Discovery and Preliminary Characterization of Lactose-Transforming Enzymes in Ewingella americana L47: A Genomic, Biochemical, and In Silico Approach

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors identified Ewingella americana L47 from Antarctica as a bacterium producing tagatose from lactose and determined its whole genome sequence. They then confirmed the presence of one L-arabinose isomerase (AraA) gene and three beta-galactosidase (LacZ, BglY, BgaA) genes in the genome. Furthermore, they attempted to heterogeneously produce these enzymes in E. coli and confirm their activities. The activity of the recombinant AraA was confirmed. The beta-galactosidase activities were confirmed using the refolded enzymes. Among these refolded enzymes, activity was detected for BgaA, but not for LacZ or BglY.

While the discovery of industrially useful bacteria and the determination of their genome sequences are positively evaluated, the current manuscript is difficult to understand and incomplete. Several figures are missing, and the discussion includes points based on unclear results, making it unlikely to be published in its current form.

 

1.L496-512: β-galactosidase activities were measured using washed pellets or refolded enzymes. Therefore, it is difficult to determine whether the enzymatic properties reflect the inherent nature of the enzyme or the properties of the recombinant enzyme produced by E. coli. Consequently, it is difficult to determine whether these enzymes function as beta-galactosidase or not. As it stands, the sections describing beta-galactosidase largely consist of redundant descriptions of unclear results. Furthermore, the authors discuss these unclear results at length, making the text itself unclear. It should be stated concisely that β-galactosidase could not be successfully expressed in E. coli, and the manuscript should be rewritten to be more comprehensible overall, including the title.

2. The authors wrote that the recombinant enzymes were refolded, but activities were little. It would be better to confirm whether they were properly refolded by measuring CD spectra or similar methods.

3. 2.3 In Silico Structural Analysis and 2.4 Molecular Dynamics and Binding Affinity Predictions: These sections present simulation results based on the predicted structures and do not provide particularly significant insights, as comparisons with purified enzymes are lacking.

Except for the sentences explaining amino acid sequence conservation, they are entirely unnecessary. They should be summarized concisely.

4. To verify whether L-arabinose isomerase and three types of β-galactosidase are actually functional within the bacteria, at least the expression of mRNA should be confirmed. It remains unclear whether the enzymatic properties of β-galactosidase reflect the inherent characteristics of the enzyme itself or are merely attributes of the recombinant enzyme. As it stands, it remains unclear whether these enzymes are even functionally active within the bacterial cells, let alone whether they are acting as beta-galactosidases. This ambiguity renders the entire paper unclear.

5. Discussion: It is meaningless to discuss results obtained using enzymes that have not been properly refolded and purified. The authors must discuss the results of enzymes confirmed to be correctly folded.

6. Supplementary Table S1 is not attached.

7. The overall three-dimensional structures of the proteins in Figures 2A and 2B appear identical.

8. L477, Figure 5A-B: There is no SDS-PAGE image of the purified enzyme.

 

Others

L25-27: Affinity with the substrate should be measured through binding experiments rather than simulation.

L472, Figure 5A: It is more helpful to readers to show all masses for markers. Specifying which band corresponds to AraA aids reader comprehension. Due to the low protein loading in lane 1, it is impossible to determine whether AraA is actually expressed.

L1000, “primary beta-garalactosidase”: The meaning of “primary” is unclear.

Author Response

To Reviewers´

 

We sincerely thank the reviewers for their thorough, insightful, and constructive evaluation of our manuscript. Their comments were carefully considered and proved highly valuable in helping us refine the focus, coherence, and overall scientific quality of the work. As a result of this process, we believe the revised manuscript is now more clearly framed, methodologically robust, and conceptually focused.

In response to the reviewers’ suggestions, the manuscript has been extensively revised to improve clarity, methodological accuracy, and consistency between experimental data and interpretation. Major revisions include the removal of all references to protein refolding, a complete restructuring and rewriting of the Discussion section, clarification of inclusion body–based enzymatic assays, correction and expansion of figures, incorporation of genome-based regulatory analyses, and the addition of new experimental data in the Supplementary Materials and Appendix.

Overall, we feel that the reviewers’ feedback has significantly strengthened the manuscript and helped sharpen its scientific message. Detailed, point-by-point responses to each comment are provided below.

