Genome-Guided Identification of an OTA-Degrading Amidohydrolase AMH2102 from Acinetobacter kookii AK4 with Enhanced Soluble Expression in Escherichia coli

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Genome-guided identification of an OTA-degrading amidohydrolase AMH2102 from Acinetobacter kookii AK4 with enhanced soluble expression in Escherichia coli

 

The manuscript is reviewed for consideration. The topic is of interest. The methodology adopted is appropriate. However, there are a few suggestions/ comments that should be adopted.

 

Introduction

There should be more details about the health hazards of Ochratoxin A in animals or animal models,

 

Discussion

No images of SEM were provided.

Conclusion

The conclusion part must be brief and only focused on the main objectives.

 

Author Response

Reviewer 1

1. Introduction

There should be more details about the health hazards of Ochratoxin A in animals or animal models.

 

Author response: Thank you for the reviewer’s comment. We have supplemented the Introduction with additional information on the health hazards of OTA in animals and animal models, including studies in donkeys[1], juvenile grass carp (Ctenopharyngodon idella)[2], and wild boars[3]. The added text emphasizes OTA accumulation in the body and its oxidative stress-related effects, and further highlights muscle as a newly recognized target tissue of OTA, together with its potential underlying mechanisms. The added content can be found in the Introduction at line 31-36.

 

Revised text: Owing to its efficient absorption and slow elimination, OTA can persist in the body and cause prolonged systemic exposure in animals[5], thereby triggering oxidative stress and multi-organ pathological damage, particularly in the liver and kidneys[6]. In addition, recent studies have shown that OTA can impair muscle growth by inhibiting myogenesis and inducing ferroptosis, further highlighting its broad spectrum of health hazards in animals[7].

 

2. Discussion

No images of SEM were provided.

 

Author response: Thank you for this comment. We would like to clarify that the SEM images are not discussed as standalone data in the Discussion section but are presented in the Results section as Figure 1C and 1D, where they are described accordingly.

 

3. Conclusion

The conclusion part must be brief and only focused on the main objectives.

 

Author response: Thank you for the comment. We agree with this suggestion and have revised the Conclusion to be brief and focused on the main objectives (line 359-365).

 

Revised text: In this study, an OTA-degrading strain Acinetobacter kookii AK4 and its key amidohydrolase gene gene2102 were identified and characterized. The encoded enzyme AMH2102 was successfully heterologously expressed, and its soluble expression was markedly improved through codon optimization and N-terminal SUMO tagging, resulting in rapid OTA degradation. These results demonstrate an effective strategy for enhancing the soluble expression of OTA-degrading enzymes and support their potential application in enzymatic OTA detoxification.

Kang, R.; Qu, H.; Guo, Y.; Zhang, M.; Fu, T.; Huang, S.; Zhao, L.; Zhang, J.; Ji, C.; Ma, Q. Toxicokinetics of a Single Oral Dose of OTA on Dezhou Male Donkeys. Toxins 2023, 15, 88, doi:10.3390/toxins15020088.

2. Zhao, P.; Zhang, L.; Feng, L.; Jiang, W.; Wu, P.; Liu, Y.; Ren, H.; Jin, X.; Zhou, X. Novel Perspective on Mechanism in Muscle Growth Inhibited by Ochratoxin A Associated with Ferroptosis: Model of Juvenile Grass Carp (Ctenopharyngodon Idella) In Vivo and In Vitro Trials. J. Agric. Food Chem.2024, 72, 4977–4990, doi:10.1021/acs.jafc.3c08080.
3. Damiano, S.; Longobardi, C.; Di Napoli, E.; Russo, V.; Piegari, G.; Raffaele, A.; Ferrucci, F.; Rubino, A.; Ciarcia, R. Histopathological Assessment and Oxidative Biomarker Analysis of Wild Boar Tissues Affected by Ochratoxin A Contamination in the Campania Region, Southern Italy. Toxins2025, 17, 428, doi:10.3390/toxins17090428.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript involves the discovery of the gene2102 in Acinetobacter kookii, encoding an amidohydrolase (AMH2102), able to degrade OTA.  The gene was heterologously expressed in E. coli, codon use was optimized, and an N-terminal fusion to a SUMO tag was carried out, increasing solubility and reaching successful OTA degradation in a shorter time. This is a relevant study, contributing to the knowledge of mycotoxin degradation, particularly of OTA, by enzymatic means.  The methods are well thought out and performed. The results of the study are promising, achieving greater solubility and degradation rates.  The authors recognize the limitations of the study, as they are using crude instead of purified protein now, and note that that is their future goal, further optimizing this process towards a safer approach.

