A Novel Strategy for Further Enhancing Superior Properties of Thermophilic Endoglucanase from Acidomyces richmondensis

Round 1

Reviewer 1 Report

The manuscript presented by Wang and colleagues discusses a new strategy to improve the thermophilic properties of an endoglucanase from Acidomyces richmondensis. Research and development to obtain ever more high-performance enzymes for industrial processes remains an active topic. The present work is a step in the same direction. The strategy employed by the authors consisted of high-throughput screening for variant identification, followed by thermostability evaluation. The manuscript as a whole is well written, but a number of points need to be clarified before publication.

First, the specific objectives of the work are not well explained. Why are you looking for a thermostable enzyme here? Do the fermentation processes used to convert biomass into fermentable sugars require heat? The fermentation processes tend to take place at mild temperatures. The development of more robust enzymes useful in fermentation processes should not be based more on their stability in relation to pH variations? Given that the fermentation process involves considerable changes in pH. The authors should be more explicit about this in the introduction.

Secondly, the methods are not well described, making it difficult to follow the results presented. Sometimes the methods appear in the results section, or not at all in the text.  For example, the authors do not describe in the methods how thermostability was assessed. Lines 138-139 just describe how enzymatic activity was determined. And here again, we don't know which sugar was used as the standard for the DNS method. Stability studies generally require enzymes to be incubated at the temperatures investigated for a certain period of time, before residual activity is tested. Was this the case here when we see from the results that the reactions were carried out at 70 and 80°C for 30 and 10 minutes respectively?

As another example, Figure 2 shows SDS PAGE results, but there is no mention in the methods section of how the SDS PAGE was performed.

The results obtained are not explained in depth. How does the mutation from Y to C result in improved enzyme stability? What property does C contain that improves stability compared with Y? Is Gibbs free energy enough on its own to justify the enzyme's thermostability? Cysteine itself can be subject to multiple modification reactions (oxidation/reduction, dissulfide bridging, etc.) and is therefore not always stable.

Other comments

Line 23: please include the full names of the amino acids Y and C to facilitate understanding by a wide audience.

Is there a standard classification of enzyme thermostability? What exactly does the term "superior thermostability" refer to?

The quality of Table 1 and the resolution of all Figures need to be improved.

Author Response

Dec 12, 2023

Dear Editor of Fermentation,

Thank you very much for your comments on manuscript titled “A Novel Strategy for Further Enhancing Superior Properties of Thermophilic Endoglucanase from Acidomyces richmondensis” (2749410). Those comments are all valuable and helpful for revising and improving our paper. We have modified the manuscript according to your comments. The revisions are highlighted in the revised manuscript in red. The point-by-point answers to the comments and suggestions are listed below.

The manuscript has been resubmitted to your journal. Again, thank you very much. We are grateful for the time you and reviewer spent reviewing and editing our manuscript. We greatly appreciate it.

We look forward to receiving your positive response.

Sincerely yours,

Le Gao, Ph.D/ Associate Professor

Tianjin Key Laboratory for Industrial Biological Systems and Bioprocessing Engineering, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China

Email: [email protected]

Tel: +86-22-2482-8745

Fax: +86-22-8486-1926

Reviewer 1:

Comments to the Author

The manuscript presented by Wang and colleagues discusses a new strategy to improve the thermophilic properties of an endoglucanase from Acidomyces richmondensis. Research and development to obtain ever more high-performance enzymes for industrial processes remains an active topic. The present work is a step in the same direction. The strategy employed by the authors consisted of high-throughput screening for variant identification, followed by thermostability evaluation. The manuscript as a whole is well written, but a number of points need to be clarified before publication.

Q1: First, the specific objectives of the work are not well explained. Why are you looking for a thermostable enzyme here? Do the fermentation processes used to convert biomass into fermentable sugars require heat? The fermentation processes tend to take place at mild temperatures. The development of more robust enzymes useful in fermentation processes should not be based more on their stability in relation to pH variations? Given that the fermentation process involves considerable changes in pH. The authors should be more explicit about this in the introduction.

