Recombinant Expression in Bacillus megaterium and Biochemical Characterization of Exo-Mannered Glycosyl Hydrolase Family 43 α-L-Arabinofuranosidase from the Korean Black Goat Rumen Metagenome
Round 1
Reviewer 1 Report
In this manuscript, a novel Gene12 gene was screened and unmasked from constructed rumen metagenomic 11 library of a Korean black goat and expressed in a Bacillus megaterium system. Then the authors also speculate that the Gene12 protein as an exo-mannered GH43 α-L-arabinofuranosidase enzyme. However, the following issues also need to be solved before publication.
Introduced the research background at the beginning of the abstract.
Was Gene12 named by reference?
Added enzyme activity of “GH43 α- of L-arabino-3 furanosidase” in the abstract.
Introduction recommends adding some current research status of “GH43 α- of L-arabino-3 furanosidase”, such as which sources of this enzyme have been reported in the literature? What are the enzyme activity properties?
L50: A large number of lignocellulolytic enzymes have also been found in the rumen ciliate genomes, recommended to cite a recent article:
Li, Z., et al. 2022. Genomic insights into the phylogeny and biomass-degrading enzymes of rumen ciliates. ISME J 16:2775–2787. doi:10.1038/s41396-022-01306-8
Materials and Methods section 2.1, briefly introduced the key experimental steps, such as the construction process of the library.
Materials and Methods section 2.4, use artificial intelligence websites to build protein models (such as AphaFold2)
Materials and Methods section 2.5, introduced the specific culture media, culture conditions (temperature, pH, etc.), and induction conditions
Section 2.6 once again mentioned protein purification. was the process of protein purification as same as Section 2.2? If different, introduced the specific purification conditions and processes.
Section 2.7, Added reference for enzyme activity determination methods
L301 and 308, what is the abbreviation for “CFU”?
L360, “againstvarious”, please pay attention to the space.
The pixels of the full text image are too low, it is recommended to improve the clarity.
Results and Discussion Section 3.4 suggests associating structural information with enzyme activity information, or comparing it with other classic GH43 family structures. Are there any characteristics? Discuss appropriately to demonstrate the innovation of the research.
Moderate editing of English language required
Author Response
In this manuscript, a novel Gene12 gene was screened and unmasked from constructed rumen metagenomic 11 library of a Korean black goat and expressed in a Bacillus megaterium system. Then the authors also speculate that the Gene12 protein as an exo-mannered GH43 α-L-arabinofuranosidase enzyme. However, the following issues also need to be solved before publication.
Response: We appreciate the reviewer’s constructive feedback. All relevant changes in the revised manuscript are marked in yellow color.
Introduced the research background at the beginning of the abstract.
Response: We have added the background information of our research in the abstract section according to the reviewers’s recommendations. All relevant changes in the revised manuscript are marked in yellow color.
Was Gene12 named by reference?
Response: Since the year of 2015, we have been doing a research project on screening a fosmid metagenomic library constructed from rumen microbial communities of black goats to find novel different lignocellulolytic enzymes. A total of 86 novel degradation enzyme genes such as cellulase, lipase, and protease were predicted by full shotgun sequencing of 56 active fosmid clones, and their activities were evaluated by cloning into expression vectors. We are also interested in applying those novel enzymes in the agricultural and food industries. This study aims to investigate the isolation and functional characterization of a novel gene (Gene 12) from the black goat rumen metagenomic library.
Added enzyme activity of “GH43 α-L-arabinofuranosidase” in the abstract.
Response: We have added the information on the enzyme activity of “GH43 α-L-arabinofuranosidase in the abstract section according to the reviewers’s recommendations. All relevant changes in the revised manuscript are marked in yellow color.
Introduction recommends adding some current research status of “GH43 α-L-arabinofuranosidase”, such as which sources of this enzyme have been reported in the literature? What are the enzyme activity properties?
Response: We have already added the source of α-L-Arabinofuranosidase in the introduction section (lines 45-48) of the original manuscript. However, we have added additional information about the sources and biochemical properties of α-L-Arabinofuranosidase of the GH43 family in the introduction section according to the reviewers’s recommendations and all relevant changes in the revised manuscript are marked in yellow color.
L50: A large number of lignocellulolytic enzymes have also been found in the rumen ciliate genomes, recommended to cite a recent article:
Li, Z., et al. 2022. Genomic insights into the phylogeny and biomass-degrading enzymes of rumen ciliates. ISME J 16:2775–2787. doi:10.1038/s41396-022-01306-8
Response: We have added the reference in the specific line according to the reviewers’s recommendations and all relevant changes in the revised manuscript are marked in yellow color.
