- Email: [email protected]
Immobilization of Y. lipolytica lipase and the continuous synthesis of geranyl propionate
Accepted Manuscript Title: Immobilization of Y. lipolytica lipase and the continuous synthesis of geranyl propionate Authors: Jing Tang, Gang Chen, Lu Wang, Ming Miao, Bo Jiang, Biao Feng PII: DOI: Reference:
S1381-1177(17)30019-X http://dx.doi.org/doi:10.1016/j.molcatb.2017.01.019 MOLCAB 3518
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date: Revised date: Accepted date:
30-11-2016 25-1-2017 25-1-2017
Please cite this article as: Jing Tang, Gang Chen, Lu Wang, Ming Miao, Bo Jiang, Biao Feng, Immobilization of Y.lipolytica lipase and the continuous synthesis of geranyl propionate, Journal of Molecular Catalysis B: Enzymatic http://dx.doi.org/10.1016/j.molcatb.2017.01.019 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Immobilization of Y. lipolytica lipase and the continuous synthesis of geranyl propionate Jing Tanga,1 , Gang Chena,1 , Lu Wanga, Ming Miaob , Bo Jiangb, Biao Fenga,b,* a
School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122,
P.R. China b
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi,
Jiangsu 214122, P.R. China
Corresponding Author *Dr. Biao Feng, School of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu214122, P. R. China Email: [email protected]; [email protected] Tel phone: +86 13921500055 Fax number: +86 510 85910799
Graphical abstract
Highlights:
Lip2 was successfully immobilized in organic solvent.
Continuous esterification was realized in a specially designed experimental CSTR.
The reaction was preliminary optimized to obtain a stable esterification ratio.
Abstract: In this study, Y. lipolytica lipase LIP2 (Lip2) was immobilized on the macroporous adsorptive resin DA201-C in n-heptane and then used to catalyze the continuous synthesis of geranyl propionate in a continuous stirred tank reactor (CSTR). With the lipase loading of 0.01 g/g resin, 25oC and adsorption for 3 h, the immobilization efficiency can be up to 98.6% and the synthetic activity of the lipase was 690.8 U/g after immobilization, representing an increase of 28.5% compared with the free lipase (synthetic activity 537.2 U/g). The immobilized Lip2 was used to generate geranyl propionate in CSTR, the continuous production of geranyl propionate was performed with 3 g of immobilized Lip2 at 35oC. At the flow rate of 6 mL/h the process can maintain its steady state at least for 12 h, which was about three times of the average residence time, and the esterification ratio of 72.8% could be attained. Finally, FT-IR and NMR were used to identify the ester. Keywords: Lip2; immobilization; geranyl propionate; CSTR.
1. Introduction Lipases (E.C.3.1.1.3) are widely used in the hydrolysis of triglycerides, the synthesis of ester in organic mediums or the resolution of racemic mixtures[1]. However, the use of free lipases will encounter some restrictions such as easy inactivation, poor operational stability, and the fact that they are not reusable[2]. The immobilization of enzymes is commonly used to improve the thermal and chemical stability, as well as selectivity and specificity of enzymes[3]. At the same time, by immobilizing the enzyme, it is possible to operate enzymatic processes continuously[4]. The terpene ester of short-chain fatty acids, such as the acetate, propionate and butyrate of geraniol and citronella, are main components of essential oils which are important flavors and fragrance compounds in food, cosmetics and pharmaceutical applications[5]. The direct extraction of those esters from plant materials involves rather expensive and low yield processes[6]. Up to now, the chemical synthesis is the main issue for the production of these esters[7]. Compared to the chemical synthesis, the enzymatic synthesis using immobilized lipase has some advantages such as the direct use of unmodified substrates, relatively short reaction time[5] for reaching a high conversion rate, moderate reaction conditions, less by-product generation, and high region specificity of the enzymes. Hence the enzymatic production of flavors and fragrances using natural raw materials
may be useful for scientific and industrial settings, considering the ever arising demands for such products[8]. The difficulty for the enzymatic synthesis of short-chain fatty acid ester lies in strong polarity of these acids which decreases the activity of lipase. To date, the enzymatic synthesis of geraniol ester with short-chain was conducted by a number of batch reactions, although a continuous process would be preferred for the large-scale production[9] due to its higher productivity than batch process[10]. Also, few researches have concerned the continuous synthesis of geranyl propionate using immobilized lipases. Y. lipolytica lipase LIP2 (Lip2) is an important lipase as it has shown high transesterification and esterification activities, which enable it to catalyze ester synthesis, biodiesel production, and enantiomer resolution[11]. Macroporous resins are good carriers for lipase immobilization[12]. In this study, the crude Lip2 was firstly immobilized on macroporous adsorptive resin DA201-C in n-heptane, then the continuous synthesis of geranyl propionate was carried out in a continuous stirred tank reactor (CSTR). Factors influencing the immobilization and esterification ratio were examined to optimize the process. 2. Materials and methods 2.1 Materials The crude, unpurified lipase Lip2 was a generous gift from Beijing Key Laboratory of Bioprocess (Beijing, China). Geraniol (98% purity) and 1-hexanol (chromatographic grade) were purchased from Sigma. Other chemical reagents such
as n-heptane, isooctane, ethanol, octoic acid and propionic acid were purchased from Sinopharm (Shanghai, China) and of analytical grade. Macroporous adsorptive resin DA201-C was purchased from Hua Yi Technology (Zhengzhou, China). 2.2 Immobilization of Lip2 The resin was firstly washed with distilled water, then soaked in pure ethanol for 12 h, and finally washed again with distilled water. After the pretreatment, the resin was dried under vacuum for 2 h at room temperature. For the immobilization, 1.0 g of dry resin was soaked in 10 mL n-heptane for 3 h in a 50 mL bottle, and then 0.1 g crude lipase Lip2 were added and the immobilization was conducted at 25oC with the shaking speed of 200 r/min. After the immobilization, the immobilized Lip2 was separated by filtration and washed with n-heptane under vacuum, then dried at room temperature for 1 h and kept at 4oC for use. 2.3 Determination of protein content and immobilization efficiency The protein concentration was determined by the method of Bradford with BSA standards. The immobilization efficiency was calculated by DI = (Pe-Pw)/Pe, where Pe is the total amount of protein used and Pw is that of protein in the washing liquid[13]. 2.4 Assay of lipase synthetic activity The activity of lipase Lip2 in esterification reaction was measured in n-heptane according to the procedure described by Wang, Xu et al [14]with small modifications. Briefly, 1 mL substrate solution (1.2 mol/L octanoic acid and 1.2 mol/L ethanol in n-heptane with a volume of 0.5 mL respectively) was added in a 5 mL capped tube. The reaction was started by adding 10 mg free lipase ( or 110 mg immobilized lipase)
and was conducted for 30 min at 35oC with the shaking speed of 200 r/min. Samples of 100 L were drawn and mixed with 100 L of n-hexane, as internal standard, then added 0.8 mL n-heptane and analyzed by gas chromatograph. One unit of lipase synthetic activity was defined as the amount of enzyme required to produce 1 mol of ester per minute under above conditions. 2.5 Purification of geranyl propionate by HPLC The reaction mixtures resulting from lipase-catalyzed esterification were filtered under vacuum to remove molecular sieves and lipase, the filtrate was separated by HPLC with a waters X Bridge C18 column (4.6×150 mm, 5 m) at 30oCand eluted with acetonitrile/water (4:1, v/v) at a rate of 2.0 mL/min. The waters 996 PDA detector was used and the wavelength was 220 nm. The fractions containing the desired products were collected. After removing the acetonitrile and water by reduced pressure distillation, the desired product was obtained finally. 2.6 Identification of purified geranyl propionate The purified geranyl propionate was identified by Fourier transform infrared spectroscopy (FT-IR) and Nuclear Magnetic Resonance (NMR). The FT-IR measurements were performed on FT-IR spectrophotometer (Nexus 470, USA), a region from 400-4000 cm-1 was used for scanning at 4 cm-1 resolution by 32 scans. The NMR analysis was acquired on Bruker NMR spectrometer (Avance Ⅲ, Switzerland). The spectra was obtained at 400 MHz using CDCl3 as the solvent and tetramethyl silane (TMS) as internal standard. 2.7 Quantitative analysis of the product
