Synergism between Cutinase and Pectinase in the Hydrolysis of Cotton Fibers’ Cuticle

Round 1

Reviewer 1 Report

The manuscript describes efforts to examine the ability of cutinase, pectin lyase, and polygalacturonase to substitute for chemical treatment of raw cotton fibers to allow for improvements in the sustainability of cotton processing. The paper is thorough and well written, with clear illustration of result. Especially promising is the observation of the greater than additive effects of cutinase and pectin lyase in the hydrolysis of the cotton fibers’ cuticle.

My only critique is that the authors include chemical treatments in the manuscript (heating with NaOH or reflux in hot DCM), but they don't directly compare the results in terms of change in weight, wetting time, etc. Inclusion of these studies would help the reader assess the relative utility of the enzymatic processing as compared to standard chemical treatments, and also how much additional progress is needed to have a commercially viable chemical alternative for cotton treatment. 

Author Response

Responses to Reviewer 1’s comments

I thank the reviewer for investing substantial efforts, which are undoubtedly contributing to this manuscript. The remarks and suggestions improved this paper’s scientific soundness and accurateness. Your contribution is greatly appreciated.

General comments:

My only critique is that the authors include chemical treatments in the manuscript (heating with NaOH or reflux in hot DCM), but they don’t directly compare the results in terms of change in weight, wetting time, etc. Inclusion of these studies would help the reader assess the relative utility of the enzymatic processing as compared to standard chemical treatments, and also how much additional progress is needed to have a commercially viable chemical alternative for cotton treatment.

The reviewer is right; this issue should be clarified and a comparison to conventional hot NaOH alkaline scouring, common in the textile industry, should be discussed in detail.

Traditional chemical scouring of cotton fibers aims at removing the hydrophobic and non-cellulosic components and producing highly absorbent fibers that can be dyed and finished uniformly. However, these chemicals also attack the cellulose, leading to a reduction in strength and loss of fabric weight. The effect of chemical scouring on the cotton fabrics’ quality (mechanical properties), weight, wetting time, cellulose exposure, color change, and readiness for dyeing and finishing processes carried out afterward is well described in the literature in former studies carried out by us [1,2] and others [3,4].

Specifically, in this study, the best wettability score of the treated cotton fabric, 10.5 s, in the polygalacturonase treatment or 24 s, achieved by the enzyme mixture of cutinase and pectinase, was still higher than that of 8 s by NaOH reported in other studies [3]. This wetting time was more similar to wettability values received by other studies carried out with enzymes. For example, wettability time was 16.5 s following Aspergillus aculeatus endo-polygalacturonase bioscouring [5], 13 s following bioscouring with α-amylase and polygalacturonase enzymes from Trichoderma harzianum, supplemented with a non-ionic surfactant (Egyptol, 1 g/L) [6], and 10 s obtained using a mixture of recombinant pectin methylesterase and pectate lyases from Clostridium thermocellum [3]. Thus, the wettability process’s effectiveness in this study using mixed enzymes may be considered acceptable compared to chemical treatment.

Still, it should be remembered that the source of the enzymes, their properties, and their titers used may influence the time and effectiveness of the bioscouring. For example, in our previous report, cutinase from Pseudomonas mandocino achieved a wetting time of 20-30 s after incubation for almost 20 h [1]. Agrawal et al. [7] presented a similar result obtained only after 30 min with Fusarium solani f. pisi cutinase. The combined effect of cutinase and pectate lyase in their study improved the above result even more, achieving almost the same degree of wax removal as solvent extraction (n-hexane for 30 min, 75°C) within 15 min.

The fabrics’ weight loss results presented here (0.004% in the mixture of cutinase and pectinase or 0.02% in the polygalacturonase treatment) are minor compared to the conventional NaOH treatment’s results described in the literature. According to Bristi et al. (2019, [4]), the cotton fabrics’ weight loss in the textile industry is around 8-12%, which means an excessive loss of fabric weight unnecessarily (the recommended weight loss for caustic scouring is 3-8%). In their experiments, caustic soda scouring resulted in a 6.2% weight loss, while enzymatic scouring with Scourzyme L. resulted in a 7.5% weight loss. This measure is also influenced by each experiment’s specific settings, i.e., incubation time, temperature, pH, enzyme type, and the titer used, which may later affect the weight loss result. For example, Rajulapati et al. (2020, [3]) reported that bioscouring with a mixture of recombinant pectin methylesterase and pectate lyases enzymes led to a weight loss of 17.3%, while the untreated cotton fabric (negative control) showed a weight loss of only 0.3%, and the positive control, NaOH treatment, showed a weight loss of 19.1%. Cutinase alone (12.8 U/ml) led to a 4% weight loss after 20 h of incubation, while the positive control, NaOH treatment, resulted in a 14% weight loss [1].

