Preparation of Ordered Macroporous ZIF-8-Derived Magnetic Carbon Materials and Its Application for Lipase Immobilization

Round 1

Reviewer 1 Report

The aim of the manuscript entitled "Preparation of Ordered Macroporous ZIF-8-Derived Magnetic Carbon Materials and its Application for Lipase Immobilization" is to address the challenges associated with the conventional use of ZIF-8 as an immobilization carrier for enzymes, specifically lipases. The focus is on improving the practical applicability of ZIF-8 in enzyme immobilization by overcoming limitations such as poor separation and recovery efficiency, small pore size, and poor acid stability. The manuscript aims to develop an innovative approach – pre-carbonization immersion – to synthesize ordered macroporous ZIF-8-derived carbon materials with stable ferromagnetism. The goal is to enhance the chemical stability of ZIF-8 through carbonization while also addressing the issue of recyclability. The resulting magnetic carbon materials are intended to serve as effective supports for immobilizing lipases, with the ultimate objective of optimizing the enzyme loading and activity for biodiesel production. The manuscript is interesting and fits well with the scope of the Journal. It is short but well-prepared in general. Still, some issues need to be addressed in order to be published. My specific comments are given below.

While the abstract discusses the use of pre-carbonization immersion as a strategy to synthesize ordered macroporous ZIF-8-derived carbon materials, it does not clearly articulate the novelty or innovation of this approach compared to existing methods. Also, in line 12, the authors state that “ZIF-8 has great potential for immobilization of enzymes due to mild synthesis conditions, high stability …” but in line 14, they write about poor acid stability. It is contradictory.

The Introduction is not informative. It mainly consists of information on what the authors did before in their lab. It is important to give the basic knowledge and the contest of the presented research. I suggest:

·         Briefly introduce the significance of enzyme immobilization in industrial applications, particularly in the context of biodiesel production.

·         Define MOFs and their characteristics.

·         Emphasize the growing interest in MOFs as promising materials for enzyme immobilization due to their large specific surface area, high porosity, and structural designability.

·         Focus on ZIF-8 and its limitations.

·         Emphasize the need for innovative approaches to overcome these limitations and enhance the applicability of ZIF-8 in enzyme immobilization.

·         Discuss the concept of carbonization as a strategy to improve the chemical stability of ZIF-8.

·         Mention existing literature and studies supporting the idea that carbonization can enhance the performance of MOFs in various applications.

·         Present the innovative approach of pre-carbonization immersion as a method to synthesize ordered macroporous ZIF-8-derived carbon materials.

·         Explain the rationale behind choosing this strategy and its potential advantages over conventional methods.

·         Conclude the introduction by emphasizing the potential impact of the study.

·         Clarify the specific contributions and novelties expected from the manuscript.

·         Briefly outline the scope of the study and the subsequent sections of the manuscript.

Figure 3 is not useful in its present form since it is too small and visibility is poor.

The results of the porosity measurement should be given in Table, so it will be easy to compare.

Section 3.3. Immobilization of lipase: doesn’t the ethanol denature lipase? Please, explain.

The main issue is the rationale behind the carbonization of MOFs. It is not economical, so the industry will hardly be interested in this technology. This issue should be thoroughly discussed.

Minor changes are required. 

Author Response

Comment No.1: While the abstract discusses the use of pre-carbonization immersion as a strategy to synthesize ordered macroporous ZIF-8-derived carbon materials, it does not clearly articulate the novelty or innovation of this approach compared to existing methods. Also, in line 12, the authors state that “ZIF-8 has great potential for immobilization of enzymes due to mild synthesis conditions, high stability …” but in line 14, they write about poor acid stability. It is contradictory.

Response: Thank you very much for your comment.

