Site-Specific Covalent Immobilization of Methylobacterium extorquens Non-Blue Laccse Melac13220 on Fe₃O₄ Nanoparticles by Aldehyde Tag

Round 1

Reviewer 1 Report

The manuscript reported immobilization of Melac13220 with aldehyde tags using three methods, the best immobilization was with magnetic particles. The immobilized Melac13220 improved the pH, temperature and thermostability. The optimum temperature of Melac13220 was increased from 65 °C to 80 °C and confirmed by DSC. The immobilized Melac13220showed higher dye decolorization as compared to free form. Many basic experiments have been performed, but it would be great if the mechanism of immobilization could be written clearly. Overall, the manuscript is recommended for publication after major revision. The following specific comments may be considered while revising the manuscript.

 

1. The method of entrapment and crosslinking used in this study is the most basic method, and there is not much novelty. It is suggested that the title should be put into magnetic particles and aldehyde tags.

 

2. The immobilization method (immobilization of aldehyde tags Melac13220 with magnetic particles) not seen in abstract, please add. In L.15, only see " covalent immobilization of the enzyme with a site-specific tag" in the abstract which is too vague.

 

3. L.97, What is the abbreviation for FGEs? Is formylglycine-generating enzyme.

 

4. In Fig 1, which band is Melac13220? Please specify.

 

5.L.181-182, why the C-terminal tag improved protein expression, the amino terminal and double terminal aldehyde tags decreased protein expression?

 

6. In section 2.2.1, the mechanism of Site-Specific Covalent Immobilization must be written in this section. What kind of functional group of SiO₂-Fe₃O₄ reacted with aldehyde tags Melac13220?

 

7. the optimal amount of enzyme solution and sodium alginate was 1:4 (V: V), but what is the enzyme concentration? It needs to be written in the Materials and methods.

 

8. Section 2.2.3, why aldehyde tags Melac13220 add glutaraldehyde for immobilization? Is glutaraldehyde added by encapsulating the enzymes in chitosan? or chitosan and enzymes solution then added glutaraldehyde? In the materials and methods did not see which step the enzyme was added.

 

9. L.337, add “(c)” in front of thermostability of the free and im.

 

10.L.482, provide the enzyme concentration.

 

11.L.487-495, provide the enzyme concentration and in which step Melac13220 was added.

 

12.L.497, how to assay C2 the concentration of immobilized Melac13220?

 

Author Response

Thank you for helping us to improve our manuscript.

1. The method of entrapment and crosslinking used in this study is the most basic method, and there is not much novelty. It is suggested that the title should be put into magnetic particles and aldehyde tags.

Response: Many thanks for the comments. In revised paper, the title has been modified as your suggestion.

2. The immobilization method (immobilization of aldehyde tags Melac13220 with magnetic particles) not seen in abstract, please add. In L.15, only see " covalent immobilization of the enzyme with a site-specific tag" in the abstract which is too vague.

Response: Many thanks for the comments. In revised paper, the abstract has been modified as your suggestion.

3. L.97, What is the abbreviation for FGEs? Is formylglycine-generating enzyme.

Response: Thanks for the comments. The full name of FGEs —— formylglycine-generating enzyme has been added to the text as your suggestion.

     4. In Fig 1, which band is Melac13220? Please specify.

Response: Many thanks for the comments. In Figure 1, the bands of Melac13220 have been  marked in gray, which may be difficult to see clearly. It has been highlighted in red in the revised paper.

     5.L.181-182, why the C-terminal tag improved protein expression, the amino terminal and double terminal aldehyde tags decreased protein expression?

Response: Thanks for the comments. The N-terminal sequence is important for proper folding of the protein because expression and folding of the protein begins at the N-terminal of the protein. Therefore, we hypothesized that N-terminal aldehyde labeling may greatly affect the  protein folding , expression and activity. In addition, the aldehyde labeled cysteines at both ends are prone to form disulfide bonds with other cysteines of the protein, which may lead to incorrect folding of the protein. Besides, the LCTPSR peptide tags of some of these proteins may be covered during the protein folding process and unable to modified by the co-expressed FGE.