The product was analyzed by GC-FID analysis with a DB-WAX capillary column (30 m×0.25 mm). The temperatures of injector and detector were set at 250oC and 260oC, respectively. Nitrogen was used as the carrier gas at a flow rate of 1.15 mL/min. The procedure was as follows: 90oC remaining for 1min, rising to 230oC at a rate of 10oC/min and remaining for 8 min. Retention times at these conditions were 12.9 and 13.2 min for geranyl propionate and geraniol respectively, using 1-hexanol as internal standard. The esterification ratio of geranyl propionate was defined as the ratio between the real content and the theoretical content of geranyl propionate. All results were repeated for three times. 2.8 Continuous synthesis of geranyl propionate The continuous synthesis of geranyl propionate was conducted in a CSTR with an effective volume of 25 mL. The reactor, especially designed for the experiment, was equipped with two tubes at the ground glass plug for the insert and outlet liquids. The immobilized enzymes were filled in a small pocket of filter cloth, which was put at two small lags located at the lower part of the reactor. The enzyme could simply be put at the bottom of the reactor if the outlet was wrapped with filter cloth. The temperature of the reactor was kept constant by a jacket connected to a water bath (Fig. 1). The substrate solution was prepared by dissolving geraniol and propionic acid in isooctane with fixed molar ratio. At the beginning of the experiment, the reactor was filled with enzyme and substrate solution and the batch reaction was conducted until
an ideal esterification ratio was reached. Then the continuous operation was started by the feeding of substrate solution via peristaltic pump with fixed flow rate. The duration of continuous synthesis was about three times of the average residence time. Samples of 0.1 mL were withdrawn hourly and were immediately mixed with 0.1 mL internal standard mixture and 0.8 mL isooctane and analyzed by GC-FID. 3. Results and discussions 3.1 Immobilization of Lip2 3.1.1 Choice of solvent In our preliminary experiment, the immobilization of Lip2 onto ion exchange resin in aqueous solution did not give satisfactory results, the immobilization efficiency of the Lip2 was only 56.01%. Considering that the esterification would take place in organic solvents, the Lip2 was immobilized inorganic solvents. It is generally acknowledged that lipases are interfacial active enzymes with lipophilic domains and can adopt both open and close conformations. The lipophilic interactions between the hydrophobic parts of the lipase and substrates or organic solvents could fix the entrapped enzyme in a more active conformation and enhanced activity[15]. In a medium of low polarity, the lipophilic domains of lipase may interact with solvent. As a result, the effect may help to induce conformation changes of lipase to an active form [13]. Three solvents of different hydrophobicity, expressed by the values of logP were tested and the results are shown in Table 1. The highest immobilization efficiency of nearly 98.6% was obtained with n-heptane and the
synthetic activity of the lipase was 690.8 U/g after immobilization, which represented an increase of 28.5% compared with the free lipase (synthetic activity 537.2 U/g). 3.1.2 Determination of enzyme loading To evaluate the loading capacity of carrier, 1.0 g of dry resins were loaded with 10 mL n-heptane solution with different initial lipase concentrations (5, 7.5, 10, 12.5, 15 mg/mL respectively). The synthetic activity increased with the initial Lip2 amount from 5 to 10 mg/mL and reached the maximum synthetic activity of 690.8 U/g after immobilization, but the activity began to decrease with the lipase concentration from 10 to 15 mg/mL. So the concentration of 10 mg/mL was chosen as the enzyme loading for the immobilization of Lip2. 3.2 Thermal properties of immobilized Lip2 It is well known that temperature can influence the activity and stability of enzyme. A rise of temperature will increase the reaction rate. This rise can be explained by the transition state theory[16]. An increase in temperature can enhance mutual miscibility and improve the diffusion process of substrates, thus reducing mass transfer limitations and favoring interactions between enzyme particles and substrates[8]. On the other hand, at high temperature, the tertiary structure of enzyme may be disrupted, causing the denaturation [17]. Although the optimal temperature of the enzyme has been given by the supplier, we did not know whether it was modified by the immobilization. In general, the immobilization would increase the stability of enzyme and rise the optimal temperature. Relative activity was the ratio between the activity of every sample and the maximum activity of sample. Fig. 2 illustrates the
effect of temperature on the activity of immobilized Lip2, it can be seen that the optimal temperature of immobilized Lip2 is 35oC, the same as the free enzyme. Visibly the immobilization did not modify the optimal temperature of Lip2. To explore the thermal stability of immobilized Lip2, both free and immobilized enzyme were incubated in the absence of substrates from a temperature range of 40 to 80oC for 2 h. The ratios of synthetic activities before and after incubation were named as residual activity and are shown in Fig. 3. The activity of immobilized Lip2 decreased more slowly than that of free lipase. After incubation for 2 h at 80oC, the residual activity of immobilized Lip2 remained 62.9% of the original value, while that of the free lipase was only 15.6% of the original. The remarkable improvement of the thermal stability by immobilization may arise from the conformation integrity of the immobilized enzyme structure after adsorption to the carriers[18], which may prevent extensive structure changes caused by thermal denaturation[19]. 3.3 Choice of solvent for the synthesis of geranyl propionate Generally speaking, the hydrophobicity of organic solvent, evaluated by the value of logP, is a key factor for esterification due to that it could affect the activity and stability of lipases and the solubility of solvents[20]. Highly hydrophobic solvents with logP > 4 are often considered as the most suitable solvent for biocatalysis with lipases, the solvents with logP values between 2 and 4 are moderately effective, and those polar solvents with logP < 2 would be ineffective [21]. But in practice the most suitable solvent for a given reaction should be determined by experiments. For the synthesis of geranyl propionate, three solvents with different logP values were
compared to choose the best one: n-hexane (logP = 3.5), n-heptane (logP = 4.0), and isooctane (logP = 4.5). The experiments were carried out in 10 mL anhydrous solvents containing enzyme. 15 mg/mL of molecular sieves 4Å were added to remove the water generated. The reaction mixture was shaken at 200 r/min in an incubator. The results are shown in Table 2. The Table 2 shows that after 3.5 h, the esterification ratio reached over 80% with all three solvents, the maximum esterification ratio of 87.5% was obtained with isooctane which could be considered as the ideal solvents for the synthesis of geranyl propionate with Lip2. This results seem to be in accord with the general opinion mentioned above. S. Y. Huang and H. L. Chang[22] found the similar trend when using surfactant-coated lipase for the esterification of geraniol and acetic in organic solvents, n-hexane and isooctane produced the highest yield of 82.2% and 83.7%, respectively. The reason may be that the structure and function of enzyme are determined to some extent by the bound water around the enzyme. The enzyme requires water layer to maintain its activity and hydrophobic solvents can preserve the catalytic activity without stripping the water from the enzyme [21]. A high value of logP could be conducive to maintain the biocatalyst at active state. The most suitable solvent for a given esterification reaction should be determined by the interaction among substrates, enzyme and solvent. Finally, isooctane was chosen for the following experiments. 3.4 Continuous synthesis of geranyl propionate From the batch experiments it has been confirmed that Lip2 could effectively