 

Reviewer 1

The authors identified Ewingella americana L47 from Antarctica as a bacterium producing tagatose from lactose and determined its whole genome sequence. They then confirmed the presence of one L-arabinose isomerase (AraA) gene and three beta-galactosidase (LacZ, BglY, BgaA) genes in the genome. Furthermore, they attempted to heterogeneously produce these enzymes in E. coli and confirm their activities. The activity of the recombinant AraA was confirmed. The beta-galactosidase activities were confirmed using the refolded enzymes. Among these refolded enzymes, activity was detected for BgaA, but not for LacZ or BglY.

While the discovery of industrially useful bacteria and the determination of their genome sequences are positively evaluated, the current manuscript is difficult to understand and incomplete. Several figures are missing, and the discussion includes points based on unclear results, making it unlikely to be published in its current form.

 

Response: We sincerely thank the reviewer for the thorough and insightful evaluation of our manuscript. We have carefully addressed all the points raised, incorporating significant revisions to improve clarity, completeness, and scientific rigor. In particular, we have clarified ambiguous sections, added missing figures, and refined the Discussion to focus on experimentally supported results.

We believe that the manuscript has been substantially strengthened through these modifications and we hope that the revised version meets the expectations of the reviewer and will be considered suitable for publication.

 

1.L496-512: β-galactosidase activities were measured using washed pellets or refolded enzymes. Therefore, it is difficult to determine whether the enzymatic properties reflect the inherent nature of the enzyme or the properties of the recombinant enzyme produced by E. coli. Consequently, it is difficult to determine whether these enzymes function as beta-galactosidase or not. As it stands, the sections describing beta-galactosidase largely consist of redundant descriptions of unclear results. Furthermore, the authors discuss these unclear results at length, making the text itself unclear. It should be stated concisely that β-galactosidase could not be successfully expressed in E. coli, and the manuscript should be rewritten to be more comprehensible overall, including the title.

 

Response: We thank the reviewer for this important comment. Upon careful reconsideration, we acknowledge that the recombinant β-galactosidases were not subjected to proper refolding protocols. Instead, enzyme activity was assessed directly from washed inclusion body preparations. To avoid confusion, we have removed references to “refolded enzymes” throughout the manuscript and now explicitly state that β-galactosidase activity was tested using washed inclusion bodies. The revised Results section (L496–512) has been rewritten to reflect this clarification. These revisions have improved the overall comprehensibility of the Results and Discussion.

We have also revised the manuscript title to more accurately reflect the scope and limitations of the study, particularly regarding the partial functional characterization and the lack of proper refolding validation. These changes improve clarity, align the text with the experimental procedures actually performed, and address the reviewer’s concerns about the interpretability of the β-galactosidase results.

 

2. The authors wrote that the recombinant enzymes were refolded, but activities were little. It would be better to confirm whether they were properly refolded by measuring CD spectra or similar methods.

 

Response: We thank the reviewer for this critical observation. Upon review, we acknowledge that the β-galactosidase preparations used were derived from washed inclusion bodies and not subjected to formal refolding procedures. As such, we cannot confirm whether these enzymes attained proper tertiary or quaternary structure. We agree that techniques such as circular dichroism spectroscopy would be ideal to validate proper folding. Due to experimental limitations, such biophysical analyses were not performed. We have revised the manuscript to accurately describe the nature of these preparations and added a statement in the Discussion to clearly acknowledge this limitation and its impact on interpretation of enzyme activity. We appreciate the reviewer’s suggestion and have incorporated it to improve the clarity and transparency of our findings

 

3. 2.3 In Silico Structural Analysis and 2.4 Molecular Dynamics and Binding Affinity Predictions: These sections present simulation results based on the predicted structures and do not provide particularly significant insights, as comparisons with purified enzymes are lacking.

Except for the sentences explaining amino acid sequence conservation, they are entirely unnecessary. They should be summarized concisely.

 

Response: We thank the reviewer for this important observation. We fully agree that structural simulations based on predicted models cannot replace experimental validation, and we have revised the manuscript accordingly. Sections 2.3 and 2.4 have been substantially condensed to present only the most essential findings. Detailed MD analyses (e.g., RMSD and RMSF plots) have been moved to the Supplementary Information (Figures S3 and S4).

We now explicitly state that MM/GBSA binding energy calculations were used solely as qualitative, comparative tools to prioritize experimental candidates—not as definitive indicators of enzymatic performance. Additionally, the Result and Discussion has been revised to emphasize that in silico binding predictions did not correlate with enzymatic activity, which was ultimately determined by successful expression and folding in the heterologous system.

