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Aspergillus niger whole-cell catalyzed synthesis of caffeic acid phenethyl ester in ionic liquids
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Aspergillus niger whole-cell catalyzed synthesis of caffeic acid phenethyl ester in ionic liquids Govindaraju Rajapriyaa, Vivek Kumar Moryaa, Ngoc Lan Maia,b, Yoon-Mo Kooa, a b
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Department of Biological Engineering, Inha University, 100 Inharo, Nam-gu Incheon 22212, Korea Faculty of Applied Sciences, Ton Duc Thang University, Ho Chi Minh City, Vietnam
A R T I C L E I N F O
A B S T R A C T
Keywords: Caffeic acid phenethyl ester (CAPE) Ionic liquid Whole-cell Aspergillus niger HalotolerantAspergillus niger halotolerant Esterification
Synthesis of caffeic acid ester essentially requires an efficient esterification process to produce various kinds of medicinally important ester derivatives. In the present study, a comprehensive and comparative analysis of whole-cell catalyzed caffeic acid esters production in ionic liquids (ILs) media was performed. Olive oil induced mycelial mass of halotolerant Aspergillus niger (A.niger) EXF 4321 was freeze dried and used as a catalyst. To ensure maximum solubilization of caffeic acid for highest substrate loading several ILs were screened and 1ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([Emim][Tf2N]) was found to have the maximum solubility and favoured for enzymatic activity of freeze dried mycelia. The whole-cell catalyzed synthesis of caffeic acid phenethyl ester (CAPE) conditions were optimized and bioconversion up to 84% was achieved at a substrate molar ratio of 1:20 (caffeic acid:2-phenyl ethanol), 30 °C for 12 h. Results obtained during this study were encouraging and helpful to design a bioreactor system to produce caffeic acid derived esters.
1. Introduction Caffeic acid (3,4-dihydroxycinnamic) (CA) and its derivatives are members of the hydroxycinnamate and phenylpropanoid metabolites, and widely distributed in plant tissues as a common source of phenolics [1]. These phenolics are widely used as folkloric medicine especially as carcinogenic inhibitor, antioxidant and antimicrobial etc. In addition to that new findings revealed their potentials in prevention of cancer and other cardiovascular diseases, which is another area of interest in this molecules [2]. Due to the low solubility of CA in hydrophobic medium, its applications in oil based foods and cosmetics are limited. In order to maintain its hydrophobicity and activity, it is essential to esterify with fatty alcohol [3]. Among these phenolics, the caffeic acid phenethyl ester (CAPE) an ester of CA is the most important active ingredient of propolis based cosmetic products. In addition to skin bioactive properties of CAPE, it has been reported for a wide range of bioactivities such as antioxidant [4], anti-inflammatory [5], anticancer activities [6,7], neuroprotective, hepatoprotective, and cardioprotective capacities [8], cytoprotective, protective against ischemia–reperfusion (I/R) injury, and protective effects [9]. Therefore, a huge and continuous demand of the CAPE and their derivatives in industries are maintained. However, isolation and purification of CAPE from natural resources is expensive, low yielding and time consuming. Naturally CAPE coexists
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with several catechol ring containing phenolic which are major hurdle for isolation [10]. A chemical synthesis also has similar challenges due to use of toxic chemicals, low yields and product purification [11]. Therefore, a greener alternative is being required to meet the industrial demand. To overcome these environmental constraints, use of biocatalyst such as lipase (either in pure or cell bound) enzyme becomes an increasingly attractive alternative to traditional chemical methods, due to the high selectivity, mild reaction conditions and environment-friendly process. In recent decade, using of whole cell as a biocatalyst was paid great attention in bio-catalyzed reaction, to reduce the overall cost of production. Whole-cell microbe mediated bio-conversion is relatively cost effective and easy to handle than the immobilized enzyme system [12]. Among them, filamentous fungi have been arisen as a most robust whole cell biocatalyst for industrial applications [13]. Ionic liquids (ILs) are molten salt at room temperature, which have a wide range of tunable physio-chemical properties [14]. Unlike organic solvents, ILs possess wide range of temperature, able to dissolve many compounds, good biological compatibility and negligible vapor pressure [15]. Over the past decades, the use of ILs in biotacalysis for organic synthesis has been extensively studied. In the presence of ILs, the biocatalysts (isolated enzyme and whole-cell) showed enhanced catalytic activity, entioselectivity and operational stability [16,17]. However, the major drawbacks of enzyme catalysts are their very
Corresponding author. E-mail address: [email protected] (Y.-M. Koo).
http://dx.doi.org/10.1016/j.enzmictec.2017.10.005 Received 15 May 2017; Received in revised form 30 August 2017; Accepted 14 October 2017 0141-0229/ © 2017 Published by Elsevier Inc.
Please cite this article as: Rajapriya, G., Enzyme and Microbial Technology (2017), http://dx.doi.org/10.1016/j.enzmictec.2017.10.005
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Fig. 1. Halotolerant A.niger EXF 4321 mycelia catalyzed esterification of caffeic acid with alcohol as a model reaction.
2.3. Lipase assay
higher costs, since they need multiple time-consuming purification processes and in some cases, removal of an enzyme from its natural cell environment may lead to partial or even complete loss of the enzyme activity [18]. Therefore, direct use of microbial whole-cells as biocatalysts instead of isolated enzymes is considered as a potential way to reduce the cost of industrial process, since they could avoid the tedious preparation procedures of the enzymes and maintain the enzyme activity by protecting the cells [19]. This study is focusing on microbe mediated greener synthesis of CAPE in ILs using a halotolerant filamentous fungus A.niger EXF 4321, isolated from solar saltern [20]. A. niger EXF 4321 was selected as catalyst due to their highly tolerance to ILs containing media in our previous screening study (data not shown). In this study, an organic solvent free green CAPE biosynthesis was performed in ILs and to meet the goal, freeze dried mycelia was used as whole cell catalyst while caffeic acid and 2 -phenyl ethanol was used as substrates (Fig. 1). This study was the first report on such kind of phenolic acid ester synthesis using microbial whole-cell catalyst and ILs system.
