Synthesis of Ibuprofen Monoglyceride Using Novozym®435: Biocatalyst Activation and Stabilization in Multiphasic Systems
Round 1
Reviewer 1 Report
This manuscript describes the enzymatic esterification of glycerol and ibuprofen in two triphasic systems composed of toluene+ibuprofene and glycerol or glycerol-water liquid phases, and a solid phase: the immobilized enzyme. The roles of water content in glycerol, reagent concentration, temperature, and enzyme concentration on enzyme activity and, for temperature, on enzyme stability have been investigated. I consider this work technically sound and well-performed. The manuscript is well written, but I have a few questions/issues that are enumerated below:
1. The information given in Figure 1 is not very clear. According to the main text, in triphasic system 2, water will be added to the reaction mixture at the start of the reaction. However, the information in the figure indicates that the water is a product of esterification and no additional water is added.
2. The use of subscripts on the Y-axis title of the graph is inconsistent. For example, “r0” is used in Figures 2,3,4,5, and Table 1, while “r0” is used in Figures 6,8.
3. Figure 2 shows that the initial reaction rates of esterification ranged from about 0.0016 to 0.0032 mol/L/min when the enzyme concentration was between 10 and 40 g/L. But according to Figure 1 in Ravelo’s paper (Catalysts 2020, 10:76), under similar reaction conditions and enzyme concentrations (without toluene only), the initial esterification rates were between 4×104 and 10×104 mol/L/min. What is the reason for this big difference?
4. On the Y-axis of Figure 3, the “,” in the numbers should be changed to “.”.
Also, there doesn’t seem to be a separate Discussion section in the manuscript. The Results section should be “Results and Discussion”.
Author Response
Thank you indeed for your constructive and valuable suggestions and corrections (in black italic font). We have carefully revised the manuscript according to your suggestions. The itemized responses (in blue) and the added text/references in the main manuscript (in red font) are attached as follows:
Comment 1
This manuscript describes the enzymatic esterification of glycerol and ibuprofen in two triphasic systems composed of toluene+ibuprofene and glycerol or glycerol-water liquid phases, and a solid phase: the immobilized enzyme. The roles of water content in glycerol, reagent concentration, temperature, and enzyme concentration on enzyme activity and, for temperature, on enzyme stability have been investigated. I consider this work technically sound and well-performed. The manuscript is well written, but I have a few questions/issues that are enumerated below:
- The information given in Figure 1 is not very clear. According to the main text, in triphasic system 2, water will be added to the reaction mixture at the start of the reaction. However, the information in the figure indicates that the water is a product of esterification and no additional water is added.
We are grateful to the reviewer for this observation. Although we have tried to explain this by including two reaction systems (triphasic system 1 with no added water at zero time and triphasic system 2 with added water before the reaction), this seems not to be clear. Thus, we have modified the figure to make it clearer, if possible. Water, in esterification systems, as the reviewer comments, is present when the reaction takes place, as it is one of the products. However, before it takes place, the addition of water seems to be a booster of this reaction with this enzyme (it has been also observed by other authors) if limited amounts are added. Possibly the reason is the reduction of viscosity of the glycerol phase and, thus, a faster mass transfer within the pores of the immobilized enzyme.
Comment 2
The use of subscripts on the Y-axis title of the graph is inconsistent. For example, “r0” is used in Figures 2,3,4,5, and Table 1, while “r0” is used in Figures 6,8.
Thank you for the observation. We have used the last notation “r0” for all cases.
Comment 3
Figure 2 shows that the initial reaction rates of esterification ranged from about 0.0016 to 0.0032 mol/L/min when the enzyme concentration was between 10 and 40 g/L. But according to Figure 1 in Ravelo’s paper (Catalysts 2020, 10:76), under similar reaction conditions and enzyme concentrations (without toluene only), the initial esterification rates were between 4×104 and 10×104 mol/L/min. What is the reason for this big difference?
