Two-Step Purification and Partial Characterization of Keratinolytic Proteases from Feather Meal Bioconversion by Bacillus sp. P45

Round 1

Reviewer 1 Report

my comments and worries are found on the manuscript. click on each comment box attached to the highlighted lines in colours on the manuscript to view the comments

Comments for author File: Comments.pdf

Author Response

Dear Editor and Reviewers,

 

Thank you for your valuable comments and suggestions on our manuscript. We have modified the manuscript accordingly, and the detailed corrections are listed below. The manuscript was modified following the recommendations of each reviewer. The changes are marked with different text color (red) to facilitate visualization.

We hope our paper is suitable for your journal and look forward to your answer.

Sincerely,

Dr. Ailton Cesar Lemes.

 

Comments from the Editors and Reviewer:

 

REVIEWER: 1

 

Comments: (Line 17, 63, 85, 94, 204, 206) The use of the expression large capacities is cloudy. The authors should make effort to make it simple and understandable by readers.

Response: We consider the application of systems in larger volumes. Therefore, we have inserted a little explanation and modified the denomination throughout the text (Lines: 17; 63; 85; 94; 180; 183; 198; 210; 217; 225; 313; 453).

 

Comments: (Line 88) The authors have not informed readers on the source of PEG but are now talking about removing it. which one should have come first?

Response: We appreciate the observation and have modified the sentence to understand the ATPS composition and PEG removal better (Lines: 87).

 

Comments: (Line 94) The use of large capacities here is yet to be understood? What actually informed the large capacities? Large capacities of what??

Response: In the manuscript, we consider the application of systems in larger volumes. Therefore, we have inserted a little explanation and modified the denomination throughout the text (Lines: 17; 63; 85; 94; 180; 183; 198; 210; 217; 225; 313; 453).

 

Comments: (Line 115) Phosphate buffer is widely known to be best at pH range between 5.8 to 7.4 why use only two pH points of 6 and 6.5? Why exclude pH of 3 to 6.2 which could have been done with citrate buffer? Or acetate buffer 3.5 to 5.5?Phosphate 8.0 while tris is 7.0 to 9

Response: We appreciate the comment. Previously we also performed the determination of optimal pH using sodium phosphate buffer (pH 4.5 – 8.0). However, we observed the same behavior with the optimum pH being verified at 7.5. Thus, we chose to use the Tris-HCL buffer since it is already the buffer commonly used in other experimental assays.

 

Comments: (Line 123) The intervals should be stated.

Response: We acknowledge the observation. We added the maximum interval value (130 min) because each experimental condition is carried out in an adequate interval due to the denaturation that occurs in the conditions used to determine the parameters of half-life, Kd, and z value. The calculation of the parameters was based on the regression obtained with a minimum of 5 points, and only coefficients of determination (R²) above 0.93 were accepted (Line: 130-131).

 

Comments: (Line 124) As described in section 2.5.4.

Response: The sentence has been modified (Line: 132-133).

 

Comments: (Line 146) As described in section 2.5.4.

Response: The sentence has been modified (Lines: 155).

 

Comments: (Line 150) Be specific. state where it was described.

Response: The sentence has been modified (Line: 159).

 

Comments: (Line 150-151) Is Azo casein the same thing as feather meal as used in line 174?

Response: In lines, 150-151 substrate refers to the substrate used for the determination of proteolytic activity. In line 174, the substrate used by the microorganism to produce the enzyme during fermentation is mentioned. We have modified the sentence to clarify this point (Lines: 187-189).

 

Comments: (Line 152-153) Conveniently diluted enzyme is not a scientific term. Lets have the dilution fold or ratio.

Response: We removed the information, as dilution is an inherent part of the analysis (as written in published enzyme activity determination protocols), and performed for proper reaction reading in a spectrophotometer. (Line: 161).

 

Comments: (Line 153) Crosscheck this with the result of thermal stability studies.

Response: The information was verified and maintained since the parameters of the proteolytic activity are used to determine the enzyme's activity at that point. The stability results indicate the enzyme's resistance against the operational conditions for application.