We believe these changes improve clarity and ensure that the computational results provide contextual support without overstating their significance, in line with the reviewer’s suggestion.

 

4. To verify whether L-arabinose isomerase and three types of β-galactosidase are actually functional within the bacteria, at least the expression of mRNA should be confirmed. It remains unclear whether the enzymatic properties of β-galactosidase reflect the inherent characteristics of the enzyme itself or are merely attributes of the recombinant enzyme. As it stands, it remains unclear whether these enzymes are even functionally active within the bacterial cells, let alone whether they are acting as beta-galactosidases. This ambiguity renders the entire paper unclear.

 

Response: We thank the reviewer for this important comment regarding native gene expression and enzymatic activity. Due to time constraints during the revision period, we were not able to perform quantitative PCR (qPCR) to assess mRNA expression levels of the β-galactosidases and L-arabinose isomerase genes. However, we conducted a rapid transcriptional test to gain initial insights. Specifically, we grew the native L47 strain under standard culture conditions, extracted total RNA, synthesized cDNA, and performed endpoint PCR using the same primer sets that were originally used for cloning the genes (see Appendix).

Interestingly, only the araA gene yielded a clear PCR amplification product, while the β-galactosidase genes (lacZ, bgaA, bglY) did not. This lack of amplification may be due to the large size of these genes and suboptimal PCR efficiency under the rapid conditions used. Designing new, optimized primers for shorter amplicons and performing proper qPCR assays would be required for conclusive gene expression analysis, but unfortunately, this was not feasible within the short revision timeframe.

Despite the absence of direct mRNA confirmation, we highlight that cell-free extracts from the native L47 strain (prior to cloning) showed clear β-galactosidase activity—evidenced by X-Gal hydrolysis on plates (Supplementary Figure 1S) and o-NPG activity in crude extracts—as well as the ability to convert galactose into tagatose (Figure 1A). These functional assays strongly suggest that at least one native β-galactosidase and the L-arabinose isomerase are expressed and active under the tested growth conditions.

In the revised Discussion, we now explicitly acknowledge this limitation and clarify that our conclusions about in vivo enzyme expression are based on indirect functional assays rather than transcript-level data. We also note that BgaA is the most likely β-galactosidase active in L47, given its activity profile in recombinant assays, although the roles of BglY and LacZ may depend on specific regulatory or environmental conditions.

In addition, and in direct response to the reviewer’s concern regarding the physiological relevance of these enzymes, we performed an in silico analysis of the genomic context and regulatory architecture of the araA, bgaA, bglY, and lacZ loci (Figure 1D). This analysis revealed operon-like gene organizations and the presence of predicted −35/−10 promoter motifs compatible with σ⁷⁰-dependent transcription, together with locus-specific regulatory genes (AraC- and LacI-type regulators). These features are consistent with inducible expression systems responsive to carbon source availability and support the conclusion that these genes are not silent but are embedded within functional regulatory frameworks. All results from operon prediction and promoter analysis have been incorporated into the revised manuscript and discussed in detail in the Discussion section.

Taken together, the combination of genomic regulatory evidence, native enzymatic activity detected in crude extracts, and recombinant functional assays provides convergent support for the functional relevance of these enzymes in strain L47, while also clarifying the current limitations regarding direct transcript-level quantification.

 

5. Discussion: It is meaningless to discuss results obtained using enzymes that have not been properly refolded and purified. The authors must discuss the results of enzymes confirmed to be correctly folded.

 

Response: We appreciate the reviewer’s comment and fully agree that interpretations should be grounded in experimentally validated data. In response, we have revised the Discussion section to focus exclusively on the enzymes for which proper folding and activity were confirmed. Specifically, we emphasize:

• AraA, which was expressed in soluble form and purified, and
• BgaA, which retained sufficient activity in washed inclusion body preparations to permit kinetic characterization.

These enzymes are now the primary basis for our biochemical discussion.

Conversely, we have removed or reframed any speculative interpretation regarding BglY and LacZ, as their inclusion bodies could not be successfully refolded into reliably active forms. In the revised manuscript, the case of LacZ is now presented not as a functional result but as an example illustrating the challenges of expressing and folding large multimeric enzymes heterologously. We also added a clarifying statement early in the Discussion to explicitly acknowledge that inclusion body preparations do not guarantee correct folding and must be interpreted cautiously.