Minor points:

-Please revise English language, for instance” At the enzymatic level, Representative enzymes, such as carboxypeptidases and am- 56 idohydrolases, have been characterized by hydrolases that catalyze the cleavage of” in lines 56 and 57

-Auto-citing rate is high in this manuscript, it is recommended to add more content and references from other research groups in Introduction and Discussion sections.

Comments on the Quality of English Language

Some sentences need corrections

Author Response

Reviewer 2

1. Please revise English language, for instance “At the enzymatic level, Representative enzymes, such as carboxypeptidases and am- 56 idohydrolases, have been characterized by hydrolases that catalyze the cleavage of”in lines 56 and 57.

 

Author response: Thank you for the reviewer’s comment. The sentence has been revised to improve clarity and English expression, and the wording has been refined accordingly (line 63-65).

 

Revised text: At the enzymatic level, representative hydrolases such as carboxypeptidases and amidohydrolases have been characterized to catalyze the cleavage of the amide bond linking the phenylalanine moiety to the isocoumarin structure.

 

2. Auto-citing rate is high in this manuscript, it is recommended to add more content and references from other research groups in Introduction and Discussion sections.

 

Author response: Thank you for the reviewer’s comment. We have revised the Introduction and Discussion sections by adding relevant background information and including additional references from other research groups, thereby reducing the self-citation rate.

 

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript contains a very substantial, apparently well-conducted and well-written study that needs few revisions.  One concern that should be discussed is that treatment with the enzyme did not reduce to level of OTA below government regulated limits.  The study used 1 µg/mL OTA to test the enzyme and it reduced OTA levels by 95.44%, meaning it left OTA at 4.56 µg/mL, which is more than double the permissible level of OTA in wine in Europe (see EUR-Lex Document 32022R1370, https://eur-lex.europa.eu/eli/reg/2022/1370/oj), namely 2 µg/kg, and more than 9-fold the permissible level of OTA in baby food (0.5 µg/kg). 

 

Also, the following proofing errors should be addressed: 

1. 56: should be “ . . . level, representative enzymes . . . ”
2. 372: The sentence fragment “The AK4 strain was isolated and screened from wheat farmland soil . . . ” is unclear. Does “screened” mean the soil was passed through a screen (the mesh number should be given) or that many isolates were examined to find the best one?
3. 432: “Bacterial were culture in LB . . . “ should read : “Bacteria were cultured in LB . . . “. 

Author Response

Reviewer 3

1. The study used 1 µg/mL OTA to test the enzyme and it reduced OTA levels by 95.44%, meaning it left OTA at 4.56 µg/mL, which is more than double the permissible level of OTA in wine in Europe (see EUR-Lex Document 32022R1370, https://eur-lex.europa.eu/eli/reg/2022/1370/oj), namely 2 µg/kg, and more than 9-fold the permissible level of OTA in baby food (0.5 µg/kg).

 

Author response: Thank you for this detailed and insightful comment. We appreciate the reviewer’s careful consideration of OTA levels in relation to regulatory limits. We would like to clarify several points about this points.

 

First, the estimation of residual OTA was based on the degradation ratio measured at a specific sampling time point. Under our experimental conditions, strain AK4 achieved a degradation ratio of 95.44% toward 1 μg/mL OTA after 6 h, corresponding to a residual OTA concentration of 45.6 μg/L. This value was used to illustrate the degradation capacity of AK4 at a defined time point, rather than to assess compliance with regulatory maximum limits.

 

Second, the OTA degradation experiment using the bacterial strain was designed to demonstrate the feasibility of microbial degradation at a fixed time point. Importantly, the bacterial strain itself is not intended for direct application in food or feed systems, and thus its performance was not evaluated against regulatory thresholds for final product safety.

 

Third, as demonstrated in this study, OTA-degrading capacity is primarily attributed to the intracellular enzyme. The recombinant enzyme AMH2102 achieved 99.96% degradation within 15 min, resulting in a residual OTA concentration of 0.4 μg/L, and after expression optimization, complete degradation was achieved within 3 min. In these cases, the residual OTA levels were already below the relevant EU regulatory limits. Moreover, it is reasonable to expect that OTA concentrations would continue to decrease with extended reaction time.

