enzym. 2014;101:80–86. 22. Yu X, Li Y, Wu D. Enzymatic synthesis of gallic acid esters using microencapsulated tannase: Effect of organic solvents and enzyme specificity. J Mol Catal B: Enzym. 2004;30:69–73. 23. Kanwar SS, Verma ML, Maheshwari C, Chauhan S, Chimni SS, Chauhan GS. Properties of poly (AAc-co-HPMA-cl-EGDMA) hydrogel-bound lipase of Pseudomonas aeruginosa MTCC-4713 in synthesis of methyl acrylate. J Appl Polym Sci. 2007;104: 183–191.
723 24. Sim JH, Khor GK, Kamaruddin AH, Bhatia S. Thermodynamic studies on activity and stability of immobilized Thermomyces lanuginosus in producing Fatty Acid Methyl Ester (FAME). Int J Sci Res Pub. 2013;3:2250–2259. 25. Kumar S, Pahujani S, Ola RP, Kanwar SS, Gupta R. Enhanced stability of silica immobilized lipase from Bacillus coagulans BTS-3 and synthesis of ethyl propionate. Acta Microbiol Immunol Hungarica. 2005;53:217–229. 26. Kumar A, Kanwar SS. Synthesis of ethyl ferulate in organic medium using celite-immobilized lipase. Bioresour Technol. 2010;102:2162–2167. 27. Andreia A, Vitor CA, Barbara DAP, Susana JK, Carlos AVB, Janaina FMB. Enzymatic synthesis and optimization of isoamyl butyrate. J Braz Chem Soc. 2011;22:2148–2156. 28. Mustafa UU. A study on the lipase catalyzed esterification in organic solvent. Trends J Agric. 1998;22:1697–1702. Manuscript received Sept. 30, 2014, and revision received Feb. 21, 2015.
Priyanka Dogra and Ghanshyam S. Chauhan Dept. of Chemistry, Himachal Pradesh University, Summer Hill, Shimla 171 005, India DOI 10.1002/btpr.2072 Published online March 18, 2015 in Wiley Online Library (wileyonlinelibrary.com)
Gallic acid (3, 4, 5- trihydroxybenzoic acid) is an important antioxidant, anti-inflammatory, and radical scavenging agent. In the present study, a purified thermo-tolerant extra-cellular lipase of Bacillus licheniformis SCD11501 was successfully immobilized by adsorption on Celite 545 gel matrix followed by treatment with a cross-linking agent, glutaraldehyde. The celite-bound lipase treated with glutaraldehyde showed 94.8% binding/retention of enzyme activity (36 U/g; specific activity 16.8 U/g matrix; relative increase in enzyme activity 64.7%) while untreated matrix resulted in 88.1% binding/retention (28.0 U/g matrix; specific activity 8.5 U/g matrix) of lipase. The celite-bound lipase was successfully used to synthesis methyl gallate (58.2%), ethyl gallate (66.9%), n-propyl gallate (72.1%), and n-butyl gallate (63.8%) at 55oC in 10 h under shaking (150 g) in a water-free system by sequentially optimizing various reaction parameters. The low conversion of more polar alcohols such as methanol and ethanol into their respective gallate esters might be due to the ability of these alcohols to severely remove water from the protein hydration shell, leading to enzyme inactivation. Molecular sieves added to the reaction mixture resulted in enhanced yield of the alkyl ester(s). The characterization of synthesised esters was done through fourier transform infrared (FTIR) C 2015 American Institute of Chemical Engispectroscopy and 1H NMR spectrum analysis. V neers Biotechnol. Prog., 31:715–723, 2015 Keywords: gallic acid, lipase, esterification, immobilization, methanol, ethanol, propanol, butanol
Introduction Lipases are a group of enzymes naturally endowed with the property of performing reactions in aqueous as well as organic solvents.1 Lipases are attractive enzymes for preparation of some important intermediates such as chiral alcohols, carboxylic acids, and amines, and have been extensively used in food, textile, detergents, paper, chemical, pharmaceutical, and environmental industries in the last few decades.2 The enzymatic trans-esterification has been performed in solvents and solvent-free media by different immobilized lipases.3,4 From the application viewpoint, enzymatic synthesis in organic solvents is not suitable due to toxicity, flammable nature of solvent and damaging effects on the environment. Thus enzymatic solvent-free systems are being developed to make the enzymatic process competitive.5 The biocatalysts properties are being inclined by selection of different immobilization methods.6 Inorganic supports with the stability in anhydrous and oil/water medium can provide an easily separable and reusable system in continuous fabrication and the modification of inorganic material is in favour of improving its biocompatibility with enzymes, contributing to the improved enzymatic activity and stability.2 Gallic acid Correspondence concerning this article should be addressed to S. Sharma at [email protected]. C 2015 American Institute of Chemical Engineers V