We are grateful to the reviewer for the comment. In reality, when the y-axis title indicates the initial reaction rate multiplied by a factor of 104, it means that a value of 25 in the axis is, in reality, a value of 25x10-4. This is done to avoid a large number of figures in the axis, for example, 0.0025. However, and for the sake of clarity, we have commented on it on the first figure where this form of presenting the y-axis values have been used and we have expressed the initial reaction rate in it this way in all cases to facilitate an straight-forward comparison. Please, see in page 4, after Figure 4:
“It can be seen that the initial esterification rate increases steadily with enzyme concentration from 16·10-4 to 33·10-4 mol L-1 min-1, as expected in a Michaelis-Menten behavior (first order with respect to enzyme).”
Comment 4
On the Y-axis of Figure 3, the “,” in the numbers should be changed to “.”.
We have modified the values in the y-axis to avoid the use of commas, thank you.
Comment 5
Also, there doesn’t seem to be a separate Discussion section in the manuscript. The Results section should be “Results and Discussion”.
We have corrected the title of the Results section. Now it reads “Results and Discussion”, as both are combined for a better understanding of the manuscript. Thank you for your indication.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
This work describes the optimization of ibuprofen monoglyceride production by immobilized enzyme in a three-phase system. In addition to solvent and temperature optimization, mass transfer within the immobilized catalyst was also studied. A mathematical model was established that showed good agreement with the experimental data.
I propose a paper for publication with some further improvements:
General:
1. The data on the possible leaching of enzymes were not given. In general, the use of a magnetic stirrer to carry out the process with an enzyme immobilized on a solid support is not preferable, since the shear forces may affect the binding forces of enzyme and support
2. The kinetic parameters were estimated by directly fitting the model to the data collected in the batch reactor. Thus, the claim that deactivation exists in one three-phase system and can be neglected in another is questionable. Deactivation is not a problem to be verified. It is only necessary to measure the initial reaction rate of the immobilized enzyme used in the reaction
Specific:
1. The introduction is too long and the importance of the reaction performed is explained very briefly.
2. The " Results" chapter should be renamed to "Results and discussion"
3. Figure 2b and line 156; Figure 4b and line 190: Was the yield measured for all reactions at the same time, or was it measured when equilibrium was reached? Also, the thermodynamics of the reaction do not depend on the concentration of the enzyme. Reduced yield may have resulted from inactivation of the enzyme during prolonged use.
4. Line 166 and Figure 3, The volumetric ratio of Gly/Tol is not the same in the text and in the figure (text: 1/20; figure 1/10)
5. Lines 170-175, The initial esterification rate depends proportionally on the enzyme concentration. To claim this, the concentration of free enzyme and immobilized enzyme would have to be the same. The note also contradicts lines 610-612
6. Lines 204-207 - Figure 6.5 is not available. From the data given in Figure 5a, it cannot be concluded that enzyme inactivation occurs at higher temperature and ibuprofen concentration
7. Line 224 It should be explained why the water concentration of 7.4% was chosen. At a lower water concentration (e.g., 4%), the same reaction rate and yield were obtained. The water is also a reaction product, which means that its concentration increases during the reaction, and the reaction rate and yield at higher water concentration was not measured, which means that it is possible that the reaction rate decreases during the reaction due to the increased water concentration.
8. Figure 8: The particle size in the graph should be written as an interval, as indicated in Table 3
9. Line 346-347: Correct the number of equations
10. Line 375-389: There is a lack of explanation as to why the mass transfer limitation is more pronounced at higher temperatures for both triphasic systems.
11. Line 509 - Scheme 1, Chapter 5 ???
12. Line 615 -620. the sentences are not clear. How was the specific activity ratio N435/lipozyme CALB calculated and how is it calculated that the enzyme retains 7.2% of its activity after immobilization.
Author Response
Second reviewer
We are really grateful to the reviewer for his/her your constructive comments and valuable corrections and recommendations (in black italic font). We have carefully revised the manuscript according to your suggestions. The itemized responses (in blue) and the added text/references in the main manuscript (in red font) are attached in the following paragraphs.
