Mammalian Arachidonic Acid 15-Lipoxygenases: Fed-Batch Fermentation, Enzyme Purification and Functional Characterization

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Line 34: In the abstract's results sentence, “...catalytically active human and mouse ALOX15 orthologs as well as of human ALOX15B” loosely implies ALOX15B is a third ortholog in the same series, when it is in fact a paralog of ALOX15. Consider rephrasing to “human and mouse ALOX15 orthologs, together with human ALOX15B,” to avoid that implication.

Line 28–29: In the Abstract's Background sentence: “rather low” and “rather challenging” both appear in the same sentence, a repetitive use of the same intensifier. Revise it for natural reading.

Line 48: “The human genome involves 6 functional ALOX genes (ALOX15, ALOX15B, ALX12, ALOX12B, ALOX5, ALOXE3)” — “ALX12” should read "ALOX12." In addition to correcting "contains" or “includes” in place of "involves."

Line 52: “The ALOX15 gene is high-level expressed in immature red blood cells...” is awkward phrasing; “...is highly expressed in...” reads more naturally.

Line 58: “...their 3D structures are very similar [6], [7, 8], [9, 10]” places three separate bracketed citation groups after one single clause. Unless each group is meant to support a distinct part of the sentence—in which case the sentence should make that distinction explicit—these would normally be combined into a single bracket, e.g., "[6–10]."

Line 160: In §2.4, the trace element solution lists “H₃BO₄ (4.7 mg/L)”, but boric acid is H₃BO₃, not H₃BO₄; 

Lines 153–154: Also in §2.4, the fermenter medium recipe is missing a component name before “5.0 g/L." Re-check the protocol, actually.

Line 161: “The pH of the trace element solution was adjusted to pH 11” is missing the initial “a” in "adjusted."

Line 163: “Solutions of mediums, lactose, and the salts were separately sterilized by vapor at 121°C…” uses "mediums," which is non-standard (the conventional plural is “media”), and describes autoclaving as “sterilized by vapor," an unusual phrasing.

Line 168: The sentence “The maintenance range of pO₂ at 35-40% was maintained manually...” repeats “maintain” within itself. “Dissolved oxygen (pO₂) was manually kept within 35–40% by increasing the stirring speed” removes the redundancy.

Line 196: In §2.5, “BioPro SmartSeCity, IEX Q20 column” is a typographical merge error; the correct name, used in this same manuscript (lines 418, 421, and 428), is “BioPro SmartSep IEX Q20."

Line 227: In §2.7, “The assay mixture was composed of a 500 mM PBS at pH 7.2” is inconsistent with the 50 mM phosphate-based buffers used consistently elsewhere in this paper (§2.4 and §2.5); 500 mM PBS is an unusually high, non-standard buffer concentration for an enzyme assay. 

Line 269: In §2.10, double-check that the “molecular extinction coefficient 1.78 mg ml⁻¹ cm⁻¹” is consistent with the intended units convention, since the more commonly reported form is the reciprocal, mL mg⁻¹ cm⁻¹. 

Line 283: The paragraph notes that “lower expression temperatures were suboptimal since... biomass formation was not sufficiently high” without ever specifying which lower temperature or temperatures were actually tested. 

Line 355: In the paragraph reporting soluble-expression percentages, “...36.2 % of mALOX15 were expressed...” breaks the capitalization convention (“mAlox15”) used consistently everywhere else in the manuscript for the mouse ortholog.

Line 362: Table 1 does not state which induction condition — IPTG alone versus IPTG plus lactose — the reported values correspond to. 

Lines 385 and 424: “Ni SepFast FF TED” is used at both of these lines in this section, versus “Ni Seplife FF TED” in Methods §2.1, the Figure 2 legend (line 420), and the Supplementary Figure S1 legend, all of which are consistent with one another. This is very likely an accidental merge of “Ni-IMAC SepFast”—a different resin discussed in the same paragraph, at lines 380 and 383—with “Ni Seplife FF TED," and both occurrences should be corrected to “Ni Seplife FF TED."

