Expression and Characterization of a GH16 Family β-Agarase Derived from the Marine Bacterium Microbulbifer sp. BN3 and Its Efficient Hydrolysis of Agar Using Raw Agar-Producing Red Seaweeds Gracilaria sjoestedtii and Gelidium amansii as Substrates
Round 1
Reviewer 1 Report
The manuscript „Expression and characterization of a β-agarase derived from the marine bacterium Microbulbifer sp. BN3 and its efficient hydrolysis of the agar-producing red seaweeds Gracilaria sjoestedtii and Gelidium amansii” provides sequence analysis of the β-agarase gene derived from marine microorganisms. It is shown that the agarase contain a catalytic structure and two CBM domains. The agarase gene was successfully expressed in a Brevibacillus expression system. The author conducted the enzymatic characterization of the purified recombinant agarase and find the optimum activity at pH 7.0 and 50 °C. This manuscript provides new insights and intersting new findings to the scientific community.
Here are my comments:
Introduction:
- Informations about Brevibacillus expression system especially about B. brevis are missing.
- Line 76ff: “It has been reported that…” – The author should provide the reference.
Results and discussion:
- Table 1: Why are the losses (Recovery %) so high during the purification of rAga-ms-R?
- Figure 3: Please check the colour of the x and y-axis labelling of Fig. 3 (a). Maybe add the unit [°C] in the legend of Fig.3 (d) and (e).
Author Response
Reviewer 1
The manuscript „Expression and characterization of a β-agarase derived from the marine bacterium Microbulbifer sp. BN3 and its efficient hydrolysis of the agar-producing red seaweeds Gracilaria sjoestedtii and Gelidium amansii” provides sequence analysis of the β-agarase gene derived from marine microorganisms. It is shown that the agarase contain a catalytic structure and two CBM domains. The agarase gene was successfully expressed in a Brevibacillus expression system. The author conducted the enzymatic characterization of the purified recombinant agarase and find the optimum activity at pH 7.0 and 50 °C. This manuscript provides new insights and intersting new findings to the scientific community.
Here are my comments:
Introduction:
Informations about Brevibacillus expression system especially about B. brevis are missing.
Information about Brevibacillus expression system has been added in lines 66-69.
Reference
Udaka S, Tsukagoshi N, Yamagata H. Bacillus brevis, a host bacterium for efficient extracellular production of useful proteins. Biotechnol Genet Eng Rev. 1989;7:113-146. doi:10.1080/02648725.1989.10647857
Line 76ff: “It has been reported that…” – The author should provide the reference.
References have been cited in line 79.
Results and discussion:
Table 1: Why are the losses (Recovery %) so high during the purification of rAga-ms-R?
Secretions of B.brevis are complicated. In order to obtain electrophoretically pure rAga-ms-R for characterization, rAga-ms-R was purified from the fermentation broth with a recovery of about 69% (Table 1.). We will optimize and verify the fermentation and purification methods in the next large-scale preparation stage.
Figure 3: Please check the colour of the x and y-axis labelling of Fig. 3 (a). Maybe add the unit [°C] in the legend of Fig.3 (d) and (e).
Figure 3 has been improved. The unit [°C] has been added in the legend of Fig.3 (d) and (e).
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The manuscript submitted by RenKuan Li et al. describes the characterization of a b-agarase derived from Microbulbifer sp. Although the work is technically solid, its novelty is limited since many of such enzymes have already been described before. Crucially, no rationale is provided for the selection of this particular representative and why it could constitute a significant improvement. The routine characterization of a well-known specificity does not warrant publication in the journal Catalysts.
Author Response
Reviewer 2
The manuscript submitted by RenKuan Li et al. describes the characterization of a b-agarase derived from Microbulbifer sp. Although the work is technically solid, its novelty is limited since many of such enzymes have already been described before. Crucially, no rationale is provided for the selection of this particular representative and why it could constitute a significant improvement. The routine characterization of a well-known specificity does not warrant publication in the journal Catalysts.
In this study, a β-agarase gene of Aga-ms-R (GenBank Accession No. MH621334) was cloned from Microbulbifer sp. BN3. Though the amino acid sequence of Aga-ms-R shares 97% similarity to β-agarase derived from Microbulbifer pacificus (WP_105101548.1, AYV64444.1), it embodies the diversity of agarases in nature. Section 2.1, Line 86-90.
