Heterologous Expression and Biochemical Characterization of a Thermostable Endoglucanase (MtEG5-1) from Myceliophthora thermophila

Round 1

Reviewer 1 Report

The manuscript submitted by the authors reports a heterologous expression of a thermostable endoglucanase from the fungus Myceliophthora thermophila (MtEG5) in a methylotrophic yeast strain Pichia pastoris GS115. The enzyme was cloned, expressed, and characterized for its properties and functionalities, especially enzymatic activity at various environmental conditions. The enzyme was found to be relatively stable at elevated temperature as well as rather wide range of pH, salt concentration and cation types. Indication of its potential application was also given.

The work was systematic, overall well-explained, and performed using the standard techniques of the field decently. However, it lacks novelty as the same enzyme has been expressed in other hosts. Some aspects of the methods are missing and some statements need qualification. Additionally, there are minor errors as well as the need to improve language and formulation of sentences. Hence this reviewer is suggesting a minor revision to the editor before the work can be accepted.

Major comments

Lines 27-28: more tolerant than what?

Lines 166-167: Duration and temperature of biomass pretreatment were missing. Additional explanation has to be given as a rationale of choosing this rather unusual way of pretreating lignocellulosic biomass.

Lines 259-260: The adjective "strong" needs to be qualified. As it was a qualitative work using microscopy, it cannot show relative indication of enzyme activity on actual biomass. Either qualify the statement or provide a quantitative assay of the enzyme's performance on biomass (not CMC).

Lines 299-300: Additional explanations are needed to make the comparison. Are they really the same enzymes?

Lines 313-314: "catalytically efficient enzyme" needs further qualification. On which basis is this statement made?

Line 321: "good application potential" needs to be qualified. See also comments to lines 259-260. The aspect of application needs to be defined. Good activity on CMC and on pretreated biomass are not the same.

Minor comments

Lines 74, 214: Misspelling of the fungus name

Table 2: Change the term for "Above 80% ..." as it is confusing for readers

Language, typo and scientific terms need to be checked throughout the manuscript. Some examples are in lines 49, 52-53, 102, 106, 223, 256, 259.

Author Response

Please see the attachment. 

Author Response File: Author Response.pdf

Reviewer 2 Report

 

This manuscript reports on the cloning, expression and characterization of a fungal thermoestable endoglucanase (MtEG5), cloned from Myceliophthora thermophila and overexpressed in Pichia pastoris strain GS115, aiming to MtEG5 application as a tool for using cellulose as a renewable energy source. The same enzyme has been cloned and characterized earlier after its expression in Pichia pastoris strain X33 (reference [14] published in 2017) or in Aspergillus niger (reference [13] published in 2014). The authors aim to show that that their system gives rise to a better catalyst than those previously reported. However, in this matter they are not convincing, and in addition the manuscript has many weak points.

 

MAJOR ISSUES

 

1.- Table 2 summarizes the differences between the three cloning and expression systems, and the MtEG5 preparations obtained, concerning enzyme behavior with respect to temperature and pH. In the text, it is stated that MtEG5 expressed by P. pastoris Gs115 is more stable (lines 27-29, 69-73 and 300-301) but Table 2 is very difficult to understand, and the reasoning is unconvincing.

 

2.- The abstract summarizes the claimed evidence for the higher suitability of MtEG5 (as prepared in this manuscript) for practical purposes. It includes several comparative sentences (lines 23, 27 and 28) without mentioning explicitly with what is the comparison made. This makes the abstract difficult to understand in isolation, which adds to the unconvincing character of Table 2.

 

3.- The complete conversion of cellulose to glucose requires the combined action of three enzymes: cellobiohydrolase, endoglucanase and beta-glucosidase (lines 46-48). The role of each of these enzymes is unclear and a scheme should be drawn and displayed, showing the reaction catalyzed by each one.

 

4.- Such a scheme should help you to explain how the MtEG5 assay works as, according to section 2.7, MtEG5 activity is assayed measuring the release of the reducing sugar from carboxymethyl cellulose. An endoglucanase alone cannot do this. This opens an important question about the kinetic assays of MtEG5. How do you relate the rate of liberation of glucose to MtEG5? If MtEG5 is rate limiting within the system used for assay (CMC degradation), the liberation of glucose would in fact represent MtEG5 rates, but if not so, then the kinetic data shown in Figures 2d, 4e-f and 5 do not reflect only MtEG5 behavior.

 

5. There is an aspect of nomenclature/classification of protein which is not correctly reflected in the manuscript. MtEG5 is a protein of the glycoside hydrolase family 5 and so should it be described, indicating its substrate specificity, and in which of the subfamilies of this extense protein family fits (see e.g. Pubmed ID: 22992189; and the Carbohydrate-Active Enzymes (CAZy) classification at http://www.cazy.org/GH5.html).

 

6. There is also an aspect of fungus taxonomy which needs clarification. The specific name Myceliophthora thermophila is used in the manuscript and also in the cited literature (references [5,8,11,14,30]). However, this is not the only name used for the species: reference [8] mentions the synonymous Sporotrichum thermophile; protein accession number XP_003659014.1, that corresponds to the protein used in the manuscript (sequence in Figure 1a) and to ATCC 42464 (line 80), mentions Thermothelomyces thermophilus; other names are mentioned in the Thermothelomyces thermophilus entry in the NCBI Taxonomy browser at https://www.ncbi.nlm.nih.gov/taxonomy (NCBI:txid78579). In fact, if one looks in the Taxonomy browser for Myceliophthora thermophila or Sporotrichum thermophile, the entry for Thermothelomyces thermophilus is recovered. This complex situation needs a comment to avoid confusion.

 

7.- Figure 1. Protein accession number XP_003659014 must be mentioned for the sequence of Figure 1a if correct; if not, then mention the correct accession. The accession numbers of all the sequences used to prepare the phylogenetic tree of Figure 1c must be also shown. The digits shown in Figure 1b must be explained.

 

8. Figure 5 and related main text.

(a) The validity the kinetic data shown in Figure 5 is critically dependent on the question raised in point 4. If this question is satisfactorily resolved, then two additional questions come out.

(b) Km dimensions as mg/mL (lines 24, 310 and 312) or g/L (Figure 5 horizontal axis) are unusual; if it cannot be expressed in units of molar concentration, the reason should be explained. I understand molar concentration cannot be used because carboxymethyl cellulose (CMC) is a polymer of undefined size for which you cannot calculate a molecular weight but if so, explain it;

(c) Vmax is given units of µmol/mg/min in the text (lines 25 and 311). This is a far more serious problem than that with Km. From the linear regression equation y = 0.0475x + 0.0117 inserted in the plot (once the y – is changed to y =), it can be calculated a Vmax = 1/0.0117 = 85.47 with the inverse units of those used in the vertical axis. Since this axis reads min*mL/µg, the estimated Vmax would be 85.47 µg/mL/min. Therefore, either the units given in the axis or those given in lines 25 and 311 are wrong. If the units given in the text (µmol/mg/min) are correct then I suppose µmol refers to glucose liberated and mg refers to the amount of MtEG5 in the assay. In this case, you should be able to convert Vmax to the catalytic constant kcat.

