Immobilization and Kinetic Properties of ß-N-Acetylhexosaminidase from Penicillium oxalicum
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsGeneral comment: The study is well conceived and executed, and most of my comments concern the methodology part where additional details need to be included to improve clarity and ensure the reproducibility of the experiments. These comments are listed below.
Specific comments:
87 – please include means of isolation of the enzyme and the respective conditions.
91 – at which temperature was soluble Hex stored?
92 – I suggest adding a sentence that the enzyme concentration was determined using the Bradford method. Since the authors identified conditions providing long-term storage stability (6 months without activity loss), it is also very important to include here the information on enzyme concentration in the stock solution, as protein concentration can affect storage stability.
102 – the enzyme was stored in (NH4)2SO4, but it is mentioned here that immobilization was carried out by mixing the Purolite matrix with 5 mL of a solution containing 0.5 mg/mL enzyme in phosphate buffer (pH 7.0, 100 mM). Was the buffer exchanged prior to immobilization, or was the enzyme stored at a higher concentration in (NH4)2SO4 and then diluted to 0.5 mg/mL in phosphate buffer?
110 – the reaction scheme of the activity assay should be included for the clarity
114 – did the authors ensure that no more than 10% of the reaction mixture was sampled? Sampling up to 10% is a common rule of thumb to maintain the representativeness of the reaction mixture during the experiment.
124 – it would be helpful to express the column volume, not just the dimensions, as this would provide a clearer understanding of how much of the column was filled with IME particles. Alternatively, the IME filling could be expressed as a percentage.
144 – if the IME filling in the same amount was used as in section 2.5.1, it would be helpful to repeat this here for clarity. If it differs, please provide details on the column filling.
183 – I assume that crushed enzyme particles were sampled as well when sampling the reaction mixture. Although 50 µL of samples were diluted in 3 mL, I wonder if these particles interfered with the spectrophotometric assay. Were the samples filtered or centrifuged prior to measurement, or did the particles not impact the spectrophotometric assay? If additional steps were taken (filtration, centrifugation), please include them here.
219 – the term “reactant” could be interpreted as a substrate, I suppose it would be better to use the term “compound” in this context, as it can refer to both the substrate and the product of the reaction, as stated in the sentence that follows
223 – the index “b” should be defined somewhere in the text as bulk
225 – I suppose it should be written “when the substrate bulk concentration csb is cs0…”, instead of “cs0 is cs0”
245 – how come the authors did not calculate specific enzyme activities using the method of initial reaction rates (with conversions below 10%)? Since the data for six experiments are already shown in Figure 3, this approach would allow them to plot the dependence of specific enzyme activities on substrate concentrations, making it easier to detect substrate inhibition from the curve trend. While this method would not reveal product inhibition (which would require a separate set of experiments with varying product concentrations), it would allow for the estimation of the parameters from independent kinetic measurements for the substrate. The same comment applies to the results with crushed and intact enzyme particles shown in Figure 4 and Figure 5, respectively.
Figure 3 – the initial substrate concentrations should be included in the figure title, consistent with Figures 4 and 5. Also, Figure 3 could be depicted in the same way as overlapping graphs, i.e. with multiple lines on a single graph, or alternatively, the figures could be labelled (a-f) with the corresponding concentrations listed for each figure.
274 – I believe that the crushing of the immobilized enzyme particles can negatively affect enzyme activity due to the mechanical force applied. Could the authors provide some comments or insights on this?
Figure 6 – please use decimal points instead of commas on the y-axis. Additionally, the figure appears to be of low resolution, so please replace it with a higher-quality version.
Author Response
The response is in attached file.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsComments to the Authors and Editors:
This manuscript systematically focused on the kinetic properties of free and immobilized ß-N-acetylhexosaminidase. And offering promising method for application of Hex in the field of biotechnology, especially for the synthesis of bioactive carbohydrates. It’s an interesting work. But there are some concerns should be explained or offered some new evidence data. Thus, in my opinion, this manuscript should be revised before publishing by Catalysts.
Detailed comments are listed below.
1. The reaction conditions of continuous flow reactors do not see the optimization process in the article, so how do you determine such conditions? Does the dynamic performance change after changing the conditions?
2. Whether the immobilization results require visual characterization data, such as laser confocal microscopy. These articles may help you and can be chosen as references:
10.1016/j.carbpol.2023.121322;
10.1016/S1872-2067(24)60020-3;
10.1016/j.ijbiomac.2024.130381.
3. Many of the references are very old articles. It is not that these papers are not academically convincing, on the contrary, more literature in recent years is needed to illustrate the research significance of this work.
Author Response
The response is in attached file.
Author Response File: Author Response.pdf