Comments: (Line 156) What was it measured with spectrophotometer or colorimeter?

Response: We change the sentence and insert the information about the spectrophotometer (Line: 165).

 

Comments: (Line 174) Is this feather meal the same with Azo casein as used in line 151?

Response: In lines, 150-151 substrate refers to the substrate used for the determination of proteolytic activity. In line 174, the substrate used by the microorganism to produce the enzyme during fermentation is mentioned. We have modified the sentence to clarify the sentence (Lines: 187-189).

 

Comments: (Line 175-176) Is this protocol the same as the one used in line 151 (reference 8)

Response: We appreciate the observation. The protocols are different, one of them is related to the determination of proteolytic activity, and the other to purification by an aqueous two-phase system (ATPS). We have modified the sentence to avoid misinterpretations (Lines: 185-189).

 

Comments: (Line 184-185) Is this result or literature review?

Response: We have modified the sentence and indicated that it refers to a previous study by our research group (Line: 201).

 

Comments: (Line 193-197) I am not too sure that this experiment was performed under material and methods.

Response: We have modified the sentence and indicated that this is previous information from our research group (Lines: 210).

 

Comments: (Line 203) The reader is at lost on what the subscript or superscript "a"  and "A" on the values really meant?

Response: We have inserted additional information to indicate that lowercase letters refer to the 1st ATPS and capital letters refer to the 2nd ATPS in relation to Tukey’s test (Lines: 218-220).

 

Comments: (Line 223-225) Section 2.5.1 addressed the experiment conducted between pH range of 6.0 to 10.0. (lines 114 -116), how come in the result section the authors claimed that the purified keratinolytic protease from Bacillus sp. P45 was active at pH values between 6.5 and 11.0 (lines 223 -225)?

Response: We apologize for our mistake and have modified the sentences to standardize the value of the pHs evaluated throughout the text (Lines: 122; 249).

 

Comments: (Line 250, 252, 254) Optimum.

Response: We changed according to the recommendation (Lines: 247; 273; 275; 277; 279).

 

Comments: (Line 250) Maximum.

Response: We changed according to the recommendation (Line: 275).

 

Comments: (Line 252) Maximum is correct.

Response: We appreciate the observation.

 

Comments: (Line 252) Recast this statement.

Response: We have revised the sentence. (Lines: 277-278).

 

Comments: (Lines 308-310) Methodology not stated under materials and methods.

Response: We have modified the sentence to clarify that the mentioned result is a previous finding of our research group (Line: 332).

 

Comments: (Line 312): Improve the English language used in this section.

Response: We have revised the English in this section.

 

Comments: (Lines 314-315) Reference?? Reported by who?

Response: We add the appropriate references (Line: 342).

 

Comments: (Lines 321-322) Hadjidi et al., ???

Response: We have modified accordingly (Line: 349).

 

Comments: (Line 325) How??

Response: Thank you for the observation. We insert probable interactions that can affect enzymatic activity (Lines: 351-352).

 

Comments: (Lines 385-359) State the exact range involved.

Response: We insert in the sentence the concentration of solvent used (Lines: 413-414).

Author Response File: Author Response.docx

Reviewer 2 Report

In the present paper, the author's  described purification and partial characterization of keratinolytic proteases from feather meal bioconversion by Bacillus sp. P45. 

The work is well written, with clear background. I have 2 important comments for the methodology. 

1) Isolated protein must be analysed by MALDI-MS for protein identyfication

2) Analysis of pure protein Fig 1 Lane 3 shows additional band. Could the authors identify contaminant protein with MW 37 kDA?

3) PAGE gel was stained with Coomasie Blue R-250 staining solution, could the author describe staining procedure in details. 

4) PAGE analysis of Protein Purity (Fig. 1) should be done by silver steining protocol for better visualization contaminant proteins.

5) Please add references in section Section Materials and Methods. 

 

 

 

Author Response

Dear Editor and Reviewers,

 

Thank you for your valuable comments and suggestions on our manuscript. We have modified the manuscript accordingly, and the detailed corrections are listed below. The manuscript was modified following the recommendations of each reviewer. The changes are marked with different text color (red) to facilitate visualization.