As you have commented, our description here is flawed. In fact, conventional magnetic modification is mainly realized by loading magnetic nanoparticles (e.g., Fe3O4, etc.) on the surface of MOFs materials, which is a cumbersome step and prone to fall off during use due to the weak force between magnetic nanoparticles and the surface of MOFs materials. In contrast, we use pre-impregnated carbonization, which allows us to embed the magnetic species directly into the skeleton of the carbon material, which is a simple step and at the same time the magnetic modification is more solid. This is discussed in more detail in the Introduction section:

(Line 80-87) “One of the main ways to modify materials for magnetic functionalization is by loading magnetic nanoparticles on the surface of the material, and most of the time this method is effective [20,21]. However, the limitation of this method is that the magnetic nano-particles are connected to the surface of the material through weak interactions (elec-trostatic interactions, van der Waals forces, etc.), and the magnetic nanoparticles may be dislodged from the material during use, resulting in a loss of magnetism. In contrast, the strategy of allowing magnetic species to be embedded in the backbone of the material by means of covalent bonding connections may be more advantageous.”

Regarding the stability of ZIF-8, ZIF-8 actually belongs to a relatively high stability class among MOFs materials, which is one of the reasons why it is widely used. However, the structure of ZIF-8 relies heavily on the coordination bond between the deprotonated N in 2-methylimidazole and zinc ions, and is therefore susceptible to destruction by fatty acids in the application of immobilized lipase-catalyzed biodiesel production. However, our previous statement in the abstract did tend to be misunderstood, thus we decided to delete the description of the high stability of ZIF-8. The revised part of the abstract is as follows:

(Line 11-14) “Among them, ZIF-8 has great potential for immobilization of enzymes due to mild synthesis conditions and good biocompatibility. However, conventional ZIF-8 crystals have poor separation and recovery efficiency due to their small pore size and poor acid stability, limiting its application in enzyme immobilization and further application greatly.”

Comment No.2: The Introduction is not informative. It mainly consists of information on what the authors did before in their lab. It is important to give the basic knowledge and the contest of the presented research. I suggest:

Response: Thank you very much for your comment.

We have revised the introductory section as you suggested, as follows:

• Briefly introduce the significance of enzyme immobilization in industrial applications, particularly in the context of biodiesel production.

(Line 35-42) “Especially for the industrial production of biodiesel, the bio-enzyme-catalyzed method offers milder reaction conditions, higher yields, and less industrial waste than the tradi-tional acid/base-catalyzed method [6,7]. The high cost of enzyme preparations has made the enhancement of enzyme reusability a research topic of great interest, usually achieved through enzyme immobilization [8,9]. In addition, the stability and catalytic activity of the enzyme in the reaction system can be effectively improved after immobi-lizing the enzyme by choosing suitable support materials and methods.”

• Define MOFs and their characteristics.
• Emphasize the growing interest in MOFs as promising materials for enzyme immobilization due to their large specific surface area, high porosity, and structural designability.

(Line 45-53) “Metal-organic frameworks (MOFs) are a new class of porous crystalline materials that consist of metal nodes (metal ions or metal clusters) self-assembled with organic ligands through ligand bonds to form a three-dimensional ordered lattice structure. MOFs are regarded as a new type of immobilized enzyme support materials with great potential due to their large specific surface area, high porosity, and extreme designability of structure and function [10,11]. However, due to the small pore size of MOFs (usually less than 2 nm), their performance in the immobilization of larger-sized enzyme molecules (e.g., lipases) is not effective, such as hindering the diffusive mass transfer of the sub-strate and affecting the catalytic performance of the immobilized enzymes [12].”

• Focus on ZIF-8 and its limitations.
• Emphasize the need for innovative approaches to overcome these limitations and enhance the applicability of ZIF-8 in enzyme immobilization.
• Discuss the concept of carbonization as a strategy to improve the chemical stability of ZIF-8.
• Mention existing literature and studies supporting the idea that carbonization can enhance the performance of MOFs in various applications.

(Line 53-61) “On the other hand, ZIF-8 (formed by coordination of zinc ions and 2-methylimidazole) is one of the MOFs materials with mild synthesis conditions and low biotoxicity, which has been regarded as a potential immobilized enzyme carrier in recent years. However, the acid stability of ZIF-8 is poor and its crystal structure can be damaged by fatty acids and other substances, which limits the application of ZIF-8 immobilized lipases [12]. The chemical stability of the materials can be effectively enhanced by obtaining their derived porous carbon materials through high-temperature pyrolytic carbonization of MOFs, which is due to the fact that the structure of carbon materials mainly relies on covalent bonds [13,14].”

• Present the innovative approach of pre-carbonization immersion as a method to synthesize ordered macroporous ZIF-8-derived carbon materials.
• Explain the rationale behind choosing this strategy and its potential advantages over conventional methods.