These adjustments ensure that the discussion is firmly based on robust experimental evidence and avoids overinterpretation. We believe this revision fully addresses the reviewer’s concern by aligning the discussion with the quality and reliability of the data obtained.

 

6. Supplementary Table S1 is not attached.

 

Response: We apologize for the oversight. Supplementary Table S1 (the biochemical profile of isolate L47) has now been included with the revised submission. In the manuscript, we have referenced Table S1 in the context of L47’s biochemical identification. The table provides the results of the RapID™ ONE panel tests for L47, which were used to phenotypically characterize the strain. This supplementary information is now properly attached for the reader’s review.”

 

7. The overall three-dimensional structures of the proteins in Figures 2A and 2B appear identical.

 

Response: Thank you for noting the mistake. We discovered that the original Figure 2A and 2B images were inadvertently the same. In the revised manuscript, we have corrected this by providing the proper images: Figure 2A now shows the homology model of L47 AraA (L-arabinose isomerase) superimposed on its hexameric template, and Figure 2B shows the model of L47 BgaA (a GH42 β-galactosidase) with its distinct active-site features. The two panels are now clearly different, as intended. We have double-checked the figures to ensure accuracy.

 

8. L477, Figure 5A-B: There is no SDS-PAGE image of the purified enzyme.

 

 Response: We thank the reviewer for pointing out the omission. In the revised manuscript, we have replaced Figure 3A with a complete SDS-PAGE image showing the expression and purification of the E. americana L-arabinose isomerase (AraA), including the purified protein (~56 kDa) obtained using a HiTrap column. This updated panel provides a detailed view of the purification workflow and final protein purity.

Figure 3B shows the SDS-PAGE analysis of the three recombinant β-galactosidases (BgaA, BglY, LacZ) from E. americana expressed in E. coli. These were not purified but instead analyzed as washed inclusion body preparations. The presence of single bands of the expected molecular weight in the washed pellets confirms successful overexpression, although no solubilization or refolding steps were applied due to low recovery and loss of activity in preliminary attempts.

We have clarified this distinction in the revised figure legend and corresponding Results section. We hope these additions address the reviewer’s concerns by providing visual confirmation of the protein expression and purification status for the enzymes studied.

 

Others

L25-27: Affinity with the substrate should be measured through binding experiments rather than simulation.

 

Response: We fully agree with the reviewer. We have clarified in the revised manuscript that any ‘affinity’ results in our study come from in silico modeling and simulations, and we do not present them as definitive measures. Specifically, we state that the MM/GBSA binding energy calculations were used only as a qualitative guide and do not replace experimental binding assays or kinetics. Furthermore, we added language to emphasize that our experimental findings (enzyme activities and kinetics) did not necessarily match the simulation-based affinity rankings, underscoring the necessity of biochemical validation. In summary, all references to substrate affinity in the manuscript are now clearly framed in the context of predictions, and we have cautioned readers that such predictions must be confirmed with actual binding experiments in future work.

 

 

L472, Figure 5A: It is more helpful to readers to show all masses for markers. Specifying which band corresponds to AraA aids reader comprehension. Due to the low protein loading in lane 1, it is impossible to determine whether AraA is actually expressed.

 

Response: We thank the reviewer for this helpful suggestion. In the revised manuscript, we have updated the SDS-PAGE image (now Figure 3A) to clearly indicate the molecular weight marker bands with their corresponding sizes (in kDa) directly on the figure. Additionally, we have annotated the ~56 kDa band corresponding to the AraA protein in the induced lanes, as suggested. While the AraA expression level was relatively low under the tested conditions—resulting in a faint band in lane 3—the identity of the band was confirmed through subsequent purification (as shown in the final lane) and enzymatic activity assays.

We have also revised the figure legend to clearly describe which band corresponds to AraA and to clarify the induction conditions used. These improvements enhance the readability and interpretation of the gel, and we are grateful to the reviewer for encouraging this clarification.

 

L1000, “primary beta-garalactosidase”: The meaning of “primary” is unclear.

 

Response: We apologize for the confusing terminology. In the revised manuscript, we have reworded the passage to avoid the term ‘primary’ in this context. We now clearly state that BgaA appears to be the principal (most significant) β-galactosidase of the three encoded in L47 in terms of lactose hydrolysis, based on our results. Instead of “primary β-galactosidase,” we describe it as the likely physiologically relevant lactose-hydrolyzing enzyme in L47, and contrast it with BglY which might play a secondary role. This change in wording should eliminate any ambiguity.”