The above explanation was added to the Discussion (lines 433-470).

Also, The following paragraph in the Result section was edited (lines 386-396):

“The combination of these enzymes resulted in a 3.6-fold improvement in the fabrics’ water absorbency (from 87 s to 24 s). Moreover, this enzyme combination with a minute amount of cutinase (1.7 U instead of 80 U, which alone caused an insignificant change) increased the amount of pectin lyase (301 U instead of 200 U), and a shorter reaction time (4 h instead of 24 h) resulted in a two-fold improvement in scouring efficiency (Fig. 7B). However, this combination’s wettability time is still quite long (125 s) and may improve by a longer incubation time. The more intensifying enzymatic reaction (of the two enzymes combined) led to increased weight loss (0.02 mg with pectin lyase alone to 0.035 mg in the enzyme mixture) and lower residual cutinase activity (Fig. 7B, insert). The degradation products of this reaction extracted from the reaction fluid are shown in Figure 8. Thus, the results indicate a synergistic interaction between the two enzymes.”

References

1. Degani, O.; Gepstein, S.; Dosoretz, C.G. Potential use of cutinase in enzymatic scouring of cotton fiber cuticle. Applied Biochemistry and Biotechnology 2002, 102, 277-289.
2. Degani, O.; Gepstein, S.; Dosoretz, C.G. A new method for measuring scouring efficiency of natural fibers based on the cellulose-binding domain-β-glucuronidase fused protein. Journal of Biotechnology 2004, 107, 265-273.
3. Rajulapati, V.; Dhillon, A.; Kumar Gali, K.; Katiyar, V.; Goyal, A. Green bioprocess of degumming of jute fibers and bioscouring of cotton fabric by recombinant pectin methylesterase and pectate lyases from Clostridium thermocellum. Process Biochemistry 2020, 92, 93-104.
4. Bristi, U.; Pias, A.; Lavlu, F.J. A sustainable process by bioscouring for cotton knitted fabric suitable for next generation. J Textile Eng Fashion Technol 2019, 5, 41-48.
5. Abdulrachman, D.; Thongkred, P.; Kocharin, K.; Nakpathom, M.; Somboon, B.; Narumol, N.; Champreda, V.; Eurwilaichitr, L.; Suwanto, A.; Nimchua, T., et al. Heterologous expression of Aspergillus aculeatus endo-polygalacturonase in Pichia pastoris by high cell density fermentation and its application in textile scouring. BMC Biotechnology 2017, 17, 15.
6. Aly, A.S.; Sayed, S.M.; Zahran, M.J. One-step process for enzymatic desizing and bioscouring of cotton fabrics. Journal of Natural Fibers, 2010, 7, 71-92.
7. Agrawal, P.B.; Nierstrasz, V.A.; Bouwhuis, G.H.; Warmoeskerken, M.M.C.G.; Cutinase and pectinase in cotton bioscouring: an innovative and fast bioscouring process. Biocatalysis and Biotransformation 2008, 26, 412-421.

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript entitled “Synergism between cutinase and pectinase in the hydrolysis of cotton fibers’ cuticle” reported the cutinase and pectinase could combine to degrade cotton fibers’ cuticle out layer at low temperature in a more environmental-friendly way compared with the conventional NaOH bleaching method, which creates unwanted alkaline wastes. The results are clearly demonstrated. However, there are a few suggestions to the manuscript below:

1. In section 2.4, the esterase activity was estimated by the p-nitrophenyl butyrate (pNPB), however, as mentioned the method cannot distinguish between esterases and the cutinases. Can the author compare such a method with the ethyl butyrate serving as the substrate (refer to the link below) for estimating the esterase activity? https://www.sigmaaldrich.com/technical-documents/protocols/biology/enzymatic-assay-of-esterase.html
2. The figure quality of Figure 1 needs to improve. Please try to use a higher resolution of the picture or a different format to improve the resolution.

Otherwise, the manuscript is acceptable for publication.

Author Response

Responses to Reviewer 2’s comments

I would like to express my sincere appreciation to the reviewer for essential and helpful advice. The time and effort invested are greatly appreciated, and certainly contributed to the manuscript and improved it. Thank you.