(Line 80-87) “One of the main ways to modify materials for magnetic functionalization is by loading magnetic nanoparticles on the surface of the material, and most of the time this method is effective [20,21]. However, the limitation of this method is that the magnetic nano-particles are connected to the surface of the material through weak interactions (elec-trostatic interactions, van der Waals forces, etc.), and the magnetic nanoparticles may be dislodged from the material during use, resulting in a loss of magnetism. In contrast, the strategy of allowing magnetic species to be embedded in the backbone of the material by means of covalent bonding connections may be more advantageous.”

• Conclude the introduction by emphasizing the potential impact of the study.
• Clarify the specific contributions and novelties expected from the manuscript.
• Briefly outline the scope of the study and the subsequent sections of the manuscript.

(Line 97-101) “The pre-carbonization immersion strategy developed in this paper can effectively prepare ZIF-8-derived porous carbon materials with stable magnetic modifications, and at the same time solves the problems of limited stability and difficult separation of ZIF-8 in immobilized lipase applications, which makes the MOFs materials more promising for immobilized lipase applications.”

Comment No.3: Figure 3 is not useful in its present form since it is too small and visibility is poor.

Response: Thank you very much for your comment.

We are very sorry that the picture we used before was not clear enough. We have redrawn Figure 3 and it should be much clearer now.

Comment No.4: The results of the porosity measurement should be given in Table, so it will be easy to compare.

Response: Thank you very much for your comment.

We have now organized the nitrogen adsorption/desorption data in Table 1, as shown in Line 184.

Comment No.5: Section 3.3. Immobilization of lipase: doesn’t the ethanol denature lipase? Please, explain.

Response: Thank you very much for your comment.

In fact, we only used a small amount of ethanol with the purpose of making the originally hydrophobic carbon material carrier more hydrophilic, which helps the immobilization of lipase. In practice, we first added 120 μL of anhydrous ethanol together with 680 μL of PBS buffer to the centrifuge tube containing the carbon material carrier, and fully moistened the surface of the carrier by ultrasonic-assisted means, and then added 200 μL of Lipase TLL (trade name: Eversa® Transform 2.0) for immobilization. In other words, the volume fraction of ethanol in the solution after the addition of the liquid enzyme was 12%, and this lipase was not denatured at this concentration.

Comment No.6: The main issue is the rationale behind the carbonization of MOFs. It is not economical, so the industry will hardly be interested in this technology. This issue should be thoroughly discussed.

Response: Thank you very much for your comment.

The cost of the MOF carbonization process consists of two main aspects: the energy consumption due to high temperature conditions, and the treatment of exhaust gases generated by the thermal decomposition of MOF. In this paper, we chose a lower pyrolysis temperature (i.e., we chose 600 °C as the optimal pyrolysis temperature among 600, 700, and 800 °C), and one of the purposes was to consider the reduction of energy consumption. For the treatment of waste gas generated by MOF pyrolysis: In fact, the generation of industrial waste is a common problem in the synthesis of enzyme immobilized carrier materials currently used in the market. For example, the synthesis of macroporous resins, one of the most commonly used enzyme immobilized carrier materials, needs to go through the steps of polymerization reaction, washing, and activation, which will generate a large amount of wastewater, waste gas, and waste solids. In contrast, the carbonization process of MOF mainly produces exhaust gas, but hardly produces waste water and waste solids, so we believe that the carbonization process of MOF should be more advantageous.

Author Response File: Author Response.pdf

Reviewer 2 Report

- Line 12: what do you mean about "biocompatibility of carrier materials"?

- Line 15-17: can we say that carbonization is not effective?

-Can enzyme attachment arms be used on the support to increase recycling?

- Can we apply support characterization methods to the ZIF-8-Enzyme complex?

-Line 25: what is the role of magnetic support in immobilization?

- In methanolysis explain the choice of the nature àof alcohol and the ratio.

Author Response

Comment No.1: Line 12: what do you mean about “biocompatibility of carrier materials”?

Response: Thank you very much for your comment.

