Journal of Biotechnology 168 (2013) 416–420
Contents lists available at ScienceDirect
Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Kinetic and thermodynamic investigation of enzymatic l-ascorbyl acetate synthesis Dong-Hao Zhang a,b,∗ , Chao Li a , Gao-Ying Zhi c a b c
College of Pharmacy, Hebei University, Baoding 071002, China Key Laboratory of Pharmaceutical Quality Control of Hebei Province, College of Pharmacy, Hebei University, Baoding 071002, China Computer Center, Hebei University, Baoding 071002, China
a r t i c l e
i n f o
Article history: Received 17 September 2013 Received in revised form 23 October 2013 Accepted 26 October 2013 Available online 7 November 2013 Keywords: l-Ascorbic acid Kinetic model Ping-Pong Bi-Bi mechanism Substrate inhibition Activation energy
a b s t r a c t Kinetics and thermodynamics of lipase-catalyzed esterification of l-ascorbic acid in acetone were investigated by using vinyl acetate as acyl donor. The results showed that l-ascorbic acid could generate inhibition effect on lipase activity. A suitable model, Ping-Pong Bi-Bi mechanism having substrate inhibition, was thus introduced to describe the enzymatic kinetics. Furthermore, the kinetic and thermodynamic parameters were calculated from a series of experimental data according to the kinetic model. The inhibition constant of l-ascorbic acid was also obtained, which seemed to imply that enhancing reaction temperature could depress the substrate inhibition. Besides, the activation energy values of the first-step and the second-step reaction were estimated to be 37.31 and 4.94 kJ/mol, respectively, demonstrating that the first-step reaction was the rate-limiting reaction and could be easily improved by enhancing temperature. © 2013 Elsevier B.V. All rights reserved.
1. Introduction l-Ascorbic acid has been used extensively in food, pharmaceutical and cosmetic fields. However, its poor liposolubility limited the application (Liu et al., 1996; Watanabe et al., 2010). To cope with this problem, many derivatives of l-ascorbic acid have been synthesized. For example, modification of l-ascorbic acid via esterification was a useful way to alter its solubility (Reyes-Duarte et al., 2011; Watanabe et al., 2012; Karmee, 2009). Recently, there were many reports concerning the synthesis of ascorbyl esters by using lipase as catalyst in organic solvent (Lerin et al., 2012; Chang et al., 2009; Zhang et al., 2011; Treichel et al., 2010). In order to identify the optimal conditions for the lipase-catalyzed esterification, it was essential to investigate the reaction mechanism, kinetics and thermodynamics of this reaction. Lipase was widely used as a biocatalyst to catalyze multisubstrate-multiproduct reactions (Batistella et al., 2012). Moreover, complex kinetic mechanisms have been proposed to provide descriptions of the lipase-catalyzed reactions. Many studies showed that lipase-catalyzed esterification reaction could be described by a Ping-Pong kinetic model (Awang et al., 2004; Chulalaksananukul et al., 1990, 1992). Especially, several
mechanisms have been proposed to explain lipase-catalyzed reactions containing substrate inhibition (Al-Zuhair, 2005; Mestri and Pai, 1995; de Castro et al., 1997). In these studies, a deadend complex formatted by one of the substrate and lipase was introduced to explain substrate inhibition phenomenon. So far, many ascorbyl esters such as ascorbyl oleate (ReyesDuarte et al., 2011), ascorbyl palmitate (Burham et al., 2009), ascorbyl linoleate (Watanabe et al., 2008) and ascorbyl benzoate (Lv et al., 2007) have been synthesized by enzymatic esterification in organic solvent. However, the studies on the enzymatic kinetics and thermodynamics were rarely reported. In this paper, it was found that l-ascorbic acid, one of the substrate, could generate inhibition on the activity of Lipozyme TLIM lipase. Thus the kinetics of lipase-catalyzed esterification of l-ascorbic acid using vinyl acetate as acyl donor was investigated. The kinetic model of the Ping-Pong mechanism having substrate inhibition was built and the kinetic and thermodynamic parameters were evaluated. These results would provide some valuable informations in enzymatic synthesis of l-ascorbyl ester. 2. Materials and methods 2.1. Materials
∗ Corresponding author at: College of Pharmacy, Hebei University, Baoding 071002, China. Tel.: +86 312 5971107; fax: +86 312 5971107. E-mail address: [email protected] (D.-H. Zhang). 0168-1656/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jbiotec.2013.10.033
This enzyme, Lipozyme TLIM (Novo Industri, Bagsvaerd, Denmark), was a preparation of a Thermomyces lanuginosus lipase immobilized on silica gel. l-Ascorbic acid (vitamin C, Vc) was
D.-H. Zhang et al. / Journal of Biotechnology 168 (2013) 416–420
purchased from Sigma Chemical Co. (St. Louis, MO, USA). Vinyl acetate (VAc, 99%) was obtained from Fuchen Chemical Co. (Tianjin, China). Analytical grade acetone was from Kewei Chemical Co. (Tianjin, China).
E
2.2. Experimental procedure For the standard reaction, Lipozyme TLIM (20 mg) was added to a reaction mixture containing l-ascorbic acid and vinyl acetate in 1 mL acetone. The concentrations of l-ascorbic acid and vinyl acetate were, respectively, varied from 3 to 12 mmol/L and 6 to 22 mmol/L. Then the reaction mixture of different substrate molar ratio was incubated in a temperature-controlled shaker at 170 rpm at a certain temperature. After 2 h, 50 L of the reaction mixture was withdrawn and evaporated, and then the residue was dissolved in methanol/water (5/5, v/v) and analyzed by HPLC. All experiments were conducted in triplicate and the mean values were calculated.