Furthermore, β-agarase gene of Aga-ms-R was cloned from Microbulbifer sp. which is a prokaryotic microorganism. In particular, secretory proteins of eukaryotic origin typically contain S-S bonds for biological activity, and it is generally difficult to produce these proteins using other prokaryotic expression systems. Compared to E. coli and eukaryotic systems, the protein expressed by the bacillus system can better reflect its original characteristics. In this study, rAga-ms-R was expressed in the Brevibacillus expression system. To our knowledge, this is the first time to report a β-agarase expressed in the Brevibacillus expression system. The characteristic secretory rAga-ms-R of Brevibacillus allowed efficient production, sharing similar characterization of those with sequence similarity (AYV64444.1). Section 2.2 Lines 124-125, Lines 134-136.
Most importantly, the results of rAga-ms-R catalyzed hydrolysis of the substrate was different from those before. Hydrolysates of agarose catalyzed by rAga-ms-R were mainly neoagarotetraose (NA4) and neoagarohexaose (NA6) in a long intermediate stage of the reaction, and neoagarohexaose (NA6) of hydrolysates would be hydrolyzed to neoagarotetraose (NA4) and neoagarobiose (NA2) under conditions of additional rAga-ms-R. Moreover, rAga-ms-R was efficient in preparing neoagarotetraose (NA4) with raw Gracilaria sjoestedtii and Gelidium amansii as substrates.Neoagarotetraose (NA4) and neoagarohexaose (DP6) exhibit anti-inflammatory and antitumor activities. Section 2.5, Lines 222-238.
References
1) T. Takano, A. Miyauchi, H. Takagi, K. Kadowaki, K. Yamane, and S. Kobayashi. Expression of the Cyclodextrin Glucanotransferase Gene of Bacillus macerans in Bacillus brevis. Biosci Biotech Biochem. (1992) 56(5): 808-809.
2) H. Tojo, T. Asano, K. Kato, S. Udaka, R. Horinouchi, and A. Kakinuma. Production of human protein disulfide isomerase by Bacillus brevis. J Biotechnol. (1994) 33(1): 55-62.
3) H. Yamagata, K. Nakahama, Y. Suzuki, A. Kakinuma, N. Tsukakoshi, and S. Udaka. Use of Bacillus brevis for efficient synthesis and secretion of human epidermal growth factor. Proc Natl Acad Sci USA.(1989) 86: 3589-3593.
4) Y. Takimura, M. Kato, T. Ohta, H. Yamagata, and S. Udaka. Secretion of human interleukin-2 in biologically active form by Bacillus brevis directly into culture medium. Biosci Biotechnol Biochem. (1997) 61(11): 1858-1861.
5) Chen YP, Wu HT, Wang GH, et al. Inspecting the genome sequence and agarases of Microbulbifer pacificus LD25 from a saltwater hot spring. J Biosci Bioeng. 2019;127(4):403-410. doi:10.1016/j.jbiosc.2018.10.001
6) Park SH, Lee CR, Hong SK. Implications of agar and agarase in industrial applications of sustainable marine biomass. Appl Microbiol Biotechnol. 2020, 104, 2815‐2832. doi:10.1007/s00253-020-10412-6
Author Response File: Author Response.pdf
Reviewer 3 Report
The manuscript (catalysts-858567) titled “Expression and characterization of a β-agarase derived from the marine bacterium Microbulbifer sp. BN3 and its efficient hydrolysis of the agar-producing red seaweeds Gracilaria sjoestedtii and Gelidium amansii” is very interesting. In my opinion, some English issues should be corrected and the authors should explain clearly what they mean by “Effects of metal ions and chemicals”. How are the metals ions added? Were not used salts? Are these salts not chemical compounds? Why just EDTA and SDS are considered chemicals? Why were the only tested?
The part of the hydrolysis is confusing, in particular the figure 4.
Author Response
Reviewer 3
The manuscript (catalysts-858567) titled “Expression and characterization of a β-agarase derived from the marine bacterium Microbulbifer sp. BN3 and its efficient hydrolysis of the agar-producing red seaweeds Gracilaria sjoestedtii and Gelidium amansii” is very interesting. In my opinion, some English issues should be corrected and the authors should explain clearly what they mean by “Effects of metal ions and chemicals”. How are the metals ions added? Were not used salts? Are these salts not chemical compounds? Why just EDTA and SDS are considered chemicals? Why were the only tested?
A wide variety of metal ions are found in the environment. They serve many functions in proteins, the most important of which is the modification of protein structures, enhancement of the structural stability of the proteins in the conformation required for biological function, or taking part in the catalytic processes of enzymes.