(3, 4, 5-trihydroxybenzoic acid), from gallnut and green tea, is known to be antioxidant, anti-inflammatory, and radical scavenger.7 Gallic acid is an organic substance occurring in many plants either as a free molecule or as part of tannic acid molecule.8 Gallic acid has been known to induce apoptosis in tumour cell lines and renal fibroblasts9 and has proapoptotic and anti-inflammatory effects on fibroblast-like synoviocytes from patients with rheumatoid arthritis.10 Alkyl gallates are used as antibacterial and anti-viral agents11 and found to affect microbial cell viability,12 virus activity,13 and human leukaemia cell proliferation.14 Gallic acid and its esters are hydroxybenzoic derivatives which are used as antioxidant additives in food and pharmaceutical industry. Oxidation of polyunsaturated lipids by reactive oxygen species is a serious and continuing problem for the food industry. Peroxidation produces rancid odours and off-flavors, decreases shelf life, alters food texture, and appearance, and may reduce nutritional quality and food safety.15 E-310 (propyl gallate) and E-311 (octyl gallate), which are known to protect cells against oxidative damage induced by reactive oxygen species, as hydroxyl radicals or hydrogen peroxide and reactive sulphur species.16,17 Synthetic galloyl esters were found to hinder cytokine-induced nuclear translocation of nuclear factor (NF)-kappa B, as well as expression of leukocyte adhesion molecules in vascular endothelium cells.18 Gallic acid and its derivatives have 715
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been extensively evaluated for their anti-tumor activity against a variety of cell lineages.19 Gallic acid inhibits activation of NF-jB and Akt signalling pathways along with the activity of cyclooxygenase, ribonucleotide reductase and glutathione (reduced). Moreover, gallic acid activates ataxia telangiectasia mutated kinase signalling pathways to prevent the processes of carcinogenesis.20 The present study was carried out to exploit the enzymatic potential of a solvent–tolerant thermophilic Bacillus licheniformis isolate SCD1105 originally isolated from a thermal spring produced an extra cellular alkaliphilic lipase under optimal physico-chemical conditions toward the synthesis of a few useful free radical scavenger esters of gallic acid. The purified lipase of B. licheniformis strain was adsorbed on to celite matrix and immobilized enzyme was used for esterification of gallic acid with chain of alcohols. The effects incubation time, concentrations of reactant, immobilized biocatalyst; reaction temperature and presence of molecular sieves on the rate of synthesis of methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate were consecutively evaluated. The synthesized esters were characterized by fourier transform infrared (FTIR) spectroscopy and nuclear magnetic resonance (NMR). Previously we have reported the synthesis of alkyl coumarate esters from the celite-bound lipase of B. licheniformis strain21 albeit at low yields in a water-free system. The present study was conducted to explore wether celite bound-lipase of a thermophilic B. licheniformis bacterium could produce improved yields of alkyl esters of gallic acid in a water-free reaction system by manipulation of vital reaction parameters.
Materials and Methods Materials Celite 545, diethylaminoethyl (DEAE) matrix (S.D. FineChem Ltd., Hyderabad, India); p-nitrophenyl palmitate (pNPP) and glutaraldehyde (Lancaster Synthesis, England); ˚ 3 1.5 mm), methanol, ethanol, n-promolecular sieves (3A panol, and n-butanol (MERCK, Mumbai, India); Bovine serum albumin, deuterated chloroform (CDCl3) solution, Tris buffer, and gallic acid (Himedia Laboratory, Ltd., Mumbai, India) were procured from various commercial suppliers. All chemicals were of analytical grade and were used as received. A chemical reactor with stirring and heating (MiniBlockTM, China) was used to perform ester synthesis using celite-immobilized lipase of B. licheniformis SCD11501. Microorganism B. licheniformis strain SCD11501 (GenBank Accession Number JN998712.1) was isolated from hot springs of Tatapani, District: Mandi, Himachal Pradesh (India). This bacterium was identified by Xcelris Labs Ltd. Ahmedabad-380 054, India on the basis of 16S RNA sequence. Purification and immobilization of lipase of B. licheniformis strain SCD11501 A thermo-tolerant extra cellular lipase produced by B. licheniformis strain SCD11501 was purified (37.0 U/mg protein) by successive techniques of salting out using ammonium sulphate, dialysis, and DEAE anion-exchange chromatography to 10.6-fold with an overall yield of 8.4% (experimental data not provided here).
