Enzyme and Microbial Technology 64–65 (2014) 60–66
Contents lists available at ScienceDirect
Enzyme and Microbial Technology journal homepage: www.elsevier.com/locate/emt
Phospholipase A2 -catalyzed acylation of lysophospholipids analyzed by experimental design Gabriele Lux, Johanna Mansfeld, Renate Ulbrich-Hofmann ∗ Institute of Biochemistry and Biotechnology, Martin-Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3, D-06120 Halle, Germany
a r t i c l e
i n f o
Article history: Received 30 April 2014 Received in revised form 14 July 2014 Accepted 14 July 2014 Available online 22 July 2014 Keywords: Phospholipid synthesis Modified D-optimal design Plackett-Burman design Phospholipase A2 Porcine pancreas Bee venom
a b s t r a c t The catalytic potential of phospholipase A2 (PLA2 ) for the synthesis of phospholipids with defined fatty acid structure in the sn-2 position has been underestimated hitherto because of very low conversion in most organic solvents. One of the most suitable solvents for PLA2 -catalyzed phospholipid synthesis is glycerol. With the aim to analyze the effect of several interacting reaction parameters on the product yield, we studied the conversion of 1-palmitoyl-2-lyso-sn-glycero-3-phosphocholine (lyso-PC) with oleic acid as model reaction in mixtures of glycerol and methanol or ethanol by methods of experimental design. PLA2 from porcine pancreas (ppPLA2 ) and from bee venom (bvPLA2 ) were compared as catalysts. For each of the four systems, nine variables were evaluated using Plackett-Burman designs. The most significant four variables were used for subsequent modified D-optimal designs with 30 runs, yielding regression equations for describing the formation of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine as a function of the variables. In both solvent systems ppPLA2 was more appropriate for the acylation reaction than bvPLA2 . Methanol proved to be more convenient as co-solvent than ethanol. The catalysis by ppPLA2 was more sensitive toward the variables temperature and concentration of Tris–HCl, whereas the reaction time and enzyme activity were more important in the acylation by bvPLA2 . Conversion up to 87 (ppPLA2 ) and 50% (bvPLA2 ) can be anticipated. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Hydrolases such as lipases, esterases and proteases are well established tools in organic synthesis [1]. Often they are used for the catalysis of condensation or transesterification reactions, which are in the reverse direction of their natural hydrolyzing function. Water-poor media and/or the removal of reaction water are common strategies to increase product yields. Phospholipases also have a synthetic potential, which is widely exploited for the exchange of head groups in phospholipids by phospholipase D [2,3]. Phospholipases A2 (PLA2 s), which have been an important research object in the biochemical literature for many years, have only been poorly investigated with respect to their application as biocatalysts in phospholipid synthesis [4]. Secretory PLA2 from bovine
Abbreviations: bvPLA2 , phospholipase A2 from bee venom; DOPC, 1,2dioleoyl-sn-glycero-3-phosphocholine; lyso-PC, 1-palmitoyl-2-lyso-sn-glycero-3phosphocholine; PLA2 , phospholipase A2 ; POPC, 1-palmitoyl-2-oleoyl-sn-glycero3-phosphocholine; ppPLA2 , phospholipase A2 from porcine pancreas; SDS, sodium dodecylsulfate. ∗ Corresponding author. Tel.: +49 345 5524864; fax: +49 345 5527303. E-mail address: [email protected] (R. Ulbrich-Hofmann). http://dx.doi.org/10.1016/j.enzmictec.2014.07.003 0141-0229/© 2014 Elsevier Inc. All rights reserved.
