Review Reports

Round 1

Reviewer 1 Report

The manuscript describes the utilization of recombinant L-arabinose isomerase from Klebsiella pneumoniae for the production of D-tagatose, which is a significant hexose in the food industry used primarily as a sweetener.
I have only a few minor editorial comments on the manuscript. The scientific content is fully understandable and I have no concerns regarding it. 
Minor points to improve are as follows:
lines 60–63: Latin names should be in italic.
line 83: The bacterial strain ER2566 is, as far as I know, supplied by New England Biolabs. This should be acknowledged.
line 88: "Underline" might be "underlined".
figure 4b: A degree mark seems to be incorrect.
Thank you for the opportunity to review your work.  

Author Response

Q1. lines 60–63: Latin names should be in italic.

Answer) Thank you for your pointing out. We rewritten the names of the strains in italic font in the revised manuscript. (Line 60−63 of the revised manuscript)

 

Q2. line 83: The bacterial strain ER2566 is, as far as I know, supplied by New England Biolabs. This should be acknowledged.

Answer) Thank you for your concern. We added the supplier as follows: “and E. coli ER2566 strain (New England Biolabs, MA, USA)” (Line 83 of the revised manuscript)

 

Q3. line 88: "Underline" might be "underlined".

Answer) Thank you for your pointing out. We changed "underline" to "underlined" in the revised manuscript. (Line 88 of the revised manuscript)

 

Q4. figure 4b: A degree mark seems to be incorrect.

Answer) Thank you for thoughtful review. We changed the degree mark of figure 2b as well as that of figure 4b in the revised manuscript.

Reviewer 2 Report

D-tagatose is an attractive sweetener as it has a similar sweetness to sugar while its glycemic index (GI 3) is very low. Bioconversion of D-galactose into D-tagatose has attracted attention since its chemical conversion has complicated purification step and the formation of chemical waste and by-products. In this paper, the authors cloned and expressed L-arabinose isomerase derived from mesophilic bacteria Klebsiella pneumoniae. They showed that the purified L-arabinose isomerase can efficiently produce D-tagatose from D-galactose.

I think the paper is clearly written, and experiments are sufficient to show this enzyme's properties. However, it is unclear how this mesophilic enzyme is better than the thermostable enzyme. 

 

Main question

The authors claimed that thermostable L-arabinose isomerase has limited ability in showing the highest activity in industrial processes, such as fermentation and whole-cell reaction, because cells are inactivated at their optimum temperature, which is too high. However, in this manuscript, there is no comparative study with themorstable L-arabinose isomerase and mesophilic one. It would be great if they can compare at least with one of the themorstable L-arabinose isomerase in fermentation or whole-cell reaction. 

Author Response

Q1. The authors claimed that thermostable L-arabinose isomerase has limited ability in showing the highest activity in industrial processes, such as fermentation and whole-cell reaction, because cells are inactivated at their optimum temperature, which is too high. However, in this manuscript, there is no comparative study with themorstable L-arabinose isomerase and mesophilic one. It would be great if they can compare at least with one of the themorstable L-arabinose isomerase in fermentation or whole-cell reaction.

Answer) Thank you for your suggestion. We mentioned thermostable and mesophilic L-arabinose isomerases in the original manuscript. Since most thermostable enzymes have higher activity and stability than mesophilic enzymes, if the whole cells expressing thermostable L-arabinose isomerases are used for the reaction at high temperatures, even if the cell components are destroyed, the enzyme will play its own role and exhibit higher activity than whole cells expressing mesophilic one.

What we are trying to mention was not that ‘mesophilic enzymes are better than thermostable enzymes in whole-cell reactions’, but that ‘thermostable enzymes have limitations when cellular components such as other enzymes or coenzymes are required, such as in biosynthetic processes, so further studies on mesophilic isomerase are needed’.

Therefore, to reduce misunderstanding, we added the phrase “using intrinsic system of cells” to the revised manuscript. (Line 68 of the revised manuscript)

Reviewer 3 Report

In this manuscript, the authors purified and characterized L-arabinose isomerase from Klebsiella pneumoniae. The enzyme exhibited maximum activity for catalysis for the conversion of D-galactose to D-Tagatose at pH 8.0 and 40 °C in the presence of Mn2+ metal ions. Furthermore, the thermal stability of recombinant E. coli cells expressing L-arabinose isomerase from K. pneumoniae was applied and optimized for whole-cell reactions. 

Before the acceptance for publishing in Applied Sciences, the following concerns need to be addressed.

1. In section 3.2, line 209-217, please rewrite the discussion about Figure 3. 

2. In conclusion section, the L-arabinose isomerase from K. pneumoniae existed as a homodimer of 113 kDa. However, in abstract and Figure 1, the enzyme was discribed as a homotetramer of 336 kDa.