417
A
P
B
EA
FP F FB
Q
EQ
E
B EB (the dead-end complex) Scheme 2. Schematic representation of the Ping-Pong Bi-Bi mechanism having substrate inhibition.
where v is the enzymatic reaction rate of the esterification; Vmax is the maximum rate of the esterification; [A] is the concentration A and K B are the Michaelis of A; [B] is the concentration of B; Km m constants of A and B, respectively.
2.3. HPLC analysis 3.2. Ping-Pong Bi-Bi mechanism having substrate inhibition
3. Kinetic model Two kinetic mechanisms (Mukesh et al., 1997; Maugard et al., 2000) were supposed in the present study. The first was called PingPong Bi-Bi mechanism. The second model was assumed Ping-Pong Bi-Bi mechanism having substrate inhibition. They are described as follows. 3.1. Ping-Pong Bi-Bi mechanism Previous studies have shown that the reaction of lipasecatalyzed esterification could be described by the Ping-Pong kinetic model (Lima et al., 1996). To understand the Ping-Pong Bi-Bi mechanism better, a schematic representation of this reaction mechanism using the Cleland notation (Lima et al., 1996) was given in Scheme 1. As shown, acyl donor (A) is bound to the enzyme at first to form the acyl-enzyme complex. As soon as one product (P) is formed and then released, acyl acceptor (B) binds to the modified enzyme (substituted form of the free enzyme) to form the second product (Q). The reaction rate equation is given by Eq. (1) (Lima et al., 1996):
v=
Vmax [A][B]
(1)
A [B] + K B [A] + [A][B] Km m
A
P
B
Q
Several recent studies (Al-Zuhair, 2005; Mestri and Pai, 1995; de Castro et al., 1997) on enzymatic esterification containing substrate inhibition attempted to describe the reaction mechanism by a modified Ping-Pong kinetic model (Scheme 2). Compared with Scheme 1, the step of a dead-end complex formation between substrate and free enzyme has been added in Scheme 2. Based on this proposed mechanism, the expression for the reaction rate is given by Eq. (2) (Yadav and Borkar, 2008):
v=
Vmax [A][B]
(2)
A [B] + K B [A] + [A][B] + (K A [B]2 /K B ) Km m m i
where KiB is the inhibition constant of B; other notations are the same as that in Eq. (1). 4. Results and discussion 4.1. Kinetics of enzymatic esterification of l-ascorbic acid Lipase-catalyzed esterification of l-ascorbic acid was carried out at 40 ◦ C for 2 h by changing substrate concentration and the results were listed in Supplementary Information (Table S1). After that, the reaction rates were calculated and shown in Fig. 1. As can be seen, for a given vinyl acetate concentration, the reaction rate decreased strangely with the increase in l-ascorbic acid concentration, which indicated that, to some extent, l-ascorbic acid had an observable inhibition effect on the enzymatic reaction. For example, lipase
0.7 0.6 V (mmol/(L*h))
The reaction resultants were analyzed using high-performance liquid chromatography (HPLC, ChuangXinTongHeng Science & Technology Co. Ltd., China) with a C-18 column (ZORBAX 300SBC18 4.6 mm ID × 250 mm (5 m), Agilent Technologies, Palo Alto, CA) and a UV detector at 254 nm. A 20-L diluted sample was injected, and the elution was done with methanol/water/acetic acid (50/50/0.1, v/v/v) at a flow rate of 0.5 mL min−1 . Reaction conversion was calculated in terms of the mole percentage of esterification, based on the ratio of consumed l-ascorbic acid to the total amount of l-ascorbic acid before reaction.
0.5 0.4 0.3 0.2 0.1 2
E
EA
FP
F FB
EQ
E
Scheme 1. Schematic representation of the Ping-Pong Bi-Bi mechanism.
4
6 8 10 (mm ol/L) C Vc
12
Fig. 1. Effect of l-ascorbic acid (Vc) concentration on the reaction rate at different vinyl acetate (VAc) concentration at 40 ◦ C. (Vinyl acetate concentrations: () 6 mmol/L, (䊉) 10 mmol/L, () 14 mmol/L, () 18 mmol/L, () 22 mmol/L.)
