The Immobilization and Stabilization of Trypsin from the Porcine Pancreas on Chitosan and Its Catalytic Performance in Protein Hydrolysis

Round 1

Reviewer 1 Report

The authors in this paper title ‘Immobilization and stabilization of trypsin from porcine pancreas on chitosan and its catalytic performance in protein hydrolysis’ highlighted the use of immobilization of trypsin by glutaraldehyde and glycine. The created trypsin has shown to better in absorption, stability and bioactivity. The paper is well written and provides a new ways to immobilize and functionalize trypsin. However, there are some concerns need to be addressed.

 

1.       Please show a pictorial representation of trypsin structure highlighting the catalytic residues.

2.       The introduction section is way too lengthy. Please shorten it in 3-4 paragraphs.

3.       In the result and discussion section 2.1 Influence of the solution pH…… It looks like the paper is written in thesis mode and need to be cut down. For example when you provide table, you don’t need to explain it in the text, maximally you summarizes it 3-4 sentences. The writing is way explanatory and must be shorten to engage the reader.

4.       What is the influence of temperature on various immobilizations like on Chit, Chit-GA and Chit-GA-Gly. They compare soluble and Chit-GA-Gly, but other would be helpful to evaluate it stability.

5.       The same control Chit and Chit-GA need to be added for BSA hydrolysis part.

NA

Author Response

The authors in this paper title ‘Immobilization and stabilization of trypsin from porcine pancreas on chitosan and its catalytic performance in protein hydrolysis’ highlighted the use of immobilization of trypsin by glutaraldehyde and glycine. The created trypsin has shown to better in absorption, stability and bioactivity. The paper is well written and provides a new ways to immobilize and functionalize trypsin. However, there are some concerns need to be addressed.

 

1. Please show a pictorial representation of trypsin structure highlighting the catalytic residues.

 

The reviewer is correct. We have added a pictorial representation of the structure of trypsin highlighting the catalytic residues, as suggested. See Figure 1 in the revised version (Page 2).

 

2. The introduction section is way too lengthy. Please shorten it in 3-4 paragraphs.

 

The reviewer is correct. The introduction was shortened from 6 to 4 paragraphs in the revised version, as suggested. See Pages 1,2, and 3.

 

3. In the result and discussion section 2.1 Influence of the solution pH…… It looks like the paper is written in thesis mode and need to be cut down. For example when you provide table, you don’t need to explain it in the text, maximally you summarizes it 3-4 sentences. The writing is way explanatory and must be shorten to engage the reader.

 

The reviewer is correct. The discussion section 2.1. was also shortened in the revised version, as suggested. However, the discussion on the possible interactions between enzyme and support were maintained for a better elucidation of the immobilization process. See comments in Pages 3,4, and 5.

 

4. What is the influence of temperature on various immobilizations like on Chit, Chit-GA and Chit-GA-Gly. They compare soluble and Chit-GA-Gly, but other would be helpful to evaluate it stability.

 

The reviewer is correct. However, these two supports (Chit and Chit–GA supports) were excluded from subsequent studies due to the superior catalytic performance of the biocatalyst prepared using Chit–GA–Gly. We demonstrated its promising application as an alternative support in Section 2.1. (See 2nd paragraph in Page 5). Moreover, its use as support in the immobilization and stabilization of proteases has not been reported in the literature yet.

 

5. The same control Chit and Chit-GA need to be added for BSA hydrolysis part.

 

The reviewer is correct. However, these two supports (Chit and Chit–GA supports) were excluded from subsequent studies due to the superior catalytic performance of the biocatalyst prepared using Chit–GA–Gly. This study also demonstrated that the biocatayst prepared using the classical Chit–GA support exhibited strong diffusional limitations in the hydrolysis of N_α-Benzoyl-DL-arginine p-nitroanilide hydrochloride (BAPNA), a small substrate molecule with a molecular mass of 434.88 g/mol. Therefore, its use as support could strongly restrict the access of large substrates such as bovine serum albumin – BSA (molecular mass of 66,500 g/mol) to the active sites of the enzyme preferentially immobilized in its internal surface. These results show that this new support (Chit–GA–Gly) results in the preparation of an active biocatalyst to be used in a broad spectrum of substrates, including various proteins.

Thanks for your contributions to greatly the quality of the manuscript.

Author Response File: Author Response.docx

Reviewer 2 Report

This manuscript entitled "Immobilization and stabilization of trypsin from porcine pancreas on chitosan and its catalytic performance in protein hydrolysis" reports novel and interesting data.

Nevertheless, authors should revise their manuscript appropriately. In more details, the design of the research is not satisfactory; authors, in their future works should apply appropriate experimental designs in order to reach to clearly errorless results.

Furthermore, the described methods are not followed well; authors should rewrite their methods, after a carefull reading, so that to be more comprenhensive to the readers.

 In addition to the abovementioned comments and suggestions, authors should reorganize their experimental work as far as it concerns the profiles of pH, temperature and the thermal inactivation curves.

