Rational Design of a Biocatalyst Based on Immobilized CALB onto Nanostructured SiO₂

Round 1

Reviewer 1 Report

This manuscript reports the rational design of lipase immobilized by adsorption onto nanostructured SiO2. The work has been well designed with some interesting findings. Several points should be addressed before publication:

 

Essential characterization of Ns silica should be presented, such as morphology, zeta potential.

 

In line 155, the surface charge distribution of CALB should be presented.

 

The leakage of the immobilized lipase is the different media used for activity assay should be studied.

 

A more detailed storage stability test should be performed. A time difference of 220 days is too wide.

Author Response

The authors acknowledge the comments of the Reviewer that allows to improve the quality of the manuscript. The modifications are highlighted in the revised version of the manuscript. Hereby, the point to point answers of the comments are presented:

Comments and Suggestions for Authors

This manuscript reports the rational design of lipase immobilized by adsorption onto nanostructured SiO₂. The work has been well designed with some interesting findings. Several points should be addressed before publication:

 

1-Essential characterization of Ns silica should be presented, such as morphology, zeta potential.

Author’s answer: The analysis of the morphology of the silica oxide through scanning electron microscopy was added to the section 2.1. of the revised version of the manuscript. A SEM micrograph of fumed silica oxide granules after hydration and calcination at 500 ^oC with a magnification of x2500 is presented, along with the following paragraph (line 121):

“The micrograph shows the morphology of the oxide composed by aggregates of silica nanoparticules of typically of 5-50 nm in size”.

The point zero charge of the silica oxide is broadly reported in the literature and in fact, was informed in the manuscript in order to explain the enzyme-oxide interaction. This information (along with a reference) is provided in line 179 of the manuscript as follows:

“Fumed silica possesses a PZC of 4.3 therefore, it can be assumed that the hydrophilic surface of silica does not possess a net charge [28]”.   

 

 2-In line 155, the surface charge distribution of CALB should be presented.

Author’s answer: in fact, the charge distribution of CALB was presented (lines 191 to 196 of the revised version of the manuscript) in the original version of the manuscript as follows,

“Therefore, the binding of CALB onto the Ns silica is produced through a definite number of charged groups of the lipase that should be correctly oriented to interact with the charged functional groups of the oxide support. In this sense, CALB possesses 317 amino acids with 18 positive residues (9 Lys, 8 Arg and 1 His) at pH 4 that are suitable to interact with SiO^- species. Additionally, the interaction of negative residues of the enzyme with the SiOH₂⁺ through hydrogen bonds cannot be disregarded”.

 

3-The leakage of the immobilized lipase is the different media used for activity assay should be studied.

Author’s answer: In fact, we only performed the experiments of stability in the ethanol-isooctane media in order to demonstrate the suitability of the CALB/SiO₂ as a biocatalyst for a particular application that is the kinetic resolution of racemic ibuprofen. However, the reviewer is correct in the sense that a different application requires additional stability experiments.

In this context, we were able to prove the absence of active enzyme desorption after the incubation in the reaction conditions. This fact is checked by the supernatant enzyme activity measurement as is described in the procedure in the section 3.8 of the manuscript. Moreover, the protein analysis of the same samples by the Bradford method gave values of 595 nm absorbance similar to the blank assays. These analyses were not shown in the manuscript.

 

4-A more detailed storage stability test should be performed. A time difference of 220 days is too wide.

Author’s answer: the storage stability was performed over the biocatalysts containing various protein loading immobilized on the silica support up to 7 months under storage and after more than two years. The fact that the higher the protein loading the lower the activity loss (7% for 22 mg protein per 100 mg oxide support) is a pretty clear indication of the biocatalyst’s stability. Even though, a more detailed storage stability might be interesting, the conclusion would be the same as the one drawn from the experiment described before, due to the low value of activity loss found at 7 months under storage. Moreover, the beauty of this study is that even after 2 years under storage of a scaled-up synthesis of the biocatalyst, there is only a 8% activity loss. Actually, the storage stability of our biocatalyst compares to NovozymÒ 435 (see line 450).    