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D.-H. Zhang et al. / Journal of Biotechnology 168 (2013) 416–420
activity declined from 33.65 to 28.29 U/g Lipozyme TLIM with the increase in l-ascorbic acid concentration from 3 to 12 mmol/L when vinyl acetate concentration was at 22 mmol/L. (One unit of lipase activity (U) was defined as the amount of enzyme that synthesized 1 mol l-ascorbyl acetate per hour under the assay conditions.) Fig. 2 (40 ◦ C) shows the Lineweaver–Burk plot of reciprocal of the reaction rates versus reciprocal of vinyl acetate concentrations at different l-ascorbic acid concentrations at 40 ◦ C. As observed, the Lineweaver–Burk plot revealed that lipase-catalyzed esterification of l-ascorbic acid typically obeyed classical Michaelis–Menten kinetics. However, the four fitting straight lines in Fig. 2 (40 ◦ C) were found to be unparallel to each other, which suggested that the enzymatic reaction did not follow simple Ping-Pong Bi-Bi mechanism (Scheme 1) (Burham et al., 2009). Moreover, when l-ascorbic acid concentration increased, the slop of the fitting straight line was raised and the intercept was reduced. These results agreed well with the existence of substrate inhibition in lipase-catalyzed synthesis of l-ascorbyl ester, which implied that l-ascorbic acid was able to bind lipase to form a dead-end enzyme-substrate complex (Al-Zuhair, 2005; Yadav and Borkar, 2008; Goto et al., 1994). As a
consequence, the formation of product molecules would be prohibited. Taking these results together, Ping-Pong Bi-Bi mechanism with l-ascorbic acid inhibition (Scheme 2) was thus proposed to describe this reaction. According to the Ping-Pong mechanism having substrate inhibition in Scheme 2, vinyl acetate (A) bound first to free enzyme (E) and formed an enzyme-acyl complex (EA). Then EA released the modified enzyme (F) and the first product acetaldehyde (P). After that, l-ascorbic acid (B) reacted with F to give the complex FB and further released free enzyme (E) and the second product ascorbyl acetate (Q). On the other hand, l-ascorbic acid (B) could also bind to free lipase before the bonding of vinyl acetate to lipase, resulting in the formation of a dead-end complex (EB) and prohibiting the formation of product molecules. By taking the reciprocal of Eq. (2) and arranging terms, we got Eq. (3):
1
v
=
A K A [B] Km + m B Vmax Vmax Ki
1 + [A]
1+
B Km [B]
1 Vmax
(3)
Fig. 2. The Lineweaver–Burk plot of reciprocal of the reaction rates versus reciprocal of vinyl acetate (VAc) concentrations at different l-ascorbic acid (Vc) concentrations at 40, 0, 15, 25, and 50 ◦ C, respectively. (l-Ascorbic acid concentrations: () 3 mmol/L, () 6 mmol/L, () 9 mmol/L, and (䊉) 12 mmol/L.)
D.-H. Zhang et al. / Journal of Biotechnology 168 (2013) 416–420 Table 1 Kinetic parameters of lipase-catalyzed esterification of l-ascorbic acid at different temperatures.
419
Table 2 The reaction rate constant of lipase-catalyzed esterification of l-ascorbic acid at different temperatures.
T (◦ C)
Vmax (mmol/(L h))
A Km (mmol/L)
B Km (mmol/L)
KiB (mmol/L)
T (◦ C)
k1 (×10−15 L/mol h)
k2 (×10−15 L/mol h)
0 15 25 40 50
16.59 13.42 28.88 11.75 24.00
1165.82 597.99 574.59 224.50 94.74
26.60 15.23 31.26 13.92 24.62
7.07 9.20 8.36 13.25 1.50
0 15 25 40 50
1.76 3.61 3.89 10.46 25.58
77.01 141.92 71.53 168.74 98.43
From Eq. (3), Eqs. (4) and (5) were derived as follows: A Km S= [B] + Vmax Vmax KiB
I=
B Km
1 1 + Vmax [B] Vmax
40
38 (5)
4.2. Thermodynamics of enzymatic esterification of l-ascorbic acid This reaction of lipase-catalyzed l-ascorbate synthesis could be considered as a two-step reaction. The first step was that vinyl acetate (A) bound to enzyme (E) and formed the first product acetaldehyde (P). The second step was that l-ascorbic acid (B) reacted with (F) and gave the second product l-ascorbyl acetate (Q). To investigate the thermodynamics of the two steps, the reactions were carried out at different temperatures (The temperature is between 0 and 50 ◦ C because Lipozyme TLIM could exhibit its optimal activity at 50 ◦ C and a higher temperature than 50 ◦ C might cause Lipozyme TLIM inactivation.) and the results were listed in Supplementary Information (Table S2). Similarly, the reaction rates were calculated and the relationships between 1/v and the reciprocal of vinyl acetate concentration were shown in Fig. 2 (0, 15, 25, 50 ◦ C). As shown, the inhibition of l-ascorbic acid on lipase activity was also discovered in Fig. 2 (0, 15, 25, 50 ◦ C), which had been already demonstrated in Fig. 2 (40 ◦ C). Depending on these data, the values of the kinetic parameters were also calculated by means of Eqs. (4) and (5) and shown in A decreased gradually with the Table 1. As presented, the value of Km B did not increase in reaction temperature whereas the value of Km change obviously, which implied that enhancing reaction temperature could notably increase the affinity between vinyl acetate and enzyme. In addition, the values of the inhibition constant (KiB ) in Table 1 seemed to suggest that the inhibition effect of l-ascorbic acid on lipase was lower at high temperature than that at low temperature. Furthermore, the reaction rate constant could be calculated from Eq. (6) referring to absolute reaction rate theory and Eyring hypothesis (Sehgal et al., 2002; Porto et al., 2006). kB T hKm
37 36
where S is the slope in Eq. (3), I is the intercept in Eq. (3). The slope and the intercept in Fig. 2 (40 ◦ C) gave the S and I valB and V ues, respectively. According to Eq. (5), we could obtain Km max A and values from the relation between I and 1/[B]. Furthermore, Km KiB values could also be determined from the relation between S and [B] according to Eq. (4). As a consequence, the kinetic parameA was much ters were calculated and listed in Table 1. As shown, Km B larger than Km (1/Km represents the enzyme affinity to substrate), which suggested that the affinity of l-ascorbic acid to enzyme was larger than the affinity of vinyl acetate to enzyme. These results appeared to verify that l-ascorbic acid was easy to combine with enzyme and form a dead-end complex.