Line 428: “...we reach a high (>98%) degree of electrophoretic homogeneity...” is a tense inconsistency, since the surrounding paragraph is written in the past tense; “...we reached a high (>98%) degree...” would match.

Line 453: The claim that “hALOX15B exhibits similar affinities for the two substrates (KₘLA = 19.8 ± 0.6 µM and KₘAA = 4.3 ± 2.1 µM)” is not well supported by the data shown: these two Kₘ values differ by roughly 4.6-fold, which is not “similar” by normal biochemical standards, and this same Kₘ difference is what drives the roughly 4-fold difference in catalytic efficiency (kᴄᴀᴛ/Kₘ) between LA and AA for this enzyme reported in Table 2. The claim should either be revised to acknowledge the real difference in Kₘ between substrates for hALOX15B or be clarified to state precisely what sense of “similar” is intended.

Lines 461–464: The use of “targeted” implies intentional change in a naturally evolved trait. Consider replacing it with “evolutionary shift” or omitting the word altogether.

Line 487: In the opening paragraph, “...with hardly any effect of the AA oxygenase activity of this enzyme [31]” should read “effect on the AA oxygenase activity."

Line 526: The sentence describing the mixed-inhibition-model fit for hALOX15 — “best fitting... was obtained using a mixed inhibition model and a Ki of 80.4 ± 4.12 nM was calculated ( 0.64, R2= 0.980)” — contains an incomplete parenthetical: “( 0.64,” appears to be missing a parameter label. Based on the parallel construction a few lines later, at line 534 (“Ki of 5.44 µM (α=4.8)”), this is very likely missing “α=” before the 0.64, and should read “(α = 0.64, R² = 0.980)”, or whatever parameter was actually intended.

Line 529: “Similar to hALOX15, the AA oxygenase activity of mAlox15 of was strongly inhibited” contains a stray extra word “of”; the sentence should read “...the AA oxygenase activity of mAlox15 was strongly inhibited.”

Lines 532 and 534: The AA-oxygenase and LA-oxygenase Ki values for mAlox15 are nearly identical despite different substrates and inhibition models. Please confirm these are independently derived values rather than a duplicated fitting result, or briefly note this coincidence in the text.

Line 547: The paragraph closing this subsection summarizes inhibitor potency as occurring “in the low micromolar range” for the AA oxygenase activity of both orthologs, but omits that hALOX15's LA-oxygenase inhibition constant (Ki = 80.4 nM, reported at line 526) is actually in the nanomolar range — more than tenfold more potent, and arguably the single most notable number in the entire inhibition dataset. A sentence should be added to this summary explicitly noting the nanomolar potency against hALOX15's LA-oxygenase activity, alongside the micromolar figures already mentioned.

Line 557: “Human ALOX15 and ALOX15B only share a low degree of amino acid conservation (40%), but their polypeptide chains fold into a similar 3D structure [26]” nearly duplicates a sentence already given in the Introduction (line 58). A brief cross-reference back to the introduction, rather than restating the fact in full, would tighten this section.

Line 579: The Figure 4 legend states “Denatured hALOX15 and AhLOX15B were prepared...” — “AhLOX15B” is a transposition typo for “hALOX15B” and should be corrected.

Lines 585, 587: The Figure 4 legend's statistics section states the data were tested for normal distribution using the Shapiro-Wilk test (line 585) but never reports the outcome of that test, then reports comparisons “by non-parametric Mann-Whitney U test (W=0, p=0.0022)” (line 587) as a single statistic. Mann-Whitney U is a two-group test, while the figure itself appears to show four separate significant comparisons—native versus denatured and versus BSA, for both hALOX15 and hALOX15B—each marked with an asterisk pair. The Shapiro-Wilk result should be stated explicitly, i.e., whether normality was rejected, to justify the non-parametric choice, and a separate W and p value should be reported for each pairwise comparison actually marked as significant in the figure, rather than one aggregate statistic covering all of them.

