Engineered Stable 5-Hydroxymethylfurfural Oxidase (HMFO) from 8BxHMFO Variant of Methylovorus sp. MP688 through B-Factor Analysis
Round 1
Reviewer 1 Report
1) Page 1, Line 25: This introduction section needs to be rewritten with a focus on the scientific aspects of renewable materials and green technologies in the context of biocatalytic oxidation of 5-hydroxymethylfurfural. Declarations of national goals for carbon dioxide limitations and non-scientific references, such as reference 1, do not fit the topic of the manuscript.
2) Page 1, Line 33: ... could be produced ...
3) Page 1, Line 36: The authors should explain what they mean with “low carbon circular economy”.
4) Page 1, Line 44: This sentence should be reformulated and solvents are not per se non-environment-friendly.
5) Page 2, Line 48: ---complete oxidation ...
6) Page 2, Line 58: The slow reaction rate and low space-time yield should be specified here.
7) Page 2, Line 67: .... protein stabilization ...
8) Page 2, Line 69: ...., hydrophobic forces, ...
9) Page 2, Line 73: It is not entirely clear what is meant by “the residues with higher B-factor value might be the un-stabilizing point of the protein”.
10) Page 2, Line 74: ... values ...
11) Page 2, Line 77: ... a loop structure ...
12) Page 3, Line 98: ... was chosen ...
13) Page 3, Line 102: mechanism of a coupled HRP-Tyr spectrofluorimetric assay
14) Page 4, Line 116: This sentence is incomplete and something is missing.
15) Page 4, Line 126: This formulation should be improved.
16) Page 4, Line 136: Does modelling support this influence on the active site?
17) Page 5, Line 142: Have the authors also investigated higher substrate concentrations than 20 mM?
18) Page 5, Line 144: The authors should discuss the reasons for this decrease in FDCA-yields with increasing HMF concentrations.
19) Page 5, Line 151: Has this indication been tested by the corresponding stability tests under operational conditions with the HMFO supernatants as described in the variation screening or with the purified HFMO variants (by the method of His-tag purification)?
20) Page 5, Line 154: This formulation of this sentence should be improved.
21) Page 6, Line 157: (a) the influence of the substrate concentration at 35 ℃; (b) the influence of the pH value
22) Page 6, Line 160: This description “The influence on the enzyme activity on the incubation time” does not make sense and is in contradiction to the legend of figure 6.
23) Page 6, Line 170: It would be better to provide more experimental details and to describe the enzyme activity in U/mg or U/ml, instead of residual activity in %.
24) Page 7, Line 173: ... a more stable microstructure ...
25) Page 7, Line 175: This formulation should be improved.
26) Page 7, Line 176: The abbreviation RMSF, which is defined here, should be used consistently. The RMSF values in the text have no dimension, while the RMSF values in figure 8 have a dimension.
27) Page 7, Line 182: The legend to figure 7 needs to be corrected, e.g. comparison instead of comparation.
28) Page 8, Line 191: What are “molecular dynamitic simulations”?
29) Page 8, Line 196: The formulation of this sentence and the following one should be improved.
30) Page 9, Line 216: For each transformant library ....
31) Page 9, Line 238: The purities and activities of the purified HFMO variants should be described.
32) Page 9, Line 244: More details should be provided for the HMFO enzyme preparations used in the HFMO enzymatic oxidations.
33) Page 10, Line 249: ... directed evolution for improving the thermal stability of ...
Author Response
Dear reviewer,
Thank you for your review of this paper, and we do appreciate your highly valuable comments. We have carefully revised the manuscript and made changes per your recommendations. Our detailed responses are listed below and all the corrections are with track changes function in the revised manuscript.
Regards!
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Qiuyang Wu et al, reported a research article on "Engineered stable 5-hydroxymethylfurfural oxidase (HMFO) from Methylovorus sp.MP688 through B-factor analysis". stability of the protein structure was improved by employing B-factor analysis through the formation of more hydrogen bonds. This article fit the aim and scope of "catalysts". However, article can be improved after minor revision.