k=
39
(4)
ln k
A Km
(6)
where k is the reaction rate constant; kB is Boltzman constant (1.38065 × 10−24 J/K); h is Plank constant (6.6261 × 10−34 J s); T is
35 34 3
3.2
3.4 3.6 3 -1 10 ×T
3.8
Fig. 3. Arrhenius plot of ln k1 vs T−1 () and ln k2 vs T−1 ().
the absolute temperature; Km is the Michaelis constant for enzymatic reaction. Table 2 listed the reaction rate constants of the two steps (k1 and k2 ). As shown, k1 values were much lower than k2 values, indicating that the first-step reaction was slow while the second-step reaction was fast. It could be concluded that the first-step reaction was the rate-limiting reaction. Depending on these k1 and k2 values at various reaction temperatures, the activation energy Ea could be further estimated according to Arrhenius equation (7). ln k = −
Ea + ln A RT
(7)
where k is the reaction rate constant; A is the frequency factor; Ea is the activity energy; R is molar gas constant. The Arrhenius plot was made on the basis of ln k vs reciprocal of temperature (Fig. 3). As presented, the activation energy Ea could be obtained from the slop of the straight line. As a consequence, Ea1 of the first-step reaction was estimated to be 37.31 kJ/mol, while Ea2 of the second-step reaction was 4.94 kJ/mol. Ea1 was much higher than Ea2 , which further confirmed that the second-step reaction was much easier than the first-step reaction. That was, the firststep reaction was the rate-limiting reaction. In addition, this result indicated that the first-step reaction rate was more sensitive to temperature based on Arrhenius equation. 5. Conclusions The kinetics of the enzymatic esterification of l-ascorbic acid by Lipozyme TLIM in acetone had been investigated. The results showed that the reaction mechanism could be described well to follow Ping-Pong Bi-Bi mechanism having substrate inhibition. Furthermore, the kinetic and thermodynamic parameters were estimated. It seemed that the inhibition of l-ascorbic acid on Lipozyme TLIM could be depressed at high temperature. The results further showed that activation energy of the first-step reaction was higher than that of the second-step reaction, suggesting that the first-step reaction was the rate-limiting reaction and could be easily improved by enhancing temperature. In conclusion, l-ascorbic acid concentration could not be blindly raised in biosynthesis of l-ascorbyl ester in order to increase the yield.
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Acknowledgments This work was supported by the Natural Science Foundation of Hebei, China (B2011201012). The authors have no conflict of interest to declare. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.jbiotec.2013.10.033. References Al-Zuhair, S., 2005. Producation of biodiesel by lipase-catalyzed transesterification of vegetable oils: a kinetics study. Biotechnol. Prog. 21, 1442–1448. Awang, R., Basri, M., Ahmad, S., Salleh, A.B., 2004. Lipase-catalyzed esterification of palm-based 9,10-dihydroxystearic acid and 1-octanol in hexane – a kinetic study. Biotechnol. Lett. 26, 11–14. Batistella, L., Ustra, M.K., Richetti, A., Pergher, S.B.C., Treichel, H., Oliveira, J.V., Lerin, L., de Oliveira, D., 2012. Assessment of two immobilized lipases activity and stability to low temperatures in organic solvents under ultrasound-assisted irradiation. Bioprocess Biosyst. Eng. 35, 351–358. Burham, H., Rasheed, R.A.G.A., Noor, N.Md., Badruddin, S., Sidek, H., 2009. Enzymatic synthesis of palm-based ascorbyl esters. J. Mol. Catal. B: Enzym. 58, 153–157. Chang, S.-W., Yang, C.-J., Chen, F.-Y., Akoh, C.C., Shieh, C.-J., 2009. Optimized synthesis of lipase-catalyzed l-ascorbyl laurate by Novozym 435. J. Mol. Catal. B: Enzym. 56, 7–12. Chulalaksananukul, W., Condort, J.S., Delorme, P., Willemot, R.M., 1990. Kinetic study of esterification by immobilized lipase in n-hexane. FEBS Lett. 276, 181–184. Chulalaksananukul, W., Condort, J.-S., Combes, D., 1992. Kinetics of geranyl acetate synthesis by lipase catalyzed transesterification in n-hexane. Enzyme Microb. Technol. 14, 293–298. de Castro, H.F., de Oliveira, P.C., Pereira, E.B., 1997. Evaluation of different approaches for lipase catalyzed synthesis of citronellyl acetate. Biotechnol. Lett. 19, 229–232. Goto, M., Kamiya, N., Miyata, M., Nakashio, F., 1994. Enzymatic esterification by surfactant-coated lipase in organic media. Biotechnol. Prog. 10, 263–268. Karmee, S.K., 2009. Biocatalytic synthesis of ascorbyl esters and their biotechnological applications. Appl. Microbiol. Biotechnol. 81, 1013–1022. Lerin, L.A., Richetti, A., Dallago, R., Treichel, H., Mazutti, M.A., Oliveira, J.V., Antunes, O.A.C., Oestreicher, E.G., de Oliveira, D., 2012. Enzymatic synthesis of ascorbyl palmitate in organic solvents: process optimization and kinetic evaluation. Food Bioprocess Technol. 5, 1068–1076.