 

Therefore, the data presented in this study represent degradation performance at specific sampling time points to illustrate degradation efficiency and dynamics, rather than final OTA concentrations intended for direct comparison with regulatory maximum limits.

 

2. 56: should be “ . . . level, representative enzymes . . . ”

 

Author response: Thank you for the reviewer’s comment. The sentence has been revised to improve clarity and English expression, and the wording has been refined accordingly (line 63-65).

 

Revised text: At the enzymatic level, representative hydrolases such as carboxypeptidases and amidohydrolases have been characterized to catalyze the cleavage of the amide bond linking the phenylalanine moiety to the isocoumarin structure.

 

3. 372: The sentence fragment “The AK4 strain was isolated and screened from wheat farmland soil . . . ” is unclear. Does “screened” mean the soil was passed through a screen (the mesh number should be given) or that many isolates were examined to find the best one?

 

Author response: Thank you for pointing out this ambiguity. Here, “screened” refers to the evaluation of multiple isolates obtained from wheat farmland soil in order to identify the strain with the highest OTA-degrading capacity, rather than physical screening through a mesh. To avoid confusion, we have revised the original sentence to clarify this meaning (line 394-396).

 

Revised text: The AK4 strain was isolated from wheat farmland soil in Shijiazhuang City, Hebei Province, China, and selected from multiple isolates based on its OTA-degrading capacity by our laboratory.

 

4. 432: “Bacterial were culture in LB . . . “ should read : “Bacteria were cultured in LB . . . “.

 

Author response: Thank you for pointing this out. We have corrected the sentence to “Bacteria were cultured in LB …” as suggested.

 

Reviewer 4 Report

Comments and Suggestions for Authors

Lines 82-84; The methods section does not clearly describe how the individual strains were isolated and screened. Please also provide more detail on this in the results section.

Lines 96-97; Please explain the function of these genes briefly.

Line 101; Figure 1; Panel B is not labeled. The labels in panel E are too small.

Line 110; Table S2; It is not clear what 'experimental concentration' in the table means.

Line 117; 'was completely degraded' should be rephrased to express that the compound was under the detection limit.

Line 123; Specify that 'cell-free supernatant' refers to the culture medium supernatant.

Lines 131-133; The data presented so far do not suggest that the protein is a protease, please explain the reasoning.

Line 135; Figure 2; Please replace the 'degradation rate' labels with something like 'degradation' or '% degraded'.

Lines 166-168; These numbers add up to 157, not 149. Also note that all these enzymes are hydrolases, so having a separate count for hydrolases is confusing (use 'other/putative hydrolases for this category).

Lines 170-171; Also commented on in the methods section: Please describe the BLAST parameters in more detail. How many known enzymes were used for the comparison, what is their average length, what is the average length of the new genes, and was % sequence identity considered in selecting the potential candidate genes? Extra or missing domains can lead to missed hits due to selection based on sequence coverage, so please describe how domain structure was considered.

 

Mentioning the range of %identity in Table S3 here in the text would be valuable.

Line 174; Table S4; Including accession numbers for the sequences in the table would be helpful.

Line 179; Figure 3; The text in this figure is too small to read, even after zooming in. It can therefore not be reviewed.

Line 195; Please clarify that 'crude enzyme' means 'crude cell lysate'.

Line 196; This conversion is not a degradation 'rate'.

Line 206; Please start by describing the problem with soluble expression of AMH2102.

Line 210; Please define the CAI value.

Line 221; It is not clear which construct is being referred to in this line.

Line 226; Figure 5B; Please indicate the sizes of the proteins with and without SUMO tags separately because it is not obvious which band corresponds to the untagged protein. There is hardly separation between the protein bands, which makes densitometric analysis of relative protein expression questionable. Please discuss the details.

Line 228; Throughout the manuscript, 'degradation rates' are not rates but conversions.

Lines 291-311; The discussion of solubility is excessive. The authors used codon optimization and SUMO-fusion, two of the most common approaches, and summarizing the results is sufficient, a review of solubility enhancement literature would only be warranted if a novel or unusual strategy were employed.

Lines 314-316; That these solubility enhancements were deduced from Figure 5B is highly questionable since the gels do not have the resolution required for clear separation of bands. Unless supporting evidence can be provided, including increased activity in the crude cell lysates, discussion around this result should be minimised. It suffices to say that the soluble expression of the SUMO-fusion protein seems to be higher based on the SDS-PAGE analysis.