 

9. Lines 313-314: It is stated that “The kinetic properties of the enzyme indicated that MtEG5 is a catalytically efficient cellulolytic enzyme”. This assertion is much dependent on the validity of the kinetic data (see point 8a). Catalytic efficiency is defined as kcat/Km. Therefore, the assertion about catalytic efficiency should be based on kinetic parameters: either in the quotient kcat/Km or, at least, in terms of kcat. A comparison with the catalysts reported in references [13,14] in these terms would be fully relevant.

 

MINOR ISSUES

 

10.- Lines 40-50: “EGs provide an entry point for the most abandoned cellulolytic enzyme [7], by selecting a random interior to hydrolyze cellulose”. The underlined parts are not understandable. This should be rephrased.

 

11.- Lines 61-62: “When cellulase was overexpressed in M. thermophila, many endogenous enzymes of M. thermophila were overexpressed at the same time”. This need additional explanation… what other enzymes?

 

12.- Lines 104-105: This does not seem to be “Genomic” RNA.

 

13.- Lines 222-223: clarify the meaning of “thermophilic destruct”.

 

14.- Lines 267 to 268: some text is missing in between.

 

15.- Line 307: “CMC is one of the best substrates”. Explain what other substrates does the enzyme hydrolyze (see point 4, about substrate specificity among other things).

 

16.- Line 310: “catalytic constants” for Km and Vmax is not correct. Catalytic constant is the denomination reserved for kcat. “Catalytic constants” can be substituted by “Kinetic parameters”.

 

Author Response

Please see the attachment. 

Author Response File: Author Response.pdf

Reviewer 3 Report

The manuscript is well organized and all experiments are well designed. I suggest accepting the article.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

 

In the revised manuscript, several of the points raised in the first reviewer report have been solved, but not all. The main point the authors try to make about their enzyme preparation MtEG5-1 is that it is better than two other preparations from other laboratories (references 16 and 17 in the revised version). As stated in my first report, their arguments were confuse and not convincing. This is still so in the revised version.

Below (points 1-4), I am being very specific about this question to help the authors reach their conclusion based in a clear argument (point 3).

In addition, after the inclusion of the new Figure 1, it has become clear that the description of MtEG5-1 assay in section 2.7 is misleading, and that the units used to express enzyme rates in the text (Vmax) and in Figure 6 should be revised (point 5).

 

MAJOR ISSUES

(Line numbers are taken from a clean pdf copy of the manuscript)

 

Altogether, Section 2.11, Figure 5, Table 2 and text comments in lines 295–302 and 325–334 are still confuse and not convincing in favor of MtEG5-1 being “better” than the other enzyme preparations, for the following reasons (points 1-4):

1.- There is not a clear distinction between measurements of activity and tests of stability; these are different kinds of experiments (like in the case of Figure 5b and Figure 5d) but, in the text, experiments measuring activity under different conditions are used to describe or discuss stability. I have detected the following instances: line 27 (indirectly), lines 73-76, line 199, line 204, legend to Figure 5c, lines 299-300, line 315.

—These instances should be corrected to avoid the confusion between activity measurements and stability tests.

—Section 2.11 should be re-written. For inspiration, I would suggest for example reading the first paragraph of “Enzyme characterization” in page 5 of Karnaouri et al. 2017 (your reference 17 in the revised version).

—I also recommend to label the vertical axis of Figure 5d “Residual relative activity” as corresponds to a true stability-versus-temperature experiment.

— I also recommend that the experiments of Figures 5b–f be clearly described in the legend, distinguishing conditions of treatment and conditions of assay.

2.- The temperature and pH ranges at which MtEG5-1 shows relative activity higher than 80% are wrong: 50–80°C should be 60–80°C, according to the data shown in Figure 5b (relative activity at 50°C is lower than 80%); and pH 3-6 should be pH 4-6 according to Figure 5c (relative activity at pH 3 is lower than 80%).

—Therefore, 50-80 should read 60-80 in line 23, line 296, Table 2, line 328 and line 368; and 3-6 should read 4-6 in line 22, line 300, Table 2, line 332 and line 368.

—Consequently, I recommend to abandon the idea that these ranges support that MtEG5-1 is better than the other preparations, as the differences are small and probably not significant, especially because they are data obtained in different laboratories, not in parallel. Furthermore, the comparisons are made in terms of activities relative to the optimum in each case, not in absolute terms of specific activity or catalytic constant.

 

3.- The MtEG5-1 stability-versus-temperature data shown in Figure 5d are not used in Table 2 and, when mentioned in the text, they are not compared with the other enzymes, although this comparison provides the clearest difference in favor of MtEG5-1.

Consequently, I recommend the following changes:

—Include a new entry in Table 2 comparing, for instance, the stability of MtEG5-1 at 70°C (83% residual activity after 2 h), with 25% residual activity of enzyme expressed in P. pastoris X33 after 2 h at 65°C (see Figure 4 of Karnaouri et al. 2017), and with the complete inactivation of the enzyme expressed in A. niger after 2 h at 60°C (see Figure 3c, StCel5A, of Tambor et al. 2012).
—Use explicitly this comparison in the text to support that MtEG5-1 is better than the other two preparations, for instance in the Abstract (line 22), in Results and Discussion (line 302) and in Conclusions (lines 365-370).

 

4.- Concerning the response of MtEG5-1 to NaCl (with an optimum of activity at 6 g/L), which is also used as an indication that MtEG5-1 is better than the other enzymes, please note as you don’t give data about the response to NaCl by the other enzymes, neither was I able to find this in the original references by Karnaouri et al. 2017 and Tambor et al. 2012.

—For this reason, and also my comments in points 2 and 3, I recommend to change your assertion “…more tolerant to high temperature, high salt and low pH than…” (lines 27-28) to “…more tolerant to high temperature than…”

 

5.- The scheme drawn upon my request (Figure 1 in the revised version) has been indeed very useful to interpret correctly the description of the MtEG5-1 assay in section 2.7). They description is misleading and should be corrected. They state their assay is based on the “release of the reducing sugar” (line 149), and “the reducing sugar content was measured” (line 153), and that one unit enzyme “releases 1 µmol of glucose” (lines 155-156). Actually, CMC cutting by endoglucanase generates reducing ends without necessarily liberating glucose. So, to avoid misleading the readers, the following changes should be made:

—Line 149: change “release of reducing sugar” to “generation of reducing sugar ends”.

—Line 153: change “the reducing sugar content was measured” to “the amount of reducing ends”.

—Lines 155-156: “releases 1 µmol of glucose per minute” to “generates the equivalent to 1 µmol of glucose per minute”.

—Enzyme rates must be expressed in these units, i.e. µmol/min. Note as Vmax is expressed as 85.47 µmol/mg/min (line 24) and as 85.47 µg/min/mL (line 359). One of them must be wrong. On the other hand, it is strange that rates of Figure 6 are expressed as the inverse of µg/min/mL. You should use here the inverse of µmol/min/mg protein. Please revise your units, your numbers and your plotted data in Figure 6. Decide what is correct and express data in the text and in Figure 6 as µmol of glucose equivalents per minute per mg of protein. Since you are working with a purified enzyme, rates should be referred to mg of protein. From these data you should calculate the catalytic constant (k_cat) of the enzyme.