Reviewer 2 Report

Comments and Suggestions for Authors This work is significant, but several issues need to be discussed with the authors:

1. In the Results section, it is mentioned that AraA was expressed solubly and purified, but the SDS-PAGE image of the purified protein sample was not shown in Figure 5. The authors need to present this result.
2. In the Results section, it is mentioned that AraA was expressed insolubly and that refolding of inclusion body proteins was performed. However, the Methods section only describes how to wash the inclusion bodies. This part needs to be supplemented with specific refolding methods (including inclusion body solubilization and refolding conditions). Refolding such a large protein is usually very difficult.
3. I did not see that the authors have deposited the genomic sequence of strain L47 in a public database, and no relevant accession number was provided.
4. Section 4.7 mentions "Genomic Characterization of the Isolate L47," but no experimental methods for genome analysis are described.
5. It is rather puzzling that the effects of metal ions on enzyme activity are mentioned multiple times in the text, with Mn²⁺ and Ca²⁺ used in some places, and Co²⁺, Mg²⁺, Mn²⁺ used in others. Why weren't other metal ions tested, and why were certain specific metal ions selected for particular experiments? For example, Mn²⁺ was selected for enzyme kinetics assays.

Author Response

To Reviewers´

 

We sincerely thank the reviewers for their thorough, insightful, and constructive evaluation of our manuscript. Their comments were carefully considered and proved highly valuable in helping us refine the focus, coherence, and overall scientific quality of the work. As a result of this process, we believe the revised manuscript is now more clearly framed, methodologically robust, and conceptually focused.

In response to the reviewers’ suggestions, the manuscript has been extensively revised to improve clarity, methodological accuracy, and consistency between experimental data and interpretation. Major revisions include the removal of all references to protein refolding, a complete restructuring and rewriting of the Discussion section, clarification of inclusion body–based enzymatic assays, correction and expansion of figures, incorporation of genome-based regulatory analyses, and the addition of new experimental data in the Supplementary Materials and Appendix.

Overall, we feel that the reviewers’ feedback has significantly strengthened the manuscript and helped sharpen its scientific message. Detailed, point-by-point responses to each comment are provided below.

Reviewer 2

 

for Authors

This work is significant, but several issues need to be discussed with the authors:

1. In the Results section, it is mentioned that AraA was expressed solubly and purified, but the SDS-PAGE image of the purified protein sample was not shown in Figure 5. The authors need to present this result.

 

Response: We thank the reviewer for pointing this out. In the revised manuscript, we have included the SDS-PAGE image of the purified AraA protein (Figure 3A). Lane 10 now shows the AraA expressed in E. coli under induction conditions, with a distinct band at approximately 56 kDa, corresponding to the expected molecular weight. This confirms that AraA was successfully expressed and purified. The figure legend and main text have been updated accordingly to highlight the identity of the band and the purification result. We believe this addition addresses the reviewer’s concern by providing clear visual evidence of the purity and size of the AraA protein.

 

2. In the Results section, it is mentioned that AraA was expressed insolubly and that refolding of inclusion body proteins was performed. However, the Methods section only describes how to wash the inclusion bodies. This part needs to be supplemented with specific refolding methods (including inclusion body solubilization and refolding conditions). Refolding such a large protein is usually very difficult.

 

Response: We thank the reviewer for this important observation. We would like to clarify that the L-arabinose isomerase (AraA) enzyme was not expressed as inclusion bodies but rather in a soluble form in E. coli Rosetta (DE3). Therefore, no solubilization or refolding procedures were required or applied for AraA. The confusion likely arose because other enzymes in our study—specifically BgaA, BglY, and LacZ—were recovered from inclusion bodies, and washing protocols were applied to these samples to remove impurities. However, we did not perform solubilization or refolding steps for these either; enzymatic assays were conducted directly on the washed inclusion body preparations, as described in the revised Methods section.

We have revised the relevant text in the Results and Materials and Methods sections to remove references to “refolding” and to accurately describe the procedures used for each enzyme. These corrections ensure clarity regarding the expression and handling of AraA and the β-galactosidases. We appreciate the reviewer’s comment, which helped us refine this important methodological distinction.

 

3. I did not see that the authors have deposited the genomic sequence of strain L47 in a public database, and no relevant accession number was provided.

