A Novel Sucrose Isomerase Producing Isomaltulose from Raoultella terrigena

Round 1

Reviewer 1 Report

The manuscript entitled “A novel SIase producing isomaltulose from Raoultella terrigena” by Liu et al. describes the recombinant expression and biochemical characterization of a novel microbial sucrose isomerase enzyme which may have industrial significance in natural sweetener production. The enzyme possessed high activity and conversion rate in isomaltulose production. The authors carried out genetic modifications based on molecular modeling to further improve the catalytic efficiency of the enzyme. The paper is clearly written and contains detailed experiments.

 

I have the following comments and questions:

 

1. The abbreviation SIase is not obvious for general readers, therefore I would modify the title accordingly. Title such as “A novel isomaltulose producing sucrose isomerase from Raoultella terrigena” would be better.

 

2. The authors used site specific mutagenesis to generate isomaltulose variants. However, it is not indicated how the quality of the pal-2 variants was tested.

 

3. I could not find the reference for Table 2 in the text. It would fit into Section 3.1.

 

4. When I compared Figure 3c and Figure 3d panels, it was not obvious why the activity of the enzyme is higher after 2 h incubation (see panel d) than after 15 min incubation (see panel c) at pH 4.0 and in the range of pH 7.0-9.0?

 

5. In the conclusions it is written that “pal-2 showed excellent catalytic ability and conversion rate”. It should be also discussed what the advantage of pal-2 is over the other known microbial sucrose isomerases.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

The work describes a novel sucrose isomerase from a bacterium Raoultella terrigena. The manuscript is a solid piece of experimental work. Still, it has many shortcomings. Therefore, it certainly needs revision.

NB! I recommend not to use abbreviation in the title. Please replace SIase with sucrose isomerase. Also, the names of the bacteria are written so that only the name of the genus (Raoultella) begins with a capital letter.

I will give here some comments. My other remarks and suggestions are added as notes to the text of the attached manuscript.

Abstract

See the highlights (notes to the text).

Introduction

As isomaltulose (palatinose) is commercially produced in very big amounts using sucrose isomerase, this fact should certainly be mentioned in the Introduction. If information on used commercial enzymes and efficiency of the process is available, these data should also be added. My question: can the enzyme from Raoultella perform better than currently commercially used SIases? See also the highlights (notes to the text).

2.2 Gene Sequence, Plasmids, Strains

The strain NCTC 9189 sequence of which was used in current work was previously annotated as Klebsiella sp. The genome of this strain was sequenced by Sanger Institute (United Kingdom). From the text provided by the authors one may think that the genome was sequenced by the authors of the manuscript. Perhaps it was not so. Also, this strain was moved from genus Klebsiella to genus Raoultella and the name was later changed from Raoultella sp. NCTC 9189 to Raoultella terrigena in July  2020. It should be noted here.

2.8 Production of Isomaltulose

Please state how many activity units (U) of Pal-2 or how many micro- or milligrams of Pal-2 with defined specific activity (U/mg) was added to the defined volume reaction mixture.  I do not understand what you mean by 10-35U/g.

2.9.3 Construction of Mutants

The amounts (1 microliter) does not tell anything about the quantity of the primers or of the template.

Figure 3 and Figure 4.

If the data are shown in %, the value corresponding to 100% should always be presented.

3.4.1. Optimization of Enzyme Dosage for Isomaltulose Biosynthesis

Please see the notes in the manuscript. Please clarify the dosage business.

3.5.2. Expression and Purification of Mutants

Question: Were the mutations verified by DNA sequencing?

 

3.5.3. Enzyme Activity of Pal-2 mutants

This paragraph should be rewritten. The data interpreted here are just predictions. Thus, they should be addressed more prudently.

4. Conclusions

Please edit the text for more clarity. See also the notes to the text.

Comments for author File: Comments.pdf

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 3 Report

Liu et al. identified a new SIase from Raoultella Terrigena, which has not been reported before. The produced and characterized this enzyme in detail. They also used computational tools to engineer the enzyme to enhance the biotransformation of sucrose to the target product, isomaltulose. The new enzyme and its variants are of practical industrial application. The manuscript is well written in general, but it still has the following weaknesses:

 

Major:

 

1. It is not very clear how the mutants are generated. I understand that they produced them using Hotspot wizard, but in reality Hopspot Wizard only generates mutants that improve the enzyme’s stability. To my understanding, improving an enzyme’s stability does not necessarily enhance its activity. But surprisingly, seven out of eight of the variants “designed” by enhancing its stability increased the enzyme’s activity, with N498P increased by 89%. Here, I want to know what is the primary goal to design these mutants? E.g., improving activity, conversion rate (which should be rated to activity), specificity (kcat/Km), or stability?
2. In the phylogenetic tree analysis, are the values such as 100, 46, etc indicating sequence identity? To my understanding, they are not. I understand the authors want to determine the pairwise sequence identity between Pal-2 and other relevant enzymes. Actually, it is very easy to do so by using a sequence alignment program such as NWalign to calculate the sequence identity between any two sequences. The authors can remove the phylogenetic analysis, which may not be well understood and discussed in the manuscript. And instead, put a pairwise sequence identity table in Figure 1.
3. In section 3.5.1, the design of mutant sites are not discussed in the paragraph.
4. For the variants/mutants, the authors only determined the rough activity and conversion rate. However, to better understand the catalytic specificity of the enzymes toward sucrose, it is very important to determine and analyze their steady-state kinetic constants (kcat, Km and kcat/Km) vs. the wild-type. These data are missing from the current manuscript. Usually, enhanced activity is related to improved kcat/Km.

 

Minor:

1. In the title, SIase should be changed to its full name, sucrose isomerase.
2. In figure 1, the overall figure legend should not be “Multiple sequence alignments of SIases”, because figure 1a is the phylogenetic analysis. They can use “Comparison of SIases from different microorganisms” or similar descriptions.
3. Line 379, how can “Arg287 formed hydrogen bonds with Arg282” since both Arg287 and Arg282 have only hydrogen-bonding donors in their side chains? This is not coincident with our common sense.
4. In figure 3, figures a-d, the data points have error bars but in figure f, it seems that the data points were determined only once.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 4 Report

Perhaps the length of the introduction could be improved to emphasize some aspects of greater interest to the reader, however I understand that for reasons of space, it is also appropriate to show what is necessary.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

The authors have addressed all my questions properly and I have no more questions.