Line 568: “...quantification of the anti-ALOX15/ALOX15B titers may be used as diagnostic method...” is missing an article; “...may be used as a diagnostic method...” reads correctly.

Line 629: The formal abbreviations list (heading at line 629) defines only ALOX, PUFAs, AA, LA, PPAR, the three H(p)ETE/H(p)ODE terms, and SDS-PAGE. Several abbreviations used repeatedly in the main text are not defined there, including FPLC, EDTA, BSA, IPTG, CRC, and the immunoassay technique, which is referred to by its full name in the text but never abbreviated as ELISA. The list should be expanded to include every abbreviation used more than once in the main text.

Line 622–627, 639–644] As already noted in the summary, the Acknowledgments and Conflicts of Interest sections are printed twice.

[Minor, Line 680–681] Reference 15 (“Communications biology 2026, 9, (1).”) ends without an article or page number. If available, the article number should be added; if this is an in-press citation that genuinely lacks final pagination yet, a brief note to that effect would clarify why.

Line 695: Reference 20 (“Frontiers in Immunology 2026, Volume 17 1790402") is formatted inconsistently with the volume/issue/page style used for every other reference in the list and should be reformatted to match.

Comments on the Quality of English Language

Several minor grammatical and typographical errors remain (e.g., "djusted," "mAlox15 of was," "effect of" instead of "effect on," and tense inconsistencies). While they do not affect comprehension, careful proofreading is recommended.

Author Response

Our reply to the comments of the reviewers and the editor is given in the rebuttal letter.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Aksenov et al. have demonstrated a fermentation method for mammalian ALOX isoforms, highlighting the careful selection of inducers and purification resins. The paper also presents an evaluation of enzyme activity in lipoxygenation, as well as its activity in a human serum antibody assay. 

1. It has been mentioned that the column resin used for protein purification can affect its activity. On page 8, line 383, it would be good to provide more details and possible explanations for this interesting phenomenon. How do the differences between the Ni-IMAC SepFast and Ni SepFast FF TED resins, or the buffers used, contribute to the observed difference in enzyme activity?

2. In Figure 3A, 12-HETE is shown with two peaks. It would be better to provide an explanation for this peak splitting on the column. Could the peak splitting be caused by structural isomers or another chromatographic phenomenon?

3. Every capitalized acronym should be defined with its full name when it is first introduced. For example, linoleic acid (LA) is first abbreviated as "LA" on line 64, while its full name is only provided later on line 72.

Author Response

Our reply to the comments of the reviewers and the editor is given in the rebuttal letter.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