The concept of biocompatibility is widely used in the fields of materials, biology and medicine, etc. Usually, biocompatible materials can be used directly in living organisms or used to synthesize biomaterials. ZIF-8 and its derived carbon materials are a class of biocompatible materials that can be used for immobilized enzymes, immobilized cells, and slow-release of medicines.

Comment No.2: Line 15-17: can we say that carbonization is not effective?

Response: Thank you very much for your comment.

On the one hand, the structure of ZIF-8 mainly depends on the coordination bond between the N atom in 2-methylimidazole and zinc ions, which is easily destroyed under acidic conditions (free fatty acids are produced during biodiesel production). On the other hand, ZIF-8 is difficult to be separated and recovered from the reaction system for reuse due to its low density and small particle size when used as an immobilization carrier for lipase. The porous carbon material obtained by direct carbonization of ZIF-8 has a stable structure and will not be damaged by fatty acids, which solves the first problem mentioned above. However, the porous carbon material is still characterized by low density and small particle size, so the second problem cannot be solved only by carbonization. So we used the method of introducing magnetism to solve the second problem. Overall, carbonization is only a part of our synthesis strategy, and although it only solves part of the problem, it is still very effective.

Comment No.3: Can enzyme attachment arms be used on the support to increase recycling?

Response: Thank you very much for your comment.

This is indeed a way to increase the recovery rate, but this tends to cause conformational changes in the enzyme molecule, leading to a decrease in the activity of the immobilized enzyme, and the more cumbersome operation of this method introduces more cost. A balance therefore needs to be struck between preparation cost and activity recovery.

Comment No.4: Can we apply support characterization methods to the ZIF-8-Enzyme complex?

Response: Thank you very much for your comment.

Most of the characterization methods used for carrier materials, such as SEM, FTIR and VSM, etc., can be used to characterize ZIF-8-Enzyme complexes as well. However, it is usually sufficient to characterize the carrier material in order to show more accurately the intrinsic structural properties of the carrier material.

Comment No.5: Line 25: what is the role of magnetic support in immobilization?

Response: Thank you very much for your comment.

The main role of magnetic modification is reflected in the enhancement of the separation and recovery performance of the immobilized enzyme in the reaction system, while it does not cause any influence in the immobilization process of lipase.

Comment No.6: In methanolysis explain the choice of the nature àof alcohol and the ratio.

Response: Thank you very much for your comment.

In section 3.5 we mentioned:

(Line 313-314) “The molar ratio of methanol to soybean oil was 4:1, and 120 U of the immobilized lipase was added per gram of soybean oil.”

The reason we chose this ratio of 4:1 instead of 3:1 was to have an excess of methanol in the reaction system to ensure full conversion of soybean oil to biodiesel.

Author Response File: Author Response.pdf

Reviewer 3 Report

Please see attached file.

Comments for author File: Comments.pdf

Author Response

Comment No.1: The title of the paper provides information that the studied adsorbents have a macroporous texture, but there are no full analyzed information about textural characteristics of the studied materials, except for the primary data of nitrogen porosimetry (Fig. 3, e,f).

Response: Thank you very much for your comment.

Figure 2 showed the SEM images of the material, where it could be seen that there were ordered macroporous structures in the material. The diameters of these macroporous structures were in the area of 200 nm. We have also added this section in the text.

(Line 117-119) “And the particles had ordered and connected macroporous structures (~200 nm in di-ameter), which were formed by thermolysis of the PS microspheres in the temperature range described above.”

Comment No.2: Fig. 3e, f contains the first experimental data of nitrogen porosimetry. It is recommended to present the processing data, for example, in the form of a table in order to confirm the macroporous texture of the developed materials. This is not obvious from the blurry pictures and text on lines 147-148.

Response: Thank you very much for your comment.

We have organized the data related to nitrogen adsorption/desorption in Table 1 (Line 184).

Since the macroporous pore size of the materials synthesized in our paper is around 200 nm, and the pore size range of the nitrogen adsorption/desorption test is usually no more than 100 nm, the macroporous structure of the materials is mainly characterized by SEM maps.

Comment No.3: Line 54-63. The reviewer does not agree with the authors’ statement that «A major constraint to the large-scale industrial application of immobilized enzymes is the high cost introduced by their separation and recovery steps». This statement refers only to the special case of preparing biocatalysts on fine powdered supports, for which it is very difficult to select an efficient reactor. It is difficult to imagine, looking at Fig. 5b, how the process using a magnet will be structured.