or porcine pancreas (ppPLA2 ) is well-known and used industrially for the hydrolysis of phospholipids to lysophospholipids which are important emulsifiers in the food and pharmaceutical industry. The reverse reaction, which is attractive for the synthesis of phospholipids with defined fatty acid structure (Fig. 1), seems to be less efficient. Lipases are often preferred for the synthesis of phospholipids with defined fatty acid structure [5,6] because they tolerate a lower water content and do not require Ca2+ ions for catalytic activity. However, as structured phospholipids with polyunsaturated fatty acids such as docosahexaenoic acid, eicosapentaenoic or conjugated linoleic acid in the sn-2 position have received increasing attention for medical, nutritional and cosmetic purposes the targeted incorporation of specified fatty acids by PLA2 s has remained in the focus of research [7–9]. The first acylation reactions by PLA2 , which were performed with polyunsaturated fatty acids in microemulsion systems [10] or with oleic acid in benzene and toluene [11] showed very low acylation yields ( H2 O content > reaction time > ppPLA2 activity > oleic acid concentration > CaCl2 concentration. In the glycerol/EtOH system, the lyso-PC concentration also played a significant role, followed by the other factors in the order of lyso-PC concentration > reaction time > ppPLA2 activity > oleic acid
Table 2 Limits and effects of the variables in the acylation reaction with ppPLA2 screened by Plackett-Burman design. Variable
Lyso-PC (mM) Temperature (◦ C) PLA2 (U mL−1 ) Reaction time (h) Oleic acid (mM) CaCl2 (mM) Tris–HCl (mM) Glycerol content (%, v/v) H2 O (mg mL−1 )
Limits
Effects
Lower limit
Upper limit
Glycerol/MeOH
Glycerol/EtOH
5.04 30 50 24 25 2.5 5 95 2
25.22 40 250 72a ; 96b 100 30 60 60 10
−7.05* 6.85* 2.78 3.03 1.13 0.25 15.60* −15.53* 3.08
−15.03* 4.50 6.28 7.50* −5.98 −5.18 16.55* −20.08* −0.55
Factor effects were estimated as described in Section 2. The asterisk (*) indicates the variables selected for the modified D-optimal design. a In glycerol/MeOH. b In glycerol/EtOH.
G. Lux et al. / Enzyme and Microbial Technology 64–65 (2014) 60–66
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Fig. 2. Acylation of lyso-PC by ppPLA2 in glycerol/MeOH and glycerol/EtOH at different glycerol percentages. (A) The formation of POPC in glycerol/MeOH was calculated according to Eq. (1) and is shown as a function of the lyso-PC concentration and temperature. The reaction time was 18 h. (B) The formation of POPC in glycerol/EtOH was calculated according to Eq. (2) and is shown and as a function of the lyso-PC concentration and the reaction time. The temperature was 40 ◦ C. The different levels in both diagrams represent glycerol contents of 55, 65, 75, 85, and 95% (v/v) (from bottom to top). The Tris–HCl concentration was 55 mM in all cases. All other parameters were as specified in the section 2.7.
concentration > CaCl2 concentration > temperature > H2 O content (Table 2). In the second step of the experimental design analysis, the two most significant factors (MeOH or EtOH content and Tris–HCl concentration) and additionally the factors lyso-PC and temperature for the glycerol/MeOH system or lyso-PC and reaction time for the glycerol/EtOH system were selected for a series of 30 experiments according to a modified D-optimal design for four factors. Within the range of the limits (Table 2), several levels as specified in Section 2.7 were chosen for the variables. The remaining five parameters were kept constant in all the experiments. The evaluation of the results as exemplified in Table S1 (Supplementary material) yielded the following regression equations for the system glycerol/MeOH: Y = 96.14692 − 8.01833 X1 + 0.12098 X1 X3 + 0.00833 X3 X4 +0.01782(X2 )2 − 0.02246(X3 )2 ,
(1)
where Y is the POPC formation (%), X1 is the reaction temperature (◦ C), X2 is the lyso-PC concentration (mM); X3 is the content of
glycerol in the mixture of glycerol/MeOH (%, v/v), and X4 is the Tris–HCl concentration (mM). Correspondingly, for the system glycerol/EtOH the following equation was obtained: Y = 0.8474X1 + 3.8016X2 − 1.2018X4 − 0.0224X1 X2 −0.0094X1 X3 + 0.0063X1 X4 − 0.0208X2 X4 + 0.0228X3 X4 −0.0691(X2 )2 + 0.064(X3 )2 − 59.6775,
(2)
where the symbols have the same meaning as in Eq. (1) except for X1 , which is the reaction time (h). The regression equations allow the prediction of product formation within the scope of the factors considered. Moreover, they enable the illustration of interdependences in the interplay of the most significant factors on the POPC formation. Thus, the different areas in Fig. 2A and B, showing the results at different ratios of glycerol/MeOH or EtOH, demonstrate the strong negative influence of the co-solvent content on the POPC formation. In contrast, the slope and curvature of the areas show the moderate trends of effects of the lyso-PC concentration and the reaction temperature
Fig. 3. Acylation of lyso-PC by ppPLA2 in glycerol/MeOH and glycerol/EtOH at different Tris–HCl concentrations. (A) The formation of POPC in glycerol/MeOH was calculated according to Eq. (1) and is shown as a function of the lyso-PC concentration and the glycerol content. The reaction time was 18 h. (B) The formation of POPC in glycerol/MeOH was calculated according to Eq. (2) and is shown as a function of the lyso-PC concentration and the glycerol content. The reaction time was 24 h. The different levels in both diagrams represent Tris–HCl concentrations of 15, 25, 35, 45, and 55 mM (from bottom to top). The temperature was 40 ◦ C in all cases. All other parameters were as specified in the section 2.7.