Immobilization of bacterial lipase on celite gel matrix Celite 545 (3 g) was incubated with 10 ml 0.05 M Tris HCl buffer of pH 9.5 for 12 h in a glass vial. (2 ml; 60 U of enzyme) enzyme was added to the matrix for 1 h at 37 C. The celite particles were sedimented by centrifugation (5 min at 10,000g at 4 C) followed by addition of glutaraldehyde (1%; v/v) for 1 h at 37 C. The celite was extensively washed with Tris buffer (0.05 M, pH 9.5) to get rid of unbound activating agent. The supernatant was decanted and immobilized biocatalyst was retained. The weight of biocatalyst was recorded and activity was assayed using 40 mg of celite-bound lipase. The immobilized protein in matrix was determined by subtracting unbound protein in the supernatant from the total protein used for immobilization as well as increased/decreased in the total activity was calculated by adding total activity of supernatant and matrix in comparison to total enzyme units of purified lipase incubated earlier. Colorimetric method for lipase assay The lipase activity was assayed using p-NPP as a chromogenic substrate. The stock solution (20 mM) of p-NPP was prepared in HPLC grade iso-propanol. The reaction mixture contained 60 ll of p-NPP stock-solution, 40 mg of enzyme, and Tris buffer (0.05 M, pH 9.0) to make final volume 3 ml. The reaction mixture was incubated at 50oC for 10 min in a waterbath (Bangalore Genei Pvt. Ltd., Bangalore). Further enzymatic reaction was stopped by chilling the reaction mixture at 220oC for 7 min. The absorbance (A410) of heat-inactivated lipase was subtracted from the absorbance of the corresponding test sample. The A410 of p-nitrophenol released was measured (Labindia 30001 UV/vis Spectrophotometer) and expressed in millimoles (mM). Each of the assays were performed in triplicate unless otherwise stated and mean values 6 SD was presented. Stock solutions of various p-nitrophenyl esters para-nitrophenyl formate (p-NPF), para-nitrophenyl acetate (p-NPA), para-nitrophenyl caprylate (p-NPC), para-nitrophenyl palmitate (p-NPP), paranitrophenyl benzoate (p-NPB), and para-nitrophenyl laurate (p-NPL) were also prepared for use in some of the experiments. Unit of lipase activity The unit (U) of enzyme activity was defined as mmole(s) of p-nitrophenol released from p-NPP per min by one ml of free enzyme or one gram of celite-immobilized enzyme (weight of matrix included) under standard assay conditions. Esterification reaction Alkyl gallate esters were synthesized by using 1 M alcohol (methanol, ethanol, propanol or butanol), 1 M gallic acid, and celite-bound purified lipase taken in glass-vial (20 ml capacity). The reaction was performed at 55 C for 10 h in a chemical reactor. The scheme of the synthesis of the methyl gallate, ethyl gallate, propyl gallate, and butyl gallate is presented schematically (Figure 1). The effect(s) of incubation time, reaction temperature, molar concentration of reactants, biocatalyst, and molecular sieves on the rate of synthesis of methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate were separately evaluated. The formed esters, that is methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate were separated on the basis of their
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Figure 1. Scheme of the synthesis of the methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate.