catalyze the synthesis of geranyl propionate. From Table 2, it can also be seen that the reaction time of 3.5 h was enough for the batch operation. In consideration the residence time distribution in the continuous reactor, an average residence time between 4 or 4.5 h should be enough for the continuous synthesis. During the continuous reaction, samples were withdrawn hourly to measure the esterification ratio. 3.4.1 Effect of enzyme loading The enzyme loading in a given reactor is a crucial economical factor for continuous operation[23]. The reactor used in this work had an effective volume of 25 mL. Fig.4 shows evolutions of esterification ratio measured at the exit of reactor with three different enzyme loading between 2 g and 4 g. Generally, the esterification ratio would increase with the enzyme loading, but after a certain extent the reaction will reach the saturation level and no obvious increment could be observed [24]. From Fig.4, it could be seen that the esterification ratio of geranyl propionate increased with the enzyme loading from 2 g to 4 g, and the difference between the loading of 3 g and 4 g was minor. Thus the loading of 3 g was chosen for following experiments. This enzyme loading seems to be voluminous with respect to the volume of the reactor. The final esterification ratio of geranyl propionate was lower compared to that obtained in batch experiment. It should be improved by strengthening the agitation when the process was scale-up to the industrial level. 3.4.2 Effect of average residence time For a CSTR with given volume, the feed flow rate is conversely proportional to
the average residence time, which is a key parameter for the continuous operation. Fig.5 presents the evolutions of esterification ratio during the continuous synthesis of geranyl propionate at different flow rates of feed. The start-up of continuous operation, corresponding to the beginning of curves, was realized after the stable esterification ratio with batch operation. The flow rate of 4.5, 6 and 9 mL/h corresponded to the average residence time of 5.6, 4.2 and 2.8 h, respectively. The highest esterification ratio of 75.8% was obtained with a flow rate of 4.5 mL/h. It was lower than that obtained in batch experiment. The residence time distribution in CSTR seemed to be the main reason for this difference. The flow rate of 6 mL/h obtained a stable esterification ratio of 72.8%, a little lower than that of 4.5 mL/h, but not far from it. In fact, with this flow rate, the average residence time was already superior to 4 h, which was long enough according to the batch experiment. On the other hand, with the flow rate of 9 mL/h, the percentage of esterification was dramatically low due to the short residence time for the reaction[25]. In fact, at this flow rate the average residence time was inferior to 3 h which was not long enough according to batch experiments (cf. Tab. 2). 3.4.3 Effect of the substrate molar ratio The relative proportions of the various substrates in a reaction mixture is a factor that defines the physical and chemical properties of a reaction system and subsequently influence the conversion [26]. So, through proper selection of substrate molar ratio, the equilibrium of an enzymatic reaction can be manipulated to favor a high conversion [27]. But a molar ratio far from the stoichiometric molar ratio will
raise the risk of substrate inhibition and increase the cost of separation. In our preliminary batch experiments, the acid/alcohol molar ratio from 2:1 to 1:3 were assayed to determine the optimal value. It was found that the esterification ratio had an increasing trend with the acid/alcohol molar ratio varying from 2:1 to 1:1.5, and begun to decrease slowly with the molar ratio from 1:1.5 to 1:3. This may be because that short chain acids such as acetate and propionic acid usually have strong hydrophilicity and small molecular size and act as an inhibitor of lipase[28]. Because of the strong polarity of short chain acids, on the one hand, the acids may be strongly adsorbed on the enzyme active site[29], resulting an excessive quantity of substrate. On the other hand, the acids could deprive the essential water around the enzyme, resulting the loss of lipase activity in organic solvents. Paul A. Claon and Casimir C. Akoh[30]found that an increase in acetic acid concentration inhibited the activities of Candida antarctica lipase and that the conversion rate of geranyl acetate decreased significantly. So, in our continuous work, the acid/alcohol molar ratios from 1:1.5 to 1:2 were chosen to further optimize the process. The solubility of geraniol and propionic acid in isooctane is good enough to form a homogenous substrate mixture. Evolutions of esterification ratio at various molar ratios are shown in Fig.6. At the molar ratio of 1:1, the esterification ratio of geranyl propionate was the lowest at the beginning and decreased to only 53.6% after 12 h of operation. The low esterification ratio at equal proportion of substrates can be attributed to the inhibition of the Lip2 activity due to the strong acidity in the reaction media [31]. The increase of alcohol content improved the conversion. But at the molar
ratio of 1:2 the esterification ratio began to decrease, this might be attributed to the substrate inhibition. N.A. Serri [21]found the similar result when using Candida rugosa lipase to catalyze the synthesis of citronelly laurate, when the acid/alcohol molar ratio was 1:2, the conversion rate of citronelly laurate was lower than that of 1:1.5. This phenomenon was considered as familiar in bimolecular reaction catalyzed by enzyme with ping pong bi bi mechanism. 3.5 Identification and analysis of geranyl propionate 3.5.1 Purification by HPLC The reaction mixture was successively separated by HPLC. Fig.7 shows its spectra. The peak at 11.21minis that of geranyl propionate. 3.5.2 Identification by FT-IR and NMR After the separation, the isolated geranyl propionate was analyzed by FT-IR and NMR. FT-IR is principally used as a qualitative technique to identify the presence of functional groups in a particular compound[26]. The results are shown in Fig. 8 (a): the signals at 2967.43 and 2925.48, 2855.61 cm-1were the stretch vibration of CH3 or CH2, and the bending vibration at 1450.78 and 1378.9 cm-1 was an additional demonstration of CH3 or CH2, there was a strong absorption at 1738.51 cm-1, which was a symbol of C=O, the signal at 1180.56 cm-1 was the stretch vibration of C-O. The above results showed the appearance of ester bond in the new product, indicating possible formation of geranyl propionate. To further characterize the structure of geranyl propionate, the 1H NMR spectrum was used to check the geranyl propionate, the spectrum showed signals in the region
of 1.25-5.5 ppm for all hydrogen atoms. According to the 1H NMR shown in Fig. 8 (b), the results were as follows:1H NMR (400 MHz, CDCl3): δ (ppm):1.25-1.63(3H, s),1.603 (3H, s), 1.683-1.705 (6H, s),2.028-2.134 (4H, m), 2.308-2.365 (2H, m), 4.591-4.609 (2H, m),5.603-5.600 (1H, m), 5.323-5.344 (1H, m). These statistics confirmed that the product was geranyl propionate. 4. Conclusion In laboratory assays, Lip2 showed a high catalytic activity for the synthesis of geranyl propionate with an esterification ratio of 87.5%. There is a good potential for industries application which could overcome the shortcomings of chemical synthesis. For the practical application, Lip2 was immobilized on adsorptive resin DA201-C. Under the optimal conditions (lipase loading 0.01 g/g resin, 25oC, n-heptane as the medium, adsorption for 3 h), the immobilization efficiency reached 98.6%, and the synthetic activity represented an increase of 28.5% compared to that of the free lipase. The immobilization did not change the optimal temperature of Lip2. The immobilized Lip2 was used to catalyze the synthesis of geranyl propionate in CSTR. The duration of continuous assay was long enough to achieve the steady state and the stable esterification ratio of 72.8% was achieved, which was inferior to that obtained by batch operation, the reason for this different could be attributed to the residence time distribution. The residence time of 4 h would not be considered as too long for industrial realization. The process could be further improved by strengthening the agitation in industrial scale. Finally, the product was confirmed by FT-IR and NMR. Thus the continuous assays have confirmed the feasibility of geranyl
propionate synthesis by immobilized Lip2 at a larger scale. Acknowledgement This study was financially supported by the Specialized Research Fund for the Doctor Program of Higher Education (Zo.20130093110010).