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G. Lux et al. / Enzyme and Microbial Technology 64–65 (2014) 60–66
Table 3 Limits and effects of the variables in the acylation reaction with bvPLA2 screened by Plackett-Burman design. Factor effects were estimated as described in Section 2. The asterisk (*) indicates the variables selected for the modified D-optimal design. Variable
Lyso-PC (mM) Temperature (◦ C) PLA2 (U mL−1 ) Reaction time (h) Oleic acid (mM) CaCl2 (mM) Tris–HCl (mM) Glycerol content (%, v/v) H2 O (mg mL−1 )
Limits
Effects
Lower limit
Upper limit
Glycerol/MeOH
Glycerol/EtOH
5.04 30 50 24 25 5 25 100 4
25.22 40 250 144 75 30 65 90 10
−6.21* 2.04 6.96* 8.46* −4.09 1.51 3.39 −2.86* −3.36
−6.20* 1.83 6.48* 7.00* −4.58 1.20 3.18 −5.10* −4.23
on the POPC formation (in glycerol/MeOH; Fig. 2A) or of the lyso-PC concentration and the reaction time (in glycerol/EtOH; Fig. 2B). The POPC formation in the glycerol/MeOH and the glycerol/EtOH systems under similar conditions were compared (Fig. 3) and this shows that MeOH as co-solvent yielded essentially better results than EtOH. In both systems an increasing Tris–HCl concentration (15–55 mM) promoted the POPC formation as the different areas in Fig. 3A and B demonstrate. The rates of POPC formation, however, are always higher in the glycerol/MeOH system than in the glycerol/EtOH system. Under convenient conditions, e.g., 95% (v/v), glycerol/5% (v/v), methanol, 55 mM Tris–HCl, 25.2 mM lyso-PC, 40 ◦ C, 2 mg mL−1 water, 18 h, 150 U mL−1 ppPLA2 , 50 mM oleic acid, 10 mM CaCl2 ), POPC formation up to 87% can be anticipated. 3.3. Acylation of lyso-PC with bvPLA2 studied by methods of experimental design The acylation of lyso-PC by oleic acid, catalyzed by bvPLA2 , was analyzed in similar way. Foregoing experiments (not shown) had indicated that the acylation by bvPLA2 proceeds much more slowly than by ppPLA2 . Furthermore, the addition of MeOH or EtOH had a beneficial effect on the progress of the reaction. Therefore, the limits of the corresponding variables for the Plackett-Burman design were changed (Table 3). Moreover, the limits of the concentrations of oleic acid, Tris–HCl, CaCl2 and water were modified in response to the experiences with the designs described above and the results of exploratory experiments with this enzyme. Table 3 shows the results of the factor analyses. The reaction time, the PLA2 activity, and the lyso-PC concentration proved to be significant for the reaction progress in the glycerol/MeOH system as well as in the
glycerol/EtOH system. In the EtOH-containing system, the glycerol content was also significant. Therefore, these four factors were used as variables in the subsequent modified D-optimal designs. The evaluation of the results yielded the following regression equations for the system glycerol/MeOH: Y = 0.761X1 + 0.0014X2 X3 − 0.0059X2 X4 +0.0011X3 X4 − 59.1746,
(3)
where Y is the POPC formation (%), X1 is the content of glycerol in the mixture of glycerol/MeOH (%), X2 is the lyso-PC concentration (mM); X3 is the bvPLA2 activity (U mL−1 ), and X4 is the reaction time. The remaining five parameters were kept constant with the levels as specified in Section 2. Correspondingly, for the system glycerol/EtOH the following equation was obtained: Y = 0.0007X1 X3 − 0.0124X1 X2 + 0.0015X1 X4 − 0.0031X2 X3 −0.0076X2 X4 + 0.0005 X3 X4 + 0.0044(X1 )2 +0.052(X2 )2 − 35.4118,
(4)