 

In addition to the reviewer’s comments, the language was corrected all along the manuscript:

 

line 153 (modified sentence): “About 80 % of the maximum adsorbed protein is reached in 5 min of the immobilization reaching the equilibrium after 30 min contact.”

Line 204: “The maximum dispersion (adsorption) limit was reached at 0.029 µmol.m^-2, i.e., 29.4…”.    

Line 267 (the name of the chemical was corrected): “substrate p-nitrophenyl dodecanoate was…”

Lines 344 (modified sentence): “According to the technical document provided by Novozymes, it is worth noticing that the commercial crude extract of CALB L possesses a certain percentage of glycerol and sorbitol.”

Line 414: “(without the presence of rac-ibuprofen) have no effect on the activity of the CALB lipase”.

Line 427 (modified sentence): “It has been observed that the incubation of NovozymÒ 435 in toluene at 80 ^oC increased its activity up to 50 %.”

 

Additionally, the legends of the x,y axis of the graphs were homogenized. For example, the legend of the x axis of the figure 5, was modified to lower case: from TIME to Time; and also, the increment of the scale of the x axis was modified. Similarly, the legend of the x axis of the Figure 6 was change from “hr” to “h” and the span and increment of that axis were modified. In the case of the Figures 8, 9 and 10 the legends of the axis were changed to lower case. Finally, the y axis (right of the graph) corresponding to the enantiomeric excess eeS% of the Figure 7 was added.

 

Reviewer 2 Report

The manuscript by Briand et al. explores the adsorption of the CALB over nanostructured SiO₂ and the effect of the addition of polyols. Although the results are presented correctly, there are several concerns and errors that need to be addressed before the paper is in any publishable form. Furthermore, the authors fail to present the scientific advancement or novelty of this method.

 

 

Major points

 

1 The paper reports that CALB can be selectively adsorbed to Ns SiO₂. However, the SDS-PAGE analysis(Fig 3) is not enough to support this statement, the relative concentration of high molecular weight protein is higher after immobilization. Can you provide other detailed data?

 

2 In this paper, the time and temperature may be randomly selected. For example, in fig 5, 6, 8, 9, and 10, the span between temperatures and time is obviously inconsistent.

 

3 Line 23 “stability under long-term storage (more than 2 years)”, This conclusion is not supported by specific data in this paper, and the storage stability data of free CALB and Novozym ® 435 is not provided.

 

4 Characterization of CALB immobilized on Ns SiO₂ is lack, such as pH stability and operational stability. Only temperature stability and storage stability, the advantage of your method is not obvious.

 

 

Minor points

 

1 Line 52-72, Line 204-216, you have provided too many references, which may have no obvious effect and affect readers' reading. Can you reduce appropriately?

 

2 In fig 7, Is there a problem with the ordinate of the picture? The eeS% of biocatalysts is < 0.7%?

Author Response

The manuscript by Briand et al. explores the adsorption of the CALB over nanostructured SiO₂ and the effect of the addition of polyols. Although the results are presented correctly, there are several concerns and errors that need to be addressed before the paper is in any publishable form. Furthermore, the authors fail to present the scientific advancement or novelty of this method.

 

The authors acknowledge the comments of the Reviewer that allow to improve the quality of the manuscript. About the statement that says: “the authors fail to present the scientific advancement or novelty of this method”, we would like to make clear that the driving force of this investigation is based on the drawbacks of the commercial Novozym®435. In fact, some of us reported that the mechanism of immobilization (interfacial activation) that may facilitate enzyme desorption under certain conditions. Other problems are specific to the polymeric support: mechanical fragility, moderate hydrophilicity that permits the accumulation of hydrophilic compounds (e.g., water or glycerin) and the most critical one, support dissolution in some organic media. These circumstances demand the use of alternative supports such as, transition metal oxides like TiO₂ (published previously by some of us) and SiO₂, investigated in this contribution. Those supports (due to their inorganic nature) remains unaltered in the typical organic media used in biocatalysis, are thermally resistant and also have a high specific surface are with surface OH groups, allowing the interaction with the lipase. In conclusion, the scientific relevance of our present research is:

-The determination of the maximum dispersion limit of enzyme over nanostructured silica oxide for the first time in the literature: this information allows obtaining an active biocatalyst with the correct amount of lipase that the support can take, without wasting valuable enzyme.