The rhodamine B-olive oil agar (ROA) plate assay was used for identification of lipase production from organism [21]. This primary plate assay method is used to determine the lipase secretion, based on the interaction of rhodamine B with fatty acid released from olive oil by the lipase hydrolysis. The mycelia were inoculated with the plate containing ROA medium and incubated for 48 h at 30 °C. After incubation, the plates were observed under UV light (350 nm). The hydrolysis of substrate causes the formation of orange fluorescent halos which are visible upon UV irradiation. Lipase activity was also quantified by standard protocol, in brief, the absorbance at 400 nm due to the release of p-nitrophenol during the hydrolysis of 50 mM p-nitro phenyl palmitate at pH 7.2 and temperature at 37 °C [22]. To initialize the reaction, freeze dried A.niger EXF4321 mycelia was added and absorbance was measured spectrophotometrically at 400 nm. One unit of enzyme activity is defined as the amount of enzyme that catalyzes the production of 1 nmol of p-nitrophenol per minute at 30 °C.
2. Materials and methods
2.4. Solubility of caffeic acid
2.1. Materials
The solubility experiment was performed as conventional method to quantify the maximum CA solubility in various ILs. In brief, an excess amount of CA was added to a 5 mL screw-capped vial containing ILs (1 mL) and the samples were vortexed for few seconds to ensure the solubilization of CA [23]. The suspension was stirred for 24 h at 30 °C. Then, the samples were centrifuged at 10,000 rpm for 5 min. The supernatant of ILs phase was sampled and diluted with methanol. The concentration of CA was determined by HPLC analysis.
Ionic liquids 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl) imide ([Emim][Tf2N]), 1-Butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([Bmim][Tf2N], 1-Hexyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([Hmim][Tf2N]), 1-octyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([Omim][Tf2N]), 1Benzyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([Bzmim] [Tf2N]) 1-Ethyl-2,3-dimethylimidazolium bis(trifluoromethylsulfonyl) imide ([Edmim][Tf2N]) used in this study were purchased from C-TRI (South Korea) and had residual chloride content of less than 30 ppm. All ILs were dried in an oven with vacuum pressure of 90 kPa at 80 °C for 48 h before use. 2-Phenyl ethanol, isooctane, CA, CAPE, methanol, 1-propanol and n-butanol were purchased from Sigma-Aldrich. All other chemicals were of analytical grade and used without further purification. A.niger EXF 4321 was received from EX Culture Collection (University of Ljubljana, Slovenia).
2.5. Caffeic acid esters synthesis Caffeic acid ester synthesis was carried out in 5 mL screw-capped vials containing caffeic acid and fatty alcohol in a molar ratio of 1:20 placed in magnetic reaction block at 30 °C with shaking speed of 200 rpm. Reaction was initiated by the addition of freeze dried A. niger EXF 4321 mycelia. From the reaction mixture, sample was carefully withdrawn at specified time intervals and centrifuged at 8000 rpm for 5 min to remove the mycelia from the reaction mixture. The samples were diluted with methanol and quantity of the ester synthesis was analyzed by HPLC. All reactions were duplicated and mean values were presented.
2.2. Inoculum preparation The cultures of A.niger EXF 4321 were incubated on potato dextrose agar slants at 30 °C for 5 days. The spores obtained were suspended in sterile distilled water containing 0.1% (v/v) Tween 80 for the preparation of inoculums. The spore concentration was determined by using hemocytometer. The spore suspension of 107 spores/mL inoculated in 250 mL flasks containing 100 mL of the Czapek dox (CZ) consist of K2HPO4 (1.0 g L−1), KCl (0.5 g L−1), NaNO3 (3.0 g L−1), MgSO4 (0.5 g L−1), FeSO4 (0.1 g L−1) and sucrose (30.0 g L−1) in distilled water. To induce lipase activity, 3% olive oil was added to 24 h grown culture at 30 °C with shaking speed of 200 rpm. Further cultures were continuously incubated in shaker for 96 h. After incubation, mycelia was filtered and washed thoroughly with chilled acetone and dried in freeze drier at −55 °C for 24 h and kept at 4 °C for further use.
2.6. HPLC analysis Quantitative analysis of CA and esters were performed by HPLC analytical method. Separation was accomplished using a Shimadzu HPLC system (Model LC-10A, Japan) equipped with a reverse-phase C18 column (4.6 × 250 mm, Waters, USA) with 5 μm particle size. The mobile phase consists of methanol and distilled water (70:30% v/v) with flow rate of 1 mL min−1. The absorbance was detected using UV detector at 325 nm.
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Tf2N based anions with increasing alkyl chain were screened at 30 °C as shown in Fig. 3. Hydrophobic ILs typically favored the esterification reaction than hydrophilic ILs, because the hydrophilic ILs has the tendency to strip some of essential water molecules from the enzyme environment. In order to maintain the enzyme secondary structure for its activity and to overcome the mass transfer limitations, Tf2N based ionic liquids was selected [25]. Comparing to other hydrophobic anion, Tf2N based anion has low viscosity value and coordinating-strength the highest solubility of CA was 0.38 mg mL−1 in water among the tested solvents and followed by that in [Emim][Tf2N] (0.21 mg mL−1). The least solubility was found 0.05 mg mL−1 in [Omim][Tf2N]. The result revealed that CA solubility decreases with increase in carbon chain length of ILs, except [Bzmim][Tf2N] (0.18 mg mL−1). In the case of [Bzmim][Tf2N], the high solubility might be due to the structure similarity between this IL and caffeic acid [26]. [Emim][Tf2N] was used as a solvent for further studies of bioconversion of caffeic acid into its ester. For the maximum yield of esterification, it is essential to maintain hydrophobicity of reaction condition. Anionic domain of ILs is a major decisive factor for enzyme activity in various biocatalyzed reaction system in ILs including CAPE synthesis [23,27]. For instance, in our previous study, hydrophilic ILs containing anion such as trifluoromethanesulfonate (TfO) and tetrafluoroborate (BF4) completely dissolved high concentration of CA but lipase enzyme showed no catalytic activity. This is because the anions could interact with protein resulted in a loss of protein secondary structure and subsequent loss in activity. In contrast, ILs containing low coordinated strength anions such as Tf2N and PF6 maintained the secondary structure and microwater environment surrounding the lipase its catalytic activity [26]. In addition, lipase catalyzed CAPE production in Tf2N based ILs was higher than corresponding PF6 based ILs [23]. Furthermore, aprotic solvent such as DMSO or dimethyl formamide which can dissolve high amount of CA are not able to use for biocatalyzed synthesis of CA esters since the polar organic solvents strip the essential water from enzyme molecules and inactivate the biocatalyst [28]. Therefore, [Emim][Tf2N] was selected among the tested Tf2N based ILs for CA solubilization to perform the esterification reaction using freeze dried mycelia of A.niger EXF 4321 as catalyst.