Lima, F.V., Pyle, D.L., Asenjo, J.A., 1996. Reaction kinetics of the esterification of lauric acid in iso-octane using an immobilized biocatalyst. Appl. Biochem. Biotechnol. 61, 411–422. Liu, X.-Y., Guo, F.-L., Wu, L.-M., Liu, Y.-C., Liu, Z.-L., 1996. Remarkable enhancement of antioxidant activity of vitamin C in an artificial bilayer by making it lipo-soluble. Chem. Phys. Lipids 83, 39–43. Lv, L.-X., Pan, Y., Li, Y.-Q., 2007. Biosynthesis of ascorbyl benzoate in organic solvents and study of its antioxygenic and antimicrobial properties. Food Chem. 101, 1626–1632. Maugard, T., Tudella, J., Legoy, M.D., 2000. Study of vitamin ester synthesis by lipase-catalyzed transesterification in organic media. Biotechnol. Prog. 16, 358–362. Mestri, S.D., Pai, J.S., 1995. Effect of moisture on lipase catalyzed esterification of geraniol of palmarosa oil in non aqueous system. Biotechnol. Lett. 17, 459–461. Mukesh, D., Jadhav, S., Banerji, A.A., Thakkar, K., Bevinakatti, H.S., 1997. Lipasecatalysed esterification reactions—experimental and modelling studies. J. Chem. Technol. Biotechnol. 69, 179–186. Porto, T.S., Porto, C.S., Cavalcanti, M.T.H., Filho, J.L.L., Perego, P., Porto, A.L.F., Converti, A., Pessoa Jr., A., 2006. Kinetic and thermodynamic investigation on ascorbate oxidase activity and stability of a Cucurbita maxima extract. Biotechnol. Prog. 22, 1637–1642. Reyes-Duarte, D., Lopez-Cortes, N., Torres, P., Comelles, F., Parra, J.L., Pena, S., Ugidos, A.V., Ballesteros, A., Plou, F.J., 2011. Synthesis and properties of ascorbyl esters catalyzed by lipozyme TL IM using triglycerides as acyl donors. J. Am. Oil Chem. Soc. 88, 57–64. Sehgal, A.C., Tompson, R., Cavanagh, J., Kelly, R.M., 2002. Structural and catalytic response to temperature and cosolvents of carboxylesterase EST1 from the extremely thermoacidophilic archaeon Sulfolobus solfataricus P1. Biotechnol. Bioeng. 80, 784–793. Treichel, H., de Oliveira, D., Mazutti, M.A., Di Luccio, M., Oliveira, J.V., 2010. A review on microbial lipases production. Food Bioprocess Technol. 3, 182–196. Watanabe, Y., Sawahara, Y., Nosaka, H., Yamanaka, K., Adachi, S., 2008. Enzymatic synthesis of conjugated linoleoyl ascorbate in acetone. Biochem. Eng. J. 40, 368–372. Watanabe, Y., Nakanishi, H., Goto, N., Otsuka, K., Kimura, T., Adachi, S., 2010. Antioxidative properties of ascorbic acid and acyl ascorbates in ML/W Emulsion. J. Am. Oil Chem. Soc. 87, 1475–1480. Watanabe, Y., Suzuki, T., Nakanishi, H., Sakuragochi, A., Adachi, S., 2012. Effect of ascorbic acid or acyl ascorbate on the stability of catechin in oil-in-water emulsion. J. Am. Oil Chem. Soc. 89, 269–274. Yadav, G.D., Borkar, I.V., 2008. Kinetic modeling of immobilized lipase catalysis in synthesis of n-butyl levulinate. Ind. Eng. Chem. Res. 47, 3358–3363. Zhang, D.-H., Lv, Y.-Q., Zhi, G.-Y., Yuwen, L.-X., 2011. Kinetic biosynthesis of lascorbyl acetate by immobilized Thermomyces lanuginosus lipase (Lipozyme TLIM). Bioprocess Biosyst. Eng. 34, 1163–1168.
Contents lists available at ScienceDirect
Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Kinetic and thermodynamic investigation of enzymatic l-ascorbyl acetate synthesis Dong-Hao Zhang a,b,∗ , Chao Li a , Gao-Ying Zhi c a b c
College of Pharmacy, Hebei University, Baoding 071002, China Key Laboratory of Pharmaceutical Quality Control of Hebei Province, College of Pharmacy, Hebei University, Baoding 071002, China Computer Center, Hebei University, Baoding 071002, China
a r t i c l e
i n f o
Article history: Received 17 September 2013 Received in revised form 23 October 2013 Accepted 26 October 2013 Available online 7 November 2013 Keywords: l-Ascorbic acid Kinetic model Ping-Pong Bi-Bi mechanism Substrate inhibition Activation energy
a b s t r a c t Kinetics and thermodynamics of lipase-catalyzed esterification of l-ascorbic acid in acetone were investigated by using vinyl acetate as acyl donor. The results showed that l-ascorbic acid could generate inhibition effect on lipase activity. A suitable model, Ping-Pong Bi-Bi mechanism having substrate inhibition, was thus introduced to describe the enzymatic kinetics. Furthermore, the kinetic and thermodynamic parameters were calculated from a series of experimental data according to the kinetic model. The inhibition constant of l-ascorbic acid was also obtained, which seemed to imply that enhancing reaction temperature could depress the substrate inhibition. Besides, the activation energy values of the first-step and the second-step reaction were estimated to be 37.31 and 4.94 kJ/mol, respectively, demonstrating that the first-step reaction was the rate-limiting reaction and could be easily improved by enhancing temperature. © 2013 Elsevier B.V. All rights reserved.