• ALX12” was correctly fixed to “ALOX12”, but the second half of the comment was not applied: line 47 still reads “The human genome involves 6 functional ALOX genes…" Please replace “involves” with “contains” or “comprises” as requested.
• The summary was moved to the Discussion, but the specific request was to state the nanomolar potency explicitly. The text says the inhibitor is “almost two orders of magnitude” more potent for hALOX15 LA-oxygenase, yet the actual figure (Ki = 80.4 nM) is never named in the summary alongside the micromolar values. Please add the nanomolar number explicitly.
• The response letter states the Communications Biology reference was corrected to 9(1):774, but reference 13 in the manuscript still ends “Communications biology 2026, 9, (1).” with no article number. Please insert 774. 
• The response letter states the Frontiers in Immunology reference style was adapted, but reference 18 still reads “Frontiers in Immunology 2026, Volume 17 1790402", which does not match the volume/issue/article format used elsewhere. Please reformat (e.g., “2026, 17, 1790402”).
• The capitalisation of “mALOX15” was fixed, but line 345 now reads “36.2% of Alox15” — the “m” prefix is missing. Use “mAlox15” for consistency with the rest of the manuscript.
• The tags [I1], [I2], [I3] and [I4] remain in the running text at lines 79, 357, 446 and 541 respectively. These are editorial/review anchors from the previous round and must be deleted before the manuscript can be typeset.
• The full “Taken together… octyl (…)carbamate effectively inhibits the AA-oxygenase activity of both human and mouse ALOX15 orthologs… almost two orders of magnitude higher for LA-oxygenase reaction of hALOX15 when compared to mAlox15” paragraph appears twice, once at the end of Results 3.3.2 (lines 531–541) and again, word-for-word, in the Discussion (lines 636–647). This is almost certainly a by-product of the Results/Discussion split. Keep the mechanistic interpretation in the Discussion.
• The text (lines 536 and 641) states the inhibitor “did not modify the product pattern of AA oxygenation". However, Figure 3A shows the E vs. E+I ratios shifting: hALOX15 from 11% to 10% 12-HETE and mAlox15 from 9% to 6% 15-HETE. If these shifts are within experimental error, please state that explicitly (e.g., “within error”); otherwise, the blanket claim of no modification needs qualifying.
• Methods §2.9 (line 249) states sera came from patients with “colon cancer (stages 1–4),” whereas the Figure 4 legend (lines 585–586) describes “colorectal cancer (CRC) patients of early stages (I and II)” with n = 6. Please reconcile the disease term (“colon” vs “colorectal”) and, more importantly, the disease stage of the cohort actually used for Figure 4.
• Lines 445–446 state, “the affinities of mAlox15 for the two fatty acids were similar to those of the human ortholog", but Table 2 reports only kcat and kcat/KM — there is no KM column, and mAlox15 KM values are never given in the text. The implied KM values (kcat/(kcat/KM) ≈ 3 µM for LA, ≈ 5 µM for AA) are in fact appreciably lower than the hALOX15 values (KM,LA = 10.3 µM). Either add a KM column to Table 2 and support the comparison, or soften the statement.
• For hALOX15/LA the text gives KM = 10.3 µM and kcat = 27.8 s⁻¹, which yields kcat/KM ≈ 2.70; Table 2 lists 2.78 (consistent with KM = 10.0). Please make the reported KM and the tabulated ratio internally consistent.
• Methods 2.3 (line 135) induces flask cultures at OD600 = 0.8, while Results (line 268) states, "We selected an optical density OD600 of 1–3 to test expression.” Please clarify whether these refer to different experiments or reconcile the values.
• Line 268 cites ref 28 for the statement that heterologous recombinant proteins are predominantly expressed in early log phase, but ref 28 (Gonzalez-Colell & Macia) is titled “Characterization of recombinase activity across cellular growth phases” — recombinase activity, not recombinant protein expression. Please confirm this is the intended citation.
• Lines 350–351 say expression of hALOX15B and mAlox15 was “somewhat lower” than hALOX15, but Table 1 shows 34 and 15 mg/L vs 100 mg/L (roughly 3-fold and 7-fold lower). “Substantially lower” would be more accurate.
• The units were correctly fixed to mL mg⁻¹ cm⁻¹, but a coefficient in mass units is a specific (mass) extinction coefficient, not a “molecular” one. Please rename it “specific extinction coefficient.”
• Table 2 would benefit from an explicit KM column (see S2); at present KM values appear only in the prose for two of the three enzymes.
• Line 110: “Ni-TED was purchased from Sunresine (City, China)” — the city name is still the literal placeholder “City.”
• “Machery-Nagel” → “Macherey-Nagel” (line 231), the correct supplier spelling.
•  “25oC” → “25°C” (line 285): a superscript “o” is used instead of the degree symbol.
• Minor consistency: “LOX protein” (line 189) vs “ALOX” used everywhere else; “dominantly present” (line 421) reads better as “predominantly present.”

Author Response

Dear editor,

            on behalf of all co-authors, I should like to thank the editor and the two expert reviewers for evaluating our ms and for helpful and constructive advice. We re-revised the ms accordingly and hope that the re-revised version of the ms may now be suitable for publication. Our response to the comments of the editor and of the reviewers is given below on the point-to-point basis.