Materials and Methods section 2.1, briefly introduced the key experimental steps, such as the construction process of the library.
Response: We have briefly described the construction process of the metagenomic library in the specific lines in section 2.1 according to the reviewers’s recommendations, and all relevant changes in the revised manuscript are marked in yellow color.
Materials and Methods section 2.4, use artificial intelligence websites to build protein models (such as AphaFold2).
Response: We have used the multiple threading LOMETS and I-TASSER simulation program to predict and build the Gene12 tertiary structure in section 2.4 based on homology modeling of the template crystal structure of Gene12. We compare all 3D models predicted by Swiss-Model, LOMETS, AlphaFold, Modeller, and I-TASSER, and after modeling, refining, and QC, we select the best one. We have noticed some disorders in some structures that we modeled using AlphaFold and we were wondering if it is normal or not, such as a space between two amino acids which seems not logical. For structural analysis, the predicted Gene12 protein model was further visualized by SPDBV 4.0.4 from the Swiss Pdb-Viewer server and submitted to the Verify3D program (http://services.mbi.ucla.edu/Verify_3D) to evaluate the entire amino acid sequence of Gene12 protein for getting information about predicted protein fitness and model validity.
Materials and Methods section 2.5, introduced the specific culture media, culture conditions (temperature, pH, etc.), and induction conditions
Response: We have added information about specific nutrient media for bacterial culture and their compositions in the respective section according to the reviewers’s recommendations. Moreover, we have already added the specific induction condition in our original manuscript, and all relevant changes in the revised manuscript are marked in yellow color.
Section 2.6 once again mentioned protein purification. was the process of protein purification as same as Section 2.2? If different, introduced the specific purification conditions and processes.
Response: We have briefly described the protein purification process and conditions in the specific lines in section 2.6 according to the reviewers’s recommendations, and all relevant changes in the revised manuscript are marked in yellow color.
Section 2.7, Added reference for enzyme activity determination methods
Response: We have added the references for the enzyme activity methods in the respective section according to the reviewers’s recommendations and all relevant changes in the revised manuscript are marked in yellow color.
L301 and 308, what is the abbreviation for “CFU”?
Response: We have added the abbreviation of CFU in the Appendix section of the revised manuscript and the relevant change in the revised manuscript is marked in yellow color.
L360, “againstvarious”, please pay attention to the space.
Response: We have revised the manuscript accordingly.
The pixels of the full text image are too low, it is recommended to improve the clarity.
Response: We have improved the image quality according to the reviewers’s recommendations.
Results and Discussion Section 3.4 suggests associating structural information with enzyme activity information, or comparing it with other classic GH43 family structures. Are there any characteristics? Discuss appropriately to demonstrate the innovation of the research.
Response: We have already discussed the enzyme activity of Gene12, which exhibited similar enzymatic activity to the enzyme from the GH43 family. However, as the reviewer pointed out, we have added more information about the enzymatic activity of Gene12 in association with its predicted protein structure in the relevant section. We have also added the subsequent references in the specific line according to the reviewers’s recommendations and all relevant changes in the revised manuscript are marked in yellow color.
Reviewer 2 Report
Dear authors, it seems to me that this manuscript has some problems that affect the quality of the manuscript. You need to consider your objective and your hypothesis to improve all the manuscript.
Abstract: I like this writing style format; However, the summary needs to be more complete in reference to the results and conclusions obtained in the present study. Complete it.
Introduction: The introduction is not entirely clear about the problem to be solved; the only thing I understand in the introduction is that a new gene was found. Add the objective of the study in an objective writing format style and add the study hypothesis before the objective.
Lines 30-31: Rewrite these lines because it seems like plagiarism with other published manuscripts and with Wikipedia.
Lines 49-50: Are you sure with the expression? “rumen's intestinal microorganisms”
Lines 59-61: It is not clear why those animals were chosen for the study. Unique characteristics; what characteristics?
Line 64: Describe the initials (GRAS) the first time that these appear.
Lines 28-59: From lines 28 to 59, the introduction follows a sequence and tells a story; however, starting at line 59, the writing is disorganized. Rewrite this entire part following a sequence.
Line 184: “Tukey” instead of “Tykey”
Data analysis: some experimental design was follow? Add it. Some statistical model was follow? Describe it. Covariates were used? Describe it.