References [1] X. Yang, G. Chen, H. Du, M. Miao, B. Feng, J. Mol. Catal. B: Enzym. 127 (2016) 34-39. [2] Ö. Kebabci, Cihangir, N., Hacettepe J. Biol. & Chem 39(3) (2011) 283-288. [3] S.S. Kanwar, S. Gehlot, M.L. Verma, R. Gupta, Y. Kumar, G.S. Chauhan, J. Appl. Polym. Sci. 110(5) (2008) 2681-2692. [4] W. Kaewthong, S. Sirisansaneeyakul, P. Prasertsan, A. H-Kittikun, Process Biochem. 40(5) (2005) 1525-1530. [5] S.M. Radzi, W. Mustafa, S. Othman, H. Noor, World Acad. Sci. Eng. Technol. 59 (2011) 677-680. [6] M. Liaquat, R.K.O. Apenten, J. Food Sci. 65(2) (2000) 295-299. [7] N.R. Mohamad, N.A. Buang, N.A. Mahat, Y.Y. Lok, F. Huyop, H.Y. Aboul-Enein, R. Abdul Wahab, Enzyme Microb. Technol. 72 (2015) 49-55. [8] N. Paroul, L.P. Grzegozeski, V. Chiaradia, H. Treichel, R.L. Cansian, J.V. Oliveira, D. de Oliveira, J. Chem. Technol. Biotechnol. 85(12) (2010) 1636-1641. [9] Y. Watanabe, K. Kuwabara, S. Adachi, K. Nakanishi, R. Matsuno, J Agric Food Chem 51(16) (2003) 4628-4632. [10] E. Séverac, O. Galy, F. Turon, P. Monsan, A. Marty, Bioresour Technol 102(8) (2011) 4954-4961. [11] P. Fickers, A. Marty, J.M. Nicaud, Biotechnol Adv 29(6) (2011) 632-44. [12] Y. Yan, X. Zhang, D. Chen, Bioresour Technol 131 (2013) 179-87. [13] Y. Gao, T. Tan, K. Nie, F. Wang, Chin. J. Biotechnol. 22(1) (2006) 114-118. [14] D. Wang, Y. Xu, Y. Teng, Bioprocess Biosyst Eng 30(3) (2007) 147-55. [15] S.P. Bastida A, .Armisen P, Biotechnol Bioeng 58(5) (1998) 486-493. [16] M.B.A. Rahman, N. Chaibakhsh, M. Basri, R.N.Z.R.A. Rahman, A.B. Salleh, S.M. Radzi, J. Chem. Technol. Biotechnol. 83(11) (2008) 1534-1540. [17] M.D. Romero, L. Calvo, C. Alba, A. Daneshfar, H.S. Ghaziaskar, Enzyme Microb. Technol. 37(1) (2005) 42-48. [18] M.-M. Zheng, L. Dong, Y. Lu, P.-M. Guo, Q.-C. Deng, W.-L. Li, Y.-Q. Feng, F.-H. Huang, J. Mol. Catal. B: Enzym. 74(1) (2012) 16-23. [19] M.M. Zheng, Y. Lu, F.H. Huang, L. Wang, P.M. Guo, Y.Q. Feng, Q.C. Deng, J Agric Food Chem 61(1) (2013) 231-7. [20] C.J. Wensen He, Yuan Ma, Yebo Yang, Xiaoming Zhang, Biao Feng, Lin Yue, J. Mol. Catal. B: Enzym. 67 (2010) 60-65. [21] N.A. Serri, A.H. Kamaruddin, W.S. Long, Bioprocess Biosyst Eng 29(4) (2006) 253-60. [22] S. Huang, H. Chang, M. Goto, Enzyme Microb. Technol. 22(7) (1998) 552-557. [23] K.C. Badgujar, B.M. Bhanage, Process Biochem. 49(8) (2014) 1304-1313. [24] S. Chattopadhyay, R. Sen, Bioresour Technol 147 (2013) 395-400. [25] J.H. Lee, S.B. Kim, C. Park, B. Tae, S.O. Han, S.W. Kim, Appl. Biochem. Biotechnol. 161(1-8) (2010) 365-371.
[26] N. Mohamad, N.A. Buang, N.A. Mahat, J. Jamalis, F. Huyop, H.Y. Aboul-Enein, R.A. Wahab, J. Ind. Eng. Chem. 32 (2015) 99-108. [27] F.E.a.G.A. M. Trani, JAOCS 68(1) (1991). [28] S.Y. Huang, H.L. Chang, J. Chem. Technol. Biotechnol. 74(2) (1999) 183-187. [29] G.D. Yadav, P.S. Lathi, J. Mol. Catal. B: Enzym. 27(2-3) (2004) 113-119. [30] P.A. Claon, C.C. Akoh, J. Am. Oil Chem. Soc. 71(6) (1994) 575-578. [31] P. Pires-Cabral, M.M.R. da Fonseca, S. Ferreira-Dias, Biochem. Eng. J. 43(3) (2009) 327-332.
Fig.1. Schematic diagram of the CSTR for geranyl propionic A: feed reservoir, B: effluent reservoir, C: magnetic stirrer, D: reactor, E: peristaltic pump, F: equipment for immobilized Lip2, H: online checking equipment
Relative activity (%)
100
80
60
40
20 20
30
40
Temperature (oC) Fig. 2. Optimal temperature of immobilized Lip2
50
60
free enzyme
immobilized enzyme
Residual activity (%)
100 80 60 40 20 0 40
50
60 70 Temperature (oC)
80
90
Fig. 3. Thermal stability of free and immobilized Lip2
2g
3g
4g
Esterification ratio (%)
100
80
60
40 0
2
4
6 Time (h)
8
10
12
Fig. 4. Effect of enzyme loading on esterification ratio of geranyl propionate (conditions: initial concentration of acid and geraniol 20 mmol/L and 30 mmol/L respectively, 35oC, isooctane as the solvent with a flow rate of 0.10 mL/min)
6mL/h
9mL/h
4.5mL/h
Esterification ratio (%)
100 80 60 40 20 0
2
4
6 Time (h)
8
10
12
Fig. 5. Effect of flow rate on esterification ratio of geranyl propionate (conditions: initial concentration of acid and geraniol 20 mmol/L and 30 mmol/L respectively, immobilized enzyme loading 3g, 35oC)
1:1
1:1.5
1:2
Esterification ratio
100 80 60 40 20 0
2
4
6 Time (h)
8
10
12
Fig. 6. Effect of substrate molar ratio on esterification ratio of geranyl propionate (conditions: immobilized enzyme loading 3 g with a flow rate of 0.10 mL/min, 35oC)
Fig. 7. HPLC spectra of the reaction mixture
(a)
(b)
Fig. 8. FT-IR and NMR spectra of geranyl propionate
Table 1 Immobilization of Lip2 in three organic solvents Medium
immobilization
synthetic activity
efficiency (%)
(U/g enzyme)
logP
n-hexane
3.5
96.3
597.5
n-heptane
4.0
98.6
690.8
isooctane
4.5
97.3
561.3
Table 2 Effect of solvents on the synthesis of geranyl propionate Esterification ratio (%) Time (h) n-hexane
n-heptane
isooctane
1
43.3
37.3
45.0
2
69.5
60.5
73.7
2.5
74.1
68.7
79.3
3
82.4
78.9
85.9
3.5
84.3
81.3
87.5
Reaction conditions: initial concentration of acid and geraniol were 20 mmol/L and 30 mmol/L respectively, 35oC, dose of free Lip2 7 mg/mL)
S1381-1177(17)30019-X http://dx.doi.org/doi:10.1016/j.molcatb.2017.01.019 MOLCAB 3518
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date: Revised date: Accepted date:
30-11-2016 25-1-2017 25-1-2017
Please cite this article as: Jing Tang, Gang Chen, Lu Wang, Ming Miao, Bo Jiang, Biao Feng, Immobilization of Y.lipolytica lipase and the continuous synthesis of geranyl propionate, Journal of Molecular Catalysis B: Enzymatic http://dx.doi.org/10.1016/j.molcatb.2017.01.019 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Immobilization of Y. lipolytica lipase and the continuous synthesis of geranyl propionate Jing Tanga,1 , Gang Chena,1 , Lu Wanga, Ming Miaob , Bo Jiangb, Biao Fenga,b,* a
School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122,
P.R. China b
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi,
Jiangsu 214122, P.R. China
Corresponding Author *Dr. Biao Feng, School of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu214122, P. R. China Email: [email protected]; [email protected] Tel phone: +86 13921500055 Fax number: +86 510 85910799
Graphical abstract
Highlights:
Lip2 was successfully immobilized in organic solvent.