 

MINOR ISSUES

 

6.- Line 50: “hydrolyzation” should read “hydrolysis”.

 

7.- Line 52: “terminality” should read “reducing ends”.

 

8.- Line 200, “elution” should probably read “incubation”.

 

9.- Line 208, “temperature gradients” is inappropriate, as the temperatures are constant. Substitute by ”temperatures”.

Author Response

Dear Reviewer,

Thank you for taking the time to review our manuscript entitled “Heterologous expression and biochemical characterization of a thermostable endoglucanase (MtEG5-1) from Myceliophthora thermophila” and for providing us with valuable comments and suggestions. We greatly appreciate your efforts in helping us to improve the quality of our work.

We carefully reviewed your comments and suggestions and have revised our manuscript accordingly. We believe that your suggestions have significantly improved the logic and readability of our work. However, we acknowledge that due to our limited proficiency in English, we may have misunderstood some of your questions and may have faced challenges in implementing some of the corrections. Nonetheless, are grateful for your patience and meticulousness in pointing out the issues in our manuscript and providing us with detailed guidance on how to address them. As a graduate student with limited abilities, we would greatly appreciate your continued assistance in identifying areas for improvement in our work.

We have revised the Introduction, Materials and Methods, Results and Discussion, and Conclusions sections of the manuscript in response to your specific comments. We believe that these revisions have substantially improved the quality of our work, and we hope that you find our revisions satisfactory.

To provide a detailed account of the changes we have made, we have included a point-by-point response to your comments in the manuscript. We would like to thank you again for your valuable feedback and for your consideration of our work.

 

 

Kind Regards,

 

Demao Li, Ph. D/ Prof.

Tianjin Key Laboratory for Industrial Biological System and Bioprocessing Engineering, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China

Email: [email protected]

Tel.: 86-(022)-84861993

 

Response to Reviewer 2 Comments

 

Point 1:  - There is not a clear distinction between measurements of activity and tests of stability; these are different kinds of experiments (like in the case of Figure 5b and Figure 5d) but, in the text, experiments measuring activity under different conditions are used to describe or discuss stability. I have detected the following instances: line 27 (indirectly), lines 73-76, line 199, line 204, legend to Figure 5c, lines 299-300, line 315.

 

Point 1.1: —These instances should be corrected to avoid the confusion between activity measurements and stability tests.

 

Response 1.1: Thank you for your advice. We have corrected these confusing sentences. You can also read as follows: These results indicated that MtEG5-1 expressed by P. pastoris GS115 is more heat-tolerant than that expressed by A. niger and P. pastoris X33 in lines 30-31 (line 27). Although MtEG5-1 has been successfully overexpressed in Aspergillus niger and P. pastoris X33, the results indicate that MtEG5-1 has poor thermal stability and a larger molecular weight [16,17] in lines 82-84 (lines 73-76). The optimum pH for MtEG5-1 was determined by the standard assay at different pH values (3-11) using either 50 mM sodium citrate buffer pH 2-5, 50 mM phosphate buffer pH 6-8, 100 mM Tris-HCl buffer pH 9, or 50 mM NaHCO₃-NaOH buffer pH 9-11 [31]. On the other hand, the stability at different pH conditions was determined after incubation of MtEG5-1 in the above buffers at 4 °C for 24 h and measuring the remaining activity under the standard assay [17]. The optimal temperature of MtEG5-1 was estimated by using the standard assay procedure at temperatures varying from 30-90 °C for 2 h. The highest activity obtained during the assay was defined as 100% activity. To determine the temperature stability, 0.38 mg of purified MtEG5-1 was incubated at various temperatures (50, 60 and 70 °C) for different time intervals, and the residual activity was measured under the standard assay procedure. The thermostability of MtEG5-1 was tested at optimal pH. The optimal NaCl concentration of MtEG5-1 was estimated by using the standard assay procedure at NaCl concentrations varying from 1-10 g/L at 50 °C for 24 h. The influence of metal ions on purified MtEG5-1 activity was deter-mined by incorporating metal ions such as 1 mol/L Na₂SO4, MgSO₄, MnSO₄, CuSO₄, K₂SO₄, FeSO₄, and NiSO₄ solutions under the standard assay procedure at 70 °C for 2 h in lines 232-247 (line 199, line 204). Figure 5. (b) Effect of incubation with various temperatures on activity for 2 h. (c) Effect of incubation with various pH values on the stability of purified MtEG5-1 at 4 °C for 24 h. (d) Effect of incubation at various temperatures on the stability of purified MtEG5-1 for 24 h (black circle 50 °C, white circle 60 °C, black pentagram 70 °C). (e) Effect of incubation with various NaCl concentrations on the activity of purified MtEG5-1 at 50 °C for 24 h in section 3.4. Furthermore, MtEG5-1 also exhibited good thermal stability, retaining a relative activity of above 88% at all temperatures until 2 h of incubation. Subsequently, the relative activity decreased to approximately 97, 85 and 72% at temperatures of 50, 60 and 70 °C, respectively, after 3 h of incubation in lines 349-352 (lines 299-300). These results indicate that MtEG5-1 has remarkable tolerance to harsh conditions such as high salt concentrations, high temperatures, and low pH, making it an ideal candidate for cellulose degradation in lines 371-374 (line 315).

 

Point 1.2: —Section 2.11 should be re-written. For inspiration, I would suggest for example reading the first paragraph of “Enzyme characterization” in page 5 of Karnaouri et al. 2017 (your reference 17 in the revised version).

 

Response 1.2: Thank you for your advice. I have rewritten Section 2.11 “Characterization of Enzymes” according to the references in line 232-247. The optimum pH for MtEG5-1 was determined by the standard assay at different pH values (3-11) using either 50 mM sodium citrate buffer pH 2-5, 50 mM phosphate buffer pH 6-8, 100 mM Tris-HCl buffer pH 9, or 50 mM NaHCO₃-NaOH buffer pH 9-11 [31]. On the other hand, the stability at different pH conditions was determined after incubation of MtEG5-1 in the above buffers at 4 °C for 24 h and measuring the remaining activity under the standard assay [17]. The optimal temperature of MtEG5-1 was estimated by using the standard assay procedure at temperatures varying from 30-90 °C for 2 h. The highest activity obtained during the assay was defined as 100% activity. To determine the temperature stability, 0.38 mg of purified MtEG5-1 was incubated at various temperatures (50, 60 and 70 °C) for different time intervals, and the residual activity was measured under the standard assay procedure. The thermostability of MtEG5-1 was tested at optimal pH value. The optimal NaCl concentration of MtEG5-1 was estimated by using the standard assay procedure at NaCl concentrations varying from 1-10 g/L at 50 °C for 24 h. The influence of metal ions on purified MtEG5-1 activity was deter-mined by incorporating metal ions such as 1 mol/L Na₂SO4, MgSO₄, MnSO₄, CuSO₄, K₂SO₄, FeSO₄, and NiSO₄ solutions under the standard assay procedure at 70 °C for 2 h.