where the symbols have the same meaning as in Eq. (3) and the constant parameters are identical to those in the glycerol/MeOH system. Fig. 4 demonstrates that the reaction time had the most important influence on the POPC formation in both systems when working with bvPLA2 . After 24 h, the conversion was below 20% under all conditions of the range covered. A higher bvPLA2 activity or glycerol content was able to increase the POPC formation only marginally, whereas longer reaction times were able to promote
Fig. 4. Acylation of lyso-PC by bvPLA2 in glycerol/MeOH (A) and glycerol/EtOH (B). The formation of POPC was calculated according to Eqs. (3) and (4), respectively, and is shown as a function of the PLA2 activity and the glycerol content at a reaction time of 24, 48, 72, 96, 120 and 144 h (from bottom to top). The lyso-PC concentration was 5 mM. All other parameters were as specified in Section 2.7.
G. Lux et al. / Enzyme and Microbial Technology 64–65 (2014) 60–66
the conversion. The maximum POPC formation, however, did not exceed 50%, even after a reaction time of 144 h. 4. Discussion Methods of experimental design proved to be convenient to evaluate acylation reactions catalyzed by pp- and bvPLA2 and can also be recommended for similar biocatalytic systems that are governed by multiple interdependent factors. A limited set of experiments allows the thorough evaluation of the influence of several factors on the conversion. In the present study the analysis of nine factors was performed using a Plackett-Burman design with 16 experiments to quantify the effects of these factors on the conversion (Tables 2 and 3). The four most significant factors were analyzed further by a modified D-optimal design comprising 30 experiments yielding the regression equations for a quantitative description of the multifactorial system. The comparison of pp- and bvPLA2 as catalysts revealed a general superiority of ppPLA2 in the acylation of lyso-PC with oleic acid, where conversions up to 87% can be obtained within 24 h, whereas the maximum rates with bvPLA2 were 50% after 144 h only. These results suggest that ppPLA2 is principally more appropriate to catalyze acylation reactions than bvPLA2 . The lower catalytic power of bvPLA2 in the acylation reaction seems to be mainly caused by the subtle differences in the active site rendering the enzyme catalytically less effective than by a lower stability toward solvent inactivation. This is because the reaction time plays a more important role for the conversion than the co-solvent content. Interestingly, the acylation potential of ppPLA2 is strongly dependent on the Tris–HCl concentration in the system (Table 2, Fig. 3). Similar salt-activating effects have been reported for the transesterification by subtilisin [30], thermolysin [31], lipase [32], and phospholipase D [33,34], where KCl was added during lyophilization; however, the reasons for these effects are not clear. The difference in the salt effects with ppPLA2 and bvPLA2 (Table 3) supports the notion that the effects are related to the efficiency of catalysis more than to partitional or diffusional effects. Glycerol seems to be very advantageous as solvent for the reaction of lyso-PC with oleic acid. High activity coefficients of the fatty acids in this solvent as suggested by Adlercreutz et al. [5] or a stimulation of the enzyme activity as observed for membrane-bound PLA2 from bovine adrenal medullae [35] might be the reason for this beneficial solvent effect. By addition of co-solvents such as MeOH or EtOH, which reduce the viscosity of the systems and, therefore facilitate product separation, the conversions were decreased. However, at contents of these co-solvents