-The spectroscopic analysis of the silica oxide through Raman spectroscopy at three laser wavelengths which ensures the cleanest oxide support that we can have. The properties of the biocatalyst are only ascribed to the enzyme-support interaction. There is no influence of surface impurities of the silica support that might be present (without previous knowledge) if no spectroscopic analysis is performed.  

-Proved stability of the CALB/SiO₂ biocatalyst in a reaction media that is typically used in the kinetic resolution of active pharmaceuticals ingredients APIs.

Proved storage stability (2 years) of the CALB/SiO₂ biocatalyst and comparable with the commercial Novozym®435. The long-term storage investigations are not regular ones in the literature due to the rush for rapid publication.

In this context, we thought that it is worth it to include, within the conclusion section, a paragraph that compares the CALB/SiO₂, the free CALB lipase and   Novozym®435. The new paragraph from lines 652 to 665, is:

“Besides, it is possible to conclude that the material containing the lipase at the maximum dispersion limit and co-adsorbed polyols, possesses a higher conversion and enantiomeric excess (towards S-ibuprofen) than the free CALB and the commercial Novozym®435 in the kinetic resolution of rac-ibuprofen at 45 ^oC, 60 ^oC and 70 ^oC. Moreover, the oxide support (as it is the case of Novozym®435) provides and improved thermal stability compared with the free lipase, under extended contact with an ethanol-isooctane media up to 70 ^oC. Similarly, the stability under extended storage (more than 2 years) is also comparable to the commercial biocatalyst.

In conclusion, the lipase B of Candida antarctica immobilized onto Ns SiO₂ at the maximum dispersion limit with co-adsorbed polyols, is comparable in terms of activity, thermal and storage stability with the commercial one. In addition, the availability and the well-known mechanical and chemical stability of the transition metal oxides such as, silica compares with the drawbacks of polymethylmethacrylate in organic media (the polymeric support of Novozym®435) are clear advantages of the biocatalyst presented in this contribution”.

The modifications are highlighted in the revised version of the manuscript. Hereby, the point to point answers to the comments are presented:

 

Major points

1-The paper reports that CALB can be selectively adsorbed to Ns SiO₂. However, the SDS-PAGE analysis (Fig 3) is not enough to support this statement, the relative concentration of high molecular weight protein is higher after immobilization. Can you provide other detailed data?

 

Author’s answer:  In fact, other assays for analyzing the selectivity in the adsorption were not performed. The SDS-Page (figure 3) undoubtedly shows a decrease of the signal of CALB lipase (31.5-45 kDa) upon contacting the silica oxide. In contrast, the signals belonging to enzymes of higher molecular weight (above 66 kDa) remain almost unchanged suggesting that are not adsorbed as can be observed in lines 2, 3 and 4 of the SDS page analysis. Nevertheless, we modified the manuscript in line 151 by replacing the word “reveals” by “suggests” as follows (see line 165):

“This observation suggests reveals that the adsorption is selective to CALB.”

 

2-In this paper, the time and temperature may be randomly selected. For example, in fig 5, 6, 8, 9, and 10, the span between temperatures and time is obviously inconsistent.