 

Point 1.3: —I also recommend to label the vertical axis of Figure 5d “Residual relative activity” as corresponds to a true stability-versus-temperature experiment.

 

Response 1.3: Thank you for your advice. I have corrected and optimized the picture in Figure 5d. You can also read as follows:

Figure 5. (d) Effect of incubation at various temperatures on the stability of purified MtEG5-1 for 24 h (black circle 50 °C, white circle 60 °C, black pentagram 70 °C).

 

Point 1.4: — I also recommend that the experiments of Figures 5b–f be clearly described in the legend, distinguishing conditions of treatment and conditions of assay.

 

Figure 5. Effects of different factors on the activity of purified MtEG5-1. (a) Purified MtEG5-1 expression was detected by 12% SDS-PAGE. Lane M, molecular weight marker; Lane1, a purified MtEG5-1 protein. (b) Effect of incubation with various temperature on activity for 2 h. (c) Effect of incubation with various pH values on the stability of purified MtEG5-1 at 4 °C for 24 h. (d) Effect of incubation with various temperature on the stability of purified MtEG5-1 for 24 h (black circle 50 °C, white circle 60 °C, black pentagram 70 °C). (e) Effect of incubation with various NaCl concentration on the activity of purified MtEG5-1 at 50 °C for 24 h.

Response 1.4: Thank you for your advice I have clearly described the experiments in Figure 5b ~ e in the legend and changed the histogram of Figure 5f into Table 2. Furthermore, MtEG5-1 also exhibited good thermal stability, retaining a relative activity above 88% at all temperatures until 2 h of incubation. Subsequently, the relative activity decreased to approximately 97, 85 and 72% at temperatures of 50, 60 and 70 °C, respectively, after 3 h of incubation in lines 349-352 (Figure 5d).

 

Table 2. Effect of various metal ions on MtEG5-1 activity at 50 °C for 2 h.

Chemicals	Relative activity (%)	Chemicals	Relative activity (%)
None	100	Cu2+	100.2 ± 2.9
Na+	86.1 ± 2.4	K+	76.1 ± 1.6
Mg2+	97.4 ± 1.3	Fe2+	123.3 ± 3.8
Mn2+	142.6 ±2.5	Ni2+	87.6 ±2.2

Metal ions have been found to cause structural changes in enzyme [46], resulting in alterations in their activity. This study investigates the effect of Na⁺, Mg²⁺, Mn²⁺, Cu²⁺, K⁺, Fe²⁺ and Ni²⁺ on the activity of MtEG5-1, which was incubated at 50 ℃ for 2 h. The results showed that Mn²⁺ improved the activity of MtEG5-1 by 142.6%, while K⁺ inhibited its activity by 76.1% in Table 2 (lines 365-371).

 

Point 2: - The temperature and pH ranges at which MtEG5-1 shows relative activity higher than 80% are wrong: 50–80°C should be 60–80°C, according to the data shown in Figure 5b (relative activity at 50°C is lower than 80%); and pH 3-6 should be pH 4-6 according to Figure 5c (relative activity at pH 3 is lower than 80%).

 

Point 2.1: —Therefore, 50-80 should read 60-80 in line 23, line 296, Table 2, line 328 and line 368; and 3-6 should read 4-6 in line 22, line 300, Table 2, line 332 and line 368.

 

Response 2.1: Thank you for your advice. I have revised my manuscript according to your advice.  You can also read as follows: 50-80 should read 60-80 in lines 343, Table 3, 387 and 438. 3-6 should read 4-6 in lines 348, Table 3, 391 and 437.

 

Point 2.2: —Consequently, I recommend to abandon the idea that these ranges support that MtEG5-1 is better than the other preparations, as the differences are small and probably not significant, especially because they are data obtained in different laboratories, not in parallel. Furthermore, the comparisons are made in terms of activities relative to the optimum in each case, not in absolute terms of specific activity or catalytic constant.

 

Response 2.2: Thank you for your advice. According to your opinion, we carefully analyzed the data and found that the tolerance of MtEG5-1 to temperature and pH was not obvious, but the stability of MtEG5-1 after incubation at 70 °C for 2 hours was better than that of the other two enzymes, so we also modified it accordingly. These results indicated that MtEG5-1 expressed by P. pastoris GS115 is more heat-tolerant than that expressed by A. niger and P. pastoris X33 in lines 30-31. Notably, although the temperature and pH of the MtEG5-1 expressed in the three hosts were not significantly different, MtEG5-1 exhibited greater tolerance to high temperature than the enzyme expressed by P. pastoris X33 and A. niger. For instance, the thermal stability of MtEG5-1 expressed by P. pastoris GS115 still remained at 88% residual activity after 2 h at 70 °C, compared to 25% residual activity of enzyme expressed in P. pastoris X33 after 2 h at 65 °C and complete inactivation of the enzyme expressed in A. niger after 2 h at 60 °C in lines 391-397. MtEG5-1 expressed by P. pastoris GS115 is more tolerant to high temperature than the enzyme expressed by P. pastoris X33 and A. niger in lines 438-440.

 

Point 3: - The MtEG5-1 stability-versus-temperature data shown in Figure 5d are not used in Table 2 and, when mentioned in the text, they are not compared with the other enzymes, although this comparison provides the clearest difference in favor of MtEG5-1.

Consequently, I recommend the following changes:

 

Point 3.1: —Include a new entry in Table 2 comparing, for instance, the stability of MtEG5-1 at 70°C (83% residual activity after 2 h), with 25% residual activity of enzyme expressed in P. pastoris X33 after 2 h at 65°C (see Figure 4 of Karnaouri et al. 2017), and with the complete inactivation of the enzyme expressed in A. niger after 2 h at 60°C (see Figure 3c, StCel5A, of Tambor et al. 2012).

 

Response 3.1: Thank you for your advice. I have added a new entry in Table 2 and explained it in the article. Notably, although the temperature and pH of the MtEG5-1 expressed in the three hosts were not significantly different, MtEG5-1 exhibited greater tolerance to high temperature than the enzyme expressed by P. pastoris X33 and A. niger. For instance, the thermal stability of MtEG5-1 expressed by P. pastoris GS115 still remained at 88% residual activity after 2 h at 70 °C, compared to 25% residual activity of enzyme expressed in P. pastoris X33 after 2 h at 65 °C and complete inactivation of the enzyme expressed in A. niger after 2 h at 60 °C in lines 391-397.

 

Point 3.2: —Use explicitly this comparison in the text to support that MtEG5-1 is better than the other two preparations, for instance in the Abstract (line 22), in Results and Discussion (line 302) and in Conclusions (lines 365-370).

