Proteolysis of Micellar β-Casein by Trypsin: Secondary Structure Characterization and Kinetic Modeling at Different Enzyme Concentrations
, Burçin Dersu Açıkgöz 2, Günnur Güler 3,4, Andrey V. Golovanov 1 and Olga V. Sinitsyna 1Round 1
Reviewer 1 Report
Summary: beta-casein spontaneously forms micelles. Upon digestion with trypsin the micelles rearrange to nanoparticles before they fall apart into tryptic peptides. Fourier transform-infrared spectroscopy was used to measure the relative concentrations of beta-sheets, alpha-helices and hydrolysis products as a function of trypsin concentration and digestion time. Rate equations were formulated to describe three successive stages of changes in the secondary structure of casein during trypsin digestion. The results may have application to understanding how milk is transformed to curd.
Minor comments:
1. Figure 1 legend. It is suggested to write the legend as follows. Images of beta-casein nanoparticles digested with trypsin for 90 min, followed by heat inactivation of trypsin activity (a) or inaction of trypsin activity with soybean trypsin inhibitor (b).
2. Trypsin is inactivated when trypsin is stored at neutral pH because trypsin digests trypsin. I wonder why the authors do not worry about auto-hydrolysis of trypsin when they equilibrate trypsin in 20 mM potassium phosphate buffer pD 7.9 overnight at 4ËšC.
3. An interesting observation is reported, namely that trypsin digestion produces aggregates in addition to peptides. If aggregates form during trypsin digestion of most proteins, it would explain why digests often plug the extremely narrow tubing in liquid chromatography tandem mass spectrometry instruments.
Author Response
Reply to reviewer 1:
I would like to thank the reviewer for a very careful consideration of the manuscript.
Reviewer 1: 1. Figure 1 legend. It is suggested to write the legend as follows. Images of beta-casein nanoparticles digested with trypsin for 90 min, followed by heat inactivation of trypsin activity (a) or inaction of trypsin activity with soybean trypsin inhibitor (b).
Reply: This wording has been used.
Reviewer 1: 2. Trypsin is inactivated when trypsin is stored at neutral pH because trypsin digests trypsin. I wonder why the authors do not worry about auto-hydrolysis of trypsin when they equilibrate trypsin in 20 mM potassium phosphate buffer pD 7.9 overnight at 4ËšC.
Reply: According to our data, trypsin activity does not change. This is due to the rather low temperature and the fact that trypsin itself is not a sufficiently specific substrate, in contrast to low molecular weight amides or esters containing Arg or Lys residues.
Reviewer 1: 3. An interesting observation is reported, namely that trypsin digestion produces aggregates in addition to peptides. If aggregates form during trypsin digestion of most proteins, it would explain why digests often plug the extremely narrow tubing in liquid chromatography tandem mass spectrometry instruments.
Reply: We are grateful to the reviewer for this idea. We will definitely use it in the next article. An experiment is needed to substantiate this in detail.
Reviewer 2 Report
In the manuscript entitled “Proteolysis of micellar β-casein by trypsin: secondary structure characterization and kinetic modeling at different enzyme concentrations” the authors studied Tryptic proteolysis of protein micelles using β-casein (β-CN) as an example. The characterizations are well represented. But I am not an expert to decide about the novelty of this work. Though the scientific soundness is good enough to publish in this journal. Though I have some minor suggestions:
1. All the figures can be presented in a better way.
2. The introduction part can be rewritten and improved.
Author Response
Reply to reviewer 2:
I would like to thank the reviewer for a very careful consideration of the manuscript.
Reviewer 2: 1. All the figures can be presented in a better way.
Reply: The figures are redone and presented in a good way.
Reviewer 2: 2. The introduction part can be rewritten and improved.
Reply: The introductory part has been partially rewritten and improved. The following phrases were inserted:
In addition to being used as food, the various nanoforms of milk casein are promising for the targeted drug delivery and tissue engineering [11].
For this, the hydrolysis of one key peptide bond of k-casein, which destabilizes the milk casein micelle, should be considered, while the hydrolysis of other peptide bonds can be neglected.