Contents lists available at ScienceDirect
Enzyme and Microbial Technology journal homepage: www.elsevier.com/locate/emt
Phospholipase A2 -catalyzed acylation of lysophospholipids analyzed by experimental design Gabriele Lux, Johanna Mansfeld, Renate Ulbrich-Hofmann ∗ Institute of Biochemistry and Biotechnology, Martin-Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3, D-06120 Halle, Germany
a r t i c l e
i n f o
Article history: Received 30 April 2014 Received in revised form 14 July 2014 Accepted 14 July 2014 Available online 22 July 2014 Keywords: Phospholipid synthesis Modified D-optimal design Plackett-Burman design Phospholipase A2 Porcine pancreas Bee venom
a b s t r a c t The catalytic potential of phospholipase A2 (PLA2 ) for the synthesis of phospholipids with defined fatty acid structure in the sn-2 position has been underestimated hitherto because of very low conversion in most organic solvents. One of the most suitable solvents for PLA2 -catalyzed phospholipid synthesis is glycerol. With the aim to analyze the effect of several interacting reaction parameters on the product yield, we studied the conversion of 1-palmitoyl-2-lyso-sn-glycero-3-phosphocholine (lyso-PC) with oleic acid as model reaction in mixtures of glycerol and methanol or ethanol by methods of experimental design. PLA2 from porcine pancreas (ppPLA2 ) and from bee venom (bvPLA2 ) were compared as catalysts. For each of the four systems, nine variables were evaluated using Plackett-Burman designs. The most significant four variables were used for subsequent modified D-optimal designs with 30 runs, yielding regression equations for describing the formation of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine as a function of the variables. In both solvent systems ppPLA2 was more appropriate for the acylation reaction than bvPLA2 . Methanol proved to be more convenient as co-solvent than ethanol. The catalysis by ppPLA2 was more sensitive toward the variables temperature and concentration of Tris–HCl, whereas the reaction time and enzyme activity were more important in the acylation by bvPLA2 . Conversion up to 87 (ppPLA2 ) and 50% (bvPLA2 ) can be anticipated. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Hydrolases such as lipases, esterases and proteases are well established tools in organic synthesis [1]. Often they are used for the catalysis of condensation or transesterification reactions, which are in the reverse direction of their natural hydrolyzing function. Water-poor media and/or the removal of reaction water are common strategies to increase product yields. Phospholipases also have a synthetic potential, which is widely exploited for the exchange of head groups in phospholipids by phospholipase D [2,3]. Phospholipases A2 (PLA2 s), which have been an important research object in the biochemical literature for many years, have only been poorly investigated with respect to their application as biocatalysts in phospholipid synthesis [4]. Secretory PLA2 from bovine
Abbreviations: bvPLA2 , phospholipase A2 from bee venom; DOPC, 1,2dioleoyl-sn-glycero-3-phosphocholine; lyso-PC, 1-palmitoyl-2-lyso-sn-glycero-3phosphocholine; PLA2 , phospholipase A2 ; POPC, 1-palmitoyl-2-oleoyl-sn-glycero3-phosphocholine; ppPLA2 , phospholipase A2 from porcine pancreas; SDS, sodium dodecylsulfate. ∗ Corresponding author. Tel.: +49 345 5524864; fax: +49 345 5527303. E-mail address: [email protected] (R. Ulbrich-Hofmann). http://dx.doi.org/10.1016/j.enzmictec.2014.07.003 0141-0229/© 2014 Elsevier Inc. All rights reserved.