Line 386; Please describe which organisms were selected (or expected to be selected) for by this approach.

Lines 388-390; The method used for isolation of OTA-degrading organisms is not described. Please explain how this was done (the current method seems to refer to a mixed culture but lacks detail on how individual OTA-degrading organisms were isolated).

Line 428; Please describe the solvent composition.

Lines 468-469; Please describe the BLAST parameters in more detail. Was there a cutoff for sequence identity to known OTA-degrading enzymes?

 

Comments on the Quality of English Language

The English is good and understandable but would benefit from minor editing. 

Author Response

Reviewer 4

1. Lines 82-84; The methods section does not clearly describe how the individual strains were isolated and screened. Please also provide more detail on this in the results section.

 

Author response: Thank you for pointing out the aspects that could be further improved. We have refined the results section of the original manuscript with a more detailed description of the isolation and screening process of strain AK4 (line 91-99).

 

Revised text: Soil samples suspended in sterile saline were inoculated into LB medium for enrichment. After 24 h of incubation, the cultures were centrifuged, resuspended in saline, and incubated with OTA, revealing detectable OTA-degrading activity. The enriched cultures were then serially diluted and plated on LB agar, followed by repeated streaking to obtain pure isolates. The OTA-degrading capacity of each isolate was subsequently evaluated using cell suspensions incubated with 1 μg/mL OTA. In total, thirteen OTA-degrading bacterial strains were obtained, among which strain AK4 exhibited the highest degradation efficiency and was therefore selected for further characterization.

 

2. Lines 96-97; Please explain the function of these genes briefly.

 

Author response: Thank you for the reviewer’s valuable comments. In response to this issue, we have added the following content to the original manuscript, which has been highlighted in red in the revised version in line 113-117.

 

Revised text: The infC, tsf, pgk, and rpoB genes encode translation initiation factor IF-3, elongation factor Ts, phosphoglycerate kinase, and the β subunit of RNA polymerase, respectively. These genes are highly conserved in bacteria and are involved in essential cellular processes, including protein synthesis, energy metabolism, and transcription.

 

3. Line 101; Figure 1; Panel B is not labeled. The labels in panel E are too small.

 

Author response: Thank you for your careful review. The labels for panel B have been added, and panel E has been adjusted to improve readability for readers.

 

4. Line 110; Table S2; It is not clear what 'experimental concentration' in the table means.

 

Author response: Thank you for your question. The OTA concentrations used in this study are referred to as the “experimental concentration”, which corresponds to the OTA levels applied in the experiments described in Section 2.2 (Determination of the degradation capacity of the AK4 strain). The experimental concentration was set at 10- or 100-fold higher than the national standard limits. For clarity, the term “experimental concentration” in Table S2 has been revised to “concentration used in this study”.

 

5. Line 117; 'was completely degraded' should be rephrased to express that the compound was under the detection limit.

 

Author response: Thank you for your suggestion. The relevant wording has been revised accordingly: showing that OTA was below the detection limit after 6 h (line 139-140).

 

6. Line 123; Specify that 'cell-free supernatant' refers to the culture medium supernatant.

 

Author response: Thank you for the suggestion. In this study, the term “cell-free supernatant” is intentionally used to emphasize the absence of cells rather than the culture medium itself. As described in the Methods section, the supernatant was obtained by removing cells through centrifugation followed by filtration.

 

Moreover, this terminology has been widely adopted in previous studies with similar experimental designs. For example, Luo et al. used the expression “the cell-free supernatant from the culture of strain CW117 had no degradation ability” in the Results section of their study published in Applied and Environmental Microbiology[4]. Similarly, Yang et al. repeatedly used the term “cell-free supernatant” in Journal of Hazardous Materials, including statements such as “As there was almost no OTA degradation activity in the cell-free supernatant”[5]. In addition, Cai et al. employed the same terminology multiple times in Environmental Pollution, for example in “Cell-free supernatant, cells, and cell lysate degradation of AFB1”[6].

 

Therefore, we have retained the original wording for clarity and consistency with the literature, as well as to clearly highlight the cell-free nature of the assay.