1. Introduction l-Ascorbic acid has been used extensively in food, pharmaceutical and cosmetic fields. However, its poor liposolubility limited the application (Liu et al., 1996; Watanabe et al., 2010). To cope with this problem, many derivatives of l-ascorbic acid have been synthesized. For example, modification of l-ascorbic acid via esterification was a useful way to alter its solubility (Reyes-Duarte et al., 2011; Watanabe et al., 2012; Karmee, 2009). Recently, there were many reports concerning the synthesis of ascorbyl esters by using lipase as catalyst in organic solvent (Lerin et al., 2012; Chang et al., 2009; Zhang et al., 2011; Treichel et al., 2010). In order to identify the optimal conditions for the lipase-catalyzed esterification, it was essential to investigate the reaction mechanism, kinetics and thermodynamics of this reaction. Lipase was widely used as a biocatalyst to catalyze multisubstrate-multiproduct reactions (Batistella et al., 2012). Moreover, complex kinetic mechanisms have been proposed to provide descriptions of the lipase-catalyzed reactions. Many studies showed that lipase-catalyzed esterification reaction could be described by a Ping-Pong kinetic model (Awang et al., 2004; Chulalaksananukul et al., 1990, 1992). Especially, several
mechanisms have been proposed to explain lipase-catalyzed reactions containing substrate inhibition (Al-Zuhair, 2005; Mestri and Pai, 1995; de Castro et al., 1997). In these studies, a deadend complex formatted by one of the substrate and lipase was introduced to explain substrate inhibition phenomenon. So far, many ascorbyl esters such as ascorbyl oleate (ReyesDuarte et al., 2011), ascorbyl palmitate (Burham et al., 2009), ascorbyl linoleate (Watanabe et al., 2008) and ascorbyl benzoate (Lv et al., 2007) have been synthesized by enzymatic esterification in organic solvent. However, the studies on the enzymatic kinetics and thermodynamics were rarely reported. In this paper, it was found that l-ascorbic acid, one of the substrate, could generate inhibition on the activity of Lipozyme TLIM lipase. Thus the kinetics of lipase-catalyzed esterification of l-ascorbic acid using vinyl acetate as acyl donor was investigated. The kinetic model of the Ping-Pong mechanism having substrate inhibition was built and the kinetic and thermodynamic parameters were evaluated. These results would provide some valuable informations in enzymatic synthesis of l-ascorbyl ester. 2. Materials and methods 2.1. Materials
∗ Corresponding author at: College of Pharmacy, Hebei University, Baoding 071002, China. Tel.: +86 312 5971107; fax: +86 312 5971107. E-mail address: [email protected] (D.-H. Zhang). 0168-1656/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jbiotec.2013.10.033
This enzyme, Lipozyme TLIM (Novo Industri, Bagsvaerd, Denmark), was a preparation of a Thermomyces lanuginosus lipase immobilized on silica gel. l-Ascorbic acid (vitamin C, Vc) was
D.-H. Zhang et al. / Journal of Biotechnology 168 (2013) 416–420
purchased from Sigma Chemical Co. (St. Louis, MO, USA). Vinyl acetate (VAc, 99%) was obtained from Fuchen Chemical Co. (Tianjin, China). Analytical grade acetone was from Kewei Chemical Co. (Tianjin, China).
E
2.2. Experimental procedure For the standard reaction, Lipozyme TLIM (20 mg) was added to a reaction mixture containing l-ascorbic acid and vinyl acetate in 1 mL acetone. The concentrations of l-ascorbic acid and vinyl acetate were, respectively, varied from 3 to 12 mmol/L and 6 to 22 mmol/L. Then the reaction mixture of different substrate molar ratio was incubated in a temperature-controlled shaker at 170 rpm at a certain temperature. After 2 h, 50 L of the reaction mixture was withdrawn and evaporated, and then the residue was dissolved in methanol/water (5/5, v/v) and analyzed by HPLC. All experiments were conducted in triplicate and the mean values were calculated.
417
A
P
B
EA
FP F FB
Q
EQ
E
B EB (the dead-end complex) Scheme 2. Schematic representation of the Ping-Pong Bi-Bi mechanism having substrate inhibition.
where v is the enzymatic reaction rate of the esterification; Vmax is the maximum rate of the esterification; [A] is the concentration A and K B are the Michaelis of A; [B] is the concentration of B; Km m constants of A and B, respectively.