Lines 188-193: Those two ideas have no correlation. Separate them into two paragraphs or rewrite them to find a correlation.
Line 191: I don't know what (name, definition and/or concept) those initials (GH43 and CAZy) are about. Describe it.
Line 192: Change “gastrointestinal tract (GIT)” instead of “gastrointestinal (GI) tracts”
Lines 188-202: This is a good review; however, what about your results? What data was obtained and what is the discussion of this data? What happened for you to get those results?
This same comment should be applied to the other subtopics of the results and discussion topic.
Author Response
Dear authors, it seems to me that this manuscript has some problems that affect the quality of the manuscript. You need to consider your objective and your hypothesis to improve all the manuscript.
Response: We appreciate the reviewer’s constructive feedback. We have revised the manuscript accordingly. All relevant changes in the revised manuscript are marked in yellow color.
Abstract: I like this writing style format; However, the summary needs to be more complete in reference to the results and conclusions obtained in the present study. Complete it.
Response: We have added information concisely about the findings of our investigation in the abstract according to the reviewers’s recommendations and all relevant changes in the revised manuscript are marked in yellow color.
Introduction: The introduction is not entirely clear about the problem to be solved; the only thing I understand in the introduction is that a new gene was found. Add the objective of the study in an objective writing format style and add the study hypothesis before the objective.
Response: We have added information briefly about the study hypothesis and objective of our investigation in the relevant sections of the introduction according to the reviewers’s recommendations. We have also added additional information about the sources and biochemical properties of α-L-Arabinofuranosidase of the GH43 family in the introduction section and all relevant changes in the revised manuscript are marked in yellow color.
Lines 30-31: Rewrite these lines because it seems like plagiarism with other published manuscripts and with Wikipedia.
Response: We have revised the manuscript accordingly.
Lines 49-50: Are you sure with the expression? “rumen's intestinal microorganisms”
Response: We have revised the manuscript accordingly.
Lines 59-61: It is not clear why those animals were chosen for the study. Unique characteristics; what characteristics?
Response: We have revised the manuscript accordingly.
Line 64: Describe the initials (GRAS) the first time that these appear.
Response: We have already added the abbreviation of the “GRAS” in the Appendix section.
Lines 28-59: From lines 28 to 59, the introduction follows a sequence and tells a story; however, starting at line 59, the writing is disorganized. Rewrite this entire part following a sequence.
Response: We have revised the manuscript accordingly.
Line 184: “Tukey” instead of “Tykey”
Response: We have revised the manuscript accordingly.
Data analysis: some experimental design was follow? Add it. Some statistical model was follow? Describe it. Covariates were used? Describe it.
Response: We have added the information about the statistical analysis according to the reviewers’s recommendations and all relevant changes in the revised manuscript are marked in yellow color.
Lines 188-193: Those two ideas have no correlation. Separate them into two paragraphs or rewrite them to find a correlation.
Response: We have revised the manuscript accordingly.
Line 191: I don't know what (name, definition and/or concept) those initials (GH43 and CAZy) are about. Describe it.
Response: We have already added the abbreviations of the “GH43 and CAZy” in the Appendix section.
Line 192: Change “gastrointestinal tract (GIT)” instead of “gastrointestinal (GI) tracts”
Response: We have revised the manuscript accordingly.
Lines 188-202: This is a good review; however, what about your results? What data was obtained and what is the discussion of this data? What happened for you to get those results?
Response: The isolation and characterization of lignocellulolytic enzymes from different sources have been conducted by using different screening methods. Among them, culture-dependent screening approaches were used vastly for screening the lignocellulolytic enzymes. However, those traditional approaches were not successfully screening for more than 99% of the lignocellulolytic microbial communities of rumens because they were unculturable. To overcome this technical problem, we have used metagenomics-based approaches in our experiment for screening and characterization of novel functional lignocellulolytic genes from the unculturable microbial communities of rumens. Among the rumens of various ruminants, our study focused on Korean goat rumen because of their unique digestibility of lignocellulosic biomasses. Our study was conducted by screening a fosmid metagenomic library constructed from rumen microbial communities of black goats to find a novel α-L-arabino-furanosidases enzyme, which can be used as an effective enzyme in agricultural and food industries.
This same comment should be applied to the other subtopics of the results and discussion topic.
Response: We have revised the manuscript accordingly.
Round 2
Reviewer 1 Report
All my questions have been answered
Reviewer 2 Report
Dear authors, it seems to me that this manuscript can be approved in current form.