6. In section 2.2.1, the mechanism of Site-Specific Covalent Immobilization must be written in this section. What kind of functional group of SiO2-Fe3O4 reacted with aldehyde tags Melac13220?

Response: Many thanks for the comments. In revised paper , the mechanism of this immobilization mehod has been written in section 2.2.1. The site-specific immobilization refers to the introduction of some special functional group into the enzyme protein through rational design of protein. The use of these functional group make the enzyme specifically bind to the specific site of the carrier. This method has become a study hotpot because of its ability to improve enzyme activity, stability or catalytic specificity. In this study, we used site-specific covalent immobilization method. Through the rational design and modification of the enzyme, a specific functional group ,aldehyde group, was introduced into the enzyme protein, and the functional group was covalently immobilized with the amino carrier. The combination of an aldehyde group with an amino group can produce a Schiff base, which is used to covalently immobilize a protein containing an aldehyde tag to a carrier containing an amino group.

7. the optimal amount of enzyme solution and sodium alginate was 1:4 (V: V), but what is the enzyme concentration? It needs to be written in the Materials and methods.

Response: Thanks for the comments. In revised paper, the enzyme concentration has been added in the Materials and methods section 3.2.2

8. Section 2.2.3, why aldehyde tags Melac13220 add glutaraldehyde for immobilization? Is glutaraldehyde added by encapsulating the enzymes in chitosan? or chitosan and enzymes solution then added glutaraldehyde? In the materials and methods did not see which step the enzyme was added.

Response: Many thanks for the comments. In the crosslinking mehod was not used aldehyde tags Melac13220. The expression in this part refers to the reaction of aldehyde group in glutaraldehyde as cross-linking agent with the amino group in the enzyme . When glutaraldehyde reacts with chitosan, the aldehyde group on the surface of the carrier may react with the amino group of the enzyme and other functional groups on the surface such as phenols, thiols and imidazole.

The chitosan was dissolved in acetic acid solution, then dropped into KOH solution, and the high-quality beads were collected and put into glutaraldehyde solution, and finally incubated and immobilized with laccase. In revised version, this part of the experiment has been explained in more detail.

9. L.337, add “(c)” in front of thermostability of the free and im.

Response: Thanks for the suggestion. In revised version, “(c)” has been added in front of the free and immobilized Melac13220.

    10.L.482, provide the enzyme concentration.

Response: Thanks for the comments. In revised version , the enzyme concentration has been added to this part.

    11.L.487-495, provide the enzyme concentration and in which step Melac13220 was added.

Response: Many thanks for the comments. The enzyme concentration and more details have been added in revised version.

    12.L.497, how to assay C2 the concentration of immobilized Melac13220?

Response: C₂ is the concentration of supernatant after immobilization, the concentration was determined by BCA method.

Reviewer 2 Report

- Can you use a commercial enzyme.

- Can we generalize this method to other enzymes.

- Line 460: lac-case or laccase.

- Line 480: can you specify the ratio enzyme/alginate.

- You must develop the characterizations of the immobilized enzyme.

- It is desirable to take photos of the immobilized enzymes.

- Can we determine the performance/price ratio of the three methods.

- The efficiency of the biocatalyst goes to the immobilization method or to different compounds used.

- Have you tested the pH effect on colors (decolorization) without using biocatalysts.

- Line 557: we may apply these biocatalysts in the case of mixture of dyes (industrial wastewater).

Author Response

Many thanks for your valuable question.

1.Can you use a commercial enzyme.

Response: Many thanks for the comments. This method cannot be used for commercial enzymes, for we cannot add tags on an enzyme expressed  already .

2. Can we generalize this method to other enzymes.

Response: Thanks for the comments. We've previously used this method to immobilize haloacid dehalogenase ST2570 (EC 3.8.1.2) from Sulfolobus tokodaii, Lipase A (EC 3.1.1.3) from Bacillus subtilis (BsLA), acyl aminopeptidase (EC 3.4.19.1) from Sulfolobus tokodaii (ST0779). But none of them can increase the stability of enzyme like this study.