solubility in hot water using a separating funnel. The gallic acid was soluble in hot water while the formed esters (methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate formed in different test tubes) were insoluble in hot water and were separated out using separating funnel. The amount of ester synthesized was determined and represented as % yield. The synthetic reactions in water-free medium were performed in duplicate mean values were presented. Effect of incubation time on synthesis of methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate The reaction mixture (2 g) comprised celite-immobilized lipase (10 mg), gallic acid (1 M): methanol (1 M) or gallic acid (1 M): ethanol (1 M) or gallic acid (1 M): n-propanol (1 M) or gallic acid (1 M): n-butanol (1 M) taken in Teflon-coated capped glass vials (5 ml capacity) in a solvent-free system, respectively. The glass vials were incubated at 55oC in a chemical reactor for 4, 6, 8, 10, and 12 h under shaking (150g). Effect of reaction temperature on the synthesis of methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate The effects of reaction temperature (40, 45, 50, 55, and 60 C) on the synthesis of methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate by the immobilizedlipase (10 mg) were studied at incubation time of 10 h under continuous shaking (150g). Effect of relative molar concentration of reactants on the synthesis of methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate The concentration of one of the reactants (gallic acid) was kept constant at 1 M and varying the concentration of the other reactant (methanol, ethanol, n-propanol, and n-butanol; 1–4 M) in the reaction mixture (2 g) in a solvent-free system. The esterification was carried out at 55 C for optimized time (10 h) under continuous shaking using 10 mg celite-bound lipase. Effect of amount of biocatalyst on synthesis of methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate The synthesis of ethyl gallate, methyl gallate, n-propyl gallate, and n-butyl gallate were studied by taking different
amount of immobilized lipase (0.5–3% of acid weight) in reaction mixture (2 g) containing 1 M of gallic acid: 1 M methanol; 1 M of gallic acid: 1 M ethanol; 1 M of gallic acid: 1 M n-propanol; and 1 M of gallic acid: 1 M n-butanol in a solventfree system at 55 C for 10 h under shaking (150g). Effect of molecular sieves on synthesis of methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate Molecular sieves were added to the reaction system to study their effect on the synthesis of methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate by celiteimmobilized lipase. To the reaction mixture, varying amounts (1–5% of reaction volume of 2 g) of molecular sieves were added. The esterification was carried out at 55oC under shaking (150g) for 10 h. Characterization of methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate Synthesized esters were characterized by Fourier transform infrared spectroscopy and NMR to get evidence of the formation of reaction product. FTIR spectrum was recorded on Perkin Elmer spectrophotometer in transmittance mode in KBr and NMR spectroscopy (INOVA 400 MHz spectrophotometer) using tetramethyl silane (TMS) as internal standard in CHCl3.
Results Effect of incubation time on synthesis of methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate using celite-bound lipase of B. licheniformis SCD11501 The effect of reaction time on synthesis of alcoholic esters of gallic acid (methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate) by celite-immobilized lipase (10 mg) was studied at 55 C in a chemical reactor under continuous shaking (Figure 2) up to 12 h in a solvent-/water-free system. The yield of methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate were 55.7%, 63.0%, 67.6%, and 60.4%, respectively after 10 h. The maximum yield was recorded for n-propyl gallate (67.6%) while minimum was observed for methyl gallate (55.7%). It seems that the low conversion with more polar alcohols such as methanol and
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Figure 2. Effect of incubation time on the synthesis of methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate.
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Figure 4. Effect of relative molar concentration of reactants on the synthesis of methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate.
Figure 3. Effect of reaction temperature on the synthesis of methyl gallate, ethyl gallate, n-propyl gallate, and nbutyl gallate.
Figure 5. Effect of amount of biocatalyst on synthesis of methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate.