 

7. Lines 131-133; The data presented so far do not suggest that the protein is a protease, please explain the reasoning.

 

Author response: Thank you for this insightful comment. We agree that the current data do not provide direct evidence that the active protein is a protease. The conclusion was mainly based on the proteinaceous nature of the activity and its sensitivity to SDS, which indicates a strong dependence on protein structural integrity. However, the limited effects of proteinase K, PMSF, and EDTA suggest that the active component does not correspond to a typical protease.

 

Accordingly, we have revised the description to indicate that the OTA-degrading activity is mainly attributed to a heat-sensitive, non-metal-dependent intracellular protein, rather than explicitly referring to it as a protease.

 

8. Line 135; Figure 2; Please replace the 'degradation rate' labels with something like 'degradation' or '% degraded'.

 

Author response: Thank you for the suggestion. The replacement has been completed. To ensure terminological accuracy, particularly when describing degradation percentages at fixed time points, all instances of “degradation rate” have been revised to “degradation ratio” throughout the manuscript.

 

9. Lines 166-168; These numbers add up to 157, not 149. Also note that all these enzymes are hydrolases, so having a separate count for hydrolases is confusing (use 'other/putative hydrolases for this category).

 

Author response: Thank you for the comment. Following your suggestion, the category “hydrolases” has been revised to “other hydrolases”. Because some enzymes fall into both broader and more specific functional categories, overlapping counts occurred, leading to apparent discrepancies between category numbers and the total number of unique enzymes. To avoid ambiguity, the original description has been revised accordingly (line 190-193).

 

Revised text: annotation and screening against the NR database identified 18 proteases, 34 peptidases, 6 amidases, 3 amidohydrolases, 1 β-lactamase, 4 carboxypeptidases and 83 other hydrolases, resulting in 149 candidate enzyme genes.

 

10. Lines 170-171; Also commented on in the methods section: Please describe the BLAST parameters in more detail. How many known enzymes were used for the comparison, what is their average length, what is the average length of the new genes, and was % sequence identity considered in selecting the potential candidate genes? Extra or missing domains can lead to missed hits due to selection based on sequence coverage, so please describe how domain structure was considered.

 

Author response: We appreciate the reviewer’s comment. In this study, BLASTP was indeed used to identify candidate genes by comparing the predicted proteins with previously reported OTA-degrading enzymes. All BLASTP searches were performed using the default parameters with  a BLOSUM62 matrix, an E-value cutoff of 0.05.

 

The enzymes used for BLASTP comparison are listed in detail in Table S3 and include a total of 20 enzymes, comprising 7 carboxypeptidases, 2 peptidases, and 11 hydrolases. The average length of the reference enzymes was 454.35 amino acids, whereas the average length of the candidate genes was 933.18 bp. Candidate selection was based on a comprehensive evaluation of overall sequence similarity, alignment length, and percentage identity, rather than the application of a single strict cutoff.

 

We acknowledge that differences in domain architecture, such as the presence of additional or missing domains, may potentially lead to missed hits in BLASTP-based analyses. While this aspect was not explicitly addressed in the initial candidate selection, the use of relatively permissive criteria was intended to reduce the likelihood of excluding potential candidates due to partial alignments. We believe that the current approach remains appropriate for the scope and objectives of this study.

 

Revised text: The proteins encoded by the candidate genes were compared with reported OTA-degrading enzymes using BLASTP with default NCBI parameters (BLOSUM62 matrix, E-value cutoff of 0.05), and proteins exhibiting a sequence coverage greater than 50% were selected as potential OTA-degrading genes. The reference enzymes used for BLASTP comparison are listed in Table S3 and comprise 20 enzymes, including 7 carboxypeptidases, 2 peptidases, and 11 hydrolases. The average length of the reference enzymes was 454.35 amino acids, whereas the average length of the candidate genes was 933.18 bp.

 

11. Mentioning the range of %identity in Table S3 here in the text would be valuable.

 

Author response: Thank you for this suggestion. We have now included a brief description of the range of % identity from Table S3 in the main text (line 198-199). The added sentence reads: “the sequence identities between the candidate genes and the reference enzymes ranged from approximately 22% to 89%”.

 

12. Line 174; Table S4; Including accession numbers for the sequences in the table would be helpful.

 

Author response: Thank you for this suggestion. The GenBank accession numbers for all sequences have now been added to the table.

 

13. Line 179; Figure 3; The text in this figure is too small to read, even after zooming in. It can therefore not be reviewed.

