A Rapid Method for the Selection of Amidohydrolases from Metagenomic Libraries by Applying Synthetic Nucleosides and a Uridine Auxotrophic Host

Round 1

Reviewer 1 Report

I liked the approach taken by this paper with its attempt to use a neat selection system to obtain novel genes and their enzyme products from metagenomic libraries.

In several ways they were successful in this goal but I think that the paper needs to be considerably improved. Most of my comments are in the form of annotated comments in situ in the attached Pdf. Some of these only relate to minor matters but others require more attention - please respond to these. Here, I just stress the most important general points.

1. From the start, they must make it clear just why there is a need (either academic and/or applied) for a new set of the sorts of enzymes that they seek. And then at the end, there needs to be some synthesis rather than just a list.
2. Similarly, when it comes to the screening of the enzymes with different potential substrates, they need to justify the choice of the particular molecules.
3. I was genuinely confused in the section that deals with the sequence relatedness of the different clones. THe handling of D8 RL was particularly troublesome to me. Some of the other conclusions also did not seem to make sense to me. (See my comments in the relevant section in the attached Pdf.) I think that it would be very useful if you showed directly the sequences of the relevant enzymes, together with a more direct comparison with their nearest known homologues.
4. In the section on D8 RL, why not show other deduced features such as hydrophobicity, predicted secondary structure etc?

Comments for author File: Comments.pdf

Author Response

Please see the attachment.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

The article may be published as it is, and does not need revision. The author could correct some parts in the introduction (lines 49-53, page 2) to make the sentences more understandable. 

Author Response

On behalf of all co-authors I would like to thank you for your efforts to improve our manuscript. Below, please find our responses to comment.

1 Comment:

The author could correct some parts in the introduction (lines 49-53, page 2) to make the sentences more understandable.

1 Answer:

The 49-53 sentences are rewritten:

Also, the acyl transfer activity of amidase in the presence of hydroxylamine, resulting in spectrophotometric quantification of hydroxamic acid/iron(III) complexes as well as the whole-cell methods that use the chemical properties of one of the products formed in the amidohydrolase enzymatic reaction to form coloured complexes with stable iron salts have been proposed [23][24].

Reviewer 3 Report

Authors present system for selection of amidohydrolases from metagenomic samples.

In the work authors have used auxotrofic organism and substrate selection to capture wanted functionalities, tested and sequenced positive transformants and performed homology and phylogenetic analysis of sequences.

All selected enzymes were assayed for number of substrates to determine their respective substrate specificities and activities. And in detail analysis of one enzyme deemed by the authors interesting based on homology and representation in databases.

Don't have any major complaint on part of results and discussion - it was performed and presented correctly.

But would like to see more detailed description in materials about samples used to create metagenomic libraries as authors only state soil and sediment samples. Were there any specificities for those samples/environments to choose them specifically.

 

Minor corrections:

Ln 21-24 The sentences are nearly identical, duplication.. combine them into one.

Ln 24 delete allow

Ln 27-28 Clarify sentence.. e.g. currently the function of 30-40% of the genes in these genomes is still unknown.

Ln 111 has-> have

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Thank you for dealing with the first round of comments in such a satisfactory way.