Functional Study of Different Lignocellulases from Trichoderma guizhouence NJAU4742 in the Synergistic Degradation of Natural Straw

Round 1

Reviewer 1 Report

In this study, the authors describe a characterization of the hydrolytic enzymes (cellulases and hemicellulases) of the filamentous fungus Trichoderma guizhouence NJAU4742, produced heterologously, and then their application in the treatment of lignocellulosic substrates. Although the topic addressed is of interest due to the potential application of hydrolytic enzymes in biomass treatment, the manuscript needs revision in its current form.

The Introduction is too general and does not focus on the topic of interest, which is fungal cellulase and hemicellulase enzymes, the advantages of heterologous production of these enzymes using expression vectors, and the synergistic mode of action of these enzymes in the degradation of lignocellulosic substrates. There is also no clear justification in the introduction for the use of enzymes from T. guizhouence NJAU4742. Why was this species/strain chosen? Is there any previous information to support its use in this study? This information should be provided here.

The Materials & Methods section lacks relevant methodological information, making some assays difficult to understand. The assays to characterize enzyme properties are not clear. At what temperature was the optimal pH determined? And at what pH was the optimal temperature determined? It is stated that acetate and Tris-HCl buffers were used, but other buffer solutions appear in the results (Fig. 2d-f). How was the metal ion test conducted? At what time and at what concentration were the metal ions applied? Were they all used at the same concentration? At what pH and temperature were these tests performed? What pH and temperature conditions were used to determine the kinetic parameters Vmax and K_M for each enzyme?  For enzymatic treatments of the lignocellulosic substrates, the pH, temperature, incubation time, and agitated or static conditions under which the treatments were performed must be reported. What was the enzyme dosage applied? All of these data should be included in the methodology as they make it easier to understand the tests and the results shown in Fig. 3. In what proportion or concentration were the lignocellulosic substrates used? What was the particle size of the lignocellulosic substrates?    

The Results & Discussion has a good description of the results, but the discussion has weaknesses. What are the pH values and optimum temperature of these enzymes compared to the same enzymes from other Trichoderma species? How do the authors explain the different effect of metal ions on the activity of EGL, BGL, and XYN? What explains the dramatic effect of Fe and Cu on BGL and XYN? What does CK in Fig. 2g-i mean? This must be described in the figure legend.  The results of the kinetic parameters (Vmax and K_M) of the enzymes would be better organized in a table for easy visualization.  In this regard, there is no clarity in the concentration units used to express Vmax and K_M values; some are expressed in µg.mL^-1.min^-1 and others in µmol.mL^-1.min^-1. These should be expressed in the same units, preferably in µmol.mL^-1.min^-1. The paragraph on lines 246-248 states that the results of the kinetic parameters suggest that the enzymes have a potential for practical applications. This is a very ambiguous sentence. Please revise this statement.

Regarding the effects of enzymes on lignocellulosic substrates, the use of the enzyme doses given in the legend to Fig. 3 is unclear. What does 0.1M XYN mean? A molar (mol. L^-1) concentration of the XYN enzyme? How were these enzyme doses determined? This is not explained in the methodology, nor is how the different proportions of each enzyme combination were chosen. The pH and temperature chosen for these tests are important because each enzyme has a different optimum pH and temperature according to the results shown in this manuscript. This is not discussed here. On the other hand, could adsorption of the enzymes on the lignocellulosic substrates have occurred and could this have affected the action of the enzymes on the substrates?

When evaluating functional groups, crystallinity, and surface changes by SEM on lignocellulosic substrates after enzymatic treatment, there is no indication of the ratio of EGL, BGL, and XYN enzymes used and why only this combination was chosen for these analyses. Is the combination of the three enzymes EGL + BGL + XYN responsible for the changes observed in Fig. 4-6? Could the combination of only two enzymes EGL + XYN or EGL + BGL produce the same effects? Were these tests not performed? This is not addressed here.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript titled "Functional study of different lignocellulases from Trichoderma guizhouence NJAU4742 in the synergistic degradation of natural straw" describes the heterologous expression in yeast and enzymatic characterisation of three enzymes (endoglucanase, B-glucosidase and xylanase) from Trichoderma guizhouence , with the end purpose of processing wheat straw towards valuable end products such as biofuels. The manuscript shows promise and the current results can serve as valuable foundational theory for further practical developments. 

Here are line-by-line comments that will lead to the improvement of the current version of the manuscript:

Line 5: the words in the name of the institution should be capitalized

Line 21: What does "CK" mean?

In fact, there are many acronyms used in the manuscript without being defined at the time of first occurrence. Please define your acronyms. You may include a list of abbreviations for quick referral by readers.

Keywords: "Enzymatic characteristics" is too vague; list as keywords the exact characteristics that were probed here

Line 33: is (not "are")

Materials and methods: there should be a first subsection about all chemicals, buffers and kits used, including purity or grade and vendor (e.g. CMC-Na, PDA, methanol, Tris, NaCl, etc. - all chemicals in all protocols)

Line 85: provide reference to the website where the manufacturer's instructions can be retrieved from (including date accessed)

Line 90: 170 rpm corresponds to what instrumentation/device? Describe it, type/brand/manufacturer

Line 92: indicate type of centrifuge, manufacturer and its g-force (as well as rpm speed used for separation)

Line 121: the nature of ions is not enough; indicate also the concentration(s) that was/were used for each type of ion added deliberately

Line 137: Why did you leave out the pairing BGL & XYN when studying synergism? What is your rationale for this choice?

Line 174: Do not refer vaguely to Supplementary Materials, be specific and refer to exact relevant figures from there (figs. S1-S3)

Figure 2 shows relative enzymatic activities on the y-axis. Relative to what?

Line 215: "previous" (spelling issue); "Consistent with" (cross out "In" as it does not belong in this context)

Section 3.2. Indicate the specific figure, not just "Supplementary Materials". The figure is the current figure S6, but since this section precedes section 3.3 about synergism, in fact it should be renumbered as figure S4 and the synergism supplementary figures should follow in sequence

Line 241: According to the Michaelis-Menten equation shown in figure S6-b, and the units on the x-axis, I conclude that 0.22 is a molar concentration, so the text on this line should not read 0.22 micrograms per milliliter. Please correct either the figure or text, as it is not clear which one is wrong.

Section 3.3: once again, indicate the exact supplementary figures, don't just refer to Supplementary Materials at large

Conclusions: Add a phrase about the future prospects and potential industrial applications of the findings of this work

References: Journal names should be abbreviated and DOI numbers should be added.

The level of English is good, in general. A few misspellings and plural noun-singular verb incorrect pairings, otherwise it is fine.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

In this version of the manuscript, the authors have incorporated most of my observations. They have also answered most of my questions and doubts.