ethanol might be due to their capacity to severely remove water from the protein hydration shell, there by leading to enzyme inactivation. Thus in the subsequent esterification reactions, a reaction time of 10 h for ethyl gallate, methyl gallate, n-propyl gallate, and n-butyl gallate at 55 C was considered optimum using celite-bound lipase. After 10 h, there might be a gradual loss of lipase activity at 55oC on account of heat denaturation and/or aggregation/precipitation of protein/enzyme in water-free solvent system. Such an adverse effect on the biocatalyst might have lead to decrease in yield(s) of esters. Previously, maximum yield of n-propyl gallate was recorded at 12 h incubation time.22 In an another study, immobilization of Pseudomonas aeruginosa lipase onto a synthetic poly (AAc-co-HPMA-cl-EGDMA) hydrogel catalysed the esterification of methanol and acrylic acid into methyl acrylate in a relatively short period of 6 h at 55 C.23
after which the yield of the synthesized ester(s) decreased. This decline in ester yield might be on account of rapid denaturation of the matrix-bound lipase for an extended period of time at 55oC. A more or less a similar yields of methyl gallate (56.3%), ethyl gallate (63.9%), n-propyl gallate (68.1%), and n-butyl gallate (61.0%) were achieved at 55oC in 10 h under shaking. In a previous study, 40 C was the most desirable operating temperature for enzymatic trans-esterification reaction where the process attained 100% fatty acid methyl ester yield at 4 h.24 A reaction temperature of 55 C was also found to be optimum for the synthesis of ethyl propionate using a commercial lipase, Steapsin.25 In a previous study the optimal temperature for the synthesis of ethyl ferulate using celite-bound commercial lipase was found to be 45 C.26
Effect of reaction temperature on the synthesis of methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate by celite-bound lipase The effect of change in the reaction temperature on the synthesis of methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate by celite-immobilized biocatalyst (10 mg) was also studied (Figure 3) in a water-free system. The B. licheniformis SCD11501 possesses a GC content of 55% establishing a thermo-tolerant nature of enzyme. A maximum increase in yield of esters of gallic acid was recorded at a temperature of 55 C in 10 h under shaking
Effect of relative molar concentration of reactants on the synthesis of methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate When esterification using celite-bound biocatalyst (10 mg) was performed by keeping the concentration of one of the reactants (gallic acid) constant at 1 M and varying the concentration of the other reactant (methanol, ethanol, n-propanol, and n-butanol; 1–4 M) in the water-free system (Figure 4). The molar ratio of 1:1 (acid: alcohol) was found to be optimum for the synthesis of methyl gallate (56.3%) while maximum ester yield for ethyl gallate (64%), n-propyl gallate (68.8%), and n-butyl gallate (61.8%) was observed at
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1:2 molar concentrations. Earlier, molar ratio of 1: 1 was recorded to be optimum for the synthesis of isoamyl butyrate using Lipozyme TL IM.27In another study, a molar ratio of 1:3 (ethanol: propionic acid) was optimum for the synthesis of ethyl propionate in hexane.25 Generally it is seen that either an equimolar ratio of acid and alcohol or a molar excess in favour of alcohol results in higher yield of ester in a water free or water-restricted system.26,27
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Effect of amount of biocatalyst on synthesis of methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate The synthesis of methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate was studied by employing different amounts of immobilized lipase (0.5–3%, w/v) at 55oC for 10 h under shaking. The maximum synthesis of methyl gallate (57.4%) and ethyl gallate (65.2%) was obtained with 2.5% (w/v) of immobilized lipase while maximum yield of n-propyl gallate (70.1%) and n-butyl gallate (62.6%) were obtained with 1.5% of celiteimmobilized lipase (Figure 5). As reported in the synthesis of methyl acrylate 12.5 mg/ml immobilized lipase from Pseudomonas aeruginosa was used to get maximum yield of ester.23 Effect of addition of molecular sieves to the reaction system on synthesis of methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate
Figure 6. Effect of molecular sieves on synthesis of methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate.
Molecular sieves were added in the reaction mixture to which the celite-bound lipase was introduced as a biocatalyst and the ester synthesis was performed at 10 h at 55oC. The solvent-free reaction mixture (gallic acid and alcohol viz. methanol, ethanol, n-propanol, or n-butanol) was treated with varying amounts (1–5% of reaction volume of 2 g) of molecular sieves to remove traces of water molecules, if any (Figure 6). The maximum amount of methyl
Figure 7. FTIR spectrum (a) methyl gallate, (b) ethyl gallate, (c) n-propyl gallate, and (d) n-butyl gallate.
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Figure 8.
NMR spectrum of (a) methyl gallate, (b) ethyl gallate, (c) n-propyl gallate, and (d) n-butyl gallate.
gallate (58.2%), ethyl gallate (66.8%), n-propyl gallate (72.1%), and n-butyl gallate (63.8%) was recorded when the molecular sieves were used at a concentration of 3%, (w/v) of reaction mixture 55 C for 10 h under shaking. Any further addition of molecular sieves to the reaction mixture had an adverse affect on the amount of ester synthesized.
An improvement in rate of esterification has been previously reported for esterification of lauric acid and methanol in the presence of molecular sieves.28 The excess of molecular sieves often over-scavenge the water content in the water-free reaction system there by rendering lipase molecules inactive.
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Figure 8.