Comment 1
This work describes the optimization of ibuprofen monoglyceride production by immobilized enzyme in a three-phase system. In addition to solvent and temperature optimization, mass transfer within the immobilized catalyst was also studied. A mathematical model was established that showed good agreement with the experimental data.
I propose a paper for publication with some further improvements:
General:
- The data on the possible leaching of enzymes were not given. In general, the use of a magnetic stirrer to carry out the process with an enzyme immobilized on a solid support is not preferable, since the shear forces may affect the binding forces of enzyme and support
Thank you for your appreciations, they help us to further explain our procedures and, in general, our approach. In this research, we were looking for the effect of cosolvents on the kinetics for this esterification reaction (water and toluene). Although we agree with the reviewer’s observation, during this, and previous, research batch runs with magnetic stirring were performed but only after ensuring no mechanical damage to the immobilized enzyme (the shape of the bottom of the flask and the type of stirrer –a disk with double cross- to minimize contact between the magnet and the flask bottom). We have added some more information in this respect in section 3.2.1., in Methods.
“The mixture, contained in a 50 mL round flask, was agitated with a circular double-cross magnet in the bottom to avoid attrition of the biocatalyst and set on an aluminum jacket”
As for the enzyme leaching, runs performed in this manuscript were not designed to study leaching in depth, although, from our experience, in case of a massive protein leaching (Novozym®435 has a very high concentration of CALB), a very stable emulsion should be formed, which was not the case (and it was the case when using the free enzyme Lypozyme®CALB-L). It is evident that further work needs to be done as indicated at the end of the manuscript regarding the in-flow or continuous use of these systems to have a clear understanding of the stability of the enzyme in diverse compositional and thermal conditions. However, considering the added insights and the notable amount of experimentation required to study in depth the stability of the catalyst under flow conditions, this will be the subject of another manuscript we are working in. In any case, we have added further comments in this sense at the end of the discussion section.
“Continuous production of drugs in small-sized factories maintain the flexibility of batch processing while optimizing process time use and drug safe handling and manufacture [41]. However, further studies working with the improved multiphasic system here addressed under continuous or flowing conditions are needed to ensure the absence of enzyme leaching or reduce it by added enhancement of the biocatalyst immobilization (for example ,by entrapment techniques). Enzyme leaching is a particular effect that affects Novozym®435 due to the hydrophobic nature of the bonds of the enzyme CALBL and the ion exchanger that serves as support: Lewatit VP OC 1600 [8]. This industrial preparation suffers from enzyme leaching at high temperatures in non-polar solvents, especially when using triglycerides as substrates, even if the fatty acids are short (acetic, butyric), because tri- and diglycerides usually act as biosurfactans, disrupting the hydrophobic bonds that links the enzymes among them and to the support surface [8]. Although in this work, the main phase is very polar (glycerol with a notable percentage of water), long-term in-flow experiments in fixed-bed or basket stirred tank reactors under a diversity of operational conditions should be the subject of further researchs in view of the process insights here reported.”
Comment 2
The kinetic parameters were estimated by directly fitting the model to the data collected in the batch reactor. Thus, the claim that deactivation exists in one three-phase system and can be neglected in another is questionable. Deactivation is not a problem to be verified. It is only necessary to measure the initial reaction rate of the immobilized enzyme used in the reaction.