Metal ions play important roles in the biological function of many enzymes. The various modes of metal-protein interaction include metal-, ligand-, and enzyme-bridge complexes. Metals can serve as electron donors or acceptors, Lewis acids or structural regulators. Those that participate directly in the catalytic mechanism usually exhibit anomalous physicochemical characteristics reflecting their entatic state.
In this study, we described methods as reported before. Various metal ions, calculated as salt concentration, were added in 50 mmol/L NaH2PO4-citric acid buffer (pH 7.0) in salt form. Also, they all are chemicals. EDTA and SDS are chemicals different from metal ions. Sentences have been improved in lines 172,175 and 313. Testing the effects of metal ions and other chemicals on enzyme activity serves only to characterize the rAga-ms-R.
Reference:
Riordan JF. The role of metals in enzyme activity. Ann Clin Lab Sci. 1977;7(2):119-129.
The part of the hydrolysis is confusing, in particular the figure 4.
Section 2.5 and legend of figure 4 have been improved.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
In their revised manuscript, the authors have tried to emphasize the novelty of their work but I am still not convinced. Indeed, simply switching to a different (and well-known) expression system is merely a technical issue and represents a routine that does not warrant publication in a high-impact journal. The product profile of the enzyme could be the most interesting aspect but that should be elaborated in much more detail. In fact, I doubt that it will turn out to be really "new" when compared under exactly the same conditions with related enzymes (some of which show a similarity of no less than 97%!). And even if that was the case, the determinants of their product profile should then be explored (in sequence and/or structure) to actually report some new insights to the scientific community. On a final note: the language certainly needs to be improved.
Author Response
Reviewer 2 In their revised manuscript, the authors have tried to emphasize the novelty of their work but I am still not convinced. Indeed, simply switching to a different (and well-known) expression system is merely a technical issue and represents a routine that does not warrant publication in a high-impact journal. The product profile of the enzyme could be the most interesting aspect but that should be elaborated in much more detail. In fact, I doubt that it will turn out to be really "new" when compared under exactly the same conditions with related enzymes (some of which show a similarity of no less than 97%!). And even if that was the case, the determinants of their product profile should then be explored (in sequence and/or structure) to actually report some new insights to the scientific community. On a final note: the language certainly needs to be improved.
Authors’ response: The language of manuscript has been improved again. The following has been added to the end of RESULTS AND DISCUSSION.
In order to adapt to various living environments, differences in the same enzyme represent a consequence of natural evolution. Constantly enriching genetic resources is conducive to the study of its function and evolution. The intent of trying out different expression systems is to better obtain the protein encoded by the gene to facilitate the study of function, characteristics and application.
The hydrolysis of β-glycosidic linkages of agarose can yield neoagarooligosaccharides (NAOs) of diverse sizes, and the hydrolysis products produced by the β-agarase-catalyzed reaction vary depending on the type of β-agarase employed. The bulk of the products formed by hydrolysis of the substrate of the GH16 family agarase was neoagarotetraose (NA4). Amino acid sequence alignment (Figure.1a) of Aga-ms-R indicates that it belongs to the GH16 family of β-agarases, with up to 97% homology to β-agarase derived from Microbulbifer pacificus (WP_105101548.1, AYV64444.1). To our knowledge, β-agarase (WP_105101548.1) has not been characterized whereas β-agarase (AYV64444.1) hydrolyzed agarose to mainly neoagarobiose (NA2).
In our study, rAga-ms-R showed the difference in degrading substrate as those reported before. Hydrolysis of agarose catalyzed by rAga-ms-R yielded mainly neoagarotetraose (NA4) and neoagarohexaose (NA6) in a long intermediate stage of the reaction, and neoagarohexaose (NA6) of hydrolysates will be further hydrolyzed to neoagarotetraose (NA4) and neoagarobiose (NA2) under conditions of additional rAga-ms-R. Moreover, rAga-ms-R was efficient in producing neoagarotetraose (NA4) with raw Gracilaria sjoestedtii and Gelidium amansii as substrates. However, the relationship between amino acid sequence of agarase and pattern of agarose hydrolysis has to await further studies.
References:
1) Chen YP, Wu HT, Wang GH, et al. Inspecting the genome sequence and agarases of Microbulbifer pacificus LD25 from a saltwater hot spring. J Biosci Bioeng. 2019;127(4):403-410. doi:10.1016/j.jbiosc.2018.10.001
2) Park SH, Lee CR, Hong SK. Implications of agar and agarase in industrial applications of sustainable marine biomass. Appl Microbiol Biotechnol. 2020, 104, 2815‐2832. doi:10.1007/s00253-020-10412-6
Author Response File: 
Author Response.docx