Fig. 2. Rhodamine-B assay for detection of lipase secretion by halotolerant filamentous fungi A.niger EXF 4321.
3. Results and discussion 3.1. Lipase activity of A.niger EXF 4321 Fig. 2 shows the lipase activity of A. niger EXF 4321 as determined by Rhodamine-B assay. The orange fluorescent halos were formed around the colonies indicating that the halotolerant A. niger EXF 4321was able to produce the lipase enzyme. The lipase produced in CZ supplemented olive oil media was quantitatively evaluated by hydrolysis of p-nitrophenol hydrolysis and the results are presented in Table 1. This analysis allows distinguishing between the intra and extracellular lipase activity of A.niger EXF 4321. It is acknowledged that various lipase inducers such as soybean, olive, sunflower and cotton oils used as carbon source could affect the production of lipase in microorganism [24]. For comparison, the culture of A. niger EXF 4321 was grown in medium containing soybean as well as olive as lipase inducer. After 96 h incubation, the supernatant and mycelia were freeze dried and then subjected to lipolytic assay. The lipolytic activities of supernatant and mycelia with soy bean treatment showed approximately 0.23 and 2.05 U mg−1 respectively. The enzyme activities were about 0.14 and 3.95 U mg−1, respectively, when olive was used as an inducer (Table 1). Oleic acid and linolenic acid are the major fatty acids present in the vegetable oils. For induction of lipase production in microbial source, contribution of oleic acid is greater than linolenic acid. In case of olive oil the concentration of oleic acid is higher than soybean oil, it is to be thought that, the efficiency of lipase induction related to the concentration of oleic acid in the oil which we used [24]. Comparing to supernatant, the mycelia lipase showed higher enzyme activity and it was used as biocatalyst for further experiments.
3.3. Effect of temperature Enzyme or biocatalyst has its optimum temperature range where maximum enzyme activity and stability are achieved. In case of phenolic acid ester conversion, the temperature is also one of the key factors for the synthesis of caffeic acid phenethyl ester [11]. Fig. 4 showed the effect of temperature on the CAPE synthesis in [Emim] [Tf2N] between 25 and 50 °C using freeze dried mycelia of A.niger EXF 4321. As expected, higher reaction temperature until 30 °C resulted in higher conversion. The increase of conversion from 25 to 30 °C could be combined results of both the increase in enzyme activity and the increase of CA solubility in ILs. Furthermore, lower viscosity of [Emim] [Tf2N] at higher temperature has been shown to enhance the rate of diffusion of substrate molecules to enzyme as well as promote collisions between enzyme and substrate molecules at accelerated reaction rates [29]. The highest conversion was obtained at 30 °C. Further increase in temperature resulted in a decrease in reaction conversion due to thermal inactivation of mycelial bound enzyme at temperature higher than 30 °C. Therefore, further reactions were performed at of 30 °C.
3.2. Solubility of caffeic acid in ILs The solubility of caffeic acid in various hydrophobic ILs containing Table 1 Intra and extracellular lipase activity of A. niger EXF 4321.
3.4. Effect of substrate ratio
Lipase inducer
Sample
Enzyme activity (U mg−1)
Olive oil
Mycelia Supernatant Mycelia Supernatant
3.95 0.14 2.05 0.23
Soybean oil
The bioconversion efficiency of mycelia was evaluated at different concentration ratio of 2-phenyl alcohol to CA as shown in Fig. 5. The CA conversion increased with increasing molar ratio of 2-phenyl ethanol to CA up to 20:1. Because of the presence of two OH groups on the benzene ring of CA, it is difficult for alcohol to attack highly resonance stabilized configuration of CA if equal amounts of phenethyl alcohol 3
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Fig. 3. Solubility of caffeic acid in different ionic liquids. Solubility experiments were carried out at 30 °C for 24 h.
3.5. Effect of catalyst load
and CA (1:1) are used. Therefore, excess alcohol might attribute to thermodynamical shift of the equilibrium in favor of the synthesis of CAPE [23]. However higher the concentration of alcohol could inactivate the enzyme cascade as observed when the molar ratio of phenethyl alcohol to CA is higher than 30:1. The similar pattern was observed in Novozyme 435 when the excess of 2-phenyl ethanol was used [26]. It is possible that at higher concentration, alcohol may prevent the binding of acid molecule to the enzyme active site by means of reversible competition that leads to unavailability enzyme for binding of acid molecules [26,28]. Therefore, substrate molar ratio of 20:1 was used for further experiments. In addition, due to the limited solubility of CA in ILs (ca. 12 mM at 30oC), the possible amount of water could be produced is relative low (ca. 12 mM of water corresponding to 0.01 wt% of water content in reaction media). This amount of water is considered to have no negative effect on the enzyme catalyzed esterification reactions in ILs [30].
Fig. 6 shows the effect of catalyst (freeze dried mycelia) loaded on CAPE synthesis. The freeze dried mycelia of A.niger EXF 4321 of different weight was added to the reaction media. It was found that conversion of CA increased with increase in mass of lyophilized mycelia in reaction mixture from 5 to 20 mg. However, there was no significant increase in conversion yield with increasing mass of lyophilized mycelia from 20 to 30 mg. The correlation between catalyst amount and reaction conversion yield suggested that this system was limited by mass transfer of substrate. Moreover, when catalyst load was higher than 20 mg, the difficulty in maintain the uniform suspension of mycelia in reaction mixture might have negative effect on the reaction conversion. In addition, a decrease in specific enzyme activity was observed when catalyst amount was increased higher than 20 mg. Therefore, 20 mg catalyst loading was considered as optimal condition in this reaction.
Fig. 4. Effect of temperature on the conversion of caffeic acid. Reaction conditions: 12 mM CA, 360 mM 2-phenyl ethanol, 15 mg freeze dried mycelia, 30 °C, and 200 rpm. Conversion was determined at 24 h.