Continuous esterification was realized in a specially designed experimental CSTR.
The reaction was preliminary optimized to obtain a stable esterification ratio.
Abstract: In this study, Y. lipolytica lipase LIP2 (Lip2) was immobilized on the macroporous adsorptive resin DA201-C in n-heptane and then used to catalyze the continuous synthesis of geranyl propionate in a continuous stirred tank reactor (CSTR). With the lipase loading of 0.01 g/g resin, 25oC and adsorption for 3 h, the immobilization efficiency can be up to 98.6% and the synthetic activity of the lipase was 690.8 U/g after immobilization, representing an increase of 28.5% compared with the free lipase (synthetic activity 537.2 U/g). The immobilized Lip2 was used to generate geranyl propionate in CSTR, the continuous production of geranyl propionate was performed with 3 g of immobilized Lip2 at 35oC. At the flow rate of 6 mL/h the process can maintain its steady state at least for 12 h, which was about three times of the average residence time, and the esterification ratio of 72.8% could be attained. Finally, FT-IR and NMR were used to identify the ester. Keywords: Lip2; immobilization; geranyl propionate; CSTR.
1. Introduction Lipases (E.C.3.1.1.3) are widely used in the hydrolysis of triglycerides, the synthesis of ester in organic mediums or the resolution of racemic mixtures[1]. However, the use of free lipases will encounter some restrictions such as easy inactivation, poor operational stability, and the fact that they are not reusable[2]. The immobilization of enzymes is commonly used to improve the thermal and chemical stability, as well as selectivity and specificity of enzymes[3]. At the same time, by immobilizing the enzyme, it is possible to operate enzymatic processes continuously[4]. The terpene ester of short-chain fatty acids, such as the acetate, propionate and butyrate of geraniol and citronella, are main components of essential oils which are important flavors and fragrance compounds in food, cosmetics and pharmaceutical applications[5]. The direct extraction of those esters from plant materials involves rather expensive and low yield processes[6]. Up to now, the chemical synthesis is the main issue for the production of these esters[7]. Compared to the chemical synthesis, the enzymatic synthesis using immobilized lipase has some advantages such as the direct use of unmodified substrates, relatively short reaction time[5] for reaching a high conversion rate, moderate reaction conditions, less by-product generation, and high region specificity of the enzymes. Hence the enzymatic production of flavors and fragrances using natural raw materials
may be useful for scientific and industrial settings, considering the ever arising demands for such products[8]. The difficulty for the enzymatic synthesis of short-chain fatty acid ester lies in strong polarity of these acids which decreases the activity of lipase. To date, the enzymatic synthesis of geraniol ester with short-chain was conducted by a number of batch reactions, although a continuous process would be preferred for the large-scale production[9] due to its higher productivity than batch process[10]. Also, few researches have concerned the continuous synthesis of geranyl propionate using immobilized lipases. Y. lipolytica lipase LIP2 (Lip2) is an important lipase as it has shown high transesterification and esterification activities, which enable it to catalyze ester synthesis, biodiesel production, and enantiomer resolution[11]. Macroporous resins are good carriers for lipase immobilization[12]. In this study, the crude Lip2 was firstly immobilized on macroporous adsorptive resin DA201-C in n-heptane, then the continuous synthesis of geranyl propionate was carried out in a continuous stirred tank reactor (CSTR). Factors influencing the immobilization and esterification ratio were examined to optimize the process. 2. Materials and methods 2.1 Materials The crude, unpurified lipase Lip2 was a generous gift from Beijing Key Laboratory of Bioprocess (Beijing, China). Geraniol (98% purity) and 1-hexanol (chromatographic grade) were purchased from Sigma. Other chemical reagents such
as n-heptane, isooctane, ethanol, octoic acid and propionic acid were purchased from Sinopharm (Shanghai, China) and of analytical grade. Macroporous adsorptive resin DA201-C was purchased from Hua Yi Technology (Zhengzhou, China). 2.2 Immobilization of Lip2 The resin was firstly washed with distilled water, then soaked in pure ethanol for 12 h, and finally washed again with distilled water. After the pretreatment, the resin was dried under vacuum for 2 h at room temperature. For the immobilization, 1.0 g of dry resin was soaked in 10 mL n-heptane for 3 h in a 50 mL bottle, and then 0.1 g crude lipase Lip2 were added and the immobilization was conducted at 25oC with the shaking speed of 200 r/min. After the immobilization, the immobilized Lip2 was separated by filtration and washed with n-heptane under vacuum, then dried at room temperature for 1 h and kept at 4oC for use. 2.3 Determination of protein content and immobilization efficiency The protein concentration was determined by the method of Bradford with BSA standards. The immobilization efficiency was calculated by DI = (Pe-Pw)/Pe, where Pe is the total amount of protein used and Pw is that of protein in the washing liquid[13]. 2.4 Assay of lipase synthetic activity The activity of lipase Lip2 in esterification reaction was measured in n-heptane according to the procedure described by Wang, Xu et al [14]with small modifications. Briefly, 1 mL substrate solution (1.2 mol/L octanoic acid and 1.2 mol/L ethanol in n-heptane with a volume of 0.5 mL respectively) was added in a 5 mL capped tube. The reaction was started by adding 10 mg free lipase ( or 110 mg immobilized lipase)
and was conducted for 30 min at 35oC with the shaking speed of 200 r/min. Samples of 100 L were drawn and mixed with 100 L of n-hexane, as internal standard, then added 0.8 mL n-heptane and analyzed by gas chromatograph. One unit of lipase synthetic activity was defined as the amount of enzyme required to produce 1 mol of ester per minute under above conditions. 2.5 Purification of geranyl propionate by HPLC The reaction mixtures resulting from lipase-catalyzed esterification were filtered under vacuum to remove molecular sieves and lipase, the filtrate was separated by HPLC with a waters X Bridge C18 column (4.6×150 mm, 5 m) at 30oCand eluted with acetonitrile/water (4:1, v/v) at a rate of 2.0 mL/min. The waters 996 PDA detector was used and the wavelength was 220 nm. The fractions containing the desired products were collected. After removing the acetonitrile and water by reduced pressure distillation, the desired product was obtained finally. 2.6 Identification of purified geranyl propionate The purified geranyl propionate was identified by Fourier transform infrared spectroscopy (FT-IR) and Nuclear Magnetic Resonance (NMR). The FT-IR measurements were performed on FT-IR spectrophotometer (Nexus 470, USA), a region from 400-4000 cm-1 was used for scanning at 4 cm-1 resolution by 32 scans. The NMR analysis was acquired on Bruker NMR spectrometer (Avance Ⅲ, Switzerland). The spectra was obtained at 400 MHz using CDCl3 as the solvent and tetramethyl silane (TMS) as internal standard. 2.7 Quantitative analysis of the product
