Review Reports

Round 1

Reviewer 1 Report

In this manuscript, authors compared the structural features and mechanisms of thermoinactivation of hyperthermophilic L-asparaginase (L-ASNase) from archaea Thermococcus sibiricus (TsA) with bacterial L-ASNases from Melioribacter roseus (MrA) and Rhodospirillum rubrum (RrA). The catalytic parameters of L-asparagine hydrolysis for these enzymes were determined by circular dichroism (CD) spectroscopy.

 Changes in the tertiary structure of the enzymes during heating were monitored by fluorescence spectroscopy. Based on this results, they 

speculated that the increased entropy of the denaturation process for TsA may indicate an insufficiently stable intermediate state of the enzyme during denaturation. However, there are little experiments was designed and carried out to confirm their guess. It is this reviewer's opinion that authors should design some mutants on some amino acid residues which have direct correlation about α-helix and β-structures of these L-ASNases.

Some minor points:

The descriptions about Fogure 8 and Table 3 do not belong in discussion section, but in the results.

Please check the newly written text in the discussion and the result part to remove repetitions.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

In this manuscript, authors describe the thermal denaturation behavior of 3 different L-asparaginases, which are medically and biotechnologically important enzymes. They provide detailed thermal denaturation kinetics and unfolding pattern based on various spectroscopic techniques as well as structural (amino acid) composition analysis, which can affect thermal stability. The study renders a nice methodology for comparing enzymes of different thermal stability that can also be used for other biotechnologically relevant enzymes. I recommend publication of the manuscript in Catalysts after the below minor changes are taken care of:

-          The title of the article is too long. A concise title will be much better.

-          It will be very informative for the reader if the authors include a reaction scheme for the enzymatic reaction catalyzed by asparaginases in the introduction.

-          Page 2, 1^st paragraph, 1^st sentence – typo between species name should be corrected.

-          Page 2, 1^st paragraph, last sentence:  “…………..to increase the thermal stability of enzymes due to mutagenesis or moidification” – typo at “modification. Also “due to” does not seem to be the best word choice.

-          Page 3, last paragraph: “Furthermore, it is assumed that these forms of the mentioned L-ASNases have activity.” This sentence is not very clear.

-          Table 1: Replace “amount of amino acids” by “number of amino acids”.

-          Page 11, 1^st sentence: “QD” or “CD”?

-          Figure 2a: 2,5 mM should be “2.5 mM”

-          The authors should clearly define the activity unit (IU/mg), especially with regards to steady-state parameters. Ideally, they should report kcat value in terms of reciprocal time units.

-          Page 6, 1^st paragraph: “….the melting temperature was was 50 °C…..” , “was” written twice.

-          Authors can explain better the differences in Tm values obtained by CD method vs. fluorescence method, and which is better for a more reliable Tm value.

-          In the discussion part, where deltaH and deltaS are mentioned, deltaG values can also be elaborated.

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The revised version is acceptable for publication.

Author Response

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Author Response File: Author Response.docx