Characterization of methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate Characterization of the synthesized esters was carried out by FTIR spectroscopy, 1H NMR spectra were recorded on in deuterated chloroform (CDCl3) solution with internal standard TMS (0 ppm), and chemical shifts were reported in parts
(Continued)
per million (d/ppm). NMR is one of the most important techniques for the estimation of product (ester) as it deals with the protons vicinity of the ester/compound. This technique explained the change of environment of proton when the one reactant is converted into desired product. FTIR spectrum of methyl gallate has a sharp peak at 1730 cm21 which was
Table 1. Characteristic Peaks of Various Esters Formed From Gallic Acid 1
FTIR Characteristic Peaks S. No. 1. 2. 3. 4. 5.
Compound Gallic acid Methylgallate Ethylgallate Propyllgallate Butylgallate
Functional Groups AC@O, AC@O, AC@O, AC@O, AC@O,
AOH, ACH AOACAO, ACH AOACAO, ACH AOACAO, ACH AOACAO
21
H NMR Peaks Value in
Absorption Values (cm )
Functional Groups
Values in ppm
1693, 3500, 2929.1 1730, 1215.5, 2900 1719.2, 1220, 2965 1730.9, 1245, 2900 1726.3, 1245, 2999
ACOOH, Benzene ring ACH3, Benzene ring ACH2CH3, Benzene ring ACH2CH2CH3, Benzene ring ACH2CH2CH2CH3, Benzene ring
12.74, 7.7, 7.5 3.89, 6.95 3.92, 2.3, 6.95 3.92, 2.2, 2.3, 6.95 1.5, 2.2, 2.3, 3.92, 6.95
ascribed to AC@O stretching of ester group and when compared with the spectrum of gallic acid this peak of ester was not present in it that clearly confirmed that the lipasecatalysed esterification reaction had occurred. The other peak at 2939.5 cm21 was due to the C-H stretching of methyl group present in methyl gallate (Figure 7a). Three spectra of ethyl gallate, n-propyl gallate, and n-butyl gallate also had the same characteristic peak due to the AC@O stretching of the ester group at 1719.2, 1730.9, or 1726.3 cm21, respectively, along with the peaks at 2900–2700 cm21 due to CAH stretching (Figure 7b–d). 1 H NMR spectra of gallate series (methyl, ethyl, n-propyl, and n-butyl) were compiled (Figure 8 and Table 1). Spectrum of methyl gallate had peaks with (d/ppm) 3.95 (singlet, 3H of ester group attached with CH3) and 6.95 (doublet, 1 H of benzene ring; Figure 8a). As the chain length of the ester increased, the corresponding peaks due to the methylene and methyl protons of the alkyl group also appeared with d/ppm values in the range 3.2–1.9 thereby confirming the synthesis of different esters with various alcohols. For ethyl gallate the signals due to AC2H5 and benzene ring attained value at 2.3 (ACH3), 3.92 (ACH2), and 6.95 ppm (Figure 8b). Similarly, for n-propyl and n-butyl gallate the peaks due to the absorption by different protons of AC3H7 and AC4H9 d values at 2.3(ACH3), 2.2(ACH2), 3.92(ACH2), and 6.95 ppm and 1.5(ACH2), 2.2(ACH2), 2.3(ACH2), 3.92(ACH2), 6.95, respectively, were observed (Figure 8c,d).
Conclusion The study explored wether improved yields of alkyl esters of gallic acid (methyl gallate, ethyl gallate, n-propyl gallate, and n-butyl gallate) could be achieved by manipulation of vital reaction parameters by employing a celite-immobilized purified lipase of a thermophilic B. licheniformis SCD11501 strain in a water-free system. Esterification reactions were carried out by reacting gallic acid and methanol, ethanol, n-propanol, or nbutanol in different ratio in the presence of celite-bound lipase at selected temperatures for different time periods in a chemical reactor. Attractive yields of methyl gallate (58.2%), ethyl gallate (66.9%), n-propyl gallate (72.1%), and n-butyl gallate (63.8%) in the presence of celite-bound lipase could be achieved after sequentially optimizing various reaction parameters. Maximum ester synthesis was recorded for n-propyl gallate (72.1%) at 55 C in 10 h at 1.5% biocatalyst concentration in the presence of molecular sieves. The low conversion seen with more polar alcohols such as methanol and ethanol might be because of over-scavenging of water from the protein hydration shell leading to enzyme inactivation.
Acknowledgments The financial support from Department of Biotechnology, Ministry of Science and Technology, Government of India to Department of Biotechnology, Himachal Pradesh Univer-
sity, Shimla (India) is thankfully acknowledged. Moreover, the authors do not have any conflict of interest at their place of work.
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