 

Author response: Thank you for pointing this out. We have replaced the figure with a higher-resolution version to improve readability.

 

14. Line 195; Please clarify that 'crude enzyme' means 'crude cell lysate'.

 

Author response: Thank you for this comment. In this study, the term “crude enzyme” is used to emphasize that the enzyme preparation was not purified and was analyzed in comparison with the purified enzyme, which is consistent with its usage in related studies in this field.

 

For example, Abrunhosa et al. reported the degradation of OTA using crude enzyme from Aspergillus niger in the title of their article published in Food Biotechnology. In that study, crude enzymes were prepared by low-temperature extraction of A. niger cultures with buffer, followed by filtration and centrifugation; the supernatant was then lyophilized, subjected to acetone precipitation, washed, and resuspended to obtain crude enzyme[7]. Similarly, Chang et al. described the preparation of crude enzyme in Food Additives & Contaminants by ultrasonic disruption of cell followed by suspension in Tris-HCl buffer (pH 7.0). In that work, both crude enzyme and purified proteins were used to evaluate OTA degradation, as stated in the original text: “The HPLC analysis indicated that OTA decreased by 41% and 72% when co-cultivated with the supernatant of the crude enzyme and the purified protein of carboxypeptidase, respectively”[8]. In addition, Fu et al. reported in Journal of Hazardous Materials that crude enzymes were obtained by resuspending washed cells in washing buffer, disrupting them by ultrasonication, and collecting the supernatant by centrifugation at 12,000 × g for 10 min[9].

 

To avoid any ambiguity, we have now clarified in the revised manuscript that “crude enzyme” specifically refers to the crude cell lysate containing unpurified enzymes (line 529-530).

 

15. Line 196; This conversion is not a degradation 'rate'.

 

Author response: Thank you for your further reminder. Following your previous suggestion, we have revised all relevant descriptions throughout the manuscript and replaced the term “degradation rate” with “degradation ratio”. This terminology has also been adopted in previous studies (Fig 3 and Fig 4 in article [4], Fig 2 and Fig 3 in article [10]) and more appropriately emphasizes the extent of OTA degradation at a given time point rather than a kinetic rate.

 

16. Line 206; Please start by describing the problem with soluble expression of AMH2102.

 

Author response: Thank you very much for this helpful suggestion. We agree that the original text did not clearly describe the motivation for performing expression optimization.

 

In preliminary experiments, AMH2102 exhibited a relatively low expression level in Escherichia coli, with only weak target protein signals detected by Western blot analysis, particularly in the soluble fraction (see Figure 4B in article). This limited expression restricted the availability of active enzyme for subsequent characterization.

 

Therefore, we have revised the beginning of Section 2.5 to explicitly describe this issue and to clarify the rationale for applying a series of optimization strategies aimed at improving the effective soluble expression of AMH2102. The corresponding text has been added in the revised manuscript (line 235-238).

 

Revised text: In preliminary expression experiments, AMH2102 was expressed at a relatively low level in Escherichia coli, with only weak target protein signals detected by Western blot analysis (Figure 4B). This limited expression prompted us to further optimize the expression strategy to improve the effective soluble production of AMH2102.

 

17. Line 210; Please define the CAI value.

 

Author response: Thank you for this comment. We agree that the CAI value should be clearly defined for clarity.

 

We have now added a brief explanation in the revised manuscript to indicate that CAI is a quantitative measure reflecting the degree of codon usage compatibility between a gene and the expression host. The corresponding definition has been added in the revised text (line 242-244).

 

Revised text: The CAI value reflects the degree of codon usage compatibility between a target gene and the host organism.

 

18. Line 221; It is not clear which construct is being referred to in this line.

 

Author response: Thank you for pointing this out. We agree that the original text did not clearly specify which construct was being referred to.

 

We have now revised the sentence to explicitly indicate that this result was obtained using the crude enzyme extract from E. coli BL21(DE3) expressing the SUMO-tagged, codon-optimized AMH2102(gene B) construct. This clarification has been added to the revised manuscript (line 253-256).

 

Revised text: Notably, among all tested constructs, the crude enzyme obtained from BL21(DE3) expressing SUMO-tagged, codon-optimized AMH2102 (gene B) achieved complete (100%) degradation of OTA within 3 min (Figure 5D),

 

19. Line 226; Figure 5B; Please indicate the sizes of the proteins with and without SUMO tags separately because it is not obvious which band corresponds to the untagged protein. There is hardly separation between the protein bands, which makes densitometric analysis of relative protein expression questionable. Please discuss the details.