or porcine pancreas (ppPLA2 ) is well-known and used industrially for the hydrolysis of phospholipids to lysophospholipids which are important emulsifiers in the food and pharmaceutical industry. The reverse reaction, which is attractive for the synthesis of phospholipids with defined fatty acid structure (Fig. 1), seems to be less efficient. Lipases are often preferred for the synthesis of phospholipids with defined fatty acid structure [5,6] because they tolerate a lower water content and do not require Ca2+ ions for catalytic activity. However, as structured phospholipids with polyunsaturated fatty acids such as docosahexaenoic acid, eicosapentaenoic or conjugated linoleic acid in the sn-2 position have received increasing attention for medical, nutritional and cosmetic purposes the targeted incorporation of specified fatty acids by PLA2 s has remained in the focus of research [7–9]. The first acylation reactions by PLA2 , which were performed with polyunsaturated fatty acids in microemulsion systems [10] or with oleic acid in benzene and toluene [11] showed very low acylation yields ( H2 O content > reaction time > ppPLA2 activity > oleic acid concentration > CaCl2 concentration. In the glycerol/EtOH system, the lyso-PC concentration also played a significant role, followed by the other factors in the order of lyso-PC concentration > reaction time > ppPLA2 activity > oleic acid
Table 2 Limits and effects of the variables in the acylation reaction with ppPLA2 screened by Plackett-Burman design. Variable
Lyso-PC (mM) Temperature (◦ C) PLA2 (U mL−1 ) Reaction time (h) Oleic acid (mM) CaCl2 (mM) Tris–HCl (mM) Glycerol content (%, v/v) H2 O (mg mL−1 )
Limits
Effects
Lower limit
Upper limit
Glycerol/MeOH
Glycerol/EtOH
5.04 30 50 24 25 2.5 5 95 2
25.22 40 250 72a ; 96b 100 30 60 60 10
−7.05* 6.85* 2.78 3.03 1.13 0.25 15.60* −15.53* 3.08
−15.03* 4.50 6.28 7.50* −5.98 −5.18 16.55* −20.08* −0.55
Factor effects were estimated as described in Section 2. The asterisk (*) indicates the variables selected for the modified D-optimal design. a In glycerol/MeOH. b In glycerol/EtOH.
G. Lux et al. / Enzyme and Microbial Technology 64–65 (2014) 60–66
63
Fig. 2. Acylation of lyso-PC by ppPLA2 in glycerol/MeOH and glycerol/EtOH at different glycerol percentages. (A) The formation of POPC in glycerol/MeOH was calculated according to Eq. (1) and is shown as a function of the lyso-PC concentration and temperature. The reaction time was 18 h. (B) The formation of POPC in glycerol/EtOH was calculated according to Eq. (2) and is shown and as a function of the lyso-PC concentration and the reaction time. The temperature was 40 ◦ C. The different levels in both diagrams represent glycerol contents of 55, 65, 75, 85, and 95% (v/v) (from bottom to top). The Tris–HCl concentration was 55 mM in all cases. All other parameters were as specified in the section 2.7.
concentration > CaCl2 concentration > temperature > H2 O content (Table 2). In the second step of the experimental design analysis, the two most significant factors (MeOH or EtOH content and Tris–HCl concentration) and additionally the factors lyso-PC and temperature for the glycerol/MeOH system or lyso-PC and reaction time for the glycerol/EtOH system were selected for a series of 30 experiments according to a modified D-optimal design for four factors. Within the range of the limits (Table 2), several levels as specified in Section 2.7 were chosen for the variables. The remaining five parameters were kept constant in all the experiments. The evaluation of the results as exemplified in Table S1 (Supplementary material) yielded the following regression equations for the system glycerol/MeOH: Y = 96.14692 − 8.01833 X1 + 0.12098 X1 X3 + 0.00833 X3 X4 +0.01782(X2 )2 − 0.02246(X3 )2 ,
(1)
where Y is the POPC formation (%), X1 is the reaction temperature (◦ C), X2 is the lyso-PC concentration (mM); X3 is the content of
glycerol in the mixture of glycerol/MeOH (%, v/v), and X4 is the Tris–HCl concentration (mM). Correspondingly, for the system glycerol/EtOH the following equation was obtained: Y = 0.8474X1 + 3.8016X2 − 1.2018X4 − 0.0224X1 X2 −0.0094X1 X3 + 0.0063X1 X4 − 0.0208X2 X4 + 0.0228X3 X4 −0.0691(X2 )2 + 0.064(X3 )2 − 59.6775,
(2)
where the symbols have the same meaning as in Eq. (1) except for X1 , which is the reaction time (h). The regression equations allow the prediction of product formation within the scope of the factors considered. Moreover, they enable the illustration of interdependences in the interplay of the most significant factors on the POPC formation. Thus, the different areas in Fig. 2A and B, showing the results at different ratios of glycerol/MeOH or EtOH, demonstrate the strong negative influence of the co-solvent content on the POPC formation. In contrast, the slope and curvature of the areas show the moderate trends of effects of the lyso-PC concentration and the reaction temperature
Fig. 3. Acylation of lyso-PC by ppPLA2 in glycerol/MeOH and glycerol/EtOH at different Tris–HCl concentrations. (A) The formation of POPC in glycerol/MeOH was calculated according to Eq. (1) and is shown as a function of the lyso-PC concentration and the glycerol content. The reaction time was 18 h. (B) The formation of POPC in glycerol/MeOH was calculated according to Eq. (2) and is shown as a function of the lyso-PC concentration and the glycerol content. The reaction time was 24 h. The different levels in both diagrams represent Tris–HCl concentrations of 15, 25, 35, 45, and 55 mM (from bottom to top). The temperature was 40 ◦ C in all cases. All other parameters were as specified in the section 2.7.