 

Authors’ answer: About the temperature selection, it is worth mentioning that 45 ^oC is the optimum for the kinetic resolution of racemic ibuprofen with ethanol catalyzed with the commercial catalyst Novozym®435, according to our previous investigation published in: J. Chem. Technol. Biot. 84 (2009) 1461-1473. In fact, that study indicated that the activity diminished at 55 ^oC. More recently, the Chiaradia et al. investigated the thermal stability of free CALB at 40 ^oC, 60 ^oC and 80 ^oC (Appl. Biochem. Biotechnol. 180 (2016) 558-575)). In fact, 60 ^oC has been reported as the upper limit for lipases to be active since above that temperature, a certain lipase denaturation is expected. Regarding the time span investigated in the present contribution, again it is based on our previous investigations that allowed concluding that the optimum conversion of substrate in the kinetic resolution of rac-ibuprofen (and rac-ketoprofen) is reached at 12 h of reaction or above. Nevertheless, the enantiomeric ratio E of the kinetic resolution of rac-ibuprofen catalyzed with CALB/SiO₂, has no change above 4 h of reaction meaning that there are not meaningful changes in the rate of reaction and therefore, the slope of the conversion of R-ibuprofen in the ethyl ester is steady. In this context, the Figures 8, 9 and 10 compare the activity of free CALB, Novozym®435 and CALB lipase immobilized on Ns SiO₂ in the esterification of rac-ibuprofen with ethanol at various temperatures within the steady state of the reaction.  

The figures named by the Reviewer were improved in terms of the legends and span. For instance, the figure 5 shows the conversion, enantiomeric excess and ratio as a function of time. The increment of the scale of the x axis was modified for an easier relationship between time and the data. Additionally, the legend of this figure was modified from upper case (TIME) to lower case (Time).

Similarly, the legend of the x axis of the Figure 6 was change from “hr” to “h” and the span and increment of that axis were modified.

In the case of the Figures 8, 9 and 10 the legends of the axis were changed to lower case.

 

3-Line 23 “stability under long-term storage (more than 2 years)”, This conclusion is not supported by specific data in this paper, and the storage stability data of free CALB and Novozym® 435 is not provided.

 

Author’s answer: we investigated the stability under storage (up to 2 years) of the biocatalyst synthesized in our contribution. Nevertheless, Novozymes Co. provides an inform regarding the storage stability of Novozym®435 at various temperatures (from 10 ^oC towards 40 ^oC) for 26, 52, 78, 104 and 120 weeks. In addition, Battiston et al. reported the investigation of the stability of free CALB up to 90 days at room temperature and 3-5 ^oC. In this context, the revised version of the manuscript provides a comparison between the stability of the CALB immobilized over silica oxide and the published data of the commercial Novozym® 435 and the free lipase.

The paragraph that compares the stability of the free CALB lipase, the commercial Novozym®435 and our biocatalyst, the following (see line 445-453):

“According to the investigations of Batistton et al., the free CALB possesses 20 % of residual activity (80 % activity loss in the synthesis of ethyl oleate) after storage for 90 days at 3-5 ^oC [38]. The authors demonstrated that the immobilization of the lipase over a mesoporous molecular sieve MCM-48 improves the stability of the lipase, reaching a 51 % of residual activity in the same time period of storage.

A report provided by the Novozyme Co. about the storage stability of the commercial Novozym®435 indicates no activity loss after storage for 120 weeks (about 2 years) at 10 ^oC [39]. In this context, it become important to somehow extend the investigation of the stability of CALB immobilized over the silica support. In fact, the activity of a biocatalyst containing a 0.028 mmol.m^-2 of lipase loading…..”.

 

4- Characterization of CALB immobilized on Ns SiO₂ is lack, such as pH stability and operational stability. Only temperature stability and storage stability, the advantage of your method is not obvious.

 

Authors’ comments: The pH stability, besides the pH of the reaction media (pH ~2.6-2.6) containing rac-ibuprofen, was not investigated. The operational stability of the biocatalyst is somehow proved through the extended contact (15 h) with the reaction media (ethanol in isooctane) and further determination of the activity in the kinetic resolution of rac-ibuprofen. Even though, it is not the typical investigation of reuse, those experiments mimic the exposure of the biocatalyst with the reaction media that is the major cause of deactivation. In fact, previous investigation of some of use (Ortiz, C.; Ferreira, M.L.; Barbosa, O.C.S.; dos Santos, J.; Rodrigues, R.C.; Berenguer-Murcia, A.; Briand L.E.; Fernandez-Lafuente, R. Novozym 435: the “perfect” lipase immobilized biocatalyst?, Catal Sci Technol 2019, 9, 2380-2420) demonstrated that the exposure of NovozymÒ435 to ethanol is the main caused of the loss of physical integrity and activity loss of that biocatalyst.  In this context, we decided that was worth it to investigate the operation stability of CALB/SiO₂ upon extended exposure and various temperature to the alcohol containing media.