 

Author response: Thank you for this valuable comment. We agree that the molecular weights of AMH2102 with and without the SUMO tag should be more clearly indicated.

 

In the revised Figure 5B’s legend, we have now explicitly labeled the theoretical molecular weights of untagged AMH2102 (~52.35 kDa) and SUMO-tagged AMH2102 (~62.45 kDa), to clarify the correspondence between the observed bands and the respective constructs.

 

Regarding the concern about band separation, we acknowledge that the molecular weight difference between the SUMO-tagged and untagged proteins is relatively small, resulting in limited separation on SDS-PAGE. However, the densitometric analysis was performed based on the Western blot signals rather than the Coomassie-stained SDS-PAGE gel. Western blotting enables specific detection of the target protein using an anti-His antibody, thereby minimizing interference from co-migrating host proteins.

 

Quantification was restricted to the bands within the expected molecular weight regions of untagged and SUMO-tagged AMH2102, and comparisons were made only within the same construct to evaluate relative soluble versus insoluble expression. Therefore, the analysis is appropriate for assessing relative expression trends rather than absolute protein quantities.

 

We have added a clarification in the figure legend and revised the text accordingly to address this point (line 260-262 and line 546-549).

 

Revised text 1: The theoretical molecular weights of untagged AMH2102 and SUMO-tagged AMH2102 are approximately 52.35 kDa and 62.45 kDa, respectively.

 

Revised text 2: The expression levels of the target proteins were analyzed by densitometric quantification of Western blot band intensities using ImageJ software within the expected molecular weight regions.

 

20. Line 228; Throughout the manuscript, 'degradation rates' are not rates but conversions.

 

Author response: Thank you for this comment. We have revised the manuscript and replaced “degradation rates” with “degradation ratio” throughout the text to accurately describe the extent of OTA conversion at a given time point. All relevant sections have been updated accordingly.

 

21. Lines 291-311; The discussion of solubility is excessive. The authors used codon optimization and SUMO-fusion, two of the most common approaches, and summarizing the results is sufficient, a review of solubility enhancement literature would only be warranted if a novel or unusual strategy were employed.

 

Author response: We thank the reviewer for this helpful comment. We agree that the discussion on soluble expression was overly extensive. As the strategies codon optimization and SUMO fusion applied in this study are commonly used approaches, a detailed review of solubility enhancement literature is not necessary. Following the suggestion, the discussion of protein solubility in Lines 291-311 has been substantially condensed. Non-essential background information have been removed.

 

We retained two representative examples of solubility optimization applied to mycotoxin-degrading enzymes, as they are directly relevant to this study and illustrate that commonly used strategies such as codon optimization and fusion tags have been effective in related systems. To the best of our knowledge, however, there are currently no reports in the field of OTA-degrading enzymes demonstrating improved heterologous expression in Escherichia coli through the combined use of codon optimization and solubility-enhancing fusion tags. Therefore, this discussion was intended to emphasize the novelty of applying these established strategies specifically to OTA-degrading enzymes, with brief supporting examples rather than an extensive literature review.

 

The corresponding text has been streamlined in the revised manuscript (line 327-334).

 

Revised text: Soluble expression is a major bottleneck in recombinant protein production, especially for enzymes, as overexpression in Escherichia coli often leads to inactive inclusion bodies. Optimization strategies, such as codon optimization and fusion with solubility-enhancing tags, are therefore commonly employed. Similar approaches have successfully improved soluble expression of other mycotoxin-degrading enzymes. For example, codon optimization enhanced the lactonohydrolase ZHD101[45], and codon optimization combined with cold-shock-induced expression facilitated soluble expression of PsMnp while reducing inclusion bodies[46].

 

22. Lines 314-316; That these solubility enhancements were deduced from Figure 5B is highly questionable since the gels do not have the resolution required for clear separation of bands. Unless supporting evidence can be provided, including increased activity in the crude cell lysates, discussion around this result should be minimised. It suffices to say that the soluble expression of the SUMO-fusion protein seems to be higher based on the SDS-PAGE analysis.

