Review Reports

Round 1

Reviewer 1 Report

This manuscript presents the isolation, partial purification and initial biochemical characterization of a new laccase from a high-altitude fungal strain. It is a well organized and documented study that should be of interest to the laccase community.  The English is bimodal: to the end of Results, the English is excellent; in the Discussion, there are a number of common mistakes (e.g. line 329, should be ...due to proper...).  It is recommended that the manuscript be accepted, subject to some copy editing and addressing the following points,.

- line 24, and throughout: attention should be given to the number of significant figures used for reporting results. Here, the activity should be as 51±25; of the four activities given in the following two lines, only the 57.3 arguably warrants three significant figures, the rest, two.  such changes should be effected throughout the manuscript.

- line 39, replace Roman numerals for reference citations with Arabic numerals.  The smaller font used in the reference list makes the superscript Roman numerals illegible in any case. 

- line 42:  superscript for ref 1 citation.

- line 47 needs a space between "of" and "lignocellulosic"; there are many dozens of such elisions in the manuscript that should be fixed.

- line 61:  the phrase "laccase enzyme(s)" is redundant, use one word or the other; fix this throughout

- lines 71 and 72: gallic acid is missing from the materials list;  elsewhere, gallic should not be capitalized

- lines 247 and 248:  given the uncertainties in the two activities, gallic acid and vanillin (not capitalized) are equally inducing

- line 257:  the plural word "media" should be replaced by the singular "medium; there are many places throughout the manuscript where this should be done

- Table 2:  delete "Total" from the heading of the second column; is the culture medium filtrate, as stated in the Experimental section 100 ml--we need this to calculate % recoveries;  likewise are the ammonium sulfate and acetone pptn fractions 10 ml? Adjust numerical entries to 3 significant figures.

-line 280: 94.8%

- line 287:  comment on the apparent activation shown by the pH 6 line in fig. 6 d

- line 290: how does this 43 kDa compare with other laccases (comment here or in discussion)?

- line 297:  what are the uncertainties in the Km and Vmax values?  Should have some kinetics for lignin model substrates.

- line 329: …..due to proper...

- line 333:  ….caused by agitation....

- line 347:  Bollag mis-spelled

- line 361:  .. Further, acetone-precipitated laccase resulted in better activity and yield than....

- line 371:  ...purification are necessary...

- line 373:  ...also been reported...

- line 384:  ...whereas...

- Conclusions:  this section is too long and repetitive summary, rather than a succinct statement of the significant findings

Author Response

- line 24, and throughout: attention should be given to the number of significant figures used for reporting results. Here, the activity should be as 51±25; of the four activities given in the following two lines, only the 57.3 arguably warrants three significant figures, the rest, two.  such changes should be effected throughout the manuscript.

Thank you for the suggestion. All enzyme activities are now reported with out decimals.  Mathematical rounding off rules are used to adjust the numbers.  The means the enzyme activities are reported to 2 sig figs.

- line 39, replace Roman numerals for reference citations with Arabic numerals.  The smaller font used in the reference list makes the superscript Roman numerals illegible in any case. 

Thank you for the due diligence. All references have been verified and any Roman numerals citations are now written with Arabic numbers.

- line 42:  superscript for ref 1 citation.

This has been changed.  The citation now appears as [1].

- line 47 needs a space between "of" and "lignocellulosic"; there are many dozens of such elisions in the manuscript that should be fixed.

Fusing of the words appears to happen because of some software glitch and have noticed this happening in a number of places.  We have fixed the above and number of other fused words.  We are submitting a pdf and doc version of the manuscript. 

- line 61:  the phrase "laccase enzyme(s)" is redundant, use one word or the other; fix this throughout

The word ‘Enzyme’ deleted  and laccase is now written as “laccase(s) to signify the fact that there laccase isozymes important in the process described.

- lines 71 and 72: gallic acid is missing from the materials list;  elsewhere, gallic should not be capitalized

It is now included.

- lines 247 and 248:  given the uncertainties in the two activities, gallic acid and vanillin (not capitalized) are equally inducing

This is now included

- line 257:  the plural word "media" should be replaced by the singular "medium; there are many places throughout the manuscript where this should be done

“Media” is changed “medium”.