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Fig. 5. Effect of caffeic acid: 2-phenyl ethanol molar ratio on the conversion of caffeic acid. Reaction conditions: 12 mM CA, 15 mg freeze dried mycelia, 30 °C, and 200 rpm. Conversion was determined at 24 h.
Fig. 6. Effect of freeze dried mycelia load on the conversion of caffeic acid. Reaction conditions: 12 mM CA, 360 mM 2-phenyl ethanol, 30 °C, and 200 rpm. Conversion was determined at 24 h.
3.7. Comparison with previous studies on the biocatalyzed synthesis of CAPE in ionic liquids
3.6. Effect of alcohols in caffeic acid ester synthesis The effect of alcohol chain length on the synthesis of CA ester was also investigated. Aliphatic alcohols such as methanol, propanol and butanol were used in the whole-cell catalyzed synthesis of corresponding CA ester in [Emim][Tf2N]. Conversion of CA was observed to increase with increase in alkyl chain of aliphatic alcohol. In this study caffeic acid butyl ester (CABE) yield was higher, comparing to other CA esters with shorter chain alcohols (Fig. 7). Comparing to CAPE synthesis, the aliphatic ester synthesis was lower in ILs using whole cell catalyst due to negative effect of aliphatic alcohol towards the enzyme. They can strip off the structural water of enzyme, hence modifying the structure of enzyme which could lead to the loss of enzyme activity [31]. In addition, the aliphatic alcohol can bind to polar groups of amino acid residues within the active site of enzymes, prevent the acyl transfer process. As a result, the use of short chain alcohol can lower the yield of esterification reaction [32].
Table 2 shows the comparison of previous studies on the biocatalyzed synthesis of CAPE in ILs. It is worth mentioned that, all the previous studies employed purify lipase (Novozym 435, immobilized Candida antartica type B lipase) as catalyst and the reaction was carried out at relative high temperature, ranging from 70 to 84 °C. High reaction temperature would be favorable for the solubility of CA, which considering as important factors for the lipase-catalyzed synthesis of CAPE in ILs. However, the activity of lipase was significantly influenced by the high reaction temperature. For instance, only 31% enzyme activity of Novozym 435 was maintained after five cycles of uses for CAPE synthesis [28]. On the other hand, the optimal reaction temperature of whole-cell catalyzed synthesis of CAPE is much lower as compared to that of lipase catalyzed reactions. In addition, the direct use of microbial whole-cells as biocatalysts could reduce the cost of the synthesis 5
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Fig. 7. Effect of the structure of alcohol on the conversion of caffeic acid. Reaction conditions: 12 mM CA, 20 mg freeze dried mycelia, alcohol to CA molar ratio (20:1), 30 °C, and 200 rpm. Conversion was determined at 24 h.
Table 2 Comparison of biocatalyzed synthesis of CAPE in ionic liquids. Entry
Reaction conditions
Conversion (%)
Ref.
1 2. 3 4
12 mM of CA, CA:2-phenylethanol molar ratio = 1:20, 16.2 mg Novozym 435 in 0.5 mL [Emim][TF2N] at 70 °C 12 mM of CA, CA:2-phenylethanol molar ratio = 1:27.1, 17.8 mg Novozym 435 in 0.5 mL [Emim][TF2N] at 73.7 °C 65 mM of CA, CA:2-phenylethanol molar ratio = 1:16, mass ratio of CA to Novozym 435 = 1:14 in [Emim][TF2N] at 84 °C 65 mM of CA, CA:2-phenylethanol molar ratio = 1:30, mass ratio of CA to Novozym 435 = 1:18 in 1 mL [Bmim][TF2N] containing 2% DMSO at 80 °C 12 m mM, CA:2-phenylethanol molar ratio = 1:20, mass ratio of CA to catalyst = 1:20 (c.a 20 mg), in 0.5 mL [Emim][Tf2N] at 30 °C
92% at 48 h 96.6% at 60 h 98.76% at 72 h 96.23% at 24 h
[23] [29] [26] [28]
84.1% at 24 h
This study
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process, since they could avoid the tedious preparation procedures of the purified enzymes is considered as advantages of this study process. 4. Conclusion We developed a novel whole-cell mediated bioconversion process for synthesis of CAPE using [Emim][Tf2N] as a solvent. This process will be beneficial for producing CAPE and their analogue in a cost-effective manner. Using whole-cell as a biocatalyst has several advantages with ease in catalyst preparation as one of them. In terms of bioconversion, more than 84% was obtained at 30 °C within 12 h. Based upon experimental results obtained, production of CAPE and its analogues using this method can be a strong alternative to the conventional methods. For short aliphatic chain CA ester, further studies are needed for better productivity. Conflict of interest The authors declare that they have no competing interests. Acknowledgement This work was supported by an Inha University Research Grant. References [1] C. Magnani, V.L.B. Isaac, M.A. Correa, H.R.N. Salgado, Caffeic acid: a review of its potential use in medications and cosmetics, Anal Methods 6 (10) (2014) 3203–3210. [2] P. Greenwald, Clinical trials in cancer prevention: current results and perspectives for the future, J. Nutr. 134 (12) (2004) 3507S–3512S. [3] M.-C. Figueroa-Espinoza, P. Villeneuve, Phenolic acids enzymatic lipophilization, J. Agric. Food Chem. 53 (8) (2005) 2779–2787.