 

Author response: We thank the reviewer for the comment and appreciate the concern regarding the resolution of SDS-PAGE for accurately assessing soluble protein levels. We would like to clarify that, in this study, the assessment of soluble expression was not solely based on SDS-PAGE. Western blot analysis was performed (Bottom in Figure 5B), providing a more sensitive and specific detection of the target protein, and the results consistently showed a substantial increase in soluble expression following codon optimization and SUMO fusion. Furthermore, enzymatic activity assays of the crude enzyme confirmed that this increase in soluble protein corresponded to higher functional enzyme activity (completely degrade 1 μg/mL of OTA within 3 minutes) (Figure 5D). Taken together, these complementary lines of evidence support the conclusion that the soluble expression of the codon optimization and SUMO-fusion protein was significantly enhanced, and the manuscript has been revised to clarify this point (line340-341).

 

Revised text: These results were confirmed by Western blot analysis (Figure 5C) and acitivity assays of the crude emzyme (Figure 5D).

 

23. Line 386; Please describe which organisms were selected (or expected to be selected) for by this approach.

 

Author response: We thank the reviewer for the comment. The enrichment approach was designed to favor organisms capable of metabolizing OTA under the given conditions. The expected outcome was to select for OTA-degrading strains, regardless of their specific taxonomic identity. During the enrichment and screening process, several isolates were obtained, including Acinetobacter baumannii, Acinetobacter calcoaceticus, Bacillus anthracis, Bacillus cereus and others. However, the focus of this study was on the strain exhibiting the highest OTA-degrading activity, Acinetobacter kookii AK4, and the manuscript reports results for this strain exclusively. The other isolates were not discussed in detail, as the study primarily aimed to characterize and optimize the OTA-degrading enzyme from the most active strain.

 

24. Lines 388-390; The method used for isolation of OTA-degrading organisms is not described. Please explain how this was done (the current method seems to refer to a mixed culture but lacks detail on how individual OTA-degrading organisms were isolated).

 

Author response: Thanks for the comment. To isolate individual OTA-degrading organisms from the enriched culture, serial dilutions of the culture were plated onto LB agar plates. Colonies showing OTA-degrading activity were identified through OTA degradation assays, and these colonies were repeatedly streaked on fresh LB plates until morphologically uniform single colonies were obtained. This process ensured that each isolate used in further experiments represented a pure culture. The manuscript has been updated to include these details (line 412-418).

 

Revised text: The OTA-degrading activity of the culture was assessed using thin-layer chromatography (TLC). To obtain pure OTA-degrading isolates, the active culture was subjected to serial dilution and plated on LB agar. Colonies were screened for OTA-degrading activity by TLC, and those showing activity were repeatedly streaked on fresh LB plates until morphologically uniform single colonies were obtained. These purified isolates were then used for subsequent experiments.

 

25. Line 428; Please describe the solvent composition.

 

Author response:We thank the reviewer for the comment. The detailed composition of the HPLC solvents has been added to the manuscript (line 457-459).

 

Revised text: The mobile phase consisted of acetonitrile containing 0.1%(v/v) formic acid (A) and water containing 0.1% (v/v) formic acid (B) with a flow rate of 1mL/min (A: 0.7 mL/min; B: 0.3 mL/min).

 

26. Lines 468-469; Please describe the BLAST parameters in more detail. Was there a cutoff for sequence identity to known OTA-degrading enzymes?

 

Author response: We thank the reviewer for the comment. In this study, BLASTP searches were conducted using the default parameters of the NCBI BLASTP tool. Candidate proteins were initially selected based on their functional annotations and subsequently compared with reported OTA-degrading enzymes using BLASTP with default settings (BLOSUM62 matrix, E-value cutoff of 0.05). BLASTP analysis was used to evaluate sequence similarity, without applying a strict sequence identity threshold, and conserved domain analysis was not included as a selection criterion. This clarification has been added to the manuscript (line 498-505).

 

Revised text: The proteins encoded by the candidate genes were compared with reported OTA-degrading enzymes using BLASTP with default NCBI parameters (BLOSUM62 matrix, E-value cutoff of 0.05), and proteins exhibiting a sequence coverage greater than 50% were selected as potential OTA-degrading genes. The reference enzymes used for BLASTP comparison are listed in Table S3 and comprise 20 enzymes, including 7 carboxypeptidases, 2 peptidases, and 11 hydrolases. The average length of the reference enzymes was 454.35 amino acids, whereas the average length of the candidate genes was 933.18 bp.

 

 

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