Response: Thank you very much for your comment.

In view of the porous properties of MOF materials and their derivatives, they have obvious advantages in the application of immobilized enzymes; however, MOF materials and their derivatives are usually difficult to obtain large particle sizes and are mostly in powder form. Therefore, in order to apply them to the large-scale industrial production of immobilized enzymes, the problem of their separation and recovery difficulties has to be solved. Magnetic separation is a suitable solution, since the separation of powders by filtration is industrially inefficient and centrifugation is more costly. Magnetic separation is feasible in industry and can be achieved by installing an electromagnet module outside the reactor.

Comment No.4: Line 21,23,76. For clarity of presentation and understanding, аabbreviations of CZ-x and CZ-x-M-y should be deciphered earlier than on the lines 83-86 and 246-256.

Response: Thank you very much for your advice.

We have added an explanation of the meaning of CZ-x and CZ-x-M-y on line 19-20.

(Line 17-21) “Herein, we developed a strategy of pre-carbonization immersion (immersion in aqueous FeSO₄ solution before carbonization) to synthesize ordered macroporous ZIF-8-derived carbon materials with stable ferromagnetism (denoted as CZ-x-M-y, where x denotes the carbonization temperature and y denotes the concentration of the impregnated FeSO₄ solution) and used them to immobilize lipases for biodiesel production.”

Comment No.5: Line 95-96, 98. It should be explained what the «decomposition degree of ZIF-8” is and how to evaluate it? In SEM images (Fig. 2,d,g and Fig. 2, e,f,h), significant changes are difficult to detect. It should be clarified what undergoes pyrolysis in the ZIF-8 and how to evaluate “the degree of pyrolysis”?

Response: Thank you very much for your comment.

The “decomposition degree of ZIF-8” we mentioned here is a qualitative description of the index, which will change with different temperatures and time. Generally speaking, when the time is the same, when the pyrolysis temperature is higher, the mass of ZIF-8 remaining after the pyrolysis process is less, which can be regarded as one of the indications of a high degree of decomposition. In our article, we attempted to determine the degree of decomposition by comparing the changes in the structure of ZIF-8 at the same time but different pyrolysis temperatures. Therefore, based on the differences in the absorption peaks of CZ-600, CZ-700 and CZ-800 in the FTIR spectra (Fig. 3a), we concluded that the decomposition of 2-methylimidazole in ZIF-8 gradually occurs at increasing pyrolysis temperatures, but there is a difference in the proportion of its decomposition and the products obtained.

Comment No.6: Fig. 3a is very blurry and unclear, the all inscriptions are difficult to read.

Response: Thank you very much for your comment.

Apologies for the lower resolution image we were using before. We have updated Figure 3 and it should be clear enough now.

Comment No.7: It is necessary to clearly identify the absorption peaks in FTIR spectra in the region of 3000 and 1500 cm^–1, and explain the disappearance of some and the broadening of others.

Response: Thank you very much for your advice.

We have explained the absorption peaks at 3000 and 1500 cm^–1 in the FTIR spectrum in the revised manuscript and explained the reason for the disappearance of some absorption peaks and the broadening of others.

(Line 119-132) “A further verification of this process could be obtained from the FTIR spectra (Figure 3a): the series of infrared absorption peaks belonging to PS with the wavenumber in the vicinity of 3000 cm⁻¹ disappeared when SOM-ZIF-8@PS was calcined to CZ-600. In addition, comparing the FTIR spectra of CZ-600, CZ-700 and CZ-800, it could be found that the absorption peaks near 1500 cm⁻¹ gradually disappeared as the pyrolysis tem-perature increased from 600 to 800 °C, which mainly originated from the C=C bond of the imidazole aromatic ring in 2-methylimidazole, implying that the degree of decom-position of ZIF-8 gradually increased. The fact that there were still numerous absorption peaks below the wave number 1300 cm⁻¹ in the FTIR spectra of CZ-600 proved that the degree of pyrolysis of ZIF-8 at 600 °C was low, resulting in a large number of groups still existing in CZ-600, which led to the generation of these absorption peaks. In contrast, the FTIR spectra of CZ-700 and CZ-800 were almost left with only a few broad absorption peaks, indicating a reduction in the number and type of groups in the framework of the materials.”