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[19] M. Yang, H. Wu, Y. Lian, X. Li, Y. Ren, F. Lai, G. Zhao, Using ionic liquids in wholecell biocatalysis for the nucleoside acylation, Microb. Cell Fact. 13 (2014) 143. [20] N. Gunde-Cimerman, J. Ramos, A. Plemenitaš, Halotolerant and halophilic fungi, Mycol. Res. 113 (11) (2009) 1231–1241. [21] G. Kouker, K.-E. Jaeger, Specific and sensitive plate assay for bacterial lipases, Appl. Environ. Microbiol. 53 (1) (1987) 211–213. [22] A. Rajan, D.S. Kumar, A.J. Nair, Isolation of a novel alkaline lipase producing fungus Aspergillus fumigatus MTCC 9657 from aged and crude rice bran oil and quantification by HPTLC, Int. J. Biol. Chem. 5 (11) (2011). [23] S.H. Ha, T. Van Anh, S.H. Lee, Y.-M. Koo, Effect of ionic liquids on enzymatic synthesis of caffeic acid phenethyl ester, Bioprocess Biosyst. Eng. 35 (1–2) (2012) 235–240. [24] V.M. Lima, N. Krieger, M.I.M. Sarquis, D.A. Mitchell, L.P. Ramos, J.D. Fontana, Effect of nitrogen and carbon sources on lipase production by Penicillium aurantiogriseum, Food Technol. Biotechnol. 41 (2) (2003) 105–110. [25] S.P. Ventura, L.D. Santos, J.A. Saraiva, J.A. Coutinho, Concentration effect of hydrophilic ionic liquids on the enzymatic activity of Candida antarctica lipase B, World J. Microbiol. Biotechnol. 28 (6) (2012) 2303–2310. [26] W. Jun, L. Jing, L. Zhang, G. Shuangshuang, W. Fuan, Lipase-catalyzed synthesis of caffeic acid phenethyl ester in ionic liquids: effect of specific ions and reaction
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Enzyme and Microbial Technology journal homepage: www.elsevier.com/locate/enzmictec
Aspergillus niger whole-cell catalyzed synthesis of caffeic acid phenethyl ester in ionic liquids Govindaraju Rajapriyaa, Vivek Kumar Moryaa, Ngoc Lan Maia,b, Yoon-Mo Kooa, a b
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Department of Biological Engineering, Inha University, 100 Inharo, Nam-gu Incheon 22212, Korea Faculty of Applied Sciences, Ton Duc Thang University, Ho Chi Minh City, Vietnam
A R T I C L E I N F O
A B S T R A C T
Keywords: Caffeic acid phenethyl ester (CAPE) Ionic liquid Whole-cell Aspergillus niger HalotolerantAspergillus niger halotolerant Esterification
Synthesis of caffeic acid ester essentially requires an efficient esterification process to produce various kinds of medicinally important ester derivatives. In the present study, a comprehensive and comparative analysis of whole-cell catalyzed caffeic acid esters production in ionic liquids (ILs) media was performed. Olive oil induced mycelial mass of halotolerant Aspergillus niger (A.niger) EXF 4321 was freeze dried and used as a catalyst. To ensure maximum solubilization of caffeic acid for highest substrate loading several ILs were screened and 1ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([Emim][Tf2N]) was found to have the maximum solubility and favoured for enzymatic activity of freeze dried mycelia. The whole-cell catalyzed synthesis of caffeic acid phenethyl ester (CAPE) conditions were optimized and bioconversion up to 84% was achieved at a substrate molar ratio of 1:20 (caffeic acid:2-phenyl ethanol), 30 °C for 12 h. Results obtained during this study were encouraging and helpful to design a bioreactor system to produce caffeic acid derived esters.
1. Introduction Caffeic acid (3,4-dihydroxycinnamic) (CA) and its derivatives are members of the hydroxycinnamate and phenylpropanoid metabolites, and widely distributed in plant tissues as a common source of phenolics [1]. These phenolics are widely used as folkloric medicine especially as carcinogenic inhibitor, antioxidant and antimicrobial etc. In addition to that new findings revealed their potentials in prevention of cancer and other cardiovascular diseases, which is another area of interest in this molecules [2]. Due to the low solubility of CA in hydrophobic medium, its applications in oil based foods and cosmetics are limited. In order to maintain its hydrophobicity and activity, it is essential to esterify with fatty alcohol [3]. Among these phenolics, the caffeic acid phenethyl ester (CAPE) an ester of CA is the most important active ingredient of propolis based cosmetic products. In addition to skin bioactive properties of CAPE, it has been reported for a wide range of bioactivities such as antioxidant [4], anti-inflammatory [5], anticancer activities [6,7], neuroprotective, hepatoprotective, and cardioprotective capacities [8], cytoprotective, protective against ischemia–reperfusion (I/R) injury, and protective effects [9]. Therefore, a huge and continuous demand of the CAPE and their derivatives in industries are maintained. However, isolation and purification of CAPE from natural resources is expensive, low yielding and time consuming. Naturally CAPE coexists
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with several catechol ring containing phenolic which are major hurdle for isolation [10]. A chemical synthesis also has similar challenges due to use of toxic chemicals, low yields and product purification [11]. Therefore, a greener alternative is being required to meet the industrial demand. To overcome these environmental constraints, use of biocatalyst such as lipase (either in pure or cell bound) enzyme becomes an increasingly attractive alternative to traditional chemical methods, due to the high selectivity, mild reaction conditions and environment-friendly process. In recent decade, using of whole cell as a biocatalyst was paid great attention in bio-catalyzed reaction, to reduce the overall cost of production. Whole-cell microbe mediated bio-conversion is relatively cost effective and easy to handle than the immobilized enzyme system [12]. Among them, filamentous fungi have been arisen as a most robust whole cell biocatalyst for industrial applications [13]. Ionic liquids (ILs) are molten salt at room temperature, which have a wide range of tunable physio-chemical properties [14]. Unlike organic solvents, ILs possess wide range of temperature, able to dissolve many compounds, good biological compatibility and negligible vapor pressure [15]. Over the past decades, the use of ILs in biotacalysis for organic synthesis has been extensively studied. In the presence of ILs, the biocatalysts (isolated enzyme and whole-cell) showed enhanced catalytic activity, entioselectivity and operational stability [16,17]. However, the major drawbacks of enzyme catalysts are their very
Corresponding author. E-mail address: [email protected] (Y.-M. Koo).
http://dx.doi.org/10.1016/j.enzmictec.2017.10.005 Received 15 May 2017; Received in revised form 30 August 2017; Accepted 14 October 2017 0141-0229/ © 2017 Published by Elsevier Inc.
Please cite this article as: Rajapriya, G., Enzyme and Microbial Technology (2017), http://dx.doi.org/10.1016/j.enzmictec.2017.10.005
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Fig. 1. Halotolerant A.niger EXF 4321 mycelia catalyzed esterification of caffeic acid with alcohol as a model reaction.