 

Response 3.2: Thank you for your advice. I have explicitly used this comparison in the text to support the superiority of MtEG5-1 over the other two formulations, such as the abstract in lines 21-22, the results and discussion in line 302 (deleted), and the conclusions in lines 398-402. We found that the purified MtEG5-1 exhibited optimum activity at pH 5 and 70 °C, with 88% thermal stability after incubation at 70 °C for 2 hours in lines 22-23. We deleted lines 355-358 because it is not useful in the article. The recombinant protein MtEG5-1 was stable in extreme environments, such as acidic pH 4-6, high temperature (60-80 °C), and salt (6 g/L) in our study. MtEG5-1 expressed by P. pastoris GS115 is more tolerant to high temperature than the enzyme expressed by P. pastoris X33 and A. niger in lines 435-440.

 

Point 4: - Concerning the response of MtEG5-1 to NaCl (with an optimum of activity at 6 g/L), which is also used as an indication that MtEG5-1 is better than the other enzymes, please note as you don’t give data about the response to NaCl by the other enzymes, neither was I able to find this in the original references by Karnaouri et al. 2017 and Tambor et al. 2012.

—For this reason, and also my comments in points 2 and 3, I recommend to change your assertion “…more tolerant to high temperature, high salt and low pH than…” (lines 27-28) to “…more tolerant to high temperature than…”

 

Response 4: Thank you for your advice. I have made corrections in the Abstracts, Results and Discussions, and Conclusions in lines 30-31, 391-397 and 438-440. These results indicated that MtEG5-1 expressed by P. pastoris GS115 is more heat-tolerant than that expressed by A. niger and P. pastoris X33 in lines 30-31. Notably, although the temperature and pH of the MtEG5-1 expressed in the three hosts were not significantly different, MtEG5-1 exhibited greater tolerance to high temperature than the enzyme expressed by P. pastoris X33 and A. niger. For instance, the thermal stability of MtEG5-1 expressed by P. pastoris GS115 still remained at 88% residual activity after 2 h at 70 °C, compared to 25% residual activity of enzyme expressed in P. pastoris X33 after 2 h at 65 °C and complete inactivation of the enzyme expressed in A. niger after 2 h at 60 °C in lines 391-397. MtEG5-1 expressed by P. pastoris GS115 is more tolerant to high temperature than the enzyme expressed by P. pastoris X33 and A. niger in lines 438-440.

 

Point 5: - The scheme drawn upon my request (Figure 1 in the revised version) has been indeed very useful to interpret correctly the description of the MtEG5-1 assay in section 2.7). They description is misleading and should be corrected. They state their assay is based on the “release of the reducing sugar” (line 149), and “the reducing sugar content was measured” (line 153), and that one unit enzyme “releases 1 µmol of glucose” (lines 155-156). Actually, CMC cutting by endoglucanase generates reducing ends without necessarily liberating glucose. So, to avoid misleading the readers, the following changes should be made:

Point 5.1: —Line 149: change “release of reducing sugar” to “generation of reducing sugar ends”.

 

Response 5.1: Thank you for your advice. I have revised my manuscript according to your advice in lines 160-162. You can also read as follows: The activity of MtEG5-1 was determined by the DNS method, which is based on the generation of reducing sugar ends from carboxymethyl cellulose (CMC) as a substrate and its reaction with dinitro salicylic acid.

 

Point 5.2: —Line 153: change “the reducing sugar content was measured” to “the amount of reducing ends”.

 

Response 5.2: Thank you for your advice. I have revised my manuscript according to your advice in lines 164-166. You can also read as follows: The resulting mixture was placed in boiling water for 5 min for color development, and the amount of reducing ends was determined by absorbance at 540 nm.

 

Point 5.3: —Lines 155-156: “releases 1 µmol of glucose per minute” to “generates the equivalent to 1 µmol of glucose per minute”.

 

Response 5.3: Thank you for your advice. I have revised my manuscript according to your advice in lines 168-169. You can also read as follows: An endoglucanase unit (IU) is defined as the amount of enzyme that generates the equivalent to 1 μmol of glucose per minute per milliliter in the above reaction conditions.

 

Point 5.4: —Enzyme rates must be expressed in these units, i.e. µmol/min. Note as Vmax is expressed as 85.47 µmol/mg/min (line 24) and as 85.47 µg/min/mL (line 359). One of them must be wrong. On the other hand, it is strange that rates of Figure 6 are expressed as the inverse of µg/min/mL. You should use here the inverse of µmol/min/mg protein. Please revise your units, your numbers and your plotted data in Figure 6. Decide what is correct and express data in the text and in Figure 6 as µmol of glucose equivalents per minute per mg of protein. Since you are working with a purified enzyme, rates should be referred to mg of protein. From these data you should calculate the catalytic constant (kcat) of the enzyme.

 

Figure 6. Lineweaver‒Burk plot for MtEG5-1 with CMC as substrate.

Response 5.4: Thank you for your advice. I have revised my manuscript according to your advice in lines 425-429. You can also read as follows: Based on the Lineweaver‒Burk double inverse method, the values of Km and Vmax for purified MtEG5-1 were approximately 4.06 mg/mL and 85.47 μmol/min/mg at 70 °C (pH 5), respectively Figure 6). Kcat for purified was found to be 224.92 /s, whereas the catalytic efficiency (Kcat/Km) for the hydrolysis of CMC was calculated to be 55.39 mL/s/mg.

 

Point 6: - Line 50: “hydrolyzation” should read “hydrolysis”.

 

Response 6: Thank you for your advice. I have revised my manuscript according to your advice in lines 57-58. You can also read as follows: As the most important rate-limiting enzyme in cellulose degradation, EGs focus on the hydrolysis of β-1, 4-glucosidic glucan linkages.

 

Point 7: - Line 52: “terminality” should read “reducing ends”.

 

Response 7: Thank you for your advice. I have revised my manuscript according to your advice in lines 58-60. You can also read as follows: resulting in the conversion of long cellulase chains into short chains, thereby increasing the amount of reducing ends exposed in the cellulose chain.

 

Point 8: - Line 200, “elution” should probably read “incubation”.

 

Response 8: Thank you for your advice. I rewrote Section 2.11 and revised it in full.

 

Point 9: - Line 208, “temperature gradients” is inappropriate, as the temperatures are constant. Substitute by ”temperatures”.

 

Response 9: Thank you for your advice. I have revised my manuscript according to your advice in lines 239-242. You can also read as follows: To determine the temperature stability, 0.38 mg of purified MtEG5-1 was incubated at various temperatures (50, 60 and 70 °C) for different time intervals, and the residual activity was measured under the standard assay procedure.

 

Author Response File: Author Response.pdf

Round 3

Reviewer 2 Report

 

Some aspects of the manuscript have been improved, but in the new parts new problems appear. Altogether, the manuscript still presents with major issues and many minor issues. In addition, I would like to comment that the cover letter is signed by Demao Li, PhD/Prof, but the signer identifies (himself?) “As a graduate student with limited abilities”. Probably this is also, directly or indirectly, the consequence of authors´ difficulties with English language, but it should be explained in the new cover letter.