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G. Lux et al. / Enzyme and Microbial Technology 64–65 (2014) 60–66
Table 3 Limits and effects of the variables in the acylation reaction with bvPLA2 screened by Plackett-Burman design. Factor effects were estimated as described in Section 2. The asterisk (*) indicates the variables selected for the modified D-optimal design. Variable
Lyso-PC (mM) Temperature (◦ C) PLA2 (U mL−1 ) Reaction time (h) Oleic acid (mM) CaCl2 (mM) Tris–HCl (mM) Glycerol content (%, v/v) H2 O (mg mL−1 )
Limits
Effects
Lower limit
Upper limit
Glycerol/MeOH
Glycerol/EtOH
5.04 30 50 24 25 5 25 100 4
25.22 40 250 144 75 30 65 90 10
−6.21* 2.04 6.96* 8.46* −4.09 1.51 3.39 −2.86* −3.36
−6.20* 1.83 6.48* 7.00* −4.58 1.20 3.18 −5.10* −4.23
on the POPC formation (in glycerol/MeOH; Fig. 2A) or of the lyso-PC concentration and the reaction time (in glycerol/EtOH; Fig. 2B). The POPC formation in the glycerol/MeOH and the glycerol/EtOH systems under similar conditions were compared (Fig. 3) and this shows that MeOH as co-solvent yielded essentially better results than EtOH. In both systems an increasing Tris–HCl concentration (15–55 mM) promoted the POPC formation as the different areas in Fig. 3A and B demonstrate. The rates of POPC formation, however, are always higher in the glycerol/MeOH system than in the glycerol/EtOH system. Under convenient conditions, e.g., 95% (v/v), glycerol/5% (v/v), methanol, 55 mM Tris–HCl, 25.2 mM lyso-PC, 40 ◦ C, 2 mg mL−1 water, 18 h, 150 U mL−1 ppPLA2 , 50 mM oleic acid, 10 mM CaCl2 ), POPC formation up to 87% can be anticipated. 3.3. Acylation of lyso-PC with bvPLA2 studied by methods of experimental design The acylation of lyso-PC by oleic acid, catalyzed by bvPLA2 , was analyzed in similar way. Foregoing experiments (not shown) had indicated that the acylation by bvPLA2 proceeds much more slowly than by ppPLA2 . Furthermore, the addition of MeOH or EtOH had a beneficial effect on the progress of the reaction. Therefore, the limits of the corresponding variables for the Plackett-Burman design were changed (Table 3). Moreover, the limits of the concentrations of oleic acid, Tris–HCl, CaCl2 and water were modified in response to the experiences with the designs described above and the results of exploratory experiments with this enzyme. Table 3 shows the results of the factor analyses. The reaction time, the PLA2 activity, and the lyso-PC concentration proved to be significant for the reaction progress in the glycerol/MeOH system as well as in the
glycerol/EtOH system. In the EtOH-containing system, the glycerol content was also significant. Therefore, these four factors were used as variables in the subsequent modified D-optimal designs. The evaluation of the results yielded the following regression equations for the system glycerol/MeOH: Y = 0.761X1 + 0.0014X2 X3 − 0.0059X2 X4 +0.0011X3 X4 − 59.1746,
(3)
where Y is the POPC formation (%), X1 is the content of glycerol in the mixture of glycerol/MeOH (%), X2 is the lyso-PC concentration (mM); X3 is the bvPLA2 activity (U mL−1 ), and X4 is the reaction time. The remaining five parameters were kept constant with the levels as specified in Section 2. Correspondingly, for the system glycerol/EtOH the following equation was obtained: Y = 0.0007X1 X3 − 0.0124X1 X2 + 0.0015X1 X4 − 0.0031X2 X3 −0.0076X2 X4 + 0.0005 X3 X4 + 0.0044(X1 )2 +0.052(X2 )2 − 35.4118,
(4)