The advantages of the method described in this contribution have been detailed at the very beginning of this letter.

 

Minor points

 

1 Line 52-72, Line 204-216, you have provided too many references, which may have no obvious effect and affect readers' reading. Can you reduce appropriately?

 

The text within lines 52 to 72 (lines 65 to 86 in the revised version of the manuscript) was modified by selecting the most relevant and recent investigations. The new paragraph in the revised manuscript is the following:

 

“The immobilization of CALB onto silica-based supports has deserved plenty of attention in the literature. For instance, Cruz et al. esterified acetic acid with geraniol using a biocatalyst prepared through the adsorption of a crude extract of CALB onto fumed silica [14]. Serra et al. immobilized a crude extract in different types of silica and they obtained the best results in the tributyrin hydrolysis when they used periodic mesoporous organosilica [15]. Using chemically modified silica oxide, Cassimjee et al. selectively extracted and immobilized a genetically modified CALB, generating an effective biocatalyst in the transesterification of ethyloctanoate with hexanol [16]. A few years ago, Gandomkar et al. immobilized CALB onto epoxy-functionalized silica gel for the enantioselective hydrolysis of ibuprofen esters [17]. The commercial crude extract Lipozyme® CALB L and silica gel have been used by Mittersteiner et al. to prepare biocatalysts active in the enantioselective esterification of (R, S)-2-methylbutyric acid with various aliphatic alcohols [18]. In recent work, Vesoloski et al. reported an original method of immobilization of commercial CALB by the sol-gel technique using silica from rice husk ash as a source or silicon [19]”.

 

 The text within lines 204 to 216 (line 219 in the revised version of the manuscript) was modified as follows:

“Nevertheless, the achieved protein loading is significantly higher (294 mg.g^-1) than the ones reported in previous articles [14, 15, 17, 18]. In fact, Vesolovski et al. reported a CALB loading onto a silica-based support of 148 mg.g^-1 that corresponds to the highest published in the literature until the present contribution [19]”.

 

2- In fig 7, Is there a problem with the ordinate of the picture? The eeS% of biocatalysts is < 0.7%?

 

Authors’ answers: The Reviewer is absolutely correct, the y axis corresponding to the percentage of enantiomeric excess was missing. The Figure 7 was corrected accordingly.

 

In addition to the reviewer’s comments, the language was corrected all along the manuscript:

line 153 (modified sentence): “About 80 % of the maximum adsorbed protein is reached in 5 min of the immobilization reaching the equilibrium after 30 min contact.”

Line 204: “The maximum dispersion (adsorption) limit was reached at 0.029 µmol.m^-2, i.e., 29.4…”.    

Line 267 (the name of the chemical was corrected): “substrate p-nitrophenyl dodecanoate was…”

Lines 344 (modified sentence): “According to the technical document provided by Novozymes, it is worth noticing that the commercial crude extract of CALB L possesses a certain percentage of glycerol and sorbitol.”

Line 414: “(without the presence of rac-ibuprofen) have no effect on the activity of the CALB lipase”.

Line 427 (modified sentence): “It has been observed that the incubation of Novozym® 435 in toluene at 80 ^oC increased its activity up to 50 %.”

Reviewer 3 Report

Key Information for the Rational Design of a Biocatalyst Based on Immobilized Calb onto Nanostructured SiO2. the following need to be considered.