 

We hope our paper is suitable for your journal and look forward to your answer.

Sincerely,

Dr. Ailton Cesar Lemes.

 

REVIEWER: 2

 

In the present paper, the author's described purification and partial characterization of keratinolytic proteases from feather meal bioconversion by Bacillus sp. P45.

 

The work is well written, with clear background. I have 2 important comments for the methodology.

 

Comments:

1) Isolated protein must be analysed by MALDI-MS for protein identification.

Response: We really appreciate the suggestion, but we don't have equipment available in our structure to identify the isolated protein, as recommended.

 

Comments

2) Analysis of pure protein Fig 1 Lane 3 shows additional band. Could the authors identify contaminant protein with MW 37 kDA?

Response: Unfortunately, due to available structure and limited time, it is impossible to determine what contaminating protein appears in the purified extract through ATPS and diafiltration. However, this should be deepened in future work to characterize the extract better. Additionally, it is important to highlight that the enzymatic extract obtained was adequately applied by our research group in several processes, including the enzymatic coagulation of milk and the production of hydrolysates, among others.

 

Comments

3) PAGE gel was stained with Coomasie Blue R-250 staining solution, could the author describe staining procedure in details.

Response: The protocol for staining the gel with Coomassie Blue R-250 was described in detail in section 2.4 (Lines: 115-118).

 

Comments

4) PAGE analysis of Protein Purity (Fig. 1) should be done by silver staining protocol for better visualization contaminant proteins.

Response: We stained the gel using silver and included it as a supplementary material file.

 

 

Comments

5) Please add references in section Materials and Methods.

Response: We appreciate the suggestion and have included the appropriate references in sections “2.5.1. Optimum pH and temperature for purified protease activity” and “2.5.2. Thermal stability of purified protease and deactivation kinetics”. (Lines 126; 129).

Author Response File: Author Response.docx

Reviewer 3 Report

This manuscript reported a keratinolytic protease from Bacillus sp. P45. The paper provided some new information, can be published after add some important data.

 1. As the main work of this paper is about purification and partial characterization, the specific activity should be calculated and given in the paper, not just just mentioned.

 2. It is necessary to compare this enzyme with other proteases，so it’s better to list the specific activity and molecular weight of each enzyme in table 2.

 3. The yield of the enzyme should be given, that is how much enzyme we can get for given number of culture medium.

 4. Since many parameters of this keratinolytic protease are different from reference 9, I suggest to check the substrate specific activity of keratinolytic protease from Bacillus sp. P45.

Author Response

Dear Editor and Reviewers,

 

Thank you for your valuable comments and suggestions on our manuscript. We have modified the manuscript accordingly, and the detailed corrections are listed below. The manuscript was modified following the recommendations of each reviewer. The changes are marked with different text color (red) to facilitate visualization.

 

We hope our paper is suitable for your journal and look forward to your answer.

Sincerely,

Dr. Ailton Cesar Lemes.

 

Reviewer: 3

This manuscript reported a keratinolytic protease from Bacillus sp. P45. The paper provided some new information, can be published after add some important data.

 

Comments

1. As the main work of this paper is about purification and partial characterization, the specific activity should be calculated and given in the paper, not just mentioned.

Response: We appreciate the feedback and have added specific activity values in the work, as well as a better definition of this parameter (Lines: 103-104; 220-221).

 

Comments

2. It is necessary to compare this enzyme with other proteases so it’s better to list the specific activity and molecular weight of each enzyme in Table 2.

Response: We appreciate the suggestion. We added the specific activity available in the mentioned studies, as well as the molecular weight of the purified proteases (Table 1 and 2).

 

Comments

3. The yield of the enzyme should be given, that is how much enzyme we can get for given number of culture medium.

Response: We have added to the text information about yields obtained through the ATPS sequence and diafiltration (I.e., volume, enzyme activity and specific activity yields). (Lines: 196-199).

 

Comments

4. Since many parameters of this keratinolytic protease are different from reference 9, I suggest to check the substrate specific activity of keratinolytic protease from Bacillus sp. P45.