 

Comment 1 of reviewer 1:

ALX12” was correctly fixed to “ALOX12”, but the second half of the comment was not applied: line 47 still reads “The human genome involves 6 functional ALOX genes…" Please replace “involves” with “contains” or “comprises” as requested.

Response of authors: We thank the reviewer for this comment and revised the text accordingly (line 47)

 

Comment 2 of reviewer 1:

The summary was moved to the Discussion, but the specific request was to state the nanomolar potency explicitly. The text says the inhibitor is “almost two orders of magnitude” more potent for hALOX15 LA-oxygenase, yet the actual figure (Ki = 80.4 nM) is never named in the summary alongside the micromolar values. Please add the nanomolar number explicitly.

Response of authors: We thank the reviewer for this comment and revised the text of the discussion section accordingly (lines 639-641). Now it reads: “The potency of the inhibitor was almost two orders of magnitude higher for LA-oxygenase reaction of hALOX15 (Ki = 80.4 nM) when compared to mAlox15 (Ki = 5.4 µM).“

 

Comment 3 of reviewer 1:

The response letter states the Communications Biology reference was corrected to 9(1):774, but reference 13 in the manuscript still ends “Communications biology 2026, 9, (1).” with no article number. Please insert 774.

Response of authors: We thank the reviewer for this comment. We inserted the article number to the reference (line 739).

 

Comment 4 of reviewer 1:

The response letter states the Frontiers in Immunology reference style was adapted, but reference 18 still reads “Frontiers in Immunology 2026, Volume 17 1790402", which does not match the volume/issue/article format used elsewhere. Please reformat (e.g., “2026, 17, 1790402”).

Response of authors: We thank the reviewer for this comment. We have reformatted the reference (line 754).

 

Comment 5 of reviewer 1:

The capitalization of “mALOX15” was fixed, but line 345 now reads “36.2% of Alox15” — the “m” prefix is missing. Use “mAlox15” for consistency with the rest of the manuscript.

Response of authors: Corrected as suggested by the reviewer (line 350). The “m” was inserted.

 

Comment 6 of reviewer 1:

The tags [I1], [I2], [I3] and [I4] remain in the running text at lines 79, 357, 446 and 541 respectively. These are editorial/review anchors from the previous round and must be deleted before the manuscript can be typeset.

Response of authors: The tags were removed in the revised version of the manuscript

 

Comment 7 of reviewer 1:

The full “Taken together… octyl (…)carbamate effectively inhibits the AA-oxygenase activity of both human and mouse ALOX15 orthologs… almost two orders of magnitude higher for LA-oxygenase reaction of hALOX15 when compared to mAlox15” paragraph appears twice, once at the end of Results 3.3.2 (lines 531–541) and again, word-for-word, in the Discussion (lines 636–647). This is almost certainly a by-product of the Results/Discussion split. Keep the mechanistic interpretation in the Discussion.

Response of authors: The double appearance of this paragraph was corrected.

 

Comment 8 of reviewer 1:

The text (lines 536 and 641) states the inhibitor “did not modify the product pattern of AA oxygenation". However, Figure 3A shows the E vs. E+I ratios shifting: hALOX15 from 11% to 10% 12-HETE and mAlox15 from 9% to 6% 15-HETE. If these shifts are within experimental error, please state that explicitly (e.g., “within error”); otherwise, the blanket claim of no modification needs qualifying.

Response of authors: We thank the reviewer for this comment. Now the sentence reads: “Within a small error range, the inhibitor did not modify the product pattern of AA oxygenation by the two enzymes.” (lines 630-631)

 

Comment 9 of reviewer 1:

Methods §2.9 (line 249) states sera came from patients with “colon cancer (stages 1–4),” whereas the Figure 4 legend (lines 585–586) describes “colorectal cancer (CRC) patients of early stages (I and II)” with n = 6. Please reconcile the disease term (“colon” vs “colorectal”) and, more importantly, the disease stage of the cohort actually used for Figure 4.

