Production of Daidzein and Genistein from Seed and Root Extracts of Korean Wild Soybean (Glycine soja) by Thermostable β-Galactosidase from Thermoproteus uzoniensis

Round 1

Reviewer 1 Report

It is the most rare manuscript that crosses this Reviewer's desk so thoroughly and excellently prepared that it is difficult to find flaw.  In the Reviewer's opinion the paper under current peer-analysis rises to that level.  Lucid, saliently described, thoroughly prepared, 'this' is how basic science should be done.  

Author Response

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

Reviewer 2 Report

please see the uploaded file 

Comments for author File: Comments.docx

Author Response

Q1. Line 96-102: Could you please mention primer annealing temperature and the product size of the amplicon detected by primer used?

Answer) Thank you for your suggestion. As you suggested, we newly added annealing temperature and size of amplicons in the revised manuscript as follows: “The DNA fragments of the β-galactosidase gene and vector were synthesized at an annealing temperature of 55 °C using the Phusion High-Fidelity DNA polymerase (Thermo Fisher Scientific) by PCR. The synthesized DNA fragments, which consisted of amplicons with 60 bp of overhangs for the gene (1,539 bp) and vector (5,299 bp), were ligated using Master Mix for Gibson Assembly (New England Biolabs, Ipswich, MA, USA), and the ligated DNA was transformed into E. coli BL21.” (Line 106−112 of the revised manuscript)

Q2. Line 145,147: Please mention the brand name of the devices used.

Answer) Thank you for your pointing out. We newly added the brand name of electric grinder as follows: “Each part of the Korean wild soybean was ground using an electric grinder (BioloMix, Guangdong, China).” (Line 150−151 of the revised manuscript)

Q3. Line 154-155: why you said “or” NOT “and”?

Answer) As you pointed out, we changed "or" to "and" in the revised manuscript. (Line 161 of the revised manuscript)

Q4. Line 197-199: Do you have reference to this method in calculations?

Answer) Thank you for your question. This method is commonly used in quantitative analysis using HPLC. We added a new reference to the quantitative analysis of isoflavones in the revised manuscript. (Line 207 of the revised manuscript)

Q5. In all figures in the manuscript, please put letter of figure parts above the figure NOT below.

Answer) Thank you for your concern. As you mentioned, we revised the placement of the letters in the overall figure in the revised manuscript.

Q6. Figure 260: “Data represent the means of three separate experiments, and error bars represent the standard deviation” …do you have multiple replicates in each separate experiment or only one replicate?......the standard deviation across three separate experiments?

Answer) Thank you for your suggestion. For better understanding, we revised the related sentences as follows: “Data are represented as the means of triplicate experiments, and error bars represent the standard deviation.” (Line 267−268, 273−275, 334−335, and 380−381 of the revised manuscript)

Q7. In Table 2: the data presented is about using three tissues (stem, root, and seed) to analyse the levels of six components (daidzin, daidzein, genistin, genistein, glycitin , glycitein). Statistcally we may need to know whether there is a significant difference among all these tissues in respect to their content of these 6 components either using, for example, parametric “ANOVA” or non-parametric “Kruskal Wallis” tests. Please indicated why you did not perform this step?

Answer) Thank you for your suggestion. The reason we presented Table 2 was only to indicate the content of isoflavone required for biotransformation rather than to analyze the significant general difference between isoflavones between tissues. Therefore, we added the following sentences in the revised manuscript: “In Table 2, statistical steps through parametric or non-parametric tests were not performed because it was only to indicate the content of isoflavones required for biotransformation.” (Line 339−341 of the revised manuscript)

Q8. The conclusion is so general, please include simple specific results.