1. Title may change please remove '' Key Information for the''

2. Importance of biocatalyst and its application in various discipline needs to be given authors may go through https://doi.org/10.3109/07388551.2014.950550; https://doi.org/10.1021/acs.chemrev.7b00203; https://doi.org/10.3390/catal13020250 etc

3. SEM or TEM image may be provided.

4. Comparison or advantages with other biocatalysts can be discussed.

5. References should be in journals format.

6. Check for english and spelling errors

Author Response

Key Information for the Rational Design of a Biocatalyst Based on Immobilized Calb onto Nanostructured SiO₂. the following need to be considered.

1-Title may change please remove '' Key Information for the''

Authors’ answer: The title was modified as: “Rational Design of a Biocatalyst Based on Immobilized Calb onto Nanostructured SiO₂”.

 

2- Importance of biocatalyst and its application in various discipline needs to be given authors may go through https://doi.org/10.3109/07388551.2014.950550; https://doi.org/10.1021/acs.chemrev.7b00203; https://doi.org/10.3390/catal13020250 etc

 

Authors’ answers: The application of biocatalysis focusing on pharmaceuticals was added to the introduction of the manuscript. This particular type of application was chosen due to the topic of the special issue: “Applications of Hydrolases in Medicinal Chemistry”. Besides, the references suggested by the Reviewer, the following one was also considered: 

3. Yi, D.; Bayer, T.; Badenhorst, C.P.S.; Wu, S.; Doerr, M.; Höhne, M.; Bornscheuer, U.T. Recent trends in biotechnology. Chem Soc. Rev. 2021, 50, 8003.

 The following paragraphs were included in the revised version of the manuscript (lines 29-38):

“Biocatalysis or enzymatic catalysis refers to the use of living systems or their parts (enzymes, cells) to speed up chemical reactions and in turn, replace conventional inorganic catalysts in order to achieve sustainable and eco-friendly biotransformations. In fact, technical grade lipases, nitrilases, nitrile hydratases and amidases have found application as bulk enzymes in the resolution of chiral compounds and the synthesis of enantiopure compounds in the pharmaceutical industry [1]. The technology for the enzymatic synthesis of chlorohydrin, the active pharmaceutical ingredient API of atorvastatin, gained commercial status in 2006 [2]. More recently, Merck & Co. developed the enzymatic synthesis of islatravir, a drug applied in the treatment of HIV, through five steps cascade type of mechanism that is exclusively catalyzed with enzymes [3]”.

Then, in line 47:

“….decades both for research and industrial applications, and as an enzyme-responsive drug-delivery nanosystem in cancer therapy”.

Line 54 to 57:

“In fact, complex drug delivery systems have been synthesized by immobilization of metalloproteinases onto mesoporous silica functionalized with amino groups and finally caped with bovine serum albumin to close the pores of the oxide support [12]”.

 

The references [1], [2] and [12] are the ones suggested by the Reviewer:

1. Jemli, S.; Ayadi-Zouari, D.; Hlima, H.B.; Bejar, S. Biocatalysts: applications and engineering for industrial purposes. Crit Rev Biotechnol 2016, 32, 246-258.

2. Sheldon, R.A.; Woodley, J.M. Role of biocatalysis in sustainable chemistry. Chem. Rev. 2018. 118, 801-828.

12.Sengupta, S.; Das, P.; Sharma, S.; Shukla, M.K.; Kumar, R.; Tonk, R.K.; Pandey, S.; Kumar, D. Role and application of biocatalysts in cancer drug discovery. Catalysts 2023, 13, 250.

 

3- SEM or TEM image may be provided.

 

Authors’ answer: The analysis of the morphology of the silica oxide through scanning electron microscopy was added to the section 2.1. of the revised version of the manuscript. A SEM micrograph of fumed silica oxide granules after hydration and calcination at 500 ^oC with a magnification of x2500 is presented, along with the following paragraph (line 121):

“The micrograph shows the morphology of the oxide composed by aggregates of silica nanoparticules of typically of 5-50 nm in size”.

 

4- Comparison or advantages with other biocatalysts can be discussed.