Specific comments:

1. In section 2.4, the esterase activity was estimated by the p-nitrophenyl butyrate (pNPB), however, as mentioned the method cannot distinguish between esterases and the cutinases. Can the author compare such a method with the ethyl butyrate serving as the substrate (refer to the link below) for estimating the esterase activity?

https://www.sigmaaldrich.com/technical-documents/protocols/biology/enzymatic-assay-of-esterase.html

Thank you for this question; this is indeed a topic that should be explained.

Cutinases hydrolyze the plant leaf cuticle by cleaving the ester bonds of cutin [8]. Therefore, cutinases belong to the esterase group, and more specifically, to a class of serine esterases that contain the catalytic triad (serine, histidine, and aspartate) with the active serine in the consensus sequence Gly-His/Tyr-Ser-X-Gly [9]. Conventionally, cutinase activity can be measured spectrophotometrically by following the absorbance change at 405 nm, which indicates the release of a yellow-colored p-nitrophenol (pNP) from p-nitrophenyl esters of short-chain fatty acids used as model substrates [8,10]. A very common substrate for this goal is the p-nitrophenyl butyrate.

An enzymatic determination of esterase activity can also be made using ethyl butyrate as a substrate, as suggested by Merck [11]. This procedure measures the butyric acid released followed the ester bond cleavage. The enzymatic reaction is conducted in a thermostated water bath using a pH meter together with a magnetic stirrer. After adding the reaction components, the reaction pH is maintained at 8.0 by adding 0.01N sodium hydroxide titrant (NaOH) approximately every 5 minutes. There should be approximately 10 additions of titrant during this time period; thus, the reaction is carried out for 50 minutes. The enzyme activity is the titration slope – the rate of consumption of a 0.1 ml aliquot of NaOH divided by the time.

Although this procedure is simple, it is time-consuming in comparison to the p-nitrophenyl esters spectrophotometric assay. Most reasonably, this is why the latest assay is so common for cutinase activity evaluation, while I couldn’t find any record of measuring cutinase activity using the ethyl butyrate titration technique.

More recently (compared to the two tests described above), our group developed a cutinase assay based on a specific cutinase substrate, 4-nitrophenyl (16-methyl sulfone ester) hexadecanoate (pNMSEH) [12,13]. However, this substrate is not commercially available; thus, the more accessible and fast p-nitrophenyl butyrate enzymatic assay was chosen for the current study.

The following explanation was added to the Materials and Methods section (lines 199-204):

“An enzymatic determination of esterase activity can also be made using ethyl butyrate as a substrate and the titration technique, as suggested earlier [11]. More recently, a cutinase assay based on a specific cutinase substrate, 4-nitrophenyl (16-methyl sulfone ester) hexadecanoate (pNMSEH) was developed [12,13]. Since the ethyl butyrate titration assay is time-consuming, and the pNMSEH-assay relies on a substrate that is not commercially available, the p-nitrophenyl butyrate spectrophotometric assay was chosen for this study.”

2. The figure quality of Figure 1 needs to improve. Please try to use a higher resolution of the picture or a different format to improve the resolution.

Figure 1 was replaced with a higher resolution image.

References

8. Kolattukudy, P.E.; Purdy, R.E.; Maiti, I.B. Cutinases from fungi and pollen. In Methods in Enzymology, John, M.L., Ed., Academic Press 1981, 71, 652-664.
9. Martinez, C.; Nicolas, A.; van Tilbeurgh, H.; Egloff, M.P.; Cudrey, C.; Verger, R.; Cambillau, C. Cutinase, a lipolytic enzyme with a preformed oxyanion hole. Biochemistry 1994, 33, 83-89.
10. Purdy, R.; Kolattukudy, P. Hydrolysis of plant cuticle by plant pathogens. Purification, amino acid composition, and molecular weight of two isoenzymes of cutinase and a nonspecific esterase from Fusarium solani f. Pisi. Biochemistry 1975, 14, 2824-2831.
11. Adler, A.J.; Kistiakowsky, G.B. Kinetics of pig liver esterase catalysis. Journal of the American Chemical Society 1962, 84, 695-703.
12. Degani, O. Production and purification of cutinase from fusarium oxysporum using modified growth media and a specific cutinase substrate. Advances in Bioscience and Biotechnology 2015, 6, 245-258.
13. Degani, O.; Salman, H.; Gepstein, S.; Dosoretz, C.G. Synthesis and characterization of a new cutinase substrate, 4-nitrophenyl (16-methyl sulfone ester) hexadecanoate. Journal of Biotechnology 2006, 121, 346-350.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The additional text and comparison between the chemical treatment and the enzymatic processing of the cotton nicely addresses my prior concern. I think the paper is now ready for publication.