Response of authors: We thank the reviewer for identifying this inconsistency. The terms “colon cancer” and “stages 1–4” in Section 2.9 were incorrect. We synchronized the information provided in Methods §2.9 and the Figure 4 legend. Now the sentence in §2.9 reads:” The blood sera of the patients suffering from colorectal cancer (stages I and II) were obtained…” (lines 254-255).

 

Comment 10 of reviewer 1:

Lines 445–446 state, “the affinities of mAlox15 for the two fatty acids were similar to those of the human ortholog", but Table 2 reports only kcat and kcat/KM — there is no KM column, and mAlox15 KM values are never given in the text. The implied KM values (kcat/(kcat/KM) ≈ 3 µM for LA, ≈ 5 µM for AA) are in fact appreciably lower than the hALOX15 values (KM,LA = 10.3 µM). Either add a KM column to Table 2 and support the comparison, or soften the statement.

Response of authors: Following the advice of the reviewer we added then Km values to the table 2.

 

Comment 11 of reviewer 1:

For hALOX15/LA the text gives KM = 10.3 µM and kcat = 27.8 s⁻¹, which yields kcat/KM ≈ 2.70; Table 2 lists 2.78 (consistent with KM = 10.0). Please make the reported KM and the tabulated ratio internally consistent.

Response of authors: We thank the reviewer for this comment and made the values consistent.

 

Comment 12 of reviewer 1:

Methods 2.3 (line 135) induces flask cultures at OD600 = 0.8, while Results (line 268) states, "We selected an optical density OD600 of 1–3 to test expression.” Please clarify whether these refer to different experiments or reconcile the values.

Response of authors: We thank the reviewer for identifying this inconsistency and made the values consistent. Indeed, OD600 = 0.8 was used (line 273).

 

Comment 13 of reviewer 1:

Line 268 cites ref 28 for the statement that heterologous recombinant proteins are predominantly expressed in early log phase, but ref 28 (Gonzalez-Colell & Macia) is titled “Characterization of recombinase activity across cellular growth phases” — recombinase activity, not recombinant protein expression. Please confirm this is the intended citation.

Response of authors: We thank the reviewer for identifying the incorrect citation. The reference 28 was substituted by the more relevant reference 29 in the revised manuscript.

 

Comment 14 of reviewer 1:

Lines 350–351 say expression of hALOX15B and mAlox15 was “somewhat lower” than hALOX15, but Table 1 shows 34 and 15 mg/L vs 100 mg/L (roughly 3-fold and 7-fold lower). “Substantially lower” would be more accurate.

Response of authors: Corrected as suggested by the reviewer (line 356).

 

Comment 15 of reviewer 1:

The units were correctly fixed to mL mg⁻¹ cm⁻¹, but a coefficient in mass units is a specific (mass) extinction coefficient, not a “molecular” one. Please rename it “specific extinction coefficient.”

Response of authors: Corrected as suggested by the reviewer (line 264).

 

Comment 16 of reviewer 1:

Table 2 would benefit from an explicit KM column (see S2); at present KM values appear only in the prose for two of the three enzymes.

Response of authors: Following the advice of the reviewer we added then Km values to the table 2

 

Comment 17 of reviewer 1:

Line 110: “Ni-TED was purchased from Sunresine (City, China)” — the city name is still the literal placeholder “City.”

Response of authors: Corrected as suggested by the reviewer (line 111).

 

Comment 18 of reviewer 1:

“Machery-Nagel” → “Macherey-Nagel” (line 231), the correct supplier spelling.

Response of authors: Corrected as suggested by the reviewer (line 236).

 

Comment 19 of reviewer 1:

“25oC” → “25°C” (line 285): a superscript “o” is used instead of the degree symbol.

Response of authors: Corrected as suggested by the reviewer (line 290).

 

Comment 20 of reviewer 1:

Minor consistency: “LOX protein” (line 189) vs “ALOX” used everywhere else; “dominantly present” (line 421) reads better as “predominantly present.”

Response of authors: Corrected as suggested by the reviewer (line 425).

Author Response File: Author Response.pdf