Comment No.8: In Fig. 3b it is also impossible to read the text and symbols. The origin of the zinc ion is unclear. In this case, it is recommended to give the chemical formula of the zeolite ZIF-8.

Response: Thank you very much for your comment.

We have redrawn Figure 3. In addition, we have added a description of the composition of ZIF-8.

(Line 53-55) “On the other hand, ZIF-8 (formed by coordination of zinc ions and 2-methylimidazole) is one of the MOFs materials with mild synthesis conditions and low biotoxicity, …”

Comment No.9: As a conclusion, Fig.3 should be redrawn.

Response: Thank you very much for your advice.

Apologies for the lower resolution image we were using before. We have updated Figure 3 and it should be clear enough now.

Comment No.10: Lines 188-189. Authors report that the enzyme loadings were changed “by varying the concentration of enzyme proteins in the initial solution during the immobilization process (Fig. 4e). But in Fig.4,e,f, the values of liquid enzyme addition volume (mL) are plotted on the axis “x”.

Response: Thank you very much for your comment.

Our previous manuscript was deficient in its presentation of this section, which has now been supplemented in the revised manuscript.

(Line 217-221) “We obtained a series of immobilized lipases CZ-600@TLL and CZ-600-M-0.5@TLL with gradually increasing enzyme loadings, respectively, by varying the concentration of enzyme proteins in the initial solution during the immobilization process (keeping the total volume constant and varying the amount of liquid enzyme added to 20, 50, 200, 500, and 700 μL, respectively) (Figure 4e).”

Comment No.11: Lines 194-198. Indeed, diffusion restrictions can lead to a decrease in the activity of immobilized lipase, under which the rate of mass transfer (diffusion) of the substrate (which one? Tributyrin?) decreases. It is not clear how the rate of mass transfer of the substrate can depend on the amount of lipase adsorption, i.e. enzyme loading? It is not clear from Fig. 4,e,f what volume of enzyme solution was added near point “0”?

Response: Thank you very much for your comment.

In this part, we would like to further illustrate the differences in the pore structure of the magnetic material CZ-600-M-0.5@TLL and the non-magnetic material CZ-600@TLL by comparing the differences in the patterns of change in the specific enzyme activity and enzyme activity recovery of the magnetic material CZ-600-M-0.5@TLL with the non-magnetic material CZ-600@TLL in the presence of different enzyme loadings. Both microporous and mesoporous structures exist in the magnetic material CZ-600-M-0.5, so we believe that at lower enzyme loadings, lipase molecules are preferentially adsorbed mainly in the microporous structures, while at higher enzyme loadings, lipase molecules fill most of the microporous structures and are further adsorbed in the mesoporous structures. Since the microporous structure hinders the diffusion of triglyceride substrate compared to the mesoporous structure, the apparent catalytic activity of CZ-600-M-0.5@TLL decreases at lower enzyme loading, which means that the enzyme activity recovery is low. When the enzyme loading was increased, the lipase molecules adsorbed in the mesoporous structure are able to fully contact with the substrate, resulting in an increase in the apparent catalytic activity of CZ-600-M-0.5@TLL as a whole, meaning that the enzyme activity recovery is high.

We have added specific data on the amount of liquid enzyme added in the revised manuscript.

(Line 217-221) “We obtained a series of immobilized lipases CZ-600@TLL and CZ-600-M-0.5@TLL with gradually increasing enzyme loadings, respectively, by varying the concentration of enzyme proteins in the initial solution during the immobilization process (keeping the total volume constant and varying the amount of liquid enzyme added to 20, 50, 200, 500, and 700 μL, respectively) (Figure 4e).”

Comment No.12: Lines 219-226. One cannot but agree that “separation and recovery of immobilized lipases from the reaction system was one of the most important factors for their large-scale industrial application”. There are many approaches to overcome this problem, for example, by using mechanically robust catalyst granules and suitable reactor design. Unfortunately, it is difficult to imagine how such separation can be done by strong magnet on an industrial scale for the production of a large-scale product such as biodiesel. How economically is it shown that this process will be economically beneficial, taking into account the multi-stage nature and cost of obtaining the adsorbent?

