Analysis of the Glycoside Hydrolase Family 1 from Wild Jujube Reveals Genes Involved in the Degradation of Jujuboside A

Round 1

Reviewer 1 Report

In the manuscript entitled “Identification of the β-Glucosidase Genes of Glycoside Hydrolase Family from Wild Jujube and Revealing Two Members Involved in Jujuboside A Catabolism”, the authors wanted to identify and characterize genes that function in jujubosides catabolic pathway in wild jujube (Z. jujuba var. spinosae). Jujubosides are important medicinal compounds found in the seeds of wild jujube. Using bioinformatics tools and publicly available wild jujube genome, the authors successfully identified 35 candidate β-glucosidase genes from the glycoside hydrolase family 1 (GH1). The conserved domains and motifs, as well as the genomic locations of the candidate genes, were also determined. The candidate genes were located on seven of the twelve wild jujube chromosomes and their functions were predicted based on their phylogenetic relationship with Arabidopsis homologs. Two of the candidate β-glycosidase genes (ZsBgl03 and ZsBgl40) were expressed as soluble proteins in E. coli cells and their activity was analyzed on jujuboside A (JuA). UPLC-MS/MS analysis revealed that, after a two-hour incubation period, the ZsBgl03 and ZsBgl40 proteins converted the JuA to jujuboside B (JuB) at a rate of 86.9 % and 78.8 %, respectively. The authors suggest that these two proteins could enhance the usage of jujubosides. Wild jujube fruit is rich in vitamin C and other nutrients and is regularly used in the preparation of beverages and jams. The data presented in this study provide new insight into the metabolism of jujubosides in wild jujube and lay the foundation for a more in-depth exploration of the candidate β-glucosidase genes in wild jujube. Furthermore, the information presented in this study could be useful in improving the cultivation and breeding of wild jujube.

 1.      What is the main question addressed by the research?

Since the release of jujube (Z. jujuba Mill.) and wild jujube (Z. jujuba var. spinosae) genomes, studies have identified and characterized genes involved in the biosynthesis, accumulation, or regulation pathways of triterpenoids and volatiles. However, the genes involved in jujuboside metabolism pathways remain explored. Thus, the main question addressed by this study was to identify and characterize genes that function in the metabolic pathway of jujubosides in wild jujube (Z. jujuba var. spinosae).

 2. Do you consider the topic original or relevant in the field?

This study is relevant to the field since genes that function in jujuboside metabolism pathways in wild jujube remain unexplored. Wild jujube jujubosides are an important medicinal compound; identifying the enzymes that function in their catabolism could prove helpful in enhancing their usefulness.

Does it address a specific gap in the field?

For the first time, 35 candidate β-glucosidase genes from the glycoside hydrolase family 1 (GH1) were identified in wild jujube. The conserved domains and motifs, as well as the genomic locations of the candidate genes, were also determined. The candidate genes were located on seven of the twelve wild jujube chromosomes and their functions were predicted based on their phylogenetic relationship with Arabidopsis homologs. Two of the candidate β-glycosidase genes (ZsBgl03 and ZsBgl40) were expressed as soluble proteins in E. coli cells and their activity was analyzed on jujuboside A (JuA). UPLC-MS/MS analysis revealed that, after a two-hour incubation period, the ZsBgl03 and ZsBgl40 proteins converted the JuA to jujuboside B (JuB) at a rate of 86.9 % and 78.8 %, respectively.

 3. What does it add to the subject area compared with other published material?

The results presented in this study suggest for the first time that ZsBgl03 and ZsBgl40 proteins can convert jujuboside A (JuA) to jujuboside B (JuB).

 4. What specific improvements should the authors consider regarding the methodology? What further controls should be considered?

The methodology is straightforward and nothing significant is lacking to replicate the analyses and experiments.

No additional controls are needed for the scope of this study.

 5. Are the conclusions consistent with the evidence and arguments presented and do they address the main question posed?

Yes, the conclusions are consistent with the results provided. All the data presented are relevant to the main question the authors are addressing.

 6. Are the references appropriate?

Yes, the references are appropriately relevant to the study.

 7. Please include any additional comments on the tables and figures.

·       All of the figures presented in this study need to be redone. The figures and labels are unreadable.  There is no clarity.

·       There are numerous grammatical mistakes throughout the manuscript. The authors need to carefully proofread the text.

·       The title of the manuscript is confusing and should be revised.

·       There are numerous grammatical mistakes throughout the manuscript. The authors need to carefully proofread the text.

·       The title of the manuscript is confusing and should be revised.

Author Response

Thank you for your opinions. We have revised the manuscript according to your advice. 1. We have revised all the figures to make them more readable. Some of the labels in the figures have been enlarged. 2. The title of the manuscript has been revised as “Analysis of the Glycoside Hydrolase Family 1 from Wild Jujube Reveals Genes Involved in the Degradation of Jujuboside A”. 3. We have carefully revised the text of the manuscript. Furthermore, this manuscript has been edited for proper English language, grammar, punctuation, spelling, and overall style by one or more of the highly qualified native English-speaking editors at NativeEE, which specializes in editing and proofreading scientific manuscripts for submission to peer-reviewed journals.

Reviewer 2 Report

I entered my comments directly into the PDF file. Please refer to the attached file.

"The authors identified 35 Jujube beta-glucosidase genes through Bioinformatics analysis, and heterologously expressed 2 of these genes in E. coli to prove that the recombinant protein converts JuA to JuB. This cloning and recombinant protein expression is a classic method, but it is significant in that it has identified the role of an enzyme that converts JuA into JuB, which has never been tried yet. However, considering the quality of the manuscript, I request a high quality figure presentation."

Any other part of manuscript is good discussion and conclusions.

 

Comments for author File: Comments.pdf

Author Response

Thank you for your evaluations and suggestions. According to your suggestions, we have carefully revised this manuscript.

1. We have checked all references and their relevance to the contents of the manuscript.
2. We have revised all the figures and enlarged some of the labels at a low resolution.
3. We have revised the sentences according to the marks in the attached PDF file.

Finally, we have submitted a revised manuscript with the “Track Changes”. Should you have any questions, please contact us without hesitation.