Answer) Thank you for your suggestion. We rewrote the conclusion including the specific results as follows: “In conclusion, a thermostable β-galactosidase from T. uzoniensis was cloned and characterized as a purified recombinant enzyme, which exhibited a maximum specific activity of 1,103 μmol/min/mg for pNP-β-D-galactopyranoside at pH 5.0 and 90 °C with a half-life of 46 h and exists as a homodimer of 113 kDa. The enzyme produced daidzein and genistein from daidzin and genistin in the seed and root extracts of Korean wild soybean with productivities of 1.86 and 3.30 mM/h, respectively. To the best of our knowledge, this enzyme exhibits the highest productivity for both daidzein and genistein of any enzyme reported to date. These results indicate that β-galactosidase from T. uzoniensis may be industrially useful for the hydrolysis of iso`flavone glycosides and that wild soybeans are valuable materials for isoflavone aglycone production.” (Line 403−412 of the revised manuscript )

Q9. Please take care of using same font type for all text in this section, check line:406.

Answer) Thank you for your attentive review. As pointed out, we revised the font type in Author Contributions. (Line 418−419 of the revised manuscript )

Q10. Please check the number of references you used as you duplicated the reference int end of manuscript. I also think as long as you did not cite references in the text, no need to mention them in the end of the manuscript even if as “Further Reading”s

Answer) Thank you for your concern. We are sorry for the editorial errors. We strictly checked and corrected the references in the revised manuscript.

Reviewer 3 Report

Converting isoflavone glycosides into isoflavone aglycones is an important process to increase the biological activity of isoflavones as it allows more quickly absorbed in the gut because of their lower molecular weight and better hydrophobicity.

In this paper, authors cloned a themostable beta-galactosidase from T. uzoniensis and characterized its properties, such as molecular weight, pH, temperature, themostability, and substrate specificity. Furthermore, they successfully demonstrated the production of daizein and genistein from daidzin and genistin in the seed and root extracts of Korean wild soybeans.

I think the paper is clearly written and experiments are sufficient enough to show the properties of this newly characterized enzyme. It is fit to publish in Appl. Sci.

Major comments:

1. It is not clear how the authors chose this enzyme to clone. Do they check other genes that might have homology with glycoside hydrolase family protein?

Author Response

Q1. It is not clear how the authors chose this enzyme to clone. Do they check other genes that might have homology with glycoside hydrolase family protein?

Answer) Thank you for your concern. Numerous genes with homology to glycoside hydrolases have not yet been cloned and characterized. Thermostable glycosidases from thermophilic microorganisms generally exhibit high hydrolytic activity toward isoflavone glycosides and β-galactosidase from the extreme thermophile Thermoproteus uzoniensis showed high activity on other glucosides such as ginsenosides. However, the enzyme had not yet been accurately characterized. Therefore, we choose the enzyme for application in the hydrolysis. Some of the related contents have already been described in the introduction of the original manuscrupt. (Line 48−58 of the original manuscript) We added the following sentences to the text for better understanding: “A glycoside hydrolase family 1 (GH1) protein from T. uzoniensis showed high hydrolytic activity on glucosides such as ginsenosides [19], but it has not yet been characterized and applied to hydrolyze isoflavone.” (Line 211−213 of the revised manuscript)

Reviewer 4 Report

The subject of the study is very interesting and important in the understanding of cloned a thermostable β-galactosidase from T. uzoniensis and characterized its properties, such as molecular weight, pH, temperature, thermostability,  and substrate specificity using seed and root extracts of Korean wild soybeans. On the other hand, the manuscript itself appears to be unfinished. Much of the data could be presented as supplementary material, some points are not sufficiently explained. The description of the methodology is incomplete. Most importantly, the results of the paper are not discussed. Perhaps as a result, the conclusions are also incomplete and sometimes not sufficiently evidenced.