Refer to the following references:

1. Jian, H.; Wang, Y.; Bai, Y.; Li, R.; Gao, R. Site-Specific, Covalent Immobilization of

Dehalogenase ST2570 Catalyzed by Formylglycine-Generating Enzymes and Its Application in Batch and Semi-Continuous Fow Reactors. Molecules 2016, 21, 895-908.

2. Lyu, J.; Li, Z.; Men, J.; Jiang, R.; Tang, G.; Zhou, Y.; Gao, R. Covalent immobilization of

Bacillus subtilis lipase A on Fe₃O₄ nanoparticles by aldehyde tag: An ideal immobilization with minimal chemical modification. Process Biochem. 2019, 81, 63–69.

3. Wang, F.; Li, R.; Jian, H.; Huang, Z.; Wang, Y.; Guo, Z.; Gao, R. Design and

Construction of an Effective Expression System with Aldehyde Tag for Site-Specifific Enzyme Immobilization. Catalysts 2020, 10, 410-424.

3. Line 460: lac-case or laccase.

Response: Many thanks for the suggestion. In revised paper, it has been modified to “laccase”.

4. Line 480: can you specify the ratio enzyme/alginate.

Response: Thanks for the comments. The ratio enzyme/alginate refers to the volume ratio of immobilized enzyme liquid volume to sodium alginate volume.

5. You must develop the characterizations of the immobilized enzyme.

Response: Thanks for the suggestion. In revised version, we have added kinetic parameters such as K_m and V_max. In the future research, we will carry out further characterization when application.

6. It is desirable to take photos of the immobilized enzymes.

Response: Many thanks for the suggestion. The photos of the immobilized enzyme have been provided in the supporting materials.

7. Can we determine the performance/price ratio of the three methods.

Response: This is a good question. Among the three methods, the entrapment and crosslinking method are cheaper than the site-specific covalent immobilization, but these two methods have many limitations in the further application, especially in the continuous-flow reaction.

8. The efficiency of the biocatalyst goes to the immobilization method or to different compounds used.

Response: Thanks for the suggestion. In revised paper, the corresponding expression has been added in section 2.2.

9. Have you tested the pH effect on colors (decolorization) without using biocatalysts.

Response: Thanks for the comments. The effect of pH on dyes has been tested in previous characterization of free enzymes. Therefore, 20mm Tris-HCl buffer (pH 7.5) was selected for Congo red in the decolorization experiment. 20mm Gly-HCl buffer (pH 1.5) was used for other dyes.

10. Line 557: we may apply these biocatalysts in the case of mixture of dyes (industrial wastewater).

Response: Many thanks for the suggestion. The corresponding expression has been added in the revised version.

Reviewer 3 Report

The article «Immobilized Methylobacterium extorquens non-blue laccase Melac13220 using different methods: Preparation, thermal stability and operational stability» is attracted to a relevant topic and has a high applied value. In recent years, with the increasing awareness of environmental protection, the restriction of relevant laws and increasing scientific knowledge, many industries have devoted themselves to developing green chemical technology by enzymatic methods. Laccase is considered one of the most powerful biocatalysts and can be used in a variety of biotechnology applications. In the present study, three methods: entrapment, cross-linking and covalent binding, were used to immobilize Melac13220 laccase to compare the immobilization effect of different methods. Understanding various immobilization methods would provide an informed choice for the immobilization of Melac13220 and an optional choice for the practical application of different immobilized laccase methods in the industrial field. Moreover, immobilization of Melac13220 led to improvement in dye decolorization.

 

However, a number of questions arise:

1. In Section 2.2.1 Optimization of Conditions for Site-Specific Covalent Immobilization authors should to add information about optimization of immobilization time. Why were 6 hours chosen? It is necessary to bring the results of own experiments or literature data.

2. In Section 2.2.1 it is necessary to explain why such an intense maximum of relative activity occurs at 20 ⁰C (Figure 2 A) and its sharp decline at 15 and 25 ⁰C.

3. From Figure 2B it is not clear whether only the pH of the buffer or also its composition affects the activity of laccase. To find this out, it is necessary to carry out additional experiments to determine the activity of laccase at the same pH values, but with different buffer compositions, using buffers containing glycine, Tris, and buffers without them. Quite often, the presence of glycine and Tris affects the enzyme activity value.