As we have not recovered the solid to perform several consecutive batches or perform in-flow or continuous runs, no direct evidence on deactivation is provided. However, in our experience and for diverse conditions in the ibuprofen and glycerol system –with free and immobilized CALB, with and without added water at zero time, with and without toluene as a solvent for ibuprofen-, it can be present if we do not obtain total ibuprofen conversion with a very high excess of glycerol. Let’s consider than glycerol concentration is ³8.56 mol L-1 taking into account the whole system volume in both cases (with an without added water before the reaction takes places, that is, before adding the enzyme), while the higher concentration of ibuprofen is 0.48 mol L-1 (100 g L-1). In the case of using 20 g L-1 of ibuprofen (0.097 mol L-1), it is difficult to explain a partial conversion of this substrate when the excess of glycerol is higher than 88, as, most probably, the esterification equilibrium should be totally shifted towards the products. Even when using 100 g L-1 ibuprofen, the excess of glycerol is more than 17, so it is difficult to explain a partial conversion of the acid to the ester. With these observations, is clear than a partial conversion means the existence of some kind of deactivation, not of a reversible system. Moreover, if we have a look at Figure 9d, showing the results at the highest operation temperature -80 °C- and several initial ibuprofen concentrations without adding water before the reaction, we can observed that the highest concentration of this substrate leads to a stable 50% conversion of ibuprofen and lower concentrations of the acid leads to much higher conversions. This could be due to a chemical equilibrium really shifted towards the reagents, if it were not for the results for the same temperature and added water at zero time (Figure 10d). There, we find that the conversion at 100 g L-1 initial ibuprofen is higher than 90%. If it is an equilibrium –one of the possibilities-, one could expect that the addition of one of the products should shift the equilibrium even more towards the reagents, finding a conversion lower than 50% when water is added at zero time. This is not the case; surprisingly, we found a much higher conversion, suggesting that the addition of water helps the enzyme and makes it more stable, and more active (if we consider initial rate values).
For the kinetic modelling, we have followed the whole reaction process in each batch (progress curve) instead of using the initial reaction rate approach; in any case, both strategies are possible in enzyme kinetics, though the approach is diverse. For example, progress curve is a good approach if one wants to look at product inhibition or enzyme deactivation with operation time (in a batch process), while the initial rate approach needs of careful experimentation in a wide range of initial conditions (in the presence and absence of product, for example, to take into account its inhibitory capacity). In any case, as commented for the previous observation of the reviewer, a final discussion has been added on the need for research in flowing conditions to deepen in the stability study and, possibly, in the need for further improvements in this catalyst to be used under continuous processing.
Specific:
Comment 1
The introduction is too long and the importance of the reaction performed is explained very briefly.
Thank you for the comment. We have reduce its size and include additional comments on the reaction, as the product importance has been explained in depth. In particular, the second paragraph in page 3 now reads:
“The administration of lower doses of the active stereoisomers (for example, S-ibuprofen) instead of the racemic mixture and the use of profen prodrugs, including their esters, allow for a slow release of the drug into the body, reducing unwanted side effects [10] [11] [12] [33]. In this sense, bioprocesses based on the esterification of glycerol, or other polyols, and profens allow for the pharmacological valorization of the polyol and the easy production of prodrugs with an increased bioavailability (by increasing the profen solubility in aqueous media) and a limited deleterious effect on health due to local effects on the gastrointestinal system and controlled pharmacokinetics [10][12][34], while the use of multiphasic reacting systems with glycerol as both solvent, substrate and stabilizer leads to high conversion to the prodrug at moderate to high temperatures [34]. “
The "Results" chapter should be renamed to "Results and discussion"
Comment 2
Done as indicated by the reviewer, thank you for the warning.
Comment 3
Figure 2b and line 156; Figure 4b and line 190: Was the yield measured for all reactions at the same time, or was it measured when equilibrium was reached? Also, the thermodynamics of the reaction do not depend on the concentration of the enzyme. Reduced yield may have resulted from inactivation of the enzyme during prolonged use.
Reactions are stopped at the same time. However, our results at 72 h indicated not further progression in most cases after 24 h; thus, this was the final time for all kinetic runs. As explained previously, a constant value of conversion much lower than 1 or 100% indicates, in this case, deactivation or inactivation. In the case of Figure 1, we find low values of conversion at 24 h for low enzyme concentration, as expected, while increasing enzyme concentration permits to reach a final 24 h yield higher than 90%, just because the system has had enough time to progress till this conversion. Thermodynamics are not affected –equilibrium position-, as expected; reaction rate, which is proportional to enzyme concentration –Figure 1.a-, is affected proportionally.