 

MAJOR ISSUES

 

1.- In the new description of Section 2.11, it is now stated that in the work there were two kinds of experiments of MtEG5-1 versus pH: one testing activity-vs-pH, another testing stability-vs-pH. However, only one of these experiments is shown (Figure 5c), which seems to be stability-vs-pH according to the legend of Figure 5 (although the vertical axis is not labeled as “residual relative activity”). The other experiment (activity-vs-pH) should be included in Figure 5 as a new panel.

 

2.- In the new description of Section 2.11, the new reference 31 (line 200) is practically unrelated to the work under review. It should be deleted. On the other hand, reference 17 can be omitted in line 203 (not in the rest of manuscript).

 

3.- The new description of protocol for metal ion effects in Section 2.11 (lines 210-213) has been changed with respect to previous versions without reviewer’s request and without an explanation in the cover letter. Formerly, it was described as an stability-vs-metal ion experiment (2 h incubation with 0.33 mol metal/L at 70°C, followed by activity assay under supposedly standard conditions, i.e. at 50°C), but now it is described as an activity assay (assay in the presence of 1 mol metal/L during 2 h at 70°C). If this is correct, please note as this is not the standard assay (compare to Section 2.7), therefore in line 213 after “standard assay procecedure” add “except that temperature was 70°C and reaction length was 2 h”. Whatever the actual experiment was, it should be confirmed and described correctly.

 

4.- In the new Table 2, the heading “Chemicals” should be changed to “metal ion”. The final concentration of metal in the assay reaction mixture should be stated (was it really 1 mol/L or 0.33 mol/L (see point 3)? The temperature used for this experiment was 50°C according to the title of the Table, but it was 70°C according Section 2.11 line 213.

 

5.- In the new legend to Figure 5, the experiments are not yet clearly described, as requested in my previous report, distinguishing conditions of treatment and conditions of assay. Therefore, please consider whether the following descriptions fit the actual protocols used (modify the descriptions if necessary according to the actual conditions used):
Panel (b). “Effect of assay temperature on MtEG5-1 activity. The standard assay was used except that reaction temperature was as shown in the graph”.

Panel (c). “Effect of pH on MtEG5-1 stability. The enzyme was incubated for 24 h at 4°C and the pH values indicated, followed by activity assay under standard conditions”.

Panel (d). “Effect of temperature on MtEG5-1 stability. The enzyme was incubated for the times plotted at the temperatures indicated and at pH 5, followed by activity assay under standard conditions”.

 

In the case of panel (e), the tipology and conditions of the experiment are unclear.

In the Figure legend, panel (e) is described as a stability experiment with 24 h incubation in different concentrations of NaCl, followed by activity assay. But in the text is described as an activity assay with either 24 h reaction length (lines 209-210) or 2 h reaction length (lines 302-304). Please, clarify, describe always in the same way, and in the legend to Figure 5c use the appropriate panel (e) description, with the style suggested above for either panel (b) or panels (c) and (d).

 

For the new panel to be added to Figure 5 (see my request in point 1), be careful to choose the right description style (possibly the same style as my suggestion for panel b)

 

In addition, note as the legend of Figure 5 is incomplete in the manuscript file (bottom of page 9).

 

6.- Several bibliographic references in the text must be wrongly formatted in the reference manager program or database used, as they appear as “Error! Reference source not found.d” (lines 252, 259, 260, 263, 268).

 

7.- The authors have changed the axis of Figure 6 previously labeled min x mL/µg to mg/min/µmol. I have two comments on this. First, the correct units should be mg x min / µmol. Second, the authors changed the axis label but all the points in the plot and the equation remain the same as in the former version. In principle, the change of units implies that all the values represented would also change accordingly. Therefore, please re-check and confirm.

 

8.- According to the current version of Figure 6, kinetic parameter k_cat is wrong. The Vmax calculated from this plot would be indeed 85.47 µmol x min^-1 x mg^-1 (as indicated in line 358). But then, the k_cat value 224.92 s^-1 (line 359) is wrong, since k_cat = Vmax / enzyme. To calculate k_cat with dimensions of s^-1, Vmax is 1.42 µmol x s^-1 x mg^-1, and the amount of enzyme corresponding to 1 mg is 0.018 µmol (1000 µg / 55,000). Therefore, kcat = 1.42 µmol x s^-1 x mg^-1 / 0.018 µmol mg^-1= 78.9 s^-1. The k_cat value should be corrected if Figure 6 is correct as shown (however this may not be so; see point 7).

 

9.- The catalytic efficiency (k_cat/K_m) calculated from a K_m value expressed in mg substrate/mL (i.e. not in molar units) is meaningless. Delete this datum (Line 360) or, if possible, compare it with values obtained with other endoglucanases.

 

MINOR ISSUES

 

10.- Line 18. The sentence within this line is incomprehensible.

11.- Line 23. Delete “smaller” or specify “smaller than…” what.

12.- Line 73. Change “Thus,” to “In contrast,”.

13.- Line 75-77. This sentence is incomprehensible. The manuscript does not describe “the enzymatic properties of… …expression and secretion”.

14.- Line 154. Delete “per milliliter”. The definition of enzyme unit does not depend on the volume of the sample.

15.- Line 227. “we have named the enzyme … … is named”; rewrite this sentence.

16.- Lines 262-265. This sentence is incomprehensible.

17.- Line 292. The highest relative activity… was the highest…”. Rewrite this sentence.

18.- Line 310. Delete “()”.

19.- Line 326. “…temperature and pH of the MtEG5-1..”. This sentence is understandable but incorrect. Insert the calificative “optimal” before “temperature and pH”.

20.- Line 332. Change “Collectively,” to “Altogether,”.

21.- Line 363-364. This sentence is a truism. It means just that MtEG5-1 is an endoglucanase. This is shown by other experiments in the manuscript and by the literature, it is not inferred from the mentioned kinetic properties.

 

Author Response

Dear Reviewer,

Thank you for taking the time to review our manuscript entitled “Heterologous expression and biochemical characterization of a thermostable endoglucanase (MtEG5-1) from Myceliophthora thermophila” and for providing us with valuable comments and suggestions. We greatly appreciate your efforts in helping us to improve the quality of our work.

We carefully reviewed your comments and suggestions and have revised our manuscript accordingly. We believe that your suggestions have significantly improved the logic and readability of our work. At the same time, we also re-validate and correct the experimental data. Thank you for patiently and carefully pointing out the problems in our manuscripts and providing us with detailed guidance on how to solve these problems.

We have revised the Introduction, Materials and Methods, Results and Discussion sections of the manuscript in response to your specific comments. We believe that these revisions have substantially improved the quality of our work, and we hope that you find our revisions satisfactory.

To provide a detailed account of the changes we have made, we have included a point-by-point response to your comments in the manuscript. We would like to thank you again for your valuable feedback and for your consideration of our work.

 

 

Kind Regards,

 

Demao Li, Ph. D/ Prof.

