Kinetic Study and Modeling of Wild-Type and Recombinant Broccoli Myrosinase Produced in E. coli and S. cerevisiae as a Function of Substrate Concentration, Temperature, and pH

Round 1

Reviewer 1 Report

The paper by Jimenez and Co-workers compares the kinetic characteristics of the same enzyme derived from three different origins (wild-type from Broccoli, recombinant from E.coli and from S. cerevisiae). The comparisons of kinetics are very nicely performed and sound (inhibition mechanism, effect of temperature, effect of pH).

The paper is lacking the experimental evidence of structural differences of these enzymes (folding in solution - CD spectroscopy for example). The glycosylation pattern could or should be analysed by Mass Spectrometry. But it is mentioned and discussed. Probably these experimental load would exceed the scope of this paper.

Here the focus is on the kinetics and this was performed very well and presented and explained very adequately.

 

Author Response

REVIEWER 1

The paper by Jimenez and Co-workers compares the kinetic characteristics of the same enzyme derived from three different origins (wild-type from Broccoli, recombinant from E.coli and from S. cerevisiae). The comparisons of kinetics are very nicely performed and sound (inhibition mechanism, effect of temperature, effect of pH).

The paper is lacking the experimental evidence of structural differences of these enzymes (folding in solution - CD spectroscopy for example). The glycosylation pattern could or should be analysed by Mass Spectrometry. But it is mentioned and discussed. Probably these experimental load would exceed the scope of this paper.

Here the focus is on the kinetics and this was performed very well and presented and explained very adequately.

R: we thank the comments and suggestions. The structural study of the three myrosinases will be conducted.

Reviewer 2 Report

Main question addressed by the research: The work addresses the Kinetic study and modeling of wild type and recombinant broccoli myrosinase produced in E. coli and S. cerevisiae. Originality and relevance of the topic: The topic is relevant to the field and it considers a suitable model (research gap). Added value of the paper:  The manuscript takes into account the study of substrate concentration, temperature and pH, however the main purpose of it is not clearly stated. The paper should include what aspects are critical for these assessments and clearly explain why they are analysing those and why they are needed at the end of the Introduction.

Quality of figures: Quality of figures should be improved. Specific improvements for the paper to be considered:

1. I think they have covered all the aspects required for the results, however the validation of the results is not clearly explained and this is essential in order to justify the reliability of the results.

2. Abstract is too short and general. It should summarize the main findings and applications of the paper. 

3. Apart for the R2, can you discuss the errors for each fitting and plot them with calculated vs experimental?

4. Figure 3c: Is this tendency suitable for the data? Why?

5. What is the inactivation energy? Could you explain it further?

6. The conclusions are poor and they would need more elaboration so they clearly match the results.

Author Response

REVIEWER 2

Main question addressed by the research: The work addresses the Kinetic study and modeling of wild type and recombinant broccoli myrosinase produced in E. coli and S. cerevisiae. Originality and relevance of the topic: The topic is relevant to the field and it considers a suitable model (research gap). Added value of the paper:  The manuscript takes into account the study of substrate concentration, temperature and pH, however the main purpose of it is not clearly stated. The paper should include what aspects are critical for these assessments and clearly explain why they are analysing those and why they are needed at the end of the Introduction.

R: Relevance of this study was highlighted in the penultimate paragraph of Introduction (lines 72-79)

Quality of figures: Quality of figures should be improved.

R: quality of figures was improved; figures were enlarged.

Specific improvements for the paper to be considered:

1. I think they have covered all the aspects required for the results, however the validation of the results is not clearly explained and this is essential in order to justify the reliability of the results.

R: The manuscript presents the adjustment of different kinetic models to kinetic data of wild type and recombinant myrosinase that was generated by Curiqueo et al. (2022). Perhaps I don’t clearly understand this suggestion, but since we adjusted models to experimental data in order to interpret mechanisms associated to myrosinase catalytic properties, I don’t find it necessary to validate with additional experimental data. This was clarified in the last paragraph of introduction. (lines 83-90).

1. Abstract is too short and general. It should summarize the main findings and applications of the paper. 

R: The abstract has exactly 200 words which is the limit accepted by the journal.

1. Apart for the R2, can you discuss the errors for each fitting and plot them with calculated vs experimental?

R: in the tables we reported RMSE in addition to R². RMSE indicates the average distance between predicted and experimental values. The lowest RMSE the better the fit of the model. We calculated the correlation between experimental and predicted values and informed this in the text. However, in my opinion, including the plots would not add significant information since models fit quality was informed as R², RMSE and it was added r.

1. Figure 3c: Is this tendency suitable for the data? Why?

R: interpretation of this observation was included in the text (lines 342 - 348).

1. What is the inactivation energy? Could you explain it further?

R: we defined activation Ea as the activation energy below optimum temperature, and inactivation Ea as the activation energy above optimum temperature. this was clarified in lines 352 – 353 and 365 - 366.

1. The conclusions are poor and they would need more elaboration so they clearly match the results.

R: conclusions were revised and completed. Speculations that were not confirmed in the manuscript were eliminated to agree better the results.

 

Reviewer 3 Report

The manuscript of a research paper entitled “Kinetic study and modeling of wild type and recombinant broccoli myrosinase produced in E. coli and S. cerevisiae as a function of substrate concentration, temperature, and pH” by Adielis Jiménez et al. submitted to Catalysts focuses on biochemical characteristics and modelling of native and heterologously expressed plant glucosinolates-hydrolyzing enzyme.