4. Important characteristics of enzymes, including immobilized ones, are their Michaelis constant (Km) and Vmax. These values must be presented for all laccase preparations obtained by the authors.

5. It is not clear why the authors write that, immobilization can improve the thermal stability of enzymes by preventing subunit dissociation, autolysis or proteolysis. Laccase is monomeric (not oligomeric) protein, so subunit dissociation is impossible for it. Besides, autolysis is also impossible, because laccase is not protease.

6. The DSC method has serious limitations in working with inhomogeneous, multicomponent samples and with samples, that are not classical solutions, which are immobilized enzymes. When describing the methods, the authors did not indicate how they overcame these limitations. In general, the results of DSC raise a serious question: how the optimal temperatures for the functioning of free and immobilized enzymes (60 and 80 ⁰C) turned out to be lower than their melting temperatures (57 and 79 ⁰C)?

7. In Figure 8, I would recommend that the y-axis start not from 40, but from 80%. So, the results will look more presentable.

Author Response

Thanks for your comments.

1. In Section 2.2.1 Optimization of Conditions for Site-Specific Covalent Immobilization authors should to add information about optimization of immobilization time. Why were 6 hours chosen? It is necessary to bring the results of own experiments or literature data.

Response: Many thanks for the suggestion. In revised paper, the relevant results have been added in section 2.2.1.

2. In Section 2.2.1 it is necessary to explain why such an intense maximum of relative activity occurs at 20 ⁰C (Figure 2 A) and its sharp decline at 15 and 25 ⁰C.

Response: Thanks for your comments. The immobilized Melac13220 on magnetic nanoparticles caused an increase in the rigidity of the protein conformation. The optimum temperature of the enzyme was increased. So, the enzyme activity decreased at low temperature. This phenomenon is quite popular in thermophilic enzyme, the higher the optimum temperature the low activity at room temperature.

Refer to the following references:

Zhang, F.; Wang, R.; Zhen, C.; Li, B.; Magnetic cellulose nanocrystals: Synthesis by electrostatic self-assembly approach and efficient use for immobilization of papain. J. Mol. Catal. B: Enzym. 2016, 134, 164-171.

3. From Figure 2B it is not clear whether only the pH of the buffer or also its composition affects the activity of laccase. To find this out, it is necessary to carry out additional experiments to determine the activity of laccase at the same pH values, but with different buffer compositions, using buffers containing glycine, Tris, and buffers without them. Quite often, the presence of glycine and Tris affects the enzyme activity value.

Response: Thanks for the suggestion. In subsequent experiments, phosphate buffer and Gly-NaOH buffer were used to measure with pH range of 7.0-9.0, and the effect on enzyme activity was consistent with previous studies.

4. Important characteristics of enzymes, including immobilized ones, are their Michaelis constant (Km) and Vmax. These values must be presented for all laccase preparations obtained by the authors.

Response: Many thanks for the suggestion. In revised version, we have added kinetic parameters such as K_m and V_max.

5. It is not clear why the authors write that, immobilization can improve the thermal stability of enzymes by preventing subunit dissociation, autolysis or proteolysis. Laccase is monomeric (not oligomeric) protein, so subunit dissociation is impossible for it. Besides, autolysis is also impossible, because laccase is not protease.

Response: Many thanks for the comments. The statements in this section come from citations, so we've listed all the possibilities mentioned in the citations one by one, and you may notice that one of them was “reducing aggregation”. Since laccase is a monomeric and not a protease, as you said, the effect of immobilization may be to reduce the aggregation of enzyme molecules.

6. The DSC method has serious limitations in working with inhomogeneous, multicomponent samples and with samples, that are not classical solutions, which are immobilized enzymes. When describing the methods, the authors did not indicate how they overcame these limitations. In general, the results of DSC raise a serious question: how the optimal temperatures for the functioning of free and immobilized enzymes (60 and 80 ⁰C) turned out to be lower than their melting temperatures (57 and 79 ⁰C)?