2.3. Lipase assay
higher costs, since they need multiple time-consuming purification processes and in some cases, removal of an enzyme from its natural cell environment may lead to partial or even complete loss of the enzyme activity [18]. Therefore, direct use of microbial whole-cells as biocatalysts instead of isolated enzymes is considered as a potential way to reduce the cost of industrial process, since they could avoid the tedious preparation procedures of the enzymes and maintain the enzyme activity by protecting the cells [19]. This study is focusing on microbe mediated greener synthesis of CAPE in ILs using a halotolerant filamentous fungus A.niger EXF 4321, isolated from solar saltern [20]. A. niger EXF 4321 was selected as catalyst due to their highly tolerance to ILs containing media in our previous screening study (data not shown). In this study, an organic solvent free green CAPE biosynthesis was performed in ILs and to meet the goal, freeze dried mycelia was used as whole cell catalyst while caffeic acid and 2 -phenyl ethanol was used as substrates (Fig. 1). This study was the first report on such kind of phenolic acid ester synthesis using microbial whole-cell catalyst and ILs system.
The rhodamine B-olive oil agar (ROA) plate assay was used for identification of lipase production from organism [21]. This primary plate assay method is used to determine the lipase secretion, based on the interaction of rhodamine B with fatty acid released from olive oil by the lipase hydrolysis. The mycelia were inoculated with the plate containing ROA medium and incubated for 48 h at 30 °C. After incubation, the plates were observed under UV light (350 nm). The hydrolysis of substrate causes the formation of orange fluorescent halos which are visible upon UV irradiation. Lipase activity was also quantified by standard protocol, in brief, the absorbance at 400 nm due to the release of p-nitrophenol during the hydrolysis of 50 mM p-nitro phenyl palmitate at pH 7.2 and temperature at 37 °C [22]. To initialize the reaction, freeze dried A.niger EXF4321 mycelia was added and absorbance was measured spectrophotometrically at 400 nm. One unit of enzyme activity is defined as the amount of enzyme that catalyzes the production of 1 nmol of p-nitrophenol per minute at 30 °C.
2. Materials and methods
2.4. Solubility of caffeic acid
2.1. Materials
The solubility experiment was performed as conventional method to quantify the maximum CA solubility in various ILs. In brief, an excess amount of CA was added to a 5 mL screw-capped vial containing ILs (1 mL) and the samples were vortexed for few seconds to ensure the solubilization of CA [23]. The suspension was stirred for 24 h at 30 °C. Then, the samples were centrifuged at 10,000 rpm for 5 min. The supernatant of ILs phase was sampled and diluted with methanol. The concentration of CA was determined by HPLC analysis.
Ionic liquids 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl) imide ([Emim][Tf2N]), 1-Butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([Bmim][Tf2N], 1-Hexyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([Hmim][Tf2N]), 1-octyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([Omim][Tf2N]), 1Benzyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([Bzmim] [Tf2N]) 1-Ethyl-2,3-dimethylimidazolium bis(trifluoromethylsulfonyl) imide ([Edmim][Tf2N]) used in this study were purchased from C-TRI (South Korea) and had residual chloride content of less than 30 ppm. All ILs were dried in an oven with vacuum pressure of 90 kPa at 80 °C for 48 h before use. 2-Phenyl ethanol, isooctane, CA, CAPE, methanol, 1-propanol and n-butanol were purchased from Sigma-Aldrich. All other chemicals were of analytical grade and used without further purification. A.niger EXF 4321 was received from EX Culture Collection (University of Ljubljana, Slovenia).
2.5. Caffeic acid esters synthesis Caffeic acid ester synthesis was carried out in 5 mL screw-capped vials containing caffeic acid and fatty alcohol in a molar ratio of 1:20 placed in magnetic reaction block at 30 °C with shaking speed of 200 rpm. Reaction was initiated by the addition of freeze dried A. niger EXF 4321 mycelia. From the reaction mixture, sample was carefully withdrawn at specified time intervals and centrifuged at 8000 rpm for 5 min to remove the mycelia from the reaction mixture. The samples were diluted with methanol and quantity of the ester synthesis was analyzed by HPLC. All reactions were duplicated and mean values were presented.
2.2. Inoculum preparation The cultures of A.niger EXF 4321 were incubated on potato dextrose agar slants at 30 °C for 5 days. The spores obtained were suspended in sterile distilled water containing 0.1% (v/v) Tween 80 for the preparation of inoculums. The spore concentration was determined by using hemocytometer. The spore suspension of 107 spores/mL inoculated in 250 mL flasks containing 100 mL of the Czapek dox (CZ) consist of K2HPO4 (1.0 g L−1), KCl (0.5 g L−1), NaNO3 (3.0 g L−1), MgSO4 (0.5 g L−1), FeSO4 (0.1 g L−1) and sucrose (30.0 g L−1) in distilled water. To induce lipase activity, 3% olive oil was added to 24 h grown culture at 30 °C with shaking speed of 200 rpm. Further cultures were continuously incubated in shaker for 96 h. After incubation, mycelia was filtered and washed thoroughly with chilled acetone and dried in freeze drier at −55 °C for 24 h and kept at 4 °C for further use.
2.6. HPLC analysis Quantitative analysis of CA and esters were performed by HPLC analytical method. Separation was accomplished using a Shimadzu HPLC system (Model LC-10A, Japan) equipped with a reverse-phase C18 column (4.6 × 250 mm, Waters, USA) with 5 μm particle size. The mobile phase consists of methanol and distilled water (70:30% v/v) with flow rate of 1 mL min−1. The absorbance was detected using UV detector at 325 nm.