 

Response: We apologize for not providing the genome accession number in the initial submission. The whole-genome sequence of Ewingella americana strain L47 has now been deposited in the NCBI GenBank database. The accession number is SAMN54554459. We have added this information to the manuscript (Section 2.2, New Line 283)

 

4. Section 4.7 mentions "Genomic Characterization of the Isolate L47," but no experimental methods for genome analysis are described.

 

Response: We thank the reviewer for this observation, which helped us to improve the clarity and organization of the Materials and Methods section. We would like to clarify that the experimental procedures related to genome analysis of strain L47 were already described in the manuscript, but were previously distributed across different subsections, which may have led to confusion.

Specifically, genomic DNA extraction, quality control, and sequencing using Oxford Nanopore technology were described in Section 4.5. Genome assembly and primary annotation were performed by the sequencing provider. In addition, Section 4.6 details the genome-based analyses performed in this study, including taxonomic assignment using Average Nucleotide Identity (ANI) via GTDB-Tk and FastANI, digital DNA–DNA hybridization (dDDH) using GGDC 3.0, as well as in silico operon prediction and promoter analysis.

To address the reviewer’s concern and avoid any ambiguity, we have revised the manuscript structure and section headings. All genome-related analyses are now consolidated under Section 4.6, entitled “Genome Relatedness (ANI/dDDH) and In Silico Operon/Promoter Prediction”, while Section 4.7 no longer refers to genomic characterization and focuses exclusively on microscopic and physiological characterization of strain L47.

We believe this reorganization improves transparency, ensures methodological completeness, and more accurately reflects the scope of each section.

 

5. It is rather puzzling that the effects of metal ions on enzyme activity are mentioned multiple times in the text, with Mn²⁺ and Ca²⁺ used in some places, and Co²⁺, Mg²⁺, Mn²⁺ used in others. Why weren't other metal ions tested, and why were certain specific metal ions selected for particular experiments? For example, Mn²⁺ was selected for enzyme kinetics assays.

 

Response: We thank the reviewer for this valuable observation and appreciate the opportunity to clarify the rationale behind our selection of metal ions.

The specific metal cofactors used in our enzymatic assays were chosen based on precedent from the literature for each enzyme class, as well as preliminary activity screens performed in our laboratory:

For L-arabinose isomerase (AraA): It is well established that AraA homologs require divalent metal ions for activity, most commonly Mn²⁺ or Co²⁺, which play both catalytic and structural roles (Feng et al., J. Agric. Food Chem. 2015, https://doi.org/10.1021/acs.jafc.5c04610). We therefore tested Mn²⁺, Co²⁺, and Mg²⁺. Mn²⁺ showed the highest activation of L47 AraA, followed by partial activation with Co²⁺ (~50% of Mn²⁺ activity), while Mg²⁺ had a minimal effect. Based on this, Mn²⁺ was selected as the standard cofactor for all subsequent kinetic assays. Although Co²⁺ is often effective, it was not pursued further due to its toxicity and unsuitability for food-related applications.

For β-galactosidases: The metal dependence of β-galactosidases can vary. GH2 enzymes like E. coli LacZ typically require Mg²⁺, but Mn²⁺ can substitute functionally. GH42 enzymes such as BgaA and BglY often contain structural Ca²⁺ and may benefit from Ca²⁺ supplementation. In our case, we tested Mn²⁺ and Ca²⁺ based on these known associations. Mn²⁺ (0.1 mM) consistently enhanced activity in o-NPG assays, so it was included in all β-galactosidase experiments to ensure cofactor availability. Ca²⁺ (1 mM) was also included in some assays to support potential structural stabilization. These comparisons are shown in Supplementary Figure S5. We did not observe strong activity boosts from Ca²⁺ alone, but it did not inhibit enzyme performance, so we retained it in selected conditions.

On the scope of metal testing: We acknowledge that not all possible metal ions were tested. Our objective was to focus on the most relevant cofactors reported for L-AI and β-galactosidases, and to prioritize conditions that would support observable activity for enzyme characterization.

We have added a brief clarification in the Materials and Methods section to explain our rationale for using Mn²⁺ and Ca²⁺, and explicitly noted that Mn²⁺ was selected for kinetic assays based on its observed effectiveness. We hope this explanation addresses the reviewer’s concern and clarifies our methodological choices.

 

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript has been well revised and greatly improved in accordance with the reviewer's comments. I recommend its publication in this journal.