The purpose of the current study was to investigate the proteolysis of b-CN by trypsin and to figure out the nanoparticle rearrangement and the changes in protein secondary structure by using AFM, FTIR and the methods of chemical kinetics.
Reviewer 3 Report
The manuscript contains interesting information, and it is well-written. However, the discussion is poor. In addition, the Conclusion can be improved. Authors mainly discuss their results without contrasting with other authors' results. Cited references are very old; few are from the last ten years.
Author Response
Reply to reviewer 3:
I would like to thank the reviewer for a very careful consideration of the manuscript.
Reviewer 3: The manuscript contains interesting information, and it is well-written. However, the discussion is poor. In addition, the Conclusion can be improved. Authors mainly discuss their results without contrasting with other authors' results. Cited references are very old; few are from the last ten years.
Reply: The following phrases were inserted in the discussion part:
An assumption also was made in which intermediate nanoparticles the secondary structure of the protein is retained, and in which it decreases.
When constructing the model, it was assumed that X particles have a smaller proportion of secondary structures as a result of the hydrolysis of b-CN polypeptide chains, similarly, for example, to thermal denaturation, which leads to a decrease in b-structures of self-assembling amphiphilic peptides [41]. We also used the assumption that the new particles Y have not a reduced, but the same fraction of b-sheets as the original micelles S.
A similar effect was observed in the current study, with the difference that we did not consider the intact protein, but a fraction of the intermediate component Y that was formed during proteolysis. In contrast to the generally accepted ideas about proteolysis, the proteolysis of b-CN by trypsin does not seem to be just a monotonous degradation of the secondary structure. It is important to note that this occurs at the low rates of peptide bond hydrolysis at S0/E0=10000 or 4000.
It has been established that various peptide bonds in β-CN are hydrolyzed by trypsin with different rates, and the quantitative methods for the measurement of the corresponding kinetic parameters have been proposed [19,44]. However, using various hydrolysis rate constants would lead to overly complex equations containing these parameters. Therefore, in this work, only two hydrolysis rate constants k1 and k3 were used. During hydrolysis with trypsin, the hydrophobic regions of the polypeptide chain of β-CN are not intensively hydrolyzed [19,24]. Our simple model takes into account the possibility of the formation of new nanoparticles based on the preserved hydrophobic centers. In this way, the model takes into account the specificity of the action of trypsin. In a more complex model, it is necessary to describe the kinetics of cleavage of the polypeptide chain regions capable of providing self-assembly processes and to use much more hydrolysis rate constants.
We have added the following 6 “fresh” articles, mostly published in 2022:
Khatun, S.; Appidi, T.; Rengan, A.K. Casein nanoformulations - Potential biomaterials in theranostics. Food Bioscience 2022, 50, 10220, doi:10.1016/j.fbio.2022.102200.
Zhu, Z.; Bassey, A.P.; Cao, Y.; Ma, Y.; Huang, M.; Yang, H. Food protein aggregation and its application. Food Res. Int. 2022, 160, 111725, doi:10.1016/j.foodres.2022.111725.
Melikishvili, S.; Dizon, M., Hianik, T. Application of high-resolution ultrasonic spectroscopy for real-time monitoring of trypsin activity in β-casein solution. Food Chem. 2021, 337, 127759, doi:10.1016/j.foodchem.2020.127759.
Buckin, V.; Altas, M.C. Ultrasonic monitoring of biocatalysis in solutions and complex dispersions. Catalysts 2017, 7, 336, doi:10.3390/catal7110336.
Genové, E.; Betriu, N.; Semino, C.E. β-Sheet to random coil transition in self-assembling peptide scaffolds promotes proteolytic degradation. Biomolecules 2022, 12, 411, doi:10.3390/biom12030411.
Deng, Y.; van der Veer, F.; Sforza, S.; Gruppen, H.; Wierenga, P.A. Towards predicting protein hydrolysis by bovine trypsin. Process Biochem. 2018, 65, 81-92, doi:10.1016/j.procbio.2017.11.006.