The product was analyzed by GC-FID analysis with a DB-WAX capillary column (30 m×0.25 mm). The temperatures of injector and detector were set at 250oC and 260oC, respectively. Nitrogen was used as the carrier gas at a flow rate of 1.15 mL/min. The procedure was as follows: 90oC remaining for 1min, rising to 230oC at a rate of 10oC/min and remaining for 8 min. Retention times at these conditions were 12.9 and 13.2 min for geranyl propionate and geraniol respectively, using 1-hexanol as internal standard. The esterification ratio of geranyl propionate was defined as the ratio between the real content and the theoretical content of geranyl propionate. All results were repeated for three times. 2.8 Continuous synthesis of geranyl propionate The continuous synthesis of geranyl propionate was conducted in a CSTR with an effective volume of 25 mL. The reactor, especially designed for the experiment, was equipped with two tubes at the ground glass plug for the insert and outlet liquids. The immobilized enzymes were filled in a small pocket of filter cloth, which was put at two small lags located at the lower part of the reactor. The enzyme could simply be put at the bottom of the reactor if the outlet was wrapped with filter cloth. The temperature of the reactor was kept constant by a jacket connected to a water bath (Fig. 1). The substrate solution was prepared by dissolving geraniol and propionic acid in isooctane with fixed molar ratio. At the beginning of the experiment, the reactor was filled with enzyme and substrate solution and the batch reaction was conducted until
an ideal esterification ratio was reached. Then the continuous operation was started by the feeding of substrate solution via peristaltic pump with fixed flow rate. The duration of continuous synthesis was about three times of the average residence time. Samples of 0.1 mL were withdrawn hourly and were immediately mixed with 0.1 mL internal standard mixture and 0.8 mL isooctane and analyzed by GC-FID. 3. Results and discussions 3.1 Immobilization of Lip2 3.1.1 Choice of solvent In our preliminary experiment, the immobilization of Lip2 onto ion exchange resin in aqueous solution did not give satisfactory results, the immobilization efficiency of the Lip2 was only 56.01%. Considering that the esterification would take place in organic solvents, the Lip2 was immobilized inorganic solvents. It is generally acknowledged that lipases are interfacial active enzymes with lipophilic domains and can adopt both open and close conformations. The lipophilic interactions between the hydrophobic parts of the lipase and substrates or organic solvents could fix the entrapped enzyme in a more active conformation and enhanced activity[15]. In a medium of low polarity, the lipophilic domains of lipase may interact with solvent. As a result, the effect may help to induce conformation changes of lipase to an active form [13]. Three solvents of different hydrophobicity, expressed by the values of logP were tested and the results are shown in Table 1. The highest immobilization efficiency of nearly 98.6% was obtained with n-heptane and the
synthetic activity of the lipase was 690.8 U/g after immobilization, which represented an increase of 28.5% compared with the free lipase (synthetic activity 537.2 U/g). 3.1.2 Determination of enzyme loading To evaluate the loading capacity of carrier, 1.0 g of dry resins were loaded with 10 mL n-heptane solution with different initial lipase concentrations (5, 7.5, 10, 12.5, 15 mg/mL respectively). The synthetic activity increased with the initial Lip2 amount from 5 to 10 mg/mL and reached the maximum synthetic activity of 690.8 U/g after immobilization, but the activity began to decrease with the lipase concentration from 10 to 15 mg/mL. So the concentration of 10 mg/mL was chosen as the enzyme loading for the immobilization of Lip2. 3.2 Thermal properties of immobilized Lip2 It is well known that temperature can influence the activity and stability of enzyme. A rise of temperature will increase the reaction rate. This rise can be explained by the transition state theory[16]. An increase in temperature can enhance mutual miscibility and improve the diffusion process of substrates, thus reducing mass transfer limitations and favoring interactions between enzyme particles and substrates[8]. On the other hand, at high temperature, the tertiary structure of enzyme may be disrupted, causing the denaturation [17]. Although the optimal temperature of the enzyme has been given by the supplier, we did not know whether it was modified by the immobilization. In general, the immobilization would increase the stability of enzyme and rise the optimal temperature. Relative activity was the ratio between the activity of every sample and the maximum activity of sample. Fig. 2 illustrates the
effect of temperature on the activity of immobilized Lip2, it can be seen that the optimal temperature of immobilized Lip2 is 35oC, the same as the free enzyme. Visibly the immobilization did not modify the optimal temperature of Lip2. To explore the thermal stability of immobilized Lip2, both free and immobilized enzyme were incubated in the absence of substrates from a temperature range of 40 to 80oC for 2 h. The ratios of synthetic activities before and after incubation were named as residual activity and are shown in Fig. 3. The activity of immobilized Lip2 decreased more slowly than that of free lipase. After incubation for 2 h at 80oC, the residual activity of immobilized Lip2 remained 62.9% of the original value, while that of the free lipase was only 15.6% of the original. The remarkable improvement of the thermal stability by immobilization may arise from the conformation integrity of the immobilized enzyme structure after adsorption to the carriers[18], which may prevent extensive structure changes caused by thermal denaturation[19]. 3.3 Choice of solvent for the synthesis of geranyl propionate Generally speaking, the hydrophobicity of organic solvent, evaluated by the value of logP, is a key factor for esterification due to that it could affect the activity and stability of lipases and the solubility of solvents[20]. Highly hydrophobic solvents with logP > 4 are often considered as the most suitable solvent for biocatalysis with lipases, the solvents with logP values between 2 and 4 are moderately effective, and those polar solvents with logP < 2 would be ineffective [21]. But in practice the most suitable solvent for a given reaction should be determined by experiments. For the synthesis of geranyl propionate, three solvents with different logP values were
compared to choose the best one: n-hexane (logP = 3.5), n-heptane (logP = 4.0), and isooctane (logP = 4.5). The experiments were carried out in 10 mL anhydrous solvents containing enzyme. 15 mg/mL of molecular sieves 4Å were added to remove the water generated. The reaction mixture was shaken at 200 r/min in an incubator. The results are shown in Table 2. The Table 2 shows that after 3.5 h, the esterification ratio reached over 80% with all three solvents, the maximum esterification ratio of 87.5% was obtained with isooctane which could be considered as the ideal solvents for the synthesis of geranyl propionate with Lip2. This results seem to be in accord with the general opinion mentioned above. S. Y. Huang and H. L. Chang[22] found the similar trend when using surfactant-coated lipase for the esterification of geraniol and acetic in organic solvents, n-hexane and isooctane produced the highest yield of 82.2% and 83.7%, respectively. The reason may be that the structure and function of enzyme are determined to some extent by the bound water around the enzyme. The enzyme requires water layer to maintain its activity and hydrophobic solvents can preserve the catalytic activity without stripping the water from the enzyme [21]. A high value of logP could be conducive to maintain the biocatalyst at active state. The most suitable solvent for a given esterification reaction should be determined by the interaction among substrates, enzyme and solvent. Finally, isooctane was chosen for the following experiments. 3.4 Continuous synthesis of geranyl propionate From the batch experiments it has been confirmed that Lip2 could effectively
