Isolation and Characterization of Low-Temperature and High-Salinity Amylase from Halomonas sp. KS41843

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript shows the partial purification of an amylase from a halophilic bacterium isolated from Antarctic waters. Authors are advised to comply with the following before being considered for publication:

- The biological, ecological, and/or evolutionary reasons why bacteria from Antarctic seas exhibit amylase activity should be explained in the introduction.

- Line 72 should indicate the method used to determine the protein concentration.

- It is suggested that the volumetric activity, i.e., the U per mL of culture medium (U/mL), be reported as the first value, in parallel with the protein concentration (mg/mL). Volumetric activity allows for a measurement of culture efficiency and, additionally, the activity value per unit of biomass (U/g), which indicates biomass efficiency, can be obtained.

- Line 85 should indicate the culture medium used.

- Line 87, the activity of the culture broth should have been measured prior to partial purification.

- The effect of temperature and pH on enzyme activity should have been measured using a synergistic effects design, that is, the activity should have been evaluated at all temperatures at each pH.

- Line 134 and Fig. 1 report the maximum activity of 110.4 U/mg. So why is the relative activity in Fig. 2B with 0 g/L of NaCl added less than 100%, since the conditions of both systems are the same? And also, why is the activity of the culture broth used for semi-purification 10 times lower (11.80 U/mg, Table 2)?.

- Line 138, was instead of is.

- Review the results reported in Fig. 2a, since at 24 h there is very little biomass and a lot of activity. Performing yield calculations (Activity/OD) at 24 h, the value is 140, and at 90 h, the value is 18. In other words, very little biomass could not produce such high activity, specifically because at that time, the biomass is in the adaptation phase.

- Line 172, What is Amplify?

- Fig. 4, It is suggested to include the activity values obtained, and if you wish to include the relative activity, then indicate which activity the relative values were based on.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript presents an interesting study on the isolation and characterization of a cold-active and halotolerant amylase from Halomonas sp. KS41843, an Antarctic marine bacterium. The findings highlight the enzyme's potential for various industrial applications under low-temperature and high-salinity conditions. While the topic is suitable for Fermentation, the manuscript requires major revision in its current form to meet the standards for publication. The experimental reporting, data interpretation, and overall discussion lack the depth expected of a full research article. The following major points must be thoroughly addressed:

1. In Section 2.2 of the Methods, the amylase activity of the six initially selected strains was quantified by incubating the reaction mixture at 37°C. However, the core objective of this study is to identify "low-temperature active" enzymes, and the cultivation and isolation of the strains were performed at 15°C. The authors must justify this choice or provide screening data at a relevant low temperature (e.g., 15°C) to validate their selection of candidate strains.
2. In the methods section (Section 2.3), it is stated that "additional NaCl at concentrations of 0, 20, 40, and 60 g/L" was added to Zobell medium, which inherently contains approximately 20 g/L NaCl. In the results (e.g., Figure 2B and related descriptions in Section 3.2), please clarify whether the reported NaCl concentrations refer to the "added" or "total" NaCl concentration. Ambiguity in salt concentration data can lead to misinterpretation of optimal conditions for cell growth and enzyme production, affecting the precise understanding of experimental results.
3.  Figure 2A shows a rapid drop in enzyme activity after 90 hours, which is not explained in the text. A thorough analysis is required. The authors should discuss potential causes, such as proteolytic degradation or pH shifts in the late-stage culture, to provide a complete picture of the enzyme's production profile.
4. According to the data in Table 2, the total recovery yield of the amylase after all purification steps is only 6.03%. While the purification fold (9.35-fold) is acceptable, such a low yield is a significant drawback. What is the reason?
5. All characterization was performed on a "partially purified" enzyme. This approach is a major limitation, as trace impurities can lead to inaccurate measurements of the enzyme's intrinsic properties. The authors must justify why a fully pure enzyme was not used and explicitly discuss this limitation's potential impact on their conclusions.

6. The abstract and Section 3.5 state that certain metal ions (Co2+, Mn2+, Mg2+, Fe2+, and Na+) enhanced amylase activity by ">1.5-fold." How to explain this?

 

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

• It is highly recommended that the authors include a comparative table summarizing cold-active amylases from the literature alongside the enzyme characterized in this study. This will enhance clarity and provide better context for readers.
• Please specify in all figure legends what the error bars represent (e.g., standard deviation).
• In the discussion section, consider adding a brief commentary on the significance of amylase stability in the presence of metal ions, particularly from an industrial application perspective.
• If feasible, conducting additional experiments to evaluate the stability of the amylase in the presence of detergents and solvents would strengthen the study.
• In Figure 3B, lane 8 appears to be cropped. Kindly provide the complete, uncropped image for accuracy.
• The Materials and Methods section currently lacks appropriate references. It is advisable to include relevant citations to support the methodologies employed.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors revised the manuscript based on the observations and suggestions made. The manuscript is therefore considered suitable for publication.

Author Response

The authors revised the manuscript based on the observations and suggestions made. The manuscript is therefore considered suitable for publication.

<Response>

We sincerely thank the reviewer for the positive evaluation and for recommending our manuscript for publication. We greatly appreciate the constructive comments and suggestions provided during the review process, which have helped improve the quality of our work.

Reviewer 2 Report

Comments and Suggestions for Authors

No comments

Author Response

No comments

<Response>

We greatly appreciate the constructive comments and suggestions provided during the review process.

Reviewer 3 Report

Comments and Suggestions for Authors

It is highly recommended that the authors include a comparative table summarizing cold-active amylases from the literature alongside the enzyme characterized in this study. This will enhance clarity and provide better context for readers.

Author Response

It is highly recommended that the authors include a comparative table summarizing cold-active amylases from the literature alongside the enzyme characterized in this study. This will enhance clarity and provide better context for readers.

<Response>

We thank the reviewer for this insightful suggestion. In recognition of the reviewer’s comment, we have included a brief comparison with a selection of representative cold-active amylases in the revised Discussion section. We hope this improves clarity and offers better context for readers.

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Table 3. Enzymatic characteristics of KS41843 and other cold-active amylases

Enzyme	Microbial source	Optimum temperature (°C)	Optimum pH	Specific activity (U/mg)	Molecular weight (kDa)	Cations activators	Reference
KS41843	Halomonas sp. KS41843	30	5.0	110.32	60	Co2+, Mn2+, Mb2+, Fe2+, Na+	This study
AmyD-1	Bacillus sp. dsh19-1	20	6.0	16.4	50.1	Na+, Ca2+	26
KS7913	Alteromonas sp. KS7913	25	7.0	200.34	70	Mn2+, Ba2+, Ca2+	27
α-Amylase	Pseudoalteromonas sp. 2-3	20	8.0	51.7	68.8	Ca2+	40
AmyZ	Zunongwangia profunda	35	7.0	284.4	66	Sr2+, Fe3+, Mg2+, Ba2+, NH4+, K+	41
Ef-Amy I	Eisenia fedida	40	5.5	174	57	-	43
Ef-Amy II	Eisenia fedida	35	5.0	65	57	Ca2+	43
amylase	Nocardiopsis sp. 7326	35	8	548	55	Ca2+, Mn2+, Mg2+, Cu2+, Co2+	44
ParAmy	Pseudoalteromonas arctica GS230	30	7.5	25.5	55	Mn2+, K+, Na+	45
GA2	Microbacterium foliorum	20	9	-	-	Mg2+	47
GA6	Bacillus cereus	20	10	-	-	Ca2+	47
wtAmy175	Pseudoalteromonas sp. M175	30	7.5	289.79	61	-	48