Comment 4
Line 166 and Figure 3: The volumetric ratio of Gly/Tol is not the same in the text and in the figure (text: 1/20; figure 1/10)
Thank you for the observation. The error is in the text and is now fixed.
Comment 5
Lines 170-177: The initial esterification rate depends proportionally on the enzyme concentration. To claim this, the concentration of free enzyme and immobilized enzyme would have to be the same. The note also contradicts lines 610-612
We are most grateful to the reviewer for the comment. We have carefully observed figure 2a to observe a slight hyperbolic trend of the activity with the amount of enzyme, and modify it accordingly. This can be expected if an interfacial reaction happens, as the substrates and the enzyme distribute in this interface where reaction happens. This effect was also observed when we used the free enzyme (reference 34): in that case the enzyme was distributed between the two liquid phases and a very stable emulsion was created during the process: but not all the enzyme could be well distributed onto the interphase, apparently, creating this hyperbolic trend of r0 with CE. Please, see the modified Figure 2 and the first paragraph in page 4 in the revised manuscript.
“It can be seen that the initial esterification rate increases steadily with enzyme concentration from 16·10-4 to 33·10-4 mol L-1 min-1, but there is a slightly hyperbolic trend, thus showing the effects of substrates distribution on the inner surface where the enzymes are immobilized. This trend was also observed for the free enzyme, which accommodates onto the interfacial interface between the apolar ibuprofen and the polar glycerol [34].”
Comment 6
Lines 204-207 - Figure 6.5 is not available. From the data given in Figure 5a, it cannot be concluded that enzyme inactivation occurs at higher temperature and ibuprofen concentration
Thank you for the observation. There was an error in the number of the Figure: it refers to Figure 5. As of the comment related to Figure 5a, the reviewer is most correct: enzyme inactivation cannot be observed with those data, but with data at long time values in Figures 9 and 10. We have modified the comment of figures 5a and 5b accordingly, removing any mention to inactivation there. Please, see last paragraph in Page 5:
“As can be seen, for any temperature considered, the initial rate of esterification increases with the initial concentration of ibuprofen in a hyperbolic manner, following the typical trend according to a Michaelis-Menten model, as occurs in systems employing the enzyme in its free form (CALB-L) in the absence and presence of a cosolvent [34, 35]. Likewise, as the temperature rises, the curves of the enzyme activity with the initial concentration of ibuprofen tend to be less hyperbolic and more linear. This behavior suggests that the value of KM trends to higher values, showing an apparent lower affinity of the enzyme for the ibuprofen at higher temperatures.”
And in page 6:
“This behavior is very similar to that found in the anhydrous system. However, the presence of a concentration of water in the reaction medium results in a higher value of the monoester yield at an ibuprofen concentration of 100 g·L-1 when compared to the value at 60 g·L-1, at all temperatures. This is due, surely, to a reduction in viscosity with the concomitant increase in mass transfer rate and, thus, in the observed enzyme activity. Moreover, more linear curve trends are appreciated in this figure, suggesting slightly lower affinities of the enzyme for ibuprofen in the presence of water at zero time.”
Comment 7
“Line 224: It should be explained why the water concentration of 7.4% was chosen. At a lower water concentration (e.g., 4%), the same reaction rate and yield were obtained. The water is also a reaction product, which means that its concentration increases during the reaction, and the reaction rate and yield at higher water concentration was not measured, which means that it is possible that the reaction rate decreases during the reaction due to the increased water concentration.”
We really appreciate this comment. The reviewer points out a question that is most correct: why not 4% w/w water added before the reaction takes place? Apparently, this is enough considering enzyme activity and final yield to the target product. We have, in any case, added more water to ensure a lower viscosity and, thus, a lower productivity limitation due to mass transfer in all conditions, but, possibly, with 4% w/w initial water is enough. We do not have observed, in any case, any deleterious effect to the enzyme activity due to adding more water, even if, considering Le Chatelier principle, the addition of one product should shift the equilibrium towards the substrates or reagents. This is probably due to the large excess of glycerol. Moreover, a more polar reaction phase should stabilize the enzyme, reducing leaching, while ensuring the enzyme hydration. If we look at Figure 10, more water not only do not pose a problem in this case, but it stabilized the enzyme. In any case, this is no limitation to study the effect of lower amounts of water but, evidently, it is out of the scope of this manuscript.