Response: Thank you very much for your comment.

For catalyst particles in powder form, centrifugation or filtration can be used in the laboratory to achieve separation. However, in large-scale industrial applications, centrifugation requires special equipment and is difficult and costly to operate, while filtration is inefficient. After magnetic modification of the catalyst particles, high efficiency separation can be achieved by installing an electromagnet module on the outside of the reactor. In contrast, the magnetic separation method is less difficult to operate and more efficient.

Comment No.13: Lines 261-262. Conditions for immobilization of lipase were described as follows: 50 mg of support + 0.8 mL of buffer with ethanol + 0.2 mL of soluble lipase at concentration of 8.3 mg/mL. In Fig. 4,e,f the volume of “liquid enzyme addition volume” was varied from 0 to 0.8 mL, any information about protein concentration was absent. It is recommended to add this information.

Response: Thank you very much for your comment.

We have added specific data on the amount of liquid enzyme added to the revised manuscript.

(Line 217-221) “We obtained a series of immobilized lipases CZ-600@TLL and CZ-600-M-0.5@TLL with gradually increasing enzyme loadings, respectively, by varying the concentration of enzyme proteins in the initial solution during the immobilization process (keeping the total volume constant and varying the amount of liquid enzyme added to 20, 50, 200, 500, and 700 μL, respectively) (Figure 4e).”

Comment No.14: The term “activity recovery (in %)” should be clearly explained and the method for calculating this parameter should be described. The experiment is structured as follows: first, adsorption and activity are measured, then the specific activity is calculated. Activity recovery (in %) is calculated parameter, is not it?

Response: Thank you very much for your comment and advice.

Activity recovery is the ratio of the specific enzyme activity of the immobilized enzyme to the specific enzyme activity of the free enzyme and is a calculated parameter. We have added it in the section 3.4 of the revised manuscript.

(Line 308-310) “Enzyme activity recovery was the ratio of the specific enzyme activity of the immobilized enzyme to the specific enzyme activity of the free enzyme.”

Comment No.15: Conclusions are short and not informative. It is required to add concrete results on adsorption, activity and stability immobilized TLL lipase.

Response: Thank you very much for your comment and advice.

We have added specific results on the properties of the immobilized TLL lipases in the Conclusion section of the revised manuscript.

(Line 343-347) “The enzyme loading of CZ-x-M-y increased with the decrease of carbonization temperature, in which the material, CZ-600-M-0.3, possessed the largest enzyme loading of 183.04 mg·g⁻¹ at the carbonization temperature of 600 °C, which was 49.7% higher compared with that of the non-magnetically modified CZ-600.”

(Line 350-351) “The immobilized lipase CZ-600-M-0.5@TLL showed the best reusability, which retained 81.9% activity after five catalytic reaction batches.”

Comment No.16: Lines 309-311. The authors did not convince the Reviewer that “magnetic support materials based on MOF reported in this study had promising potential for the industrial application of immobilized lipase” for the production of large-tonnage on demand inexpensive product such as biodiesel. It is believed that ZIF or MOF crystals in general are difficult to mass produce and apply in real-world applications due to inevitable intercrystalline defects. These materials are mainly advertised for applications such as gas separation and storage.

Response: Thank you very much for your comment.

In this study, ZIF-8 was converted into a porous carbon material by high-temperature pyrolytic carbonization, which eliminated the influence of the unavoidable intergranular defects in the original ZIF-8 structure on its stability. On the other hand, it is generally believed that MOF crystals usually require harsh synthesis conditions such as high temperature, high pressure and organic solvents, which is one of the factors limiting their large-scale industrial production. However, nowadays, more and more studies try to develop MOF materials synthesized under mild conditions (e.g., atmospheric pressure, room temperature, and aqueous systems, etc.), such as MIL-88A, which makes large-scale industrial production of MOF materials more feasible. In addition, in view of the excellent structure and surface properties of MOF materials, they are mainly used in the fields of gas separation and storage, but in recent years, some studies have found that MOF materials also perform well in the fields of catalysis and macromolecule adsorption. Overall, MOFs and their derivative materials, including the magnetic carbon materials reported in this paper, hold great promise for industrial applications of immobilized lipases.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors addressed my comments. 

Minor changes are required.