Tianjin Key Laboratory for Industrial Biological System and Bioprocessing Engineering, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China

Email: [email protected]

Tel.: 86-(022)-84861993

 

Response to Reviewer 2 Comments

 

Point 1:  - In the new description of Section 2.11, it is now stated that in the work there were two kinds of experiments of MtEG5-1 versus pH: one testing activity-vs-pH, another testing stability-vs-pH. However, only one of these experiments is shown (Figure 5c), which seems to be stability-vs-pH according to the legend of Figure 5 (although the vertical axis is not labeled as “residual relative activity”). The other experiment (activity-vs-pH) should be included in Figure 5 as a new panel.

 

Response 1: Thank you for your advice. We have added a new panel (activity-vs-pH) as shown in Figure 5b. You can also read as follows:

Figure 5. Effects of different factors on the activity of purified MtEG5-1. (a) Purified MtEG5-1 expression was detected by 12% SDS-PAGE. Lane M, molecular weight marker; Lane1, a purified MtEG5-1 protein. (b) Effects of assay pH on MtEG5-1 activity. The standard assay was used except that reaction pH was as shown in the graph. (c) Effects of assay temperature on MtEG5-1 activity. The standard assay was used except that reaction temperature was as shown in the graph. (d) Effects of pH on MtEG5-1 stability. The enzyme was incubated for 24 h at 4 °C and the pH values indicated, followed by activity assay under standard conditions. (e) Effects of temperature on MtEG5-1 stability. The enzyme was incubated for the times plotted at the temperatures indicated and at pH 5 for 24 h, followed by activity assay under standard conditions (black circle 50 °C, white circle 60 °C, black pentagram 70 °C). (f) Effects of assay NaCl concentration on MtEG5-1 activity. The enzyme was incubated for 2 h at 50 °C and various NaCl concentrations indicated, followed by activity assay under standard conditions.

 

 

Point 2:  - In the new description of Section 2.11, the new reference 31 (line 200) is practically unrelated to the work under review. It should be deleted. On the other hand, reference 17 can be omitted in line 203 (not in the rest of manuscript).

We have deleted Article 31 (Line 200) and Line 203 Reference 17 in Section 2.11.

 

Response 2: Thank you for your advice. We have deleted Reference 31 in line 199 and Reference 17 in line 202. 

 

Point 3:  - The new description of protocol for metal ion effects in Section 2.11 (lines 210-213) has been changed with respect to previous versions without reviewer’s request and without an explanation in the cover letter. Formerly, it was described as an stability-vs-metal ion experiment (2 h incubation with 0.33 mol metal/L at 70°C, followed by activity assay under supposedly standard conditions, i.e. at 50°C), but now it is described as an activity assay (assay in the presence of 1 mol metal/L during 2 h at 70°C). If this is correct, please note as this is not the standard assay (compare to Section 2.7), therefore in line 213 after “standard assay procecedure” add “except that temperature was 70°C and reaction length was 2 h”. Whatever the actual experiment was, it should be confirmed and described correctly.

 

Response 3: Thank you for your advice. We have reconfirmed the experimental data and corrected it. You can also read as follows: The influence of metal ions on purified MtEG5-1 activity was determined by incorporating metal ions such as 0.33 mol/L Na₂SO₄, MgSO₄, MnSO₄, CuSO₄, K₂SO₄, FeSO₄, and NiSO₄ solutions under the standard assay procedure expect that reaction length was 2 h in lines 210-213 and Table 2.

 

Point 4:  - In the new Table 2, the heading “Chemicals” should be changed to “metal ion”. The final concentration of metal in the assay reaction mixture should be stated (was it really 1 mol/L or 0.33 mol/L (see point 3)? The temperature used for this experiment was 50°C according to the title of the Table, but it was 70°C according Section 2.11 line 213.

 

Response 4: Thank you for your advice. We have reconfirmed the experimental data and corrected it. You can also read as follows: The influence of metal ions on purified MtEG5-1 activity was determined by incorporating metal ions such as 0.33 mol/L Na₂SO₄, MgSO₄, MnSO₄, CuSO₄, K₂SO₄, FeSO₄, and NiSO₄ solutions under the standard assay procedure expect that reaction length was 2 h in lines 210-213 and Table 2.

Table 2.  Effect of various metal ions on MtEG5-1 activity. The standard assay was used expect that reaction length was 2 h.

Metal ion	Relative activity (%)	Metal ion	Relative activity (%)
None	100	Cu2+	100.2 ± 2.9
Na+	86.1 ± 2.4	K+	76.1 ± 1.6
Mg2+	97.4 ± 1.3	Fe2+	123.3 ± 3.8
Mn2+	142.6 ±2.5	Ni2+	87.6 ±2.2

This study investigates the effect of Na^+, Mg²⁺, Mn²⁺, Cu^2+, K⁺, Fe²⁺ and Ni²⁺ on the activity of MtEG5-1, which was incubated at 50 °C for 2 h in lines 310-311.

 

Point 5:  - In the new legend to Figure 5, the experiments are not yet clearly described, as requested in my previous report, distinguishing conditions of treatment and conditions of assay. Therefore, please consider whether the following descriptions fit the actual protocols used (modify the descriptions if necessary according to the actual conditions used):

Panel (b). “Effect of assay temperature on MtEG5-1 activity. The standard assay was used except that reaction temperature was as shown in the graph”.

Panel (c). “Effect of pH on MtEG5-1 stability. The enzyme was incubated for 24 h at 4°C and the pH values indicated, followed by activity assay under standard conditions”.

Panel (d). “Effect of temperature on MtEG5-1 stability. The enzyme was incubated for the times plotted at the temperatures indicated and at pH 5, followed by activity assay under standard conditions”.

In the case of panel (e), the tipology and conditions of the experiment are unclear.

In the Figure legend, panel (e) is described as a stability experiment with 24 h incubation in different concentrations of NaCl, followed by activity assay. But in the text is described as an activity assay with either 24 h reaction length (lines 209-210) or 2 h reaction length (lines 302-304). Please, clarify, describe always in the same way, and in the legend to Figure 5c use the appropriate panel (e) description, with the style suggested above for either panel (b) or panels (c) and (d).

For the new panel to be added to Figure 5 (see my request in point 1), be careful to choose the right description style (possibly the same style as my suggestion for panel b)

In addition, note as the legend of Figure 5 is incomplete in the manuscript file (bottom of page 9).

 

Response 5: Thank you for your advice. We have modified the legend of Figure 5 and clearly described the experiment, distinguishing conditions of treatment and conditions of assay. In this experiment, MtEG5-1 was incubated at 50 °C for 2 h with NaCl concentration of 1-10 g/L, and the optimal NaCl concentration of MtEG5-1 was estimated by standard measurement method. You can also read as follows: The optimal NaCl concentration of MtEG5-1 was estimated by using the standard assay procedure at NaCl concentrations varying from 1-10 g/L at 50 °C for 2 h in lines 208-210. Effects of assay NaCl concentration on MtEG5-1 activity. The enzyme was incubated for 2 h at 50 °C and various NaCl concentrations indicated, followed by activity assay under standard conditions in Figure 5f (Corresponding to the previous manuscript Figure 5e). In addition, the relative activity of MtEG5-1 incubated with NaCl (1-10 g/L) at 50 °C for 2 h was above 80% and the optimal salt concentration of MtEG5-1 was 6 g/L in lines 304-306.