Response: We appreciate the suggestion and have inserted in Tables 1 and 2 the specific activity of the protease enzyme used in this work and in other available publications (Tables 1 and 2).

 

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Dear authors, 

Protein identification is a critical element of protein isolation experiments/processes. Your protein must be identified by MALDI -MS or western blot. 

Protein analysis by PAGE with silver staining protocol demonstrated low purity of your protein so the method for protein isolation must be improved.

Author Response

Dear Editor and Reviewers,

Thank you for your valuable comments and suggestions on our manuscript. We have modified the manuscript accordingly; the detailed corrections are listed below. The manuscript was altered following the recommendations of each reviewer. The changes are marked in the manuscript file with different text colors (red) to facilitate visualization.

We hope our paper suits your journal and look forward to your answer.

Sincerely,

 

REVIEWER: 2

 

Comments:

Dear authors, 

Protein identification is a critical element of protein isolation experiments/processes. Your protein must be identified by MALDI-MS or western blot. 

 

Response: 

Thank you for your comment and the possibility of improving our work. Our research group has already used other high-resolution purification methods to characterize and identify the keratinase from Bacillus sp. P45. The high-resolution methods used allowed the proper identification of the keratinolytic enzyme. The purification protocol used in the previous work was composed of the sequence of the following techniques: Ammonium sulfate precipitation (30% saturation); Ammonium sulfate precipitation (60% saturation); gel filtration (Sephadex G-75); ion exchange chromatography (DEAE Sepharose Fast Flow) which resulted in a purification factor of 20-fold purification factor and enzyme recovery around 18.0%.

The enzyme identification was performed using the UPLC (ultra-performance liquid chromatography) system coupled to a Q-TOF micro (Waters) tandem mass spectrometer with nano-ESI. The analysis of tryptic peptides revealed sequence homologies that characterize keratinase as a subtilisin-like serine protease. The 8 peptides identified from trypsin digestion and their amino acid sequences covered approximately 50% of the mature sequences from the comparative subtilisins (Daroit et al., 2010).

Now, the purpose of the current manuscript is to explore other purification methods that are simpler, less expensive, and easily scaled up. Even using a combination of two methods with lower resolution (ATPS and diafiltration), it was already possible to obtain an extract with a reduction of large contaminants that could interfere in its application, which allowed a suitable performance of the extract obtained at the end of the process.

          Our research group works with the line of application of biotechnological products in food, which does not require enzymatic extracts with high purity since the cost related to purification could compromise and make the process unfeasible due to the high costs of the final product and its low recovery. Thus, to know the performance of the extract obtained from the ATPS and diafiltration sequence, we now characterize the enzyme using conditions and components present in food processing, for example, providing accurate information for proper application.

It is important to highlight that the same enzymatic extract was applied as milk clotting to develop cream cheese with chia and quinoa flour (Lemes et al., 2016) and in the enzymatic hydrolysis to produce peptides and other bioactive compounds (Cunha et al., 2022).

We insert a brief description of the enzyme identification performed earlier. (Lines: 257-265).

 

References:

Cunha, I.C., Brandelli, A., Braga, A.R.C., Sala, L., Kalil, S.J. 2022. Feather Meal as a Source of Peptides with Antioxidant Activity from Enzymatic Hydrolysis. Waste and Biomass Valorization, 1, 1-9.

Daroit, D.J., Corrêa, A.P.F., Segalin, J., Brandelli, A. 2010. Characterization of a keratinolytic protease produced by the feather-degrading Amazonian bacterium Bacillus sp. P45. Biocatalysis and Biotransformation, 28(5-6), 370-379.

Lemes, A.C., Pavón, Y., Lazzaroni, S., Rozycki, S., Brandelli, A., Kalil, S.J. 2016. A new milk-clotting enzyme produced by Bacillus sp. P45 applied in cream cheese development. LWT - Food Science and Technology, 66, 217-224.

 

 

Comments:

Protein analysis by PAGE with silver staining protocol demonstrated low purity of your protein so the method for protein isolation must be improved.