A1: Thank you very much. Some description has been added in the manuscript in red. “Endo-β-1,4-glucanases have numerous applications in various industries, includ-ing brewing as well as the production of feed, paper and biofuels. For instance, the in-clusion of endo-β-1,4-glucanase can alleviate the adverse effects of barley β-glucan during the mashing process in the brewing industry, which is typically carried out at temperatures ranging from 50-60°C. Moreover, it can also improve the digestibility of β-glucan in poultry feedstuffs [13]. In other industries, it is used to hydrolyze cellulosic materials to release sugars, reduce mash viscosity and turbidity, bioremediate pulp waste, and increase β-glucan digestibility to improve feed conversion efficiency at 50°C [3]. Therefore, industrial enzymes generally must be robust and tolerate various harsh processing conditions as well as long-term usage and storage. ”

Q2: Secondly, the methods are not well described, making it difficult to follow the results presented. Sometimes the methods appear in the results section, or not at all in the text.  For example, the authors do not describe in the methods how thermostability was assessed. Lines 138-139 just describe how enzymatic activity was determined. And here again, we don't know which sugar was used as the standard for the DNS method. Stability studies generally require enzymes to be incubated at the temperatures investigated for a certain period of time, before residual activity is tested. Was this the case here when we see from the results that the reactions were carried out at 70 and 80°C for 30 and 10 minutes respectively? As another example, Figure 2 shows SDS PAGE results, but there is no mention in the methods section of how the SDS PAGE was performed.

A2:Thank you very much. Some methods have been added in the revised manuscript, including Section 2.7 and 2.8.

Q3: The results obtained are not explained in depth. How does the mutation from Y to C result in improved enzyme stability? What property does C contain that improves stability compared with Y? Is Gibbs free energy enough on its own to justify the enzyme's thermostability? Cysteine itself can be subject to multiple modification reactions (oxidation/reduction, dissulfide bridging, etc.) and is therefore not always stable.

A3: Thank you very much. To investigate the underlying mechanism of the Y299C mutation, which confers superior thermostability, we employed various computational simulation methods to elucidate the factors contributing to the significant increase in enzyme activity observed in the mutant.

Firstly, we utilized the I-mutant tool (https://folding.biofold.org/i-mutant/i-mutant2.0.html) to calculate the change of Gibbs free energy following the mutation of 299Y (ΔΔG, Kcal/mol). At pH=7.0, 50 ℃, ΔΔG>0 indicates an increase in stability, while ΔΔG<0 indicates a decrease in stability. Our calculations revealed that when the amino acid at position 299 was mutated from Y to C, the ΔΔG value was the highest, indicating that the Y299C mutation at this position is the most stable. These Gibbs free energy calculation results were in agreement with our experimental findings, suggesting that after mutating the amino acid at position 299 from Y to C, the enzyme structure become more stable, resulting in higher enzyme activity.

Secondly, the evaluation of the overall structure after mutation involves the calculation of protein dynamics. This is necessary to gain a better understanding of how ligand binding affects proteins. To achieve this, a simulated kinetic calculation was conducted using the Amber analysis tool. The 100 ns trajectories of the complexes were analyzed, and the RMSD values were calculated for 100 ns of simulation time. The RMSD value (Figure 3a) indicated that the Arcel5a-Y299C mutant exhibited lower deviation from the initial structure compared to the wild type, implying enhanced stability. This indicates that Arcel5a has a more stable overall structure that is conducive to enzymatic catalysis, resulting in better enzyme activity and thermal stability.

Finally, the RG value of the mutant (Figure 3b) was lower, confirming its improved overall structural stability. The analysis of conformational group distribution (Figure 3c-e) showed that the Arcel5a-Y299C mutant had a narrower range of conformational variations and a more concentrated distribution of conformational groups compared to the wild type. This reduced conformational diversity indicated higher structural stability in the mutant form. Additionally, the Cα distance difference matrix (Figure 3g) indicated shorter distances in several regions of the mutant compared to the wild type, indicating increased compactness and overall stability of the protein. Notably, the region surrounding the mutated residue 299 exhibited a prominent red coloration, indicating that the Arcel5a-Y299C mutation brought the nearby residues closer together. This closer arrangement likely enhanced the stability of the catalytic pocket, thereby improving the overall stability and catalytic efficiency of Arcel5a.

Q4: Line 23: please include the full names of the amino acids Y and C to facilitate understanding by a wide audience.

A4: Thank you very much for your suggestions. The full names of amino acids Y and C have been added in the revised manuscript in red.

Q5: Is there a standard classification of enzyme thermostability? What exactly does the term "superior thermostability" refer to?

A5: The enzymes from thermophiles exhibit optimal activity at 50℃, while enzymes from hyperthermophiles exhibit activity above 80℃. The endoglucanase activity of the mutant ArCel5A reached 3252 IU/ml, representing an 85.2% increase compared to wild-type ArCel5A at the fermentation time of 94 h. Significantly, the ArCel5A-Y299Cmutant showed superior thermostability, retaining 93.8% of its initial activity after 30 min at 70 °C, and 91.5% after 10 min at 80 °C.

Q6: The quality of Table 1 and the resolution of all Figures need to be improved.

A6: Thank you very much. Table and figures have been modified in revised manuscript.