catalyze the synthesis of geranyl propionate. From Table 2, it can also be seen that the reaction time of 3.5 h was enough for the batch operation. In consideration the residence time distribution in the continuous reactor, an average residence time between 4 or 4.5 h should be enough for the continuous synthesis. During the continuous reaction, samples were withdrawn hourly to measure the esterification ratio. 3.4.1 Effect of enzyme loading The enzyme loading in a given reactor is a crucial economical factor for continuous operation[23]. The reactor used in this work had an effective volume of 25 mL. Fig.4 shows evolutions of esterification ratio measured at the exit of reactor with three different enzyme loading between 2 g and 4 g. Generally, the esterification ratio would increase with the enzyme loading, but after a certain extent the reaction will reach the saturation level and no obvious increment could be observed [24]. From Fig.4, it could be seen that the esterification ratio of geranyl propionate increased with the enzyme loading from 2 g to 4 g, and the difference between the loading of 3 g and 4 g was minor. Thus the loading of 3 g was chosen for following experiments. This enzyme loading seems to be voluminous with respect to the volume of the reactor. The final esterification ratio of geranyl propionate was lower compared to that obtained in batch experiment. It should be improved by strengthening the agitation when the process was scale-up to the industrial level. 3.4.2 Effect of average residence time For a CSTR with given volume, the feed flow rate is conversely proportional to
the average residence time, which is a key parameter for the continuous operation. Fig.5 presents the evolutions of esterification ratio during the continuous synthesis of geranyl propionate at different flow rates of feed. The start-up of continuous operation, corresponding to the beginning of curves, was realized after the stable esterification ratio with batch operation. The flow rate of 4.5, 6 and 9 mL/h corresponded to the average residence time of 5.6, 4.2 and 2.8 h, respectively. The highest esterification ratio of 75.8% was obtained with a flow rate of 4.5 mL/h. It was lower than that obtained in batch experiment. The residence time distribution in CSTR seemed to be the main reason for this difference. The flow rate of 6 mL/h obtained a stable esterification ratio of 72.8%, a little lower than that of 4.5 mL/h, but not far from it. In fact, with this flow rate, the average residence time was already superior to 4 h, which was long enough according to the batch experiment. On the other hand, with the flow rate of 9 mL/h, the percentage of esterification was dramatically low due to the short residence time for the reaction[25]. In fact, at this flow rate the average residence time was inferior to 3 h which was not long enough according to batch experiments (cf. Tab. 2). 3.4.3 Effect of the substrate molar ratio The relative proportions of the various substrates in a reaction mixture is a factor that defines the physical and chemical properties of a reaction system and subsequently influence the conversion [26]. So, through proper selection of substrate molar ratio, the equilibrium of an enzymatic reaction can be manipulated to favor a high conversion [27]. But a molar ratio far from the stoichiometric molar ratio will
raise the risk of substrate inhibition and increase the cost of separation. In our preliminary batch experiments, the acid/alcohol molar ratio from 2:1 to 1:3 were assayed to determine the optimal value. It was found that the esterification ratio had an increasing trend with the acid/alcohol molar ratio varying from 2:1 to 1:1.5, and begun to decrease slowly with the molar ratio from 1:1.5 to 1:3. This may be because that short chain acids such as acetate and propionic acid usually have strong hydrophilicity and small molecular size and act as an inhibitor of lipase[28]. Because of the strong polarity of short chain acids, on the one hand, the acids may be strongly adsorbed on the enzyme active site[29], resulting an excessive quantity of substrate. On the other hand, the acids could deprive the essential water around the enzyme, resulting the loss of lipase activity in organic solvents. Paul A. Claon and Casimir C. Akoh[30]found that an increase in acetic acid concentration inhibited the activities of Candida antarctica lipase and that the conversion rate of geranyl acetate decreased significantly. So, in our continuous work, the acid/alcohol molar ratios from 1:1.5 to 1:2 were chosen to further optimize the process. The solubility of geraniol and propionic acid in isooctane is good enough to form a homogenous substrate mixture. Evolutions of esterification ratio at various molar ratios are shown in Fig.6. At the molar ratio of 1:1, the esterification ratio of geranyl propionate was the lowest at the beginning and decreased to only 53.6% after 12 h of operation. The low esterification ratio at equal proportion of substrates can be attributed to the inhibition of the Lip2 activity due to the strong acidity in the reaction media [31]. The increase of alcohol content improved the conversion. But at the molar
ratio of 1:2 the esterification ratio began to decrease, this might be attributed to the substrate inhibition. N.A. Serri [21]found the similar result when using Candida rugosa lipase to catalyze the synthesis of citronelly laurate, when the acid/alcohol molar ratio was 1:2, the conversion rate of citronelly laurate was lower than that of 1:1.5. This phenomenon was considered as familiar in bimolecular reaction catalyzed by enzyme with ping pong bi bi mechanism. 3.5 Identification and analysis of geranyl propionate 3.5.1 Purification by HPLC The reaction mixture was successively separated by HPLC. Fig.7 shows its spectra. The peak at 11.21minis that of geranyl propionate. 3.5.2 Identification by FT-IR and NMR After the separation, the isolated geranyl propionate was analyzed by FT-IR and NMR. FT-IR is principally used as a qualitative technique to identify the presence of functional groups in a particular compound[26]. The results are shown in Fig. 8 (a): the signals at 2967.43 and 2925.48, 2855.61 cm-1were the stretch vibration of CH3 or CH2, and the bending vibration at 1450.78 and 1378.9 cm-1 was an additional demonstration of CH3 or CH2, there was a strong absorption at 1738.51 cm-1, which was a symbol of C=O, the signal at 1180.56 cm-1 was the stretch vibration of C-O. The above results showed the appearance of ester bond in the new product, indicating possible formation of geranyl propionate. To further characterize the structure of geranyl propionate, the 1H NMR spectrum was used to check the geranyl propionate, the spectrum showed signals in the region
of 1.25-5.5 ppm for all hydrogen atoms. According to the 1H NMR shown in Fig. 8 (b), the results were as follows:1H NMR (400 MHz, CDCl3): δ (ppm):1.25-1.63(3H, s),1.603 (3H, s), 1.683-1.705 (6H, s),2.028-2.134 (4H, m), 2.308-2.365 (2H, m), 4.591-4.609 (2H, m),5.603-5.600 (1H, m), 5.323-5.344 (1H, m). These statistics confirmed that the product was geranyl propionate. 4. Conclusion In laboratory assays, Lip2 showed a high catalytic activity for the synthesis of geranyl propionate with an esterification ratio of 87.5%. There is a good potential for industries application which could overcome the shortcomings of chemical synthesis. For the practical application, Lip2 was immobilized on adsorptive resin DA201-C. Under the optimal conditions (lipase loading 0.01 g/g resin, 25oC, n-heptane as the medium, adsorption for 3 h), the immobilization efficiency reached 98.6%, and the synthetic activity represented an increase of 28.5% compared to that of the free lipase. The immobilization did not change the optimal temperature of Lip2. The immobilized Lip2 was used to catalyze the synthesis of geranyl propionate in CSTR. The duration of continuous assay was long enough to achieve the steady state and the stable esterification ratio of 72.8% was achieved, which was inferior to that obtained by batch operation, the reason for this different could be attributed to the residence time distribution. The residence time of 4 h would not be considered as too long for industrial realization. The process could be further improved by strengthening the agitation in industrial scale. Finally, the product was confirmed by FT-IR and NMR. Thus the continuous assays have confirmed the feasibility of geranyl
propionate synthesis by immobilized Lip2 at a larger scale. Acknowledgement This study was financially supported by the Specialized Research Fund for the Doctor Program of Higher Education (Zo.20130093110010).