2.3. HPLC analysis 3.2. Ping-Pong Bi-Bi mechanism having substrate inhibition
3. Kinetic model Two kinetic mechanisms (Mukesh et al., 1997; Maugard et al., 2000) were supposed in the present study. The first was called PingPong Bi-Bi mechanism. The second model was assumed Ping-Pong Bi-Bi mechanism having substrate inhibition. They are described as follows. 3.1. Ping-Pong Bi-Bi mechanism Previous studies have shown that the reaction of lipasecatalyzed esterification could be described by the Ping-Pong kinetic model (Lima et al., 1996). To understand the Ping-Pong Bi-Bi mechanism better, a schematic representation of this reaction mechanism using the Cleland notation (Lima et al., 1996) was given in Scheme 1. As shown, acyl donor (A) is bound to the enzyme at first to form the acyl-enzyme complex. As soon as one product (P) is formed and then released, acyl acceptor (B) binds to the modified enzyme (substituted form of the free enzyme) to form the second product (Q). The reaction rate equation is given by Eq. (1) (Lima et al., 1996):
v=
Vmax [A][B]
(1)
A [B] + K B [A] + [A][B] Km m
A
P
B
Q
Several recent studies (Al-Zuhair, 2005; Mestri and Pai, 1995; de Castro et al., 1997) on enzymatic esterification containing substrate inhibition attempted to describe the reaction mechanism by a modified Ping-Pong kinetic model (Scheme 2). Compared with Scheme 1, the step of a dead-end complex formation between substrate and free enzyme has been added in Scheme 2. Based on this proposed mechanism, the expression for the reaction rate is given by Eq. (2) (Yadav and Borkar, 2008):
v=
Vmax [A][B]
(2)
A [B] + K B [A] + [A][B] + (K A [B]2 /K B ) Km m m i
where KiB is the inhibition constant of B; other notations are the same as that in Eq. (1). 4. Results and discussion 4.1. Kinetics of enzymatic esterification of l-ascorbic acid Lipase-catalyzed esterification of l-ascorbic acid was carried out at 40 ◦ C for 2 h by changing substrate concentration and the results were listed in Supplementary Information (Table S1). After that, the reaction rates were calculated and shown in Fig. 1. As can be seen, for a given vinyl acetate concentration, the reaction rate decreased strangely with the increase in l-ascorbic acid concentration, which indicated that, to some extent, l-ascorbic acid had an observable inhibition effect on the enzymatic reaction. For example, lipase
0.7 0.6 V (mmol/(L*h))
The reaction resultants were analyzed using high-performance liquid chromatography (HPLC, ChuangXinTongHeng Science & Technology Co. Ltd., China) with a C-18 column (ZORBAX 300SBC18 4.6 mm ID × 250 mm (5 m), Agilent Technologies, Palo Alto, CA) and a UV detector at 254 nm. A 20-L diluted sample was injected, and the elution was done with methanol/water/acetic acid (50/50/0.1, v/v/v) at a flow rate of 0.5 mL min−1 . Reaction conversion was calculated in terms of the mole percentage of esterification, based on the ratio of consumed l-ascorbic acid to the total amount of l-ascorbic acid before reaction.
0.5 0.4 0.3 0.2 0.1 2
E
EA
FP
F FB
EQ
E
Scheme 1. Schematic representation of the Ping-Pong Bi-Bi mechanism.
4
6 8 10 (mm ol/L) C Vc
12
Fig. 1. Effect of l-ascorbic acid (Vc) concentration on the reaction rate at different vinyl acetate (VAc) concentration at 40 ◦ C. (Vinyl acetate concentrations: () 6 mmol/L, (䊉) 10 mmol/L, () 14 mmol/L, () 18 mmol/L, () 22 mmol/L.)
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activity declined from 33.65 to 28.29 U/g Lipozyme TLIM with the increase in l-ascorbic acid concentration from 3 to 12 mmol/L when vinyl acetate concentration was at 22 mmol/L. (One unit of lipase activity (U) was defined as the amount of enzyme that synthesized 1 mol l-ascorbyl acetate per hour under the assay conditions.) Fig. 2 (40 ◦ C) shows the Lineweaver–Burk plot of reciprocal of the reaction rates versus reciprocal of vinyl acetate concentrations at different l-ascorbic acid concentrations at 40 ◦ C. As observed, the Lineweaver–Burk plot revealed that lipase-catalyzed esterification of l-ascorbic acid typically obeyed classical Michaelis–Menten kinetics. However, the four fitting straight lines in Fig. 2 (40 ◦ C) were found to be unparallel to each other, which suggested that the enzymatic reaction did not follow simple Ping-Pong Bi-Bi mechanism (Scheme 1) (Burham et al., 2009). Moreover, when l-ascorbic acid concentration increased, the slop of the fitting straight line was raised and the intercept was reduced. These results agreed well with the existence of substrate inhibition in lipase-catalyzed synthesis of l-ascorbyl ester, which implied that l-ascorbic acid was able to bind lipase to form a dead-end enzyme-substrate complex (Al-Zuhair, 2005; Yadav and Borkar, 2008; Goto et al., 1994). As a
consequence, the formation of product molecules would be prohibited. Taking these results together, Ping-Pong Bi-Bi mechanism with l-ascorbic acid inhibition (Scheme 2) was thus proposed to describe this reaction. According to the Ping-Pong mechanism having substrate inhibition in Scheme 2, vinyl acetate (A) bound first to free enzyme (E) and formed an enzyme-acyl complex (EA). Then EA released the modified enzyme (F) and the first product acetaldehyde (P). After that, l-ascorbic acid (B) reacted with F to give the complex FB and further released free enzyme (E) and the second product ascorbyl acetate (Q). On the other hand, l-ascorbic acid (B) could also bind to free lipase before the bonding of vinyl acetate to lipase, resulting in the formation of a dead-end complex (EB) and prohibiting the formation of product molecules. By taking the reciprocal of Eq. (2) and arranging terms, we got Eq. (3):
1
v
=
A K A [B] Km + m B Vmax Vmax Ki
1 + [A]
1+
B Km [B]
1 Vmax
(3)
Fig. 2. The Lineweaver–Burk plot of reciprocal of the reaction rates versus reciprocal of vinyl acetate (VAc) concentrations at different l-ascorbic acid (Vc) concentrations at 40, 0, 15, 25, and 50 ◦ C, respectively. (l-Ascorbic acid concentrations: () 3 mmol/L, () 6 mmol/L, () 9 mmol/L, and (䊉) 12 mmol/L.)
D.-H. Zhang et al. / Journal of Biotechnology 168 (2013) 416–420 Table 1 Kinetic parameters of lipase-catalyzed esterification of l-ascorbic acid at different temperatures.
419
Table 2 The reaction rate constant of lipase-catalyzed esterification of l-ascorbic acid at different temperatures.