where the symbols have the same meaning as in Eq. (3) and the constant parameters are identical to those in the glycerol/MeOH system. Fig. 4 demonstrates that the reaction time had the most important influence on the POPC formation in both systems when working with bvPLA2 . After 24 h, the conversion was below 20% under all conditions of the range covered. A higher bvPLA2 activity or glycerol content was able to increase the POPC formation only marginally, whereas longer reaction times were able to promote
Fig. 4. Acylation of lyso-PC by bvPLA2 in glycerol/MeOH (A) and glycerol/EtOH (B). The formation of POPC was calculated according to Eqs. (3) and (4), respectively, and is shown as a function of the PLA2 activity and the glycerol content at a reaction time of 24, 48, 72, 96, 120 and 144 h (from bottom to top). The lyso-PC concentration was 5 mM. All other parameters were as specified in Section 2.7.
G. Lux et al. / Enzyme and Microbial Technology 64–65 (2014) 60–66
the conversion. The maximum POPC formation, however, did not exceed 50%, even after a reaction time of 144 h. 4. Discussion Methods of experimental design proved to be convenient to evaluate acylation reactions catalyzed by pp- and bvPLA2 and can also be recommended for similar biocatalytic systems that are governed by multiple interdependent factors. A limited set of experiments allows the thorough evaluation of the influence of several factors on the conversion. In the present study the analysis of nine factors was performed using a Plackett-Burman design with 16 experiments to quantify the effects of these factors on the conversion (Tables 2 and 3). The four most significant factors were analyzed further by a modified D-optimal design comprising 30 experiments yielding the regression equations for a quantitative description of the multifactorial system. The comparison of pp- and bvPLA2 as catalysts revealed a general superiority of ppPLA2 in the acylation of lyso-PC with oleic acid, where conversions up to 87% can be obtained within 24 h, whereas the maximum rates with bvPLA2 were 50% after 144 h only. These results suggest that ppPLA2 is principally more appropriate to catalyze acylation reactions than bvPLA2 . The lower catalytic power of bvPLA2 in the acylation reaction seems to be mainly caused by the subtle differences in the active site rendering the enzyme catalytically less effective than by a lower stability toward solvent inactivation. This is because the reaction time plays a more important role for the conversion than the co-solvent content. Interestingly, the acylation potential of ppPLA2 is strongly dependent on the Tris–HCl concentration in the system (Table 2, Fig. 3). Similar salt-activating effects have been reported for the transesterification by subtilisin [30], thermolysin [31], lipase [32], and phospholipase D [33,34], where KCl was added during lyophilization; however, the reasons for these effects are not clear. The difference in the salt effects with ppPLA2 and bvPLA2 (Table 3) supports the notion that the effects are related to the efficiency of catalysis more than to partitional or diffusional effects. Glycerol seems to be very advantageous as solvent for the reaction of lyso-PC with oleic acid. High activity coefficients of the fatty acids in this solvent as suggested by Adlercreutz et al. [5] or a stimulation of the enzyme activity as observed for membrane-bound PLA2 from bovine adrenal medullae [35] might be the reason for this beneficial solvent effect. By addition of co-solvents such as MeOH or EtOH, which reduce the viscosity of the systems and, therefore facilitate product separation, the conversions were decreased. However, at contents of these co-solvents
