Cloning, Expression and Characterization of an Alginate Lyase in Bacillus subtilis WB600

Round 1

Reviewer 1 Report

Overall, the manuscript entitled "Cloning, Expression and Characterization of an Alginate Lyase in Bacillus subtilis WB600' is written well. Although the results have been described well it lacks discussion. Kindly discuss your results properly. In addition, kindly consider the following points.

1.  L-102: Why did the authors use 6×His-tag at the C-terminus? Please explain it.

2.  L-113: purification of alg62, authors inoculated the sample in 100ml of fermentation medium for 24hours but did not mention the temperature.

3.  For the purification of alg62, what types of chromatography techniques did the authors use?

4.  Figure 2-B: Please mark the purified recombinant alg62 on SDS-PAGE

5.  For the Effects of carbon sources how much concentration of sucrose, glucose, sodium alginate, and starch were used? Please mention everything in your article including figures.

6.  Mention the concentration of different metal ions that were used to check the effect of metal ions on enzyme activity and rewrite again.

7.  L-323: Authors point out figure 5C, but I could not find the figure 5C in the whole paper, maybe it will be 5C instead of 6C, please rewrite it again.

Other comments

-L13. Please change 'construct' to 'constructed'.

-L74 Please italicize 'B. subtilis'. Please check throughout the manuscript.

-L89 Please change 'Luria Bertani' to 'Luria-Bertani'.

-L111 Please specify what the authors use either broth or agar. for eg. instead of LB medium, LB broth would be better. 

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

This work is focused on the recombinant expression of an Alginate Lyase 2 in Bacillus subtilis WB600. The authors verified the influence of the most critical parameters on the growth and characterized the enzyme activity changing physico-chemical parameters. This paper is suitable for publication in Fermentation with major revisions.

Below there are some questions and the detail revision points for the authors'

1)      authors should include in the materials and methods section the number of replicates for each activity assay during biochemical characterization

2)      report the amount of purified protein produced and the protein yield of the fermentation

3)      for a better view of the purification process would be useful show the chromatogram

4)      the authors showed a pure protein on SDS-PAGE gel after chromatography for this reason the specific activity should be reported and compared it with others reported in literature

5)      Enlarge all the images shown to improve the resolution

6)      Add because it is useful to carried out enzymatic assay in the presence of metal ions

7)      The authors investigated how different growth parameters affected protein production. From the text it is not clear whether a) every time a parameter has been optimized it has been used for the preparation of the following fermentation and in this case specify it better in the text or b) the parameters were evaluated one at a time, leaving the others unchanged and in this case report them in the text and moreover it is useful to carry out a fermentation using all the optimized parameters to understand how much the production improves

8)      report the production yield for each fermentation parameter changed

9)      the growths were carried out on a small scale, it would be indicative to carry out a feasibility test of the production process of recombinant alginate lyase by carrying out a large-scale fermentation (or at least in 3L flasks) using all the previously optimized parameters

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The manuscript has been revised well. 

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Dear authors,

Thank you for taking my advice and answering the questions properly but there would still be one unclear point.

Regarding point 7 discussed above the authors replied as follows:

Response 7: Special thanks to the reviewers for your careful examination of our research. When we study the effects of different growth parameters on the yield of protein, every optimized parameter is used in the next research, and we will rewrite it in the article to make the results more obvious. In addition, all the optimized parameters were used for fermentation, and the final enzyme activity was 1.54 times higher than the initial one.

But in the new version of the manuscript, precisely in the materials and methods, from the line 169 to 173 the authors reported as follows:

Moreover, to determine the optimal culture conditions, different temperatures (25 °C, 30 °C, 33 °C, 37 °C, 40 °C), initial pH (4, 5, 6, 7, 8, 9, 10, 11), inoculum size (0.1%, 0.5%, 1%, 2%, 4%, 6%), and the filling volume (40 mL, 70 mL, 100 mL, 130 mL, 150 mL) were assayed while keeping the other parameters unchanged. All tests were performed in triplicate, and the results are given as average values.

The underlined parts are not consistent with each other, it would be appropriate to be clearer about it and rewrite this part

Author Response

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Author Response File: Author Response.pdf