Response: Thanks for the comments. Fe₃O₄ nanoparticles were used in this study With a fast response to applied magnetic fields, and can be easily separated and recovered from the reaction system. Since the optimal temperatures determination in this experiment is carried out in a relatively short period of time, and the temperature-programmed DSC process is a relatively slow process, some enzyme molecules may become inactive conformations during this process. These results are consistent with the results of some immobilization studies.

Refer to the following references:

1. Battistel, E.; Attanasio, F.; Rialdi, G. Thermal stabibilty of immobilised α-chymotrypsinogen. J. Therm. Anal. Calorim. 2000, 61, 513-525.
2. Loch, CP.; Lima, MFS. Study of the degradation of the immobilization devices used in patients submitted to neck and head radiotherapy. Materia-Brazil. 2010, 12, 461-471.
3. Falahati, M.; Ma'mani, L.; Saboury, A.; Shafiee, A.; Foroumadi, A.; Badiei, A. Aminopropyl-functionalized cubic Ia3d mesoporous silica nanoparticle as an efficient support for immobilization of superoxide dismutase. BBA-Proteins Proteo. 2011, 1814, 1195-1202.

    7. In Figure 8, I would recommend that the y-axis start not from 40, but from 80%. So, the results will look more presentable.

Response: Many thanks for the comments. In revised paper, Figure 8 has been modified as your suggestion.

 

Round 2

Reviewer 1 Report

The author has replied to most of the queries, some of the following minor comments need to be corrected.

1. L.233, Figure 2c should be Figure 2d.

2.L.522-524, Authors use protein concentration to evaluate immobilization efficiency, protein concentration should be written clearly in the definition of C1 and C2.

Author Response

1. 233, Figure 2c should be Figure 2d.

Response: Many thanks for the comment. In revised paper, it has been modified to “Figure 2d”.

2. 522-524, Authors use protein concentration to evaluate immobilization efficiency, protein concentration should be written clearly in the definition of C1 and C2

Response: Thanks for your comment. In revised paper, the definitions of C1 and C2 have been described in more detail.

Reviewer 3 Report

The authors took into account most of my recommendations, but some questions still remained open.

1. The authors wrote, that in their experiments phosphate buffer and Gly-NaOH buffer were used to measure with pH range of 7.0-9.0, and the effect on enzyme activity was consistent with previous studies. These results should be added to the article, with the corresponding conclusion that the composition of the buffer does not affect the activity of laccase.

2. The authors obtained an interesting effect: for immobilized laccase, both Km and Vmax are higher than for the free enzyme. This effect is indeed observed under certain conditions (in most cases, Km increases and Vmax decreases). This is worth discussing in this article using literature data.

3. Everything that the authors wrote in response to my comments about the method of differential scanning calorimetry is correct, but this information needs to be added to the text of the article. I think that this will be of interest to a wide range of readers, and not just to the reviewer, because, I repeat, the application of the DSC method to inhomogeneous systems has its own characteristics and limitations. A detailed description of the DSC methodology must be added to section 3. Materials and Methods

Author Response

1. The authors wrote, that in their experiments phosphate buffer and Gly-NaOH buffer were used to measure with pH range of 7.0-9.0, and the effect on enzyme activity was consistent with previous studies. These results should be added to the article, with the corresponding conclusion that the composition of the buffer does not affect the activity of laccase.

Response: Many thanks for the suggestion. In revised paper, the results that phosphate buffer and Gly-NaOH buffer were used to measure with pH range of 7.0-9.0 have been shown in the Figure 2b.

2. The authors obtained an interesting effect: for immobilized laccase, both Km and Vmax are higher than for the free enzyme. This effect is indeed observed under certain conditions (in most cases, Km increases and Vmax decreases). This is worth discussing in this article using literature data.

Response: Thanks for your suggestion. In the revised version, the discussion and references have been added as you suggested.

1. Everything that the authors wrote in response to my comments about the method of differential scanning calorimetry is correct, but this information needs to be added to the text of the article. I think that this will be of interest to a wide range of readers, and not just to the reviewer, because, I repeat, the application of the DSC method to inhomogeneous systems has its own characteristics and limitations. A detailed description of the DSC methodology must be added to section 3. Materials and Methods.

Response: Thanks for the suggestion. In revised version, the detailed description of the DSC methodology has been added to section 3.2.4.