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Tf2N based anions with increasing alkyl chain were screened at 30 °C as shown in Fig. 3. Hydrophobic ILs typically favored the esterification reaction than hydrophilic ILs, because the hydrophilic ILs has the tendency to strip some of essential water molecules from the enzyme environment. In order to maintain the enzyme secondary structure for its activity and to overcome the mass transfer limitations, Tf2N based ionic liquids was selected [25]. Comparing to other hydrophobic anion, Tf2N based anion has low viscosity value and coordinating-strength the highest solubility of CA was 0.38 mg mL−1 in water among the tested solvents and followed by that in [Emim][Tf2N] (0.21 mg mL−1). The least solubility was found 0.05 mg mL−1 in [Omim][Tf2N]. The result revealed that CA solubility decreases with increase in carbon chain length of ILs, except [Bzmim][Tf2N] (0.18 mg mL−1). In the case of [Bzmim][Tf2N], the high solubility might be due to the structure similarity between this IL and caffeic acid [26]. [Emim][Tf2N] was used as a solvent for further studies of bioconversion of caffeic acid into its ester. For the maximum yield of esterification, it is essential to maintain hydrophobicity of reaction condition. Anionic domain of ILs is a major decisive factor for enzyme activity in various biocatalyzed reaction system in ILs including CAPE synthesis [23,27]. For instance, in our previous study, hydrophilic ILs containing anion such as trifluoromethanesulfonate (TfO) and tetrafluoroborate (BF4) completely dissolved high concentration of CA but lipase enzyme showed no catalytic activity. This is because the anions could interact with protein resulted in a loss of protein secondary structure and subsequent loss in activity. In contrast, ILs containing low coordinated strength anions such as Tf2N and PF6 maintained the secondary structure and microwater environment surrounding the lipase its catalytic activity [26]. In addition, lipase catalyzed CAPE production in Tf2N based ILs was higher than corresponding PF6 based ILs [23]. Furthermore, aprotic solvent such as DMSO or dimethyl formamide which can dissolve high amount of CA are not able to use for biocatalyzed synthesis of CA esters since the polar organic solvents strip the essential water from enzyme molecules and inactivate the biocatalyst [28]. Therefore, [Emim][Tf2N] was selected among the tested Tf2N based ILs for CA solubilization to perform the esterification reaction using freeze dried mycelia of A.niger EXF 4321 as catalyst.
Fig. 2. Rhodamine-B assay for detection of lipase secretion by halotolerant filamentous fungi A.niger EXF 4321.
3. Results and discussion 3.1. Lipase activity of A.niger EXF 4321 Fig. 2 shows the lipase activity of A. niger EXF 4321 as determined by Rhodamine-B assay. The orange fluorescent halos were formed around the colonies indicating that the halotolerant A. niger EXF 4321was able to produce the lipase enzyme. The lipase produced in CZ supplemented olive oil media was quantitatively evaluated by hydrolysis of p-nitrophenol hydrolysis and the results are presented in Table 1. This analysis allows distinguishing between the intra and extracellular lipase activity of A.niger EXF 4321. It is acknowledged that various lipase inducers such as soybean, olive, sunflower and cotton oils used as carbon source could affect the production of lipase in microorganism [24]. For comparison, the culture of A. niger EXF 4321 was grown in medium containing soybean as well as olive as lipase inducer. After 96 h incubation, the supernatant and mycelia were freeze dried and then subjected to lipolytic assay. The lipolytic activities of supernatant and mycelia with soy bean treatment showed approximately 0.23 and 2.05 U mg−1 respectively. The enzyme activities were about 0.14 and 3.95 U mg−1, respectively, when olive was used as an inducer (Table 1). Oleic acid and linolenic acid are the major fatty acids present in the vegetable oils. For induction of lipase production in microbial source, contribution of oleic acid is greater than linolenic acid. In case of olive oil the concentration of oleic acid is higher than soybean oil, it is to be thought that, the efficiency of lipase induction related to the concentration of oleic acid in the oil which we used [24]. Comparing to supernatant, the mycelia lipase showed higher enzyme activity and it was used as biocatalyst for further experiments.
3.3. Effect of temperature Enzyme or biocatalyst has its optimum temperature range where maximum enzyme activity and stability are achieved. In case of phenolic acid ester conversion, the temperature is also one of the key factors for the synthesis of caffeic acid phenethyl ester [11]. Fig. 4 showed the effect of temperature on the CAPE synthesis in [Emim] [Tf2N] between 25 and 50 °C using freeze dried mycelia of A.niger EXF 4321. As expected, higher reaction temperature until 30 °C resulted in higher conversion. The increase of conversion from 25 to 30 °C could be combined results of both the increase in enzyme activity and the increase of CA solubility in ILs. Furthermore, lower viscosity of [Emim] [Tf2N] at higher temperature has been shown to enhance the rate of diffusion of substrate molecules to enzyme as well as promote collisions between enzyme and substrate molecules at accelerated reaction rates [29]. The highest conversion was obtained at 30 °C. Further increase in temperature resulted in a decrease in reaction conversion due to thermal inactivation of mycelial bound enzyme at temperature higher than 30 °C. Therefore, further reactions were performed at of 30 °C.
3.2. Solubility of caffeic acid in ILs The solubility of caffeic acid in various hydrophobic ILs containing Table 1 Intra and extracellular lipase activity of A. niger EXF 4321.
3.4. Effect of substrate ratio
Lipase inducer
Sample
Enzyme activity (U mg−1)
Olive oil
Mycelia Supernatant Mycelia Supernatant
3.95 0.14 2.05 0.23
Soybean oil
The bioconversion efficiency of mycelia was evaluated at different concentration ratio of 2-phenyl alcohol to CA as shown in Fig. 5. The CA conversion increased with increasing molar ratio of 2-phenyl ethanol to CA up to 20:1. Because of the presence of two OH groups on the benzene ring of CA, it is difficult for alcohol to attack highly resonance stabilized configuration of CA if equal amounts of phenethyl alcohol 3
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Fig. 3. Solubility of caffeic acid in different ionic liquids. Solubility experiments were carried out at 30 °C for 24 h.
3.5. Effect of catalyst load
and CA (1:1) are used. Therefore, excess alcohol might attribute to thermodynamical shift of the equilibrium in favor of the synthesis of CAPE [23]. However higher the concentration of alcohol could inactivate the enzyme cascade as observed when the molar ratio of phenethyl alcohol to CA is higher than 30:1. The similar pattern was observed in Novozyme 435 when the excess of 2-phenyl ethanol was used [26]. It is possible that at higher concentration, alcohol may prevent the binding of acid molecule to the enzyme active site by means of reversible competition that leads to unavailability enzyme for binding of acid molecules [26,28]. Therefore, substrate molar ratio of 20:1 was used for further experiments. In addition, due to the limited solubility of CA in ILs (ca. 12 mM at 30oC), the possible amount of water could be produced is relative low (ca. 12 mM of water corresponding to 0.01 wt% of water content in reaction media). This amount of water is considered to have no negative effect on the enzyme catalyzed esterification reactions in ILs [30].