Comments:
- Authors mentioned chemical scheme for the formation of FDCA from HMF. This scheme is already reported by Marco W. Fraajje etal (from references 11&12). Reviewer suggest authors to include the references in the caption of Scheme-1.
- Authors mentioned 5-hydroxymethylfurfural oxidase as HMFO in the title. However authors used HFMO in the first line of abstract, in the scheme, figure 3, 4, 5, 6, 8 and through out the text. This typo need to be corrected through out the article.
Author Response
Dear reviewer,
Thank you for your review of this paper, and we do appreciate your highly valuable comments. We have carefully revised the manuscript and made changes per your recommendations. Our detailed responses are listed below and all the corrections are with track changes function in the revised manuscript.
Regards!
Author Response File: Author Response.pdf
Reviewer 3 Report
The manuscript described the study of the mutations of a 5-hydroxymethylfurfural oxidase (HMFO) 8BxHMFO variant from Methylovorus sp. MP688. Ten mutation sites were selected from the analysis of the B factor with the highest values. Five of them showed higher fluorescence intensity than the 8BxHMFO screened by the HRP-Tyr assay were selected for further study. Two enzyme variants, Q319K and N44G, showed better conversion rates than the 8BxHMFO were subjected for further investigation. The results indicated that both variants showed better catalytic stability in the temperature range of 25 to 40℃ and longer half-lives than the 8BxHMFO. Especially, the Q319K variant significantly increased the furan-2,5-dicarboxylic acid (FDCA) yield to 98 % at 35℃. The improvement was also demonstrated by using in silico simulation studies to prove that more hydrogen bonds may be formed around the mutated residues to increase the conformational stability of the variant proteins. The merit of this work is to advance the performance and the stability of the 8BxHMFO by incorporating the mutation Q319K or N44G for applications. It is recommended for publication after modifying the manuscript. The suggestions for the correction are listed below.
- The engineered enzyme variants were from the mutation of the 8BxHMFO variant of Methylovorus MP688. However, the fact was not reflected on the title or even in the abstract, though the description was shown in quotation for comparison.
- Lys328 was not shown in Figure 1b, the structure of 8BxHMFO.
- The full name of the label of the y-axis in Figure 2, FI, should be described in the legend like that in Figure 3.
- Why the fluorescence intensity in Figure 2a was lower than that in Figure 2b when the same concentration of H2O2 was used? The conditions for the reactions could be described.
- It was described that when the temperature was increased to 30 and 35℃, two of the single-site mutants (95-98 %) show better FDCA yields than others (around 90%) (128-130, page 4). However, it is hard to evaluate them from Figure 4. An additional table may be required to show the statistical significance of the values.
- It was described that the combinatorial mutation of N44G-Q319K showed the highest FDCA yield only at 40℃ (152-153, page 5). However, the FDCA yield at 40℃ was the lowest among all the temperature conditions. Additionally, it was explained that the improved FDCA yields in Q319K and N44G variants were due to the improvement of operational stability (150-151, page 5). It can be further described why the lower catalytic efficiencies of the two single-site mutations than the 8BxHMFO had higher FDCA yields (Table 1).
- The accurate values of the half-lives of four HMFO variants can be listed in a table or described in the text for comparison. It seems that the half-life of the N44G variant is less than 72 hours in Figure 6.
- The source of the original 8BxHMFO gene should be described (200-201, page 9). The strain is Methylovorus MP688.
- The conclusions should be modified if the description or the explanation from the suggestions listed above were changed.
Author Response
Dear reviewer,
Thank you for your review of this paper, and we do appreciate your highly valuable comments. We have carefully revised the manuscript and made changes per your recommendations. Our detailed responses are listed below and all the corrections are with track changes function in the revised manuscript.
Regards!
Author Response File: Author Response.pdf