 

Response: We appreciate the suggestion. However, our research group has already performed enzyme purification using high-resolution techniques for enzyme identification (ammonium sulfate precipitation + gel filtration + ion exchange chromatography) (Daroit et al., 2010). In this work, we propose using simple and less expensive purification techniques for industrial applications that require partially purified enzymes. We indicate in the text the importance of establishing simpler, more accessible, and less costly purification protocols and characterizing each of the extracts obtained. (Lines: 266-276).

Reviewer 3 Report

Some important information has been added to the article. But there are still some small questions need to be confirmed.

1. “Regarding the yield, for each 1000 mL of cell-free culture medium produced with a specific activity of 965 U/mg, 1680 mL of a purified extract were obtained with a specific activity of 25795 U/mg, which results in a volume yield of 168%, enzymatic activity yield of 422%, and specific activity yield of 2265%.”

I cannot understand the meaning this sentence, why the total enzyme activity of 1 L culture medium can be so high than crude extract after finished purification?  Please check it or explain the result. The enzyme is activated during purification?

 

2. In “3.4. Effects of salts and solvents on proteolytic activity”, CaO can not exist in water, maybe it’s Ca(OH)2?  Please correct it in whole paper (include figures), and also in Fig.3, correct FeCl to FeCl3.

Author Response

Dear Editor and Reviewers,

Thank you for your valuable comments and suggestions on our manuscript. We have modified the manuscript accordingly; the detailed corrections are listed below. The manuscript was altered following the recommendations of each reviewer. The changes are marked in the manuscript file with different text colors (red) to facilitate visualization.

We hope our paper suits your journal and look forward to your answer.

 

REVIEWER: 3

 

Some important information has been added to the article. But there are still some small questions need to be confirmed.

 

Comments: 1. "Regarding the yield, for each 1000 mL of cell-free culture medium produced with a specific activity of 965 U/mg, 1680 mL of a purified extract were obtained with a specific activity of 25795 U/mg, which results in a volume yield of 168%, enzymatic activity yield of 422%, and specific activity yield of 2265%." I cannot understand the meaning this sentence, why the total enzyme activity of 1 L culture medium can be so high than crude extract after finished purification?  Please check it or explain the result. The enzyme is activated during purification?

 

Response: We appreciate the observation and the possibility of correcting our mistake. We made a mistake in determining the process yield when using the second ATPS activity values - we considered the bottom phase instead of the top phase, where the enzyme partitions. We changed the values in the text, indicating that the volume yield was 27.5% and the activity yield was 134.5%. Recovery values greater than 100% for enzyme activity are expected since the diafiltration process removes contaminants that inhibit enzyme activity, but also by eliminating polyethylene glycol and salts that cause interference in activity and protein determinations (commonly reported in the literature) (Golunski et al., 2016; Rossouw et al., 2021). (Lines:197-205).

 

Comments: 2. In "3.4. Effects of salts and solvents on proteolytic activity", CaO can not exist in water, maybe it's Ca(OH)2?  Please correct it in whole paper (include figures), and also in Fig.3, correct FeCl to FeCl₃.

 

Response: We appreciate the comment. The salt used in experiments is presented as calcium oxide P.A. purchased from Qeel, São Paulo, Brazil (CaO. MW: 56.08). Thus, we chose to maintain the chemical formula on the package presentation. Regarding FeCl₃, we changed Figure 3 and inserted the correct chemical formula.

 

 

Golunski, S.M., Sala, L., Silva, M.F., Dallago, R.M., Mulinari, J., Mossi, A.J., Brandelli, A., Kalil, S.J., Di Luccio, M., Treichel, H. 2016. Interference of salts used on aqueous two-phase systems on the quantification of total proteins. Int J Biol Macromol, 83, 30-3.

 

Rossouw, S., Bendou, H., Bell, L., Rigby, J., Christoffels, A. 2021. Effect of polyethylene glycol 20 000 on protein extraction efficiency of formalin-fixed paraffin-embedded tissues in South Africa. Afr J Lab Med, 10(1), 1122.

 

Round 3

Reviewer 2 Report

Dear authors,

Thank you for MS revision.