3. Please double check the buffer system in Figure 3 and 5, it should be 50 mM phosphate buffer for pH 8.0 but not citrate buffer.

4. Please unify the the specific activity of enzyme (1.8 U/mg to 0.8 U/mL). 

5. In Table 1, the negative control for EDTA should be better due to the lowest activity.

6. In line 265, please delete "at 40 oC"

Author Response

Q1. In section 3.2, line 209-217, please rewrite the discussion about Figure 3.

Answer) Thank you for your suggestion. Since we discussed "the recombinant E. coli cells expressing L-arabinose isomerase" in section 3.4, we did not discuss it in section 3.2 in the original manuscript. However, for better understanding, we have added a discussion to section 3.2 with the following content: “However, the activity of the recombinant E. coli cells expressing l-arabinose isomerase from K. pneumoniae was more than 60% after incubation for 5 h at 50 °C (Figure 3), which indicated that the recombinant cells were more stable in producing D-tagatose from D-galactose than the enzyme.” (Line 218−221 of the revised manuscript)

 

Q2. In conclusion section, the L-arabinose isomerase from K. pneumoniae existed as a homodimer of 113 kDa. However, in abstract and Figure 1, the enzyme was discribed as a homotetramer of 336 kDa.

Answer) We are sorry for any confusion caused by editorial errors. “Homohexamer of 336 kDa” is correct, and the incorrect part has been corrected in the revised manuscript. (Line 21, 187, and 309 of the revised manuscript)

 

Q3. Please double check the buffer system in Figure 3 and 5, it should be 50 mM phosphate buffer for pH 8.0 but not citrate buffer.

Answer) Thank you for your pointing out. We changed “citrate buffer” to “phosphate buffer” in legends of Figure 3 and 5. (Line 234, 286, and 289 of the revised manuscript)

 

Q4. Please unify the specific activity of enzyme (1.8 U/mg to 0.8 U/mL).

Answer) Thank you for your concern. We think you are a bit misunderstanding about them. “1.8 U/mg is the specific activity of enzyme toward D-galactose, and “0.8 U/mL” is the concentration of the enzyme used in reactions.

 

Q5. In Table 1, the negative control for EDTA should be better due to the lowest activity.

Answer) Thank you for your question. Negative control means no EDTA treatment. If EDTA that chelates metal ions is not added, the enzyme becomes active because the unknown metal ion in the native state interacts with the enzyme. For a better understanding, we elaborate on the negative control as follows: “the negative control (not treated with EDTA)”. (Line 239−240 of the revised manuscript)

 

Q6. In line 265, please delete "at 40 oC"

Answer) Thank you for your pointing out. We deleted “at 40 °C” in the revised manuscript. (Line 270 of the revised manuscript)

Round 2

Reviewer 2 Report

Ok. I understand the point. It is still good to have a reference on the following statement.

Despite having advantages, thermostable L-arabinose isomerase has limited ability in showing the highest activity in industrial processes, such as fermentation and whole-cell reaction using intrinsic system of cells, because cells are inactivated at their optimum temperature, which is too high.

Author Response

Thank you for your suggestion. As you suggested, we newly added four references as follows.

35. Mordukhova, E.A.; Lee, H.S.; Pan, J.G. Improved thermostability and acetic acid tolerance of Escherichia coli via directed evolution of homoserine o-succinyltransferase. Appl. Environ. Microbiol. 2008, 74, 7660-7668, DOI:10.1128/aem.00654-08.
36. Gur, E.; Biran, D.; Gazit, E.; Ron, E.Z. In vivo aggregation of a single enzyme limits growth of Escherichia coli at elevated temperatures. Mol. Microbiol. 2002, 46, 1391-1397, DOI:10.1046/j.1365-2958.2002.03257.x.
37. van de Vossenberg, J.L.; Ubbink-Kok, T.; Elferink, M.G.; Driessen, A.J.; Konings, W.N. Ion permeability of the cytoplasmic membrane limits the maximum growth temperature of bacteria and archaea. Mol. Microbiol. 1995, 18, 925-932, DOI:10.1111/j.1365-2958.1995.18050925.x.
38. Ron, E.Z.; Davis, B.D. Growth rate of Escherichia coli at elevated temperatures: limitation by methionine. J. Bacteriol. 1971, 107, 391-396, DOI:10.1128/jb.107.2.391-396.1971.

Reviewer 3 Report

The authors have done all the necessary correction and now the manuscript could be accepted as the current version.

Author Response

We would like to thank you for thoughtful review of this manuscript.