Reviewer 2 Report

The manuscript developed a B-factor analysis approach that is an effective method for the improvement of endoglucanase thermostability. The paper is interesting, well organized, well written, and introduction sets clear the gap of knowledge and contribution of the manuscript. The topic is relevant and in the scope of the journal but, before it can be published, some points need attention:

1)     Please define the meaning of the abbreviation ArCel5A on its first appearance

2)     Item 2.1: please define the microorganism used, the spore concentration in the inoculum and provide a reference for this topic

3)     Line 109: there is some information underlined, please correct this

4)     Item 2.2: Should TrCel5A be ArCel5A? This section is confusing. Authors mention that evaluated 2 enzymes but which ones are not clear. Please bring part of Item 3.3 (including figure 2A) to this section to clarify the methodology.

5)     Please divide item 2.6 in two topics, to create a separate section for B factor prediction

6)     Provide reference for item 2.8

7)     Line 209: correct “was were”

8)     I believe data from figure 2C are exchanged (blue and red curves). By the text, the activity of the transformant Y299C should be higher than the wild type.

9)      Put the tables close to its mention in the text.

10) What are the deviations on data from Table 2? Please add it.

11) Table 1 is not called in the text

Author Response

Dec 12, 2023

Dear Editor of Fermentation,

Thank you very much for your comments on manuscript titled “A Novel Strategy for Further Enhancing Superior Properties of Thermophilic Endoglucanase from Acidomyces richmondensis” (2749410). Those comments are all valuable and helpful for revising and improving our paper. We have modified the manuscript according to your comments. The revisions are highlighted in the revised manuscript in red. The point-by-point answers to the comments and suggestions are listed below.

The manuscript has been resubmitted to your journal. Again, thank you very much. We are grateful for the time you and reviewer spent reviewing and editing our manuscript. We greatly appreciate it.

We look forward to receiving your positive response.

Sincerely yours,

Le Gao, Ph.D/ Associate Professor

Tianjin Key Laboratory for Industrial Biological Systems and Bioprocessing Engineering, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China

Email: [email protected]

Tel: +86-22-2482-8745

Fax: +86-22-8486-1926

Reviewer 2:

Comments to the Author

The manuscript developed a B-factor analysis approach that is an effective method for the improvement of endoglucanase thermostability. The paper is interesting, well organized, well written, and introduction sets clear the gap of knowledge and contribution of the manuscript. The topic is relevant and in the scope of the journal but, before it can be published, some points need attention.

Q1: Please define the meaning of the abbreviation ArCel5A on its first appearance.

A1: Thank you very much for your suggestions. The definition of ArCel5A has been explained when the abstract and text first appeared and marked in the revised manuscript in red.

Q2: Item 2.1: please define the microorganism used, the spore concentration in the inoculum and provide a reference for this topic.

A2: Thank you very much for your suggestions. Some descriptions including microorganism information and spore concentration in the inoculum have been added in the revised manuscript.

Q3: Line 109: there is some information underlined, please correct this.

A3: Thank you very much for your suggestions. I am very sorry for incorrect information. The method “Section 2.2” has been modified in the revised manuscript in red.

Q4: Item 2.2: Should TrCel5A be ArCel5A? This section is confusing. Authors mention that evaluated 2 enzymes but which ones are not clear. Please bring part of Item 3.3 (including figure 2A) to this section to clarify the methodology.

A4: Thank you very much for your suggestions. I am very sorry for incorrect information. The method “Section 2.2” has been modified in the revised manuscript in red.

Q5: Please divide item 2.6 in two topics, to create a separate section for B factor prediction.

A5: Thank you very much for your suggestions. A new item “B factor analysis” has been added to the revised manuscript in revised manuscript in red.

Q6: Provide reference for item 2.8.

A6: Thank you very much for your suggestions. The reference has been added in the revised manuscript in red.

Q7:  Line 209: correct “was were”.

A7: Thank you very much for your suggestions. The error has been modified in the revised manuscript in red.

Q8:   I believe data from figure 2C are exchanged (blue and red curves). By the text, the activity of the transformant Y299C should be higher than the wild type.

A8: Thank you very much for your suggestions. The errors in Fig.2 have been corrected and redrawn in the revised manuscript.

Q9: Put the tables close to its mention in the text.

A9: Thank you very much for your suggestions. Table 1 and Table 2 have already been swapped in order, close to its mention in the text.

Q10: What are the deviations on data from Table 2? Please add it.

A10: Thank you very much. Deviations on data from Table 2 have been added in the revised manuscript.

Q11: Table 1 is not called in the text.

A11: Thank you very much for your suggestions. Table 1 has been cited in section 3.5 and marked in the revised manuscript in red.