Review Reports

Round 1

Reviewer 1 Report

The authors characterized two enzymes, the transaminase from Sbv333 and the laccase from Sbv286, by expressing in E coli and purified by His-tag chromatography.  Sbv333-TA was highly thermostable as predicted by the sequence alignment with the TA from thermophile, and works as transaminase for many kinds of substrate, especially was active in the transamination of b-ketoesters which have been difficult for know TAs.  Sbv286-LAC was thermoactivated at temperature over 60 deg and showed the higher activity in the presence of the 10 % cosolvent; acetonitrile, which is suitable for the enzymatic reactions in organic-water solvent.

 

The authors propose the “combined identification approaches” are important to afford novel and functional biocatalysts, but the “combined” is very confusing. The authors should clearly state “what approaches are combined.”

 

For the functional analyses, authors only show the relative activity thorough the paper. It is strongly recommended that the activity should be clearly described by the following manner, reaction rate, or unit as shown in the material and method.

mol of product/mol (or weight) of enzyme

Because the relative activity only works in this paper, and if other researchers would express these proteins and would compare their activity with this paper, they could not.

In addition, 100 % relative activity should be identical at least for each enzyme and describe the details in the main text and figure captions.

In figure 5, the 100 % relative activity should be different between (a) and (b).  That’s another reason to show the experimental raw values for y-axes instead of the relative activity.

 

 

The followings are the comments.

• In figures 4 and 5: How the lines are described? Curve-fitting with least square deviations or by hand? Lines in Figure 4(b) and (c) are quite abnormal because the lines are curves between the temperatures 60 and 70 degs.

The description of x-axis should be identical between Figure 4(a) and 5(a).

• In figure4(c): The authors state that they measured the melting curve for Sbv333-TA by CD spectrometer. The melting curve should be an important information to show the thermal stability of Sbv333-TA, so the overlay the curve with the enzymatic activity.

In addition to the thermal stability, is the unfolding of Sbv333-TA is reversible or not? It is very important for the characterization of enzyme.

• The author emphasize that Sbv333-TA has a unique activity, transamination of b-ketoesters, Table S4 or its summary should be shown in the article, not in the supporting information.
• Line329-349: Authors discuss about their findings that Sbv333-TA and Sbv286-LAC were thermally stable and showed high activity in high temperature. It is clear that the sequence of Sbv333-TA has high identity with other TAs from thermostable bacterium and the LACs generally are described as thermophilic enzymes.Based on the bioinformatic analysis, the reason why such thermostable Sbv333-TA is found in mesophilic strain should be explained.
• Section 4.6; It should be clearly noted how much amount (mol or mg) of enzymes (Sbv333-TA or SBV286-LAC) are added to the reaction mixture to estimate the activity.

 

 

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

This article descripted a classic enzyme screening strategy, namely whole genome sequencing-software prediction-enzyme cloning and expression-characterization. The experimental results show that this is an effective strategy. It is very interesting that the two enzymes obtained in this work are thermophilic enzymes, which brings great convenience to the follow-up research and application of these enzymes. It is recommended that this article be published after supplementing the necessary data. 1. As a transaminase, its most important property is stereoselectivity. The description of stereoselectivity of SBV333-TA in this article is unclear. It is recommended to give the optical purity of the different products to confirm the stereoselectivity of this enzyme, and add the results in Table S5. 2. For laccase, the substrate specificity is the most important property. It is recommended that this article supplement the substrate specificity of Sbv286-LAC and measure the specific activity of the enzyme. 3. There are some other small mistakes, such like two 2.3.1 and the bands of FigS1 and FigS2 should be marked, please check the hole manuscript carefully. 4. The figure of the thermostable of Sbv286-LAC is missing. 5. Studying two enzymes at the same paper leads to undepth research on both enzymes. It might be a better choice to study only one enzyme and study it in detail.

Author Response

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Round 2

Reviewer 1 Report

Line248; Tm is described 85 °C in this line, but in the abstract 84 (line 30). These should be identical.

Figure 4; Top of the figure is trimmed.

              Authors should clarify which conditions are 100% activity for (a) and (b).

              For (a), what temperature the reaction was processed?

The graph shows almost 0 activity at pH 7, is that correct? If so, why no activity at pH 7? If not, how the “relative activities” are calculated? It should be described in the Methods section.

The definition of the “relative activity” should be clarified through the paper and be described at least in Methods section.

Line 253; “Sbv333-TA stability” should be “Sbv333-TA activity”

Line 413; 220 should be corrected to 210 based on the supporting information.

Line 527 & 528; “-“ after “cm” should be superscript.

Line 532; What does “amount” mean? Mol? Weight? The authors should clearly describe the unit.

 

Author Response

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Reviewer 2 Report

 

I don't think the result of Fig6a is reasonable, it contradicts Fig4c clearly.

For thermophilic enzyme, thermostability is one of its most important characteristics. Usually the thermostability curve of an enzyme is the change of enzyme activity at different temperatures and different times, at least to be able to judge the half-life of the enzyme under the highest temperature experimental conditions.

I suggest replace Fig4c and Fig6a with another one.

Author Response

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Round 3

Reviewer 1 Report

Thank you for the response.  The paper is almost ready for publication with minor modification.

In Figure 4, the axes don't have ticks although Figure 5 have. I strongly recommend the ticks are added.

Author Response

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Author Response File: Author Response.pdf