Heterologous Expression and Characterization of a Ferulic Acid Esterase from Aspergillus aculeatus with Potential Use in Sunflower Seed Processing

Round 1

Reviewer 1 Report

The manuscript describes a classical approach to heterologous protein expression and its product characteristic (enzyme). It is valuable and interesting from an application point of view therefore meets the scope of the journal. The manuscript is easy to read but needs language correction to sound less lab-jargon-like. However, some points must be corrected and revised prior to publication, especially mistaken signatures/accessions of sequences. The manuscript lacks a description or reference of the molecular cloning procedures that resulted in the generation of GME3292-pET28a(+) and GME11122-pET28a(+), especially: PCR conditions and primer sequences (!) for GME3292 and GME11122 insert amplification, restriction digestion, ligation, transformation, insert sequence verification of recombinant plasmid, etc. Please note, that ommiting description of methods by providing a reference is not acceptable as in several cases details must be given. If any patents pending covering lacking details please indicate. Also paragraph 2.6 lacks details regarding an agent used for His-tagged recombinant protein elution from Ni-resin (native, reducing or denaturing conditions?). Paragraph 2.8 lacks details such as salts used as metal ion donnors, the ranges of concentrations used, composition of buffers, pH for the analysis, and other relevant conditions at least briefly noted (as details are in referenced paper). But the paper referred to deals with different enzyme, so substrate used for esterase activity and method used for product quantitation need to be indicated.

These must be provided to allow verification of the experiments, otherwise, the manuscript cannot be considered complete.

Another major drawback of the manuscript is insufficient discussion of the results obtained, making it just a report from performed experiment. The results are not compared to similar ones from literature. Paragraph 3.1 - are obtained identity values high or low as compared to other  homologous enzyme pairs (regarding secondary metabolism) from the same organisms ? P3.2 - are the optimal conditions for expression of the recombinant proteins similar or any different from other examples of recombinant proteins expression from the same vector-host? Are optimum regarding enzyme activity the same as for recombinant protein yeld? 
P3.3 - as the difference in activity given in table 1 between reFAE1 and reFAE2 (respectively, 246 and 340 U/g) is insignificant (as denoted by 'c'), how can "...U. maydis (237.6 U/g) [24], the CGA hydrolysis activity of reFAE2 was increased by 43%." be a comment regarding increase? Please correct (either description or table). Please discuss the difference in activity between reFAE1 and reFAE2. 
P3.4 - what does "relatively stable" (line 196) mean in the context? What are stability ranges given in the literature for other industrially relevant enzymes, especially for food or feed processing. Was 8% and 12% activity increase (line 204) statistically significant? Please correct or rephrase the whole paragraph for better readability of the results given . Especially lines 198-213, what do you understand by "promote an enzyme" ( line 203) and "activate an enzyme" (line 204)? You write that the 10 metal ions (all) tested does not promote reFAE1, but Mn²⁺ and Ca²⁺ slightly activate reFAE2. Later (lines 207-208) Cu²⁺ decrease activity of reFAE1?! and inhibition by Fe³⁺...?! The description of the results are inconsistent. Please verify, rephrase the paragraph and discuss the results. 
P3.5 - what does the authors mean by the expression "...the potential value of enzyme" (line 232). Please elaborate. There is no discussion of the results, just comparison to sparse data.
P3.6 - there is no data to support the statement that "The change in color from green to milk white increased the acceptability and nutritional value of SSP." (lines 254-255). That is only the authors speculation.

The authors should also elaborate the applicability of an enzyme produced in Escherichia coli (Gram-negative bacterium) for food industry, especially from antibiotic selected plasmid.

 

Specific comments:

1. Consider changing the beginning of the first sentence ("It is well known that...") as it doesn't sound scientifically correct and give more significant reference for that e.g. review paper.
2. Introduce the SSP acronym at the beginning of the manuscript (line 31, it is first used at line 35).
3. Introduce the whole taxonomic designation of the strain SD14 used as it is already known (Aspergillus aculeatus SD14) at line 66 of the manuscript.  
4. Provide correct GenBank accession No. in line 85 of the manuscript as given signature "F0920" is rather strain no. at The Culture and Information Center of Industrial microorganisms of China Universities (Wuxi, China) - same in line 67 of the manuscript, and does not link to any Aspergillus related sequence nor protein in GenBank DB.
5. What do GME3292 and GME11122 (lines 59-60, 87) stand for? The search for the signatures at NCBI databases and even Google search do not give any results.
6. Consider changing "The centrifuged cells..." to "The cell pellet obtained after centrifugation..." in line 96.
7. Give chemical names of the substrates used: MFA, MCA, MSA, MpCA and CGA in paragraph 2.9. Reaction conditions for the assay.
8. Remove lines 134-136 as they contain manuscript preparation guidelines.
9. Change the title of paragraph 3.1 to 'Sequence comparison to known esterases'
10. In paragraph 3.1 (lines 137-148) provide accession numbers to sequences used in the alignment.
11. Correct taxonomic names to italics (lines: 144-147, 163, 175, 184, 238, ...).
12. In paragraph 3.2 (lines 152-161) provide all conditions used for the optimization of a specific condition e.g. what was the temperature, OD600, and induction duration for inducer concentration analysis, etc. as only one factor was changed and the other were constant.
13. "TM" as a trademark should be in the upper index in "His TrapTM HP" in line 165.
14. In Fig. 3, 4, and Tab. 1, 2, line 213, 236 correct descriptions of purified proteins, there are expression "recombinases" used but the manuscript is on "esterases" (or "recombinant proteins"). Recombinase is an enzyme of specific activity toward DNA (topoisomerase) not an abbreviation of recombinant enzyme.
15. In line 172, be more precise as the MW of the obtained protein included the His-tag.
16. Description in Tab. 3 is too sparse. Please provide more details especially what "L*", "a*", and "b*" stand for, even there is no such description in the text of paragraph 3.6.
17. In comments under Tables 2 and 3 letters (a, b, c) are mentioned as indicating statistical significance, however, there is no 'c' in the tables used, please correct. In case of Tab. 2 - the comparison is in rows not columns, please correct.
18. In line 258 remove coma in taxonomic name.

Author Response

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Author Response File: Author Response.docx

Reviewer 2 Report

Dear Authors 

After reviewing your current manuscript, I can say that it is well structured, well presented with good English. 

Though, I have the following comments on your work: 

1. In the Introduction, lines 41-43, it is not clear where did you get your information from. Reference(s) is/are needed to support your claim.
2. Section 2.6 lines 95-98, it is also not clear the amounts of chemicals used for separation and purification. 
3. Section 2.10, line 124, pH was adjusted to 9.0 by what?
4. Please remove the "original instructions of the journal"  lines 134-136!
5. In section 3.1, and in line 140, I think the 522 amino acids "number" is not correct. Please check!
6. The conclusion should be re-written in the correct way and should be modified so that it does not contain numbers and data. 
7. Although the manuscript is written in good English, still there are some English mistakes. I can give some of them here , but please minorly recheck the whole document: line 15: 56.6 and 78.4 times that of the .... should become:  56.6 and 78.4 times higher than that of the .... Line 32: Under the high pH... please delete "the". Line 149: The strictlyconserved .. strictly conserved (needs space). Line 195: the word "among" should be "between". Line 258: please delete the "dot" after Aspergillus. 
8. Notations and coefficients should be consistent and according to the instructions of the journal. Some examples: line 97: Na₂HPO4, ... Use the subscript format. Also the charges of the metals should be in a superscript format. Additionally, please check how should "weight per volume" term should be abbreviated in this journal (line 118: w: v or w/v). 
9. Wish you all the best. Regards

Author Response

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Author Response File: Author Response.docx

Reviewer 3 Report

The paper title “Heterologous expression and characterization of a ferulic acid esterase from Aspergillus aculeatus with potential use in sun- flower seed processing” is an interesting and valuable. I have only minor comments.

Minor comments

Line 27; “amino acid composition” abbreviation (aa)

Line 32; “byproduct” it should be check

Line 35; “SSP” -  whole name

Line 46 and 48; “Whole genome sequencing” abbreviation (WGS)

Line 53; “FAE” whole name

Line 61; “reFAEs” whole name

Line 75: like in line 27

Line 114; “MFA, MCA, MSA, MpCA and CGA” whole name

Line 101 and 122 check xg and rpm

Line 144; “aa”

Line 142; NCBI whole name

Line 144; “aa”

Line 145; Aspergillus  oryzae

Line 145; Aspergillus niger

Line 146; Aspergillus nidulans

Line 161; Figure 2 should be prepared like Figure 4

Line 175; “U. maydis” Italic like in line 145

Line 184; „Aspergillus oryzae” Italic

Line 258; Aspergillus. Aculeatus

Line 271; “Author Contributions” should be prepared according instructions:

Line 280; References should be check “Italic”

Author Response

Please see the attachment.

Author Response File: Author Response.docx