T (◦ C)
Vmax (mmol/(L h))
A Km (mmol/L)
B Km (mmol/L)
KiB (mmol/L)
T (◦ C)
k1 (×10−15 L/mol h)
k2 (×10−15 L/mol h)
0 15 25 40 50
16.59 13.42 28.88 11.75 24.00
1165.82 597.99 574.59 224.50 94.74
26.60 15.23 31.26 13.92 24.62
7.07 9.20 8.36 13.25 1.50
0 15 25 40 50
1.76 3.61 3.89 10.46 25.58
77.01 141.92 71.53 168.74 98.43
From Eq. (3), Eqs. (4) and (5) were derived as follows: A Km S= [B] + Vmax Vmax KiB
I=
B Km
1 1 + Vmax [B] Vmax
40
38 (5)
4.2. Thermodynamics of enzymatic esterification of l-ascorbic acid This reaction of lipase-catalyzed l-ascorbate synthesis could be considered as a two-step reaction. The first step was that vinyl acetate (A) bound to enzyme (E) and formed the first product acetaldehyde (P). The second step was that l-ascorbic acid (B) reacted with (F) and gave the second product l-ascorbyl acetate (Q). To investigate the thermodynamics of the two steps, the reactions were carried out at different temperatures (The temperature is between 0 and 50 ◦ C because Lipozyme TLIM could exhibit its optimal activity at 50 ◦ C and a higher temperature than 50 ◦ C might cause Lipozyme TLIM inactivation.) and the results were listed in Supplementary Information (Table S2). Similarly, the reaction rates were calculated and the relationships between 1/v and the reciprocal of vinyl acetate concentration were shown in Fig. 2 (0, 15, 25, 50 ◦ C). As shown, the inhibition of l-ascorbic acid on lipase activity was also discovered in Fig. 2 (0, 15, 25, 50 ◦ C), which had been already demonstrated in Fig. 2 (40 ◦ C). Depending on these data, the values of the kinetic parameters were also calculated by means of Eqs. (4) and (5) and shown in A decreased gradually with the Table 1. As presented, the value of Km B did not increase in reaction temperature whereas the value of Km change obviously, which implied that enhancing reaction temperature could notably increase the affinity between vinyl acetate and enzyme. In addition, the values of the inhibition constant (KiB ) in Table 1 seemed to suggest that the inhibition effect of l-ascorbic acid on lipase was lower at high temperature than that at low temperature. Furthermore, the reaction rate constant could be calculated from Eq. (6) referring to absolute reaction rate theory and Eyring hypothesis (Sehgal et al., 2002; Porto et al., 2006). kB T hKm
37 36
where S is the slope in Eq. (3), I is the intercept in Eq. (3). The slope and the intercept in Fig. 2 (40 ◦ C) gave the S and I valB and V ues, respectively. According to Eq. (5), we could obtain Km max A and values from the relation between I and 1/[B]. Furthermore, Km KiB values could also be determined from the relation between S and [B] according to Eq. (4). As a consequence, the kinetic parameA was much ters were calculated and listed in Table 1. As shown, Km B larger than Km (1/Km represents the enzyme affinity to substrate), which suggested that the affinity of l-ascorbic acid to enzyme was larger than the affinity of vinyl acetate to enzyme. These results appeared to verify that l-ascorbic acid was easy to combine with enzyme and form a dead-end complex.
k=
39
(4)
ln k
A Km
(6)
where k is the reaction rate constant; kB is Boltzman constant (1.38065 × 10−24 J/K); h is Plank constant (6.6261 × 10−34 J s); T is
35 34 3
3.2
3.4 3.6 3 -1 10 ×T
3.8
Fig. 3. Arrhenius plot of ln k1 vs T−1 () and ln k2 vs T−1 ().
the absolute temperature; Km is the Michaelis constant for enzymatic reaction. Table 2 listed the reaction rate constants of the two steps (k1 and k2 ). As shown, k1 values were much lower than k2 values, indicating that the first-step reaction was slow while the second-step reaction was fast. It could be concluded that the first-step reaction was the rate-limiting reaction. Depending on these k1 and k2 values at various reaction temperatures, the activation energy Ea could be further estimated according to Arrhenius equation (7). ln k = −
Ea + ln A RT
(7)
where k is the reaction rate constant; A is the frequency factor; Ea is the activity energy; R is molar gas constant. The Arrhenius plot was made on the basis of ln k vs reciprocal of temperature (Fig. 3). As presented, the activation energy Ea could be obtained from the slop of the straight line. As a consequence, Ea1 of the first-step reaction was estimated to be 37.31 kJ/mol, while Ea2 of the second-step reaction was 4.94 kJ/mol. Ea1 was much higher than Ea2 , which further confirmed that the second-step reaction was much easier than the first-step reaction. That was, the firststep reaction was the rate-limiting reaction. In addition, this result indicated that the first-step reaction rate was more sensitive to temperature based on Arrhenius equation. 5. Conclusions The kinetics of the enzymatic esterification of l-ascorbic acid by Lipozyme TLIM in acetone had been investigated. The results showed that the reaction mechanism could be described well to follow Ping-Pong Bi-Bi mechanism having substrate inhibition. Furthermore, the kinetic and thermodynamic parameters were estimated. It seemed that the inhibition of l-ascorbic acid on Lipozyme TLIM could be depressed at high temperature. The results further showed that activation energy of the first-step reaction was higher than that of the second-step reaction, suggesting that the first-step reaction was the rate-limiting reaction and could be easily improved by enhancing temperature. In conclusion, l-ascorbic acid concentration could not be blindly raised in biosynthesis of l-ascorbyl ester in order to increase the yield.
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