- Table 2:  delete "Total" from the heading of the second column; is the culture medium filtrate, as stated in the Experimental section 100 ml--we need this to calculate % recoveries;  likewise are the ammonium sulfate and acetone pptn fractions 10 ml? Adjust numerical entries to 3 significant figures.

“Total” delated and inserted table in editable format

-line 280: 94.8%

It is now rounded off to 95% as per the suggestion of reviewer #2.

- line 287:  comment on the apparent activation shown by the pH 6 line in fig. 6 d

This has already been included and now clarified.

- line 290: how does this 43 kDa compare with other laccases (comment here or in discussion)?

This info is now included in lines 381-385

- line 297:  what are the uncertainties in the Km and Vmax values?  Should have some kinetics for lignin model substrates.

Comparisons are now included in discussion. 

- line 329: …..due to proper...

Edited

- line 333:  ….caused by agitation....

Edited

 

- line 347:  Bollagmis-spelled

Edited

 

- line 361:  .. Further, acetone-precipitated laccase resulted in better activity and yield than....

Edited

 

- line 371:  ...purification are necessary...

Edited

 

- line 373:  ...also been reported...

Edited

 

- line 384:  ...whereas...

Edited

 

- Conclusions:  this section is too long and repetitive summary, rather than a succinct statement of the significant findings

The redundancies are removed and conclusions are succinctly described.

Reviewer 2 Report

The manuscript titled " Secretory Laccase from Pestalotiopsis species CDBT-F-G1 Fungal Strain Isolated from High Altitude: Optimization of its Production and Characterization”, by Yadav et al. reports the screening and identification of a fungus strain from high altitude of Nepal able to produce extracellular laccase enzyme which can later be used in the pretreatment of lignocellulosic biomass. Although references used are outdated, and the work do not bring novelty in what concern to methods or techniques, 53 samples were treated, and a new secretory laccase fungal strain is reported. The results appear to be valid and the followed methodology appropriate, with basic enzyme kinetic parameters also reported. In this context and provide that the above comments are addressed, the contribution could be of interest to Applied Science Journal.

Comment 1. It is very important to update literature. Referencing previous laccase work is poor. Outdated literature being cited.

Comment 2. Pag 1. Line 47:  pH range is higher than reported. The range presented is for fungal laccases. Examples in the literature report more basic values for bacterial laccases.

Comment 3. Please check the way references appear in the text. Authors use [i] or [1] without criteria. Please follow the Journal rules.

Comment 4. Please check the text. Space between words is missing too many times. There are typos in all the text.

Comment 5. Pag 3, line 103 and 106: 10,000 rpm (g ?)

Comment 6. Please use: Mg²⁺, Ca²⁺, Mn²⁺, Zn²⁺and not ++

Comment 7. Please check the writing of the references. Please see Journal rules and write as requested.

Author Response

Comment 1. It is very important to update literature. Referencing previous laccase work is poor. Outdated literature being cited.

The citations in the paper have been updated as suggested and all citations, with exception 4 papers including a technical protocol, are papers published after 2000.

Comment 2. Pag 1. Line 47:  pH range is higher than reported. The range presented is for fungal laccases. Examples in the literature report more basic values for bacterial laccases.

Reference #1, particularly focuses on “Fungal Laccases” while making comparisons to bacterial enzymes.

Comment 3. Please check the way references appear in the text. Authors use [i] or [1] without criteria. Please follow the Journal rules.

We have addressed this issue and appropriate changes have been made.  This problem occurs when endnote software is used.  Thank you for your diligence in this regard.  We have verified to make sure the references are listed as per the journal requirement.

Comment 4. Please check the text. Space between words is missing too many times. There are typos in all the text.

This seems to be a software issue.  We have noticed this happening when documents are exchanged between coauthors.  This time we will submit a pdf as well as word document version,

Comment 5. Pag 3, line 103 and 106: 10,000 rpm (g ?)

It is 10000 x g (g force) generated by the centrifuge which in turn is dependent on the rotor used.  It is not rpm.

Comment 6. Please use: Mg²⁺, Ca²⁺, Mn²⁺, Zn²⁺and not ++

Thank you for pointing out.  Consistently of listing ionic charges is now followed.

Comment 7. Please check the writing of the references. Please see Journal rules and write as requested.

As addressed under comment #3, this issue has been resolved and thank you for noticing.