To be more presice, I cannot welcome works dealing with enzymes, where their results depend upon unacceptable (and non-existed) "entities", i.e., the "Relative activity (%)" and/or "Relative activity" (ref. to Figs 2 and 3). These "entities" are meaningless, because do not correspond to any one of the formal Michaelis-Menten parameters, i.e., k_cat (or V_max), K_m, k_cat/K_m (or V_max/K_m). Therfore, authors should (firstly) re-examine their experimental work and inspect if the used substrate's concentrations ([S]) in the previously mentioned experiments correspond either to [S]<<K_m or to  [S]>>K_m. In the former case, their mesurements will correspond to the k_cat/K_m (or V_max/K_m) parameter, whereas in the later case will correspond to the k_cat (or V_max) parameter. If any of the previous conditions is satisfied, authors should declare it clearly in the text and on the figures; if not, authors should repeat these experiments as it is suggested.

I would suggest to authors to try to receive reults for both of the aforementioned conditions, in odere to report an ultimate work.

 Overall: Although this manuscript under consideration reports novel an interting data, however, authors should procced to a minor revision, according to my suggestions, before its publication in the Journal Catalysts.

A moderate editing of English language is required.

Author Response

This manuscript entitled "Immobilization and stabilization of trypsin from porcine pancreas on chitosan and its catalytic performance in protein hydrolysis" reports novel and interesting data.

 

Nevertheless, authors should revise their manuscript appropriately. In more details, the design of the research is not satisfactory; authors, in their future works should apply appropriate experimental designs in order to reach to clearly errorless results.

 

We beg to respectfully differ with the Reviewer. The objective of this study was to design a heterofunctional support derived from chitosan gel (Chit–GA–Gly) for immobilizing trypsin from porcine pancreas. The catalytic performance of this biocatalyst was compared with soluble and immobilized enzyme using classical chitosan-based supports (Chit and Chit–GA supports). We agree that the application of experimental designs could be conducted in further studies, however for optimizing the hydrolysis conditions for the production of protein hydrolysates from several sources, inclusing low-cost agroindustrial wastes.

 

Furthermore, the described methods are not followed well; authors should rewrite their methods, after a carefull reading, so that to be more comprenhensive to the readers.

 

The reviewer is correct. The methodology was rewritten and revised in several parts to be more comprenhensive to the readers. The corrections are highlighted in yellow in Section 3 in the revised version (see Pages 11, 12 and 13).

 

In addition to the abovementioned comments and suggestions, authors should reorganize their experimental work as far as it concerns the profiles of pH, temperature and the thermal inactivation curves. To be more presice, I cannot welcome works dealing with enzymes, where their results depend upon unacceptable (and non-existed) "entities", i.e., the "Relative activity (%)" and/or "Relative activity" (ref. to Figs 2 and 3). These "entities" are meaningless, because do not correspond to any one of the formal Michaelis-Menten parameters, i.e., k_cat (or V_max), K_m, k_cat/K_m (or V_max/K_m). Therfore, authors should (firstly) re-examine their experimental work and inspect if the used substrate's concentrations ([S]) in the previously mentioned experiments correspond either to [S]<<K_m or to  [S]>>K_m. In the former case, their mesurements will correspond to the k_cat/K_m (or V_max/K_m) parameter, whereas in the later case will correspond to the k_cat (or V_max) parameter. If any of the previous conditions is satisfied, authors should declare it clearly in the text and on the figures; if not, authors should repeat these experiments as it is suggested. I would suggest to authors to try to receive reults for both of the aforementioned conditions, in odere to report an ultimate work.

 

The reviewer is correct. The catalytic performance of enzymes can be expressed by formal Michaelis-Menten parameters. However, the results from the influence of certain parameters on the catalytic acticity of enzymes have also been extensively expressed in terms of relative activity. This can be confirmed by several recent studies described in the literature. In this study, maximum activity for both (expressed in nmol.min^-1.mL^-1 for soluble enzyme and nmol.min^-1.g^-1 for immobilized enzyme) was measured as 100% (Fig. 2) or 1 (Fig. 3). These experimental values were reported in each Figure legend. Free trypsin presented a maximum activity of 86.6 nmol.min^-1.mL^-1 (100% relative activity), and anything below that would be proportional to 100%. The same concept was also used for the immobilized trypsin. This information has been reported in Figures 2 and 3 (see legends).

 

Moreover, the non-linear exponential decay model proposed by Sadana and Henley (1987) utilized in this study for determining inactivation parameters of the enzyme (inactivation constant (k_d) and half-lives (t_1/2)) and, thus, apparent thermodynamic parameters requires express the catalytic activity values in terms of relative activity (a_r), as shown in Equation (7). This classical non-linear exponential decay model has also been been extensively applied to experimental results for inactivation studies of industrial enzymes.

 

Overall: Although this manuscript under consideration reports novel an interting data, however, authors should procced to a minor revision, according to my suggestions, before its publication in the Journal Catalysts.

 

The corrections were made as above described in order to improve the quality of the manuscript, as above described.

 

Comments on the Quality of English Language

A moderate editing of English language is required.

 

Sorry for these mistakes. The reviewer is correct. The manuscript was carefully revised in order to avoid grammatical errors.

Thanks for your contributions to greatly the quality of the manuscript.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

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