General remarks:

The subject of the manuscript is in accordance with the aims and scope of Catalysts as it characterizes a potentially important biological catalyst for the green chemistry. The novelty of the work is not exceptionally high as the kinetics of myrosinase from Brassica have been characterized previously by the same group. The current works adds some value regarding the modelling of kinetics depending on the reaction conditions and enzyme variant. Importantly, differences regarding the host and thermostability of the variants are valuable for future applications of the myrosinase.  

The manuscript is compact, generally adequately structured and composed, and clearly presented. Some small-scale amendments or clarifications have to be made before the manuscript can be considered for publication. The manuscript is generally written in the academic style English language.

Specific comments:

Abstract. Lines 23-25. The last sentence is not fully clear how the active site residues are in accordance with the pK values. The sentence needs to be rephrased.

Introduction. According to the Cazy database, the myrosinases are belonging to the GH 1 family. The well-characterized myrosinases that have a solved crystal structure available are from Brevicoryne brassicae and Sinapis alba. This data should be added to the section.

Line 51 and 54, also 149, 165. The sentences starting with a reference number need to be adjusted.

Enzyme kinetics models. This chapter is partly introductory and partly belonging to the Materials and methods section. As the 4.2 Modeling in Materials and methods is unacceptably compact with missing information on modelling parameters, it would be justified to split the section into two and move the literature overview to Introduction and information on equations that were actually used in the study to the Modeling subchapter.   

Results and discussion. Table 3 could be appearing in Introduction as it is fully based on the literature. Figure 3 – the substrate concentration and pH for measuring the specific activity of the tested temperatures should be given. The U (unit) has not been defined within the manuscript (shown on Figs 3 and 4, Table 7). Table 4 and 6 – please check the typos: “Schoolfied”, MYC instead of BMYC. Line 432 – S. cerevisiae should be in italics.

Materials and methods. Lines 471, 474. The final concentration (µg/ml) of protein should be stated.

Author Response

REVIEWER 3

The manuscript of a research paper entitled “Kinetic study and modeling of wild type and recombinant broccoli myrosinase produced in E. coli and S. cerevisiae as a function of substrate concentration, temperature, and pH” by Adielis Jiménez et al. submitted to Catalysts focuses on biochemical characteristics and modelling of native and heterologously expressed plant glucosinolates-hydrolyzing enzyme.

General remarks:

The subject of the manuscript is in accordance with the aims and scope of Catalysts as it characterizes a potentially important biological catalyst for the green chemistry. The novelty of the work is not exceptionally high as the kinetics of myrosinase from Brassica have been characterized previously by the same group. The current works adds some value regarding the modelling of kinetics depending on the reaction conditions and enzyme variant. Importantly, differences regarding the host and thermostability of the variants are valuable for future applications of the myrosinase.  

The manuscript is compact, generally adequately structured and composed, and clearly presented. Some small-scale amendments or clarifications have to be made before the manuscript can be considered for publication. The manuscript is generally written in the academic style English language.

R: we thank the referee for these comments.

Specific comments:

Abstract.

Lines 23-25. The last sentence is not fully clear how the active site residues are in accordance with the pK values. The sentence needs to be rephrased.

R: the sentence was rephrased. (line 24-25)

Introduction.

According to the Cazy database, the myrosinases are belonging to the GH 1 family. The well-characterized myrosinases that have a solved crystal structure available are from Brevicoryne brassicae and Sinapis alba. This data should be added to the section.

R: this information was included in the text. (lines 59-61)

Line 51 and 54, also 149, 165. The sentences starting with a reference number need to be adjusted.

R: this was corrected.

Enzyme kinetics models.

This chapter is partly introductory and partly belonging to the Materials and methods section. As the 4.2 Modeling in Materials and methods is unacceptably compact with missing information on modelling parameters, it would be justified to split the section into two and move the literature overview to Introduction and information on equations that were actually used in the study to the Modeling subchapter.   

R: We understand this observation, and we analysed this comment since it is arguable if mathematical – mechanistic models belong to theory section or to methodology section. In our opinion it is difficult to split the theoretical basis and parameters or variables definition from the equations. Also, it would result difficult to understand if the deduction of the model is partially given in theory section and partially in methods section. Accordingly, we considered this suggestion by describing the modelling procedure in Methods section (lines 490-498) and indicating que equation number in each table.

Results and discussion.

Table 3 could be appearing in Introduction as it is fully based on the literature.

R: the table was moved to Introduction as “Table 1” and mentioned in line 48.

Figure 3 – the substrate concentration and pH for measuring the specific activity of the tested temperatures should be given.

R: The conditions were pH 7.0 and 100 umol sinigrin. This was included in the figure legend.

The U (unit) has not been defined within the manuscript (shown on Figs 3 and 4, Table 7).

R: The definition of activity unit was included in section 4.2.

Table 4 and 6 – please check the typos: “Schoolfied”, MYC instead of BMYC. Line 432 – S. cerevisiae should be in italics.

R: spelling was corrected.

Materials and methods. Lines 471, 474. The final concentration (µg/ml) of protein should be stated.

R: this section was completed and corrected. In the activity assay we used 100 uL of protein extract; the total protein concentration in the extract might vary among different cultures then enzyme activity was expressed as “specific activity”. Protein concentration was assayed by BCA method using a commercial kit. (Lines 483-486).

Round 2

Reviewer 2 Report

Paper has significantly improved