Fig. 6 shows the effect of catalyst (freeze dried mycelia) loaded on CAPE synthesis. The freeze dried mycelia of A.niger EXF 4321 of different weight was added to the reaction media. It was found that conversion of CA increased with increase in mass of lyophilized mycelia in reaction mixture from 5 to 20 mg. However, there was no significant increase in conversion yield with increasing mass of lyophilized mycelia from 20 to 30 mg. The correlation between catalyst amount and reaction conversion yield suggested that this system was limited by mass transfer of substrate. Moreover, when catalyst load was higher than 20 mg, the difficulty in maintain the uniform suspension of mycelia in reaction mixture might have negative effect on the reaction conversion. In addition, a decrease in specific enzyme activity was observed when catalyst amount was increased higher than 20 mg. Therefore, 20 mg catalyst loading was considered as optimal condition in this reaction.
Fig. 4. Effect of temperature on the conversion of caffeic acid. Reaction conditions: 12 mM CA, 360 mM 2-phenyl ethanol, 15 mg freeze dried mycelia, 30 °C, and 200 rpm. Conversion was determined at 24 h.
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Fig. 5. Effect of caffeic acid: 2-phenyl ethanol molar ratio on the conversion of caffeic acid. Reaction conditions: 12 mM CA, 15 mg freeze dried mycelia, 30 °C, and 200 rpm. Conversion was determined at 24 h.
Fig. 6. Effect of freeze dried mycelia load on the conversion of caffeic acid. Reaction conditions: 12 mM CA, 360 mM 2-phenyl ethanol, 30 °C, and 200 rpm. Conversion was determined at 24 h.
3.7. Comparison with previous studies on the biocatalyzed synthesis of CAPE in ionic liquids
3.6. Effect of alcohols in caffeic acid ester synthesis The effect of alcohol chain length on the synthesis of CA ester was also investigated. Aliphatic alcohols such as methanol, propanol and butanol were used in the whole-cell catalyzed synthesis of corresponding CA ester in [Emim][Tf2N]. Conversion of CA was observed to increase with increase in alkyl chain of aliphatic alcohol. In this study caffeic acid butyl ester (CABE) yield was higher, comparing to other CA esters with shorter chain alcohols (Fig. 7). Comparing to CAPE synthesis, the aliphatic ester synthesis was lower in ILs using whole cell catalyst due to negative effect of aliphatic alcohol towards the enzyme. They can strip off the structural water of enzyme, hence modifying the structure of enzyme which could lead to the loss of enzyme activity [31]. In addition, the aliphatic alcohol can bind to polar groups of amino acid residues within the active site of enzymes, prevent the acyl transfer process. As a result, the use of short chain alcohol can lower the yield of esterification reaction [32].
Table 2 shows the comparison of previous studies on the biocatalyzed synthesis of CAPE in ILs. It is worth mentioned that, all the previous studies employed purify lipase (Novozym 435, immobilized Candida antartica type B lipase) as catalyst and the reaction was carried out at relative high temperature, ranging from 70 to 84 °C. High reaction temperature would be favorable for the solubility of CA, which considering as important factors for the lipase-catalyzed synthesis of CAPE in ILs. However, the activity of lipase was significantly influenced by the high reaction temperature. For instance, only 31% enzyme activity of Novozym 435 was maintained after five cycles of uses for CAPE synthesis [28]. On the other hand, the optimal reaction temperature of whole-cell catalyzed synthesis of CAPE is much lower as compared to that of lipase catalyzed reactions. In addition, the direct use of microbial whole-cells as biocatalysts could reduce the cost of the synthesis 5
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Fig. 7. Effect of the structure of alcohol on the conversion of caffeic acid. Reaction conditions: 12 mM CA, 20 mg freeze dried mycelia, alcohol to CA molar ratio (20:1), 30 °C, and 200 rpm. Conversion was determined at 24 h.
Table 2 Comparison of biocatalyzed synthesis of CAPE in ionic liquids. Entry
Reaction conditions
Conversion (%)
Ref.
1 2. 3 4
12 mM of CA, CA:2-phenylethanol molar ratio = 1:20, 16.2 mg Novozym 435 in 0.5 mL [Emim][TF2N] at 70 °C 12 mM of CA, CA:2-phenylethanol molar ratio = 1:27.1, 17.8 mg Novozym 435 in 0.5 mL [Emim][TF2N] at 73.7 °C 65 mM of CA, CA:2-phenylethanol molar ratio = 1:16, mass ratio of CA to Novozym 435 = 1:14 in [Emim][TF2N] at 84 °C 65 mM of CA, CA:2-phenylethanol molar ratio = 1:30, mass ratio of CA to Novozym 435 = 1:18 in 1 mL [Bmim][TF2N] containing 2% DMSO at 80 °C 12 m mM, CA:2-phenylethanol molar ratio = 1:20, mass ratio of CA to catalyst = 1:20 (c.a 20 mg), in 0.5 mL [Emim][Tf2N] at 30 °C
92% at 48 h 96.6% at 60 h 98.76% at 72 h 96.23% at 24 h
[23] [29] [26] [28]
84.1% at 24 h
This study
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process, since they could avoid the tedious preparation procedures of the purified enzymes is considered as advantages of this study process. 4. Conclusion We developed a novel whole-cell mediated bioconversion process for synthesis of CAPE using [Emim][Tf2N] as a solvent. This process will be beneficial for producing CAPE and their analogue in a cost-effective manner. Using whole-cell as a biocatalyst has several advantages with ease in catalyst preparation as one of them. In terms of bioconversion, more than 84% was obtained at 30 °C within 12 h. Based upon experimental results obtained, production of CAPE and its analogues using this method can be a strong alternative to the conventional methods. For short aliphatic chain CA ester, further studies are needed for better productivity. Conflict of interest The authors declare that they have no competing interests. Acknowledgement This work was supported by an Inha University Research Grant. References [1] C. Magnani, V.L.B. Isaac, M.A. Correa, H.R.N. Salgado, Caffeic acid: a review of its potential use in medications and cosmetics, Anal Methods 6 (10) (2014) 3203–3210. [2] P. Greenwald, Clinical trials in cancer prevention: current results and perspectives for the future, J. Nutr. 134 (12) (2004) 3507S–3512S. [3] M.-C. Figueroa-Espinoza, P. Villeneuve, Phenolic acids enzymatic lipophilization, J. Agric. Food Chem. 53 (8) (2005) 2779–2787.
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