• Although the abstract already has an introduction, problem statement, statement of a possible solution, selection, and final conclusion, no clear-cut future recommendations, and some quantitative data are provided to attract the interest of the reader. clarify these in 1-2 lines.
• Indeed, this manuscript is easy to read and understand, it highlights the importance of the isoflavone aglycones of Korean wild soybean, it needs more reasons or information about using enzymes instead of microbial hydrolysis for the hydrolysis of isoflavone glycosides!
• In the introduction part, Lee et. al. (2003) and Lee et. al. (2007) are rather old to support the authors' claim that talked about Korean wild soybeans have been reported to contain a high amount of isoflavones! Consider updating the reference, if possible. I highly encourage you to cite more up-to-date references.
• In the materials and methods part, it needs to add references about how to prepare β-galactosidase using E Coli cells and how to prepare wild soybean extracts from seed, stem, and root extracts!
• Is there any protocol or procedure list to prepare hydrolytic Activity? Clarify that in two-sentence!
• Figure 2: What is the reason to use (5 of PH) when the reactions were performed at different temperatures on the activity of β-galactosidase from T. uzoniensis?
• 90 references in this study are considered extremely too much!

Comments for author File: Comments.pdf

Author Response

Q1. Although the abstract already has an introduction, problem statement, statement of a possible solution, selection, and final conclusion, no clear-cut future recommendations, and some quantitative data are provided to attract the interest of the reader. clarify these in 1-2 lines.

Answer) Thank you for your suggestion. As you suggested, future recommendation was newly added as follows: “Our results will contribute to the industrial production of isoflavone aglycone using wild soybean” (Line 28−29 of the revised manuscript)

Q2. Indeed, this manuscript is easy to read and understand, it highlights the importance of the isoflavone aglycones of Korean wild soybean, it needs more reasons or information about using enzymes instead of microbial hydrolysis for the hydrolysis of isoflavone glycosides.

Answer) Thank you for your concern. We have already described in the original manuscript that microbial hydrolysis has several disadvantages, including lower productivity, lower yield and generation of by-products, compared to enzymatic hydrolysis. (Line 45−46 of the original manuscript). In addition, thermostable glycosidases from thermophilic microorganisms exhibit high hydrolytic activity toward isoflavone glycosides, and the thermostable glycosidases are more efficient to be used as recombinant enzymes expressed in E. coli through cloning because the growth conditions of thermophilic microorganisms are generally harsh. Therefore, we added the following for better understanding: “In order to use thermostable glycosidases from thermophilic microorganisms, it is more efficient to use recombinant enzymes expressed in E. coli through cloning because the growth conditions of thermophilic microorganisms are generally harsh.” (Line 54−57 of the revised manuscript)

Q3. In the introduction part, Lee et. al. (2003) and Lee et. al. (2007) are rather old to support the authors' claim that talked about Korean wild soybeans have been reported to contain a high amount of isoflavones! Consider updating the reference, if possible. I highly encourage you to cite more up-to-date references.

Answer) Thank you for your suggestion. As suggested, a new reference published in 2021 was added as no. 25 in the revised manuscript. (Line 71 and 502−503 of the revised manuscript) However, no further relevant references could be found.

Q4. In the materials and methods part, it needs to add references about how to prepare β-galactosidase using E. coli cells and how to prepare wild soybean extracts from seed, stem, and root extracts!

Answer) Thank you for your suggestion. As suggested, we added references about how to prepare β-galactosidase using E. coli cells and how to prepare wild soybean extracts in the revised manuscript. (Line 125 and 150 of the revised manuscript)

Q5. Is there any protocol or procedure list to prepare hydrolytic Activity? Clarify that in two-sentence!

Answer) Thank you for your question. As you mentioned, we briefly added the information about the pNP assay in one sentence as follows: “The activity was determined by the increase in absorbance at 405 nm due to the release of nitrophenol.” (Line 165−167 of the revised manuscript)

Q6. Figure 2: What is the reason to use (5 of PH) when the reactions were performed at different temperatures on the activity of β-galactosidase from T. uzoniensis?

Answer) Thank you for your question. Since the maximum activity was observed at pH 5.0 in the results for the effect of pH, the reactions for the effect of temperature were carried out with that pH condition. For better understanding we have added the following phrases: “where the maximal activity was observed” (Line 246 of the revised manuscript)

Q7. 90 references in this study are considered extremely too much!

Answer) Thank you for your concern. We are sorry for the editorial errors. We strictly checked and corrected the references in the revised manuscript.