Comment 8
Figure 8: The particle size in the graph should be written as an interval, as indicated in Table 3
Thank you for the observation. The Figure has been modified as requested.
Comment 9
Line 346-347: Correct the number of equations
Thank you for the warning. The equation numbers have been corrected.
Comment 10
Line 375-389: There is a lack of explanation as to why the mass transfer limitation is more pronounced at higher temperatures for both triphasic systems.
Thank you for the comment; it allows us to explain this important fact. We have now extended the first paragraph in page 12. Now it reads:
“As a conclusion, the values of We-Pt > 3 and effectiveness factor h<1 suggest that mass transfer limitations are significant in the system without water at zero time, while the higher values for h and lower values for the Weisz-Prater criterion indicate a certain overcoming of mass transfer limitations due to water, in particular when using the smaller size particle fraction of Novozym®435. As expected, the mass transfer limitations are higher as the temperature rises: this is due to the effect of temperature on the physical phenomenon, the mass transfer, and the chemical reaction. Mass transfer is usually explained by mass transfer coefficients, which slightly increases with temperature (apparent activation energies of 2-8 kJ mol-1). The kinetic constants of (bio)chemical reactions depends much more strongly on temperature (20-100 kg mol-1). Thus, at higher temperatures, the biochemical reaction rate increases strongly, while the mass transfer flux only accelerates slightly, being, in our case, this the slow, limiting kinetic phenomenon. As a consequence, the limitation due to mass transfer is more evident at 70 °C than at 50 °C (Table 3).”
Comment 11
Line 509 - Scheme 1, Chapter 5 ???
Sorry, it is a mistake. We have deleted what is not relevant for the manuscript.
Comment 12
Line 615 -620. The sentences are not clear. How was the specific activity ratio N435/lipozyme CALB calculated and how is it calculated that the enzyme retains 7.2% of its activity after immobilization.
Thank you for the comment. These calculations start with the values of the initial reaction rates or production rates of the monoglyceride for the free enzyme preparation (Lipozyme®CALB L) and the immobilized form of the enzyme (Novozym®435) at 50 °C (when the mass transfer limitations are smaller) and consider the amount of protein in both preparations. We can see that, in the presence of mass transfer limitations, the immobilized form of the enzyme only retains 4.8% of the specific activity of the free enzyme preparation. However, the effectiveness factor of 0.66 for the triphasic system with added water at zero time suggest that most of this low activity is not due to internal mass transfer limitations, but to the immobilization procedure itself. In view of the large amount of protein in the ion exchange resin (and similar immobilized forms of CALB), it is not wild to hypothesize that most activity loss is due to protein accumulation in the pores and on the pore surfaces, reducing the access of the substrates to the enzyme active center or avoiding it totally for the buried proteins. We have tried to clarify our point by introducing some additional consideration/information:
“The highest effectiveness factor obtained was 0.66 in the triphasic system with glycerol+7.4 % w/w water as liquid polar phase, ibuprofen+toluene as liquid apolar phase and N435 as solid biocatalyst, suggesting a 66% effectiveness in comparison to an ideal reference (runs driven at 50 °C): the immobilized enzyme in absence of any internal mass transfer limitation while the specific activity ratio N435/Lipozyme®CALB-L is 0.048, or 4.8%. This comparison suggest that the enzyme retains, upon immobilization and in the absence of mass transfer limitations, only 7.2% of its activity regarding this particular esterification reaction.”
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
The revised version is much better than the original manuscript, which was available to the reviewer for comparison. The suggestions and corrections of all reviewers have been taken into account and the revision has been carefully carried out, so that the revised version is suitable for publication.