 

Figure 5. Effects of different factors on the activity of purified MtEG5-1.

(a) Purified MtEG5-1 expression was detected by 12% SDS-PAGE. Lane M, molecular weight marker; Lane1, a purified MtEG5-1 protein.

(b) Effects of assay pH on MtEG5-1 activity. The standard assay was used except that reaction pH was as shown in the graph.

(c) Effects of assay temperature on MtEG5-1 activity. The standard assay was used except that reaction temperature was as shown in the graph.

(d) Effects of pH on MtEG5-1 stability. The enzyme was incubated for 24 h at 4 °C and the pH values indicated, followed by activity assay under standard conditions.

(e) Effects of temperature on MtEG5-1 stability. The enzyme was incubated for the times plotted at the temperatures indicated and at pH 5 for 24 h, followed by activity assay under standard conditions (black circle 50 °C, white circle 60 °C, black pentagram 70 °C). (f) Effects of assay NaCl concentration on MtEG5-1 activity. The enzyme was incubated for 2 h at 50 °C and various NaCl concentrations indicated, followed by activity assay under standard conditions.

 

Point 6:  - Several bibliographic references in the text must be wrongly formatted in the reference manager program or database used, as they appear as “Error! Reference source not found.d” (lines 252, 259, 260, 263, 268).

 

Response 6: Thank you for your advice. We have corrected the reference and legends in lines 253, 260, 264, 269.

 

Point 7:  - The authors have changed the axis of Figure 6 previously labeled min x mL/µg to mg/min/µmol. I have two comments on this. First, the correct units should be mg x min / µmol. Second, the authors changed the axis label but all the points in the plot and the equation remain the same as in the former version. In principle, the change of units implies that all the values represented would also change accordingly. Therefore, please re-check and confirm.

 

Response 7: Thank you for your advice. We have reconfirmed the experimental data and recalculated and corrected the units. You can also read as follows: The Km and Vmax values of purified MtEG5-1 were approximately 6.11 mg/mL and 91.74 μmol/min/mg at 70 °C (pH 5.0), respectively in lines 23-25. Based on the Lineweaver‒Burk double inverse method, the values of Km and Vmax for purified MtEG5-1 were approximately 6.11 mg/mL and 91.74 μmol/min/mg at 70 °C (pH 5), respectively (Figure 6). Kcat for purified was found to be 84.94 /s in lines 259-362 and Figure 6.

Figure 6. Lineweaver‒Burk plot for MtEG5-1 with CMC as substrate

 

Point 8: According to the current version of Figure 6, kinetic parameter kcat is wrong. The Vmax calculated from this plot would be indeed 85.47 µmol x min-1 x mg-1 (as indicated in line 358). But then, the kcat value 224.92 s-1 (line 359) is wrong, since kcat = Vmax / enzyme. To calculate kcat with dimensions of s-1, Vmax is 1.42 µmol x s-1 x mg-1, and the amount of enzyme corresponding to 1 mg is 0.018 µmol (1000 µg / 55,000). Therefore, kcat = 1.42 µmol x s-1 x mg-1 / 0.018 µmol mg-1= 78.9 s-1. The kcat value should be corrected if Figure 6 is correct as shown (however this may not be so; see point 7).

 

Response 8: Thank you for your advice. We recalculated and corrected Kcat according to the method you provided. Kcat for purified was found to be 84.94 /s in line 362.

 

Point 9: The catalytic efficiency (kcat/Km) calculated from a Km value expressed in mg substrate/mL (i.e. not in molar units) is meaningless. Delete this datum (Line 360) or, if possible, compare it with values obtained with other endoglucanases.

 

Response 9: Thank you for your advice. We have deleted catalytic efficiency (kcat/Km).

 

Point 10:  - Line 18. The sentence within this line is incomprehensible.

 

Response 10: Thank you for your advice. In this study, we successfully overexpressed endoglucanase (MtEG5-1) from M. thermophila in the methylotrophic yeast Pichia pastoris GS115 through electroporation in lines 18-20.

 

Point 11:  - Line 23. Delete “smaller” or specify “smaller than…” what.

 

Response 11: Thank you for your advice. We have deleted smaller in line 23.

 

Point 12:  - Line 73. Change “Thus,” to “In contrast,”.

 

Response 12: Thank you for your advice. We have changed “Thus,” to “In contrast,” in line 73.

 

Point 13:  - Line 75-77. This sentence is incomprehensible. The manuscript does not describe “the enzymatic properties of… …expression and secretion”.

 

Response 13: Thank you for your advice. I have rewritten this sentence. In this study, we used the enzyme obtained from M. thermophila and analyzed some enzymatic properties of MtEG5-1 overexpressed in P. pastoris GS115 in line 75-77.

 

Point 14:  - Line 154. Delete “per milliliter”. The definition of enzyme unit does not depend on the volume of the sample.

 

Response 14: Thank you for your advice. We have deleted “per milliliter” in line 154.

 

Point 15:  - Line 227. “we have named the enzyme … … is named”; rewrite this sentence.

 

Response 15: Thank you for your advice. We have rewritten this sentence. The enzyme expressed by mtEG5 gene in P. pastoris GS115 was named MtEG5-1 in this study in line 226-227.

 

Point 16:  - Lines 262-265. This sentence is incomprehensible.

 

Response 16: Thank you for your advice. I have rewritten this sentence. The protein bands were clearly visible from day 1 to day 7, indicating that MtEG5-1 had good stability (Figure 3c). The concentration of MtEG5-1 was 0.95 g/L on day 1 and the highest concentration was observed on day 5 at 1.15 g/L in lines 262-266.

 

 

Point 17:  - Line 292. The highest relative activity… was the highest…”. Rewrite this sentence.

 

Response 17: Thank you for your advice. We have rewritten this sentence. The recombinant enzyme exhibited a maximum activity at an optimum pH of 7 and an optimum temperature of 70 °C as shown in Figure 5b, c in lines 293-294.

 

Point 18:  - Line 310. Delete “()”.

 

Response 18: Thank you for your advice. We have deleted “()”.

 

Point 19:  - Line 326. “…temperature and pH of the MtEG5-1..”. This sentence is understandable but incorrect. Insert the calificative “optimal” before “temperature and pH”.

 

Response 19: Thank you for your advice. We have revised this sentence again. Notably, although there was not significantly different in the optimal temperature and pH of the MtEG5-1 expression in the three hosts, MtEG5-1 exhibited greater tolerance to high temperature than the enzyme expressed by P. pastoris X33 and A. niger in lines 329-332.

 

Point 20:  - Line 332. Change “Collectively,” to “Altogether,”.

 

Response 20: Thank you for your advice. We have changed “Collectively,” to “Altogether,” in line 335.

 

Point 21:  - Line 363-364. This sentence is a truism. It means just that MtEG5-1 is an endoglucanase. This is shown by other experiments in the manuscript and by the literature, it is not inferred from the mentioned kinetic properties.

 

Response 21: Thank you for your advice. We have deleted this sentence.

 

 

Author Response File: Author Response.pdf