Authors’ answers: the properties of CALB/SiO₂ with the free lipase and the commercial Novozym®435 are compared in terms of conversion, enantiomeric excess and catalytic activity at various temperatures (from lines 374 to 394). Additionally, the catalytic activity upon extended thermal treatment in contact with the reaction media (ethanol-isooctane) of CALB/SiO₂ was compared with the free lipase and Novozym®435 within the lines 413 and 432. The stability under extended storage was discussed in the revised version of the manuscript (lines 445-453) as follows, 

“According to the investigations of Batistton et al., the free CALB possesses 20 % of residual activity (80 % activity loss in the synthesis of ethyl oleate) after storage for 90 days at 3-5 ^oC [38]. The authors demonstrated that the immobilization of the lipase over a mesoporous molecular sieve MCM-48 improves the stability of the lipase, reaching a 51 % of residual activity in the same time period of storage.

A report provided by the Novozyme Co. about the storage stability of the commercial NovozymÒ435 indicates no activity loss after storage for 120 weeks (about 2 years) at 10 ^oC [39]. In this context, it become important to somehow extend the investigation of the stability of CALB immobilized over the silica support”.

 

The new references are the following:

38. Battiston, C.S.Z.; Ficanha, A.M.M.; Levandoski, K.L.D.; da Silva, B.A., Battiston, S.; Dallago, R.M.; Mignoni, M.L. Immobilization of lipase on mesoporous molecular sieve MCM-48 obtained using ionic solid as a structure director and esterification reaction on solvent-free. Quim Nova 2017, 40, 293-298.
39. Report of storage stability of NovozymÒ435 from Novozyme Co. published on January 30^th, 2018. Retrieved from: http://www.cliscent.com>.

 

In addition, we thought that is worth it to include, within the conclusion section, a paragraph that compares the CALB/SiO₂, the free CALB lipase and   Novozym®435. The new paragraph from lines 652  to 665, is:

“Besides, it is possible to conclude that the material containing the lipase at the maximum dispersion limit and co-adsorbed polyols, possesses a higher conversion and enantiomeric excess (towards S-ibuprofen) than the free CALB and the commercial Novozym®435 in the kinetic resolution of rac-ibuprofen at 45 ^oC, 60 ^oC and 70 ^oC. Moreover, the oxide support (as it is the case of Novozym®435) provides and improved thermal stability compared with the free lipase, under extended contact with an ethanol-isooctane media up to 70 ^oC. Similarly, the stability under extended storage (more than 2 years) is also comparable to the commercial biocatalyst.

In conclusion, the lipase B of Candida antarctica immobilized onto Ns SiO₂ at the maximum dispersion limit with co-adsorbed polyols, is comparable in terms of activity, thermal and storage stability with the commercial one. In addition, the availability and the well-known mechanical and chemical stability of the transition metal oxides such as, silica compares with the drawbacks of polymethylmethacrylate in organic media (the polymeric support of Novozym®435) are clear advantages of the biocatalyst presented in this contribution”.

 

5-References should be in journals format.

Authors’ answers: The references were formatted according to the journal requirements and appear highlighted in yellow color in the revised version of the manuscript.

 

6-Check for english and spelling errors

Authors’ answers: the language was corrected all along the manuscript:

line 153 (modified sentence): “About 80 % of the maximum adsorbed protein is reached in 5 min of the immobilization reaching the equilibrium after 30 min contact.”

Line 204: “The maximum dispersion (adsorption) limit was reached at 0.029 µmol.m^-2, i.e., 29.4…”.    

Line 267 (the name of the chemical was corrected): “substrate p-nitrophenyl dodecanoate was…”

Lines 344 (modified sentence): “According to the technical document provided by Novozymes, it is worth noticing that the commercial crude extract of CALB L possesses a certain percentage of glycerol and sorbitol.”

Line 414: “(without the presence of rac-ibuprofen) have no effect on the activity of the CALB lipase”.

Line 427 (modified sentence): “It has been observed that the incubation of Novozym®435 in toluene at 80 ^oC increased its activity up to 50 %.”

 

 

Round 2

Reviewer 1 Report

The reviewer is happy about the improvement.

Reviewer 3 Report

Comments incorporated