References [1] X. Yang, G. Chen, H. Du, M. Miao, B. Feng, J. Mol. Catal. B: Enzym. 127 (2016) 34-39. [2] Ö. Kebabci, Cihangir, N., Hacettepe J. Biol. & Chem 39(3) (2011) 283-288. [3] S.S. Kanwar, S. Gehlot, M.L. Verma, R. Gupta, Y. Kumar, G.S. Chauhan, J. Appl. Polym. Sci. 110(5) (2008) 2681-2692. [4] W. Kaewthong, S. Sirisansaneeyakul, P. Prasertsan, A. H-Kittikun, Process Biochem. 40(5) (2005) 1525-1530. [5] S.M. Radzi, W. Mustafa, S. Othman, H. Noor, World Acad. Sci. Eng. Technol. 59 (2011) 677-680. [6] M. Liaquat, R.K.O. Apenten, J. Food Sci. 65(2) (2000) 295-299. [7] N.R. Mohamad, N.A. Buang, N.A. Mahat, Y.Y. Lok, F. Huyop, H.Y. Aboul-Enein, R. Abdul Wahab, Enzyme Microb. Technol. 72 (2015) 49-55. [8] N. Paroul, L.P. Grzegozeski, V. Chiaradia, H. Treichel, R.L. Cansian, J.V. Oliveira, D. de Oliveira, J. Chem. Technol. Biotechnol. 85(12) (2010) 1636-1641. [9] Y. Watanabe, K. Kuwabara, S. Adachi, K. Nakanishi, R. Matsuno, J Agric Food Chem 51(16) (2003) 4628-4632. [10] E. Séverac, O. Galy, F. Turon, P. Monsan, A. Marty, Bioresour Technol 102(8) (2011) 4954-4961. [11] P. Fickers, A. Marty, J.M. Nicaud, Biotechnol Adv 29(6) (2011) 632-44. [12] Y. Yan, X. Zhang, D. Chen, Bioresour Technol 131 (2013) 179-87. [13] Y. Gao, T. Tan, K. Nie, F. Wang, Chin. J. Biotechnol. 22(1) (2006) 114-118. [14] D. Wang, Y. Xu, Y. Teng, Bioprocess Biosyst Eng 30(3) (2007) 147-55. [15] S.P. Bastida A, .Armisen P, Biotechnol Bioeng 58(5) (1998) 486-493. [16] M.B.A. Rahman, N. Chaibakhsh, M. Basri, R.N.Z.R.A. Rahman, A.B. Salleh, S.M. Radzi, J. Chem. Technol. Biotechnol. 83(11) (2008) 1534-1540. [17] M.D. Romero, L. Calvo, C. Alba, A. Daneshfar, H.S. Ghaziaskar, Enzyme Microb. Technol. 37(1) (2005) 42-48. [18] M.-M. Zheng, L. Dong, Y. Lu, P.-M. Guo, Q.-C. Deng, W.-L. Li, Y.-Q. Feng, F.-H. Huang, J. Mol. Catal. B: Enzym. 74(1) (2012) 16-23. [19] M.M. Zheng, Y. Lu, F.H. Huang, L. Wang, P.M. Guo, Y.Q. Feng, Q.C. Deng, J Agric Food Chem 61(1) (2013) 231-7. [20] C.J. Wensen He, Yuan Ma, Yebo Yang, Xiaoming Zhang, Biao Feng, Lin Yue, J. Mol. Catal. B: Enzym. 67 (2010) 60-65. [21] N.A. Serri, A.H. Kamaruddin, W.S. Long, Bioprocess Biosyst Eng 29(4) (2006) 253-60. [22] S. Huang, H. Chang, M. Goto, Enzyme Microb. Technol. 22(7) (1998) 552-557. [23] K.C. Badgujar, B.M. Bhanage, Process Biochem. 49(8) (2014) 1304-1313. [24] S. Chattopadhyay, R. Sen, Bioresour Technol 147 (2013) 395-400. [25] J.H. Lee, S.B. Kim, C. Park, B. Tae, S.O. Han, S.W. Kim, Appl. Biochem. Biotechnol. 161(1-8) (2010) 365-371.
[26] N. Mohamad, N.A. Buang, N.A. Mahat, J. Jamalis, F. Huyop, H.Y. Aboul-Enein, R.A. Wahab, J. Ind. Eng. Chem. 32 (2015) 99-108. [27] F.E.a.G.A. M. Trani, JAOCS 68(1) (1991). [28] S.Y. Huang, H.L. Chang, J. Chem. Technol. Biotechnol. 74(2) (1999) 183-187. [29] G.D. Yadav, P.S. Lathi, J. Mol. Catal. B: Enzym. 27(2-3) (2004) 113-119. [30] P.A. Claon, C.C. Akoh, J. Am. Oil Chem. Soc. 71(6) (1994) 575-578. [31] P. Pires-Cabral, M.M.R. da Fonseca, S. Ferreira-Dias, Biochem. Eng. J. 43(3) (2009) 327-332.
Fig.1. Schematic diagram of the CSTR for geranyl propionic A: feed reservoir, B: effluent reservoir, C: magnetic stirrer, D: reactor, E: peristaltic pump, F: equipment for immobilized Lip2, H: online checking equipment
Relative activity (%)
100
80
60
40
20 20
30
40
Temperature (oC) Fig. 2. Optimal temperature of immobilized Lip2
50
60
free enzyme
immobilized enzyme
Residual activity (%)
100 80 60 40 20 0 40
50
60 70 Temperature (oC)
80
90
Fig. 3. Thermal stability of free and immobilized Lip2
2g
3g
4g
Esterification ratio (%)
100
80
60
40 0
2
4
6 Time (h)
8
10
12
Fig. 4. Effect of enzyme loading on esterification ratio of geranyl propionate (conditions: initial concentration of acid and geraniol 20 mmol/L and 30 mmol/L respectively, 35oC, isooctane as the solvent with a flow rate of 0.10 mL/min)
6mL/h
9mL/h
4.5mL/h
Esterification ratio (%)
100 80 60 40 20 0
2
4
6 Time (h)
8
10
12
Fig. 5. Effect of flow rate on esterification ratio of geranyl propionate (conditions: initial concentration of acid and geraniol 20 mmol/L and 30 mmol/L respectively, immobilized enzyme loading 3g, 35oC)
1:1
1:1.5
1:2
Esterification ratio
100 80 60 40 20 0
2
4
6 Time (h)
8
10
12
Fig. 6. Effect of substrate molar ratio on esterification ratio of geranyl propionate (conditions: immobilized enzyme loading 3 g with a flow rate of 0.10 mL/min, 35oC)
Fig. 7. HPLC spectra of the reaction mixture
(a)
(b)
Fig. 8. FT-IR and NMR spectra of geranyl propionate
Table 1 Immobilization of Lip2 in three organic solvents Medium
immobilization
synthetic activity
efficiency (%)
(U/g enzyme)
logP
n-hexane
3.5
96.3
597.5
n-heptane
4.0
98.6
690.8
isooctane
4.5
97.3
561.3
Table 2 Effect of solvents on the synthesis of geranyl propionate Esterification ratio (%) Time (h) n-hexane
n-heptane
isooctane
1
43.3
37.3
45.0
2
69.5
60.5
73.7
2.5
74.1
68.7
79.3
3
82.4
78.9
85.9
3.5
84.3
81.3
87.5
Reaction conditions: initial concentration of acid and geraniol were 20 mmol/L and 30 mmol/L respectively, 35oC, dose of free Lip2 7 mg/mL)