Skip to main content
SpringerLink
Log in

Development and validation of a label-free method for measuring the collagen hydrolytic activity of protease

  • Research Paper
  • Published:
Bioprocess and Biosystems Engineering Aims and scope Submit manuscript

Abstract

Collagen is the most abundant fibrous structural protein, and therefore, the quantitative evaluation of the effect of protease on collagen has a profound influence on enzyme application. In this research, unlabeled native bovine hide powder was utilized to detect collagen hydrolytic activity of the protease. The optimum conditions of the determination method were as follows: 30 mg/mL substrate concentration, 30 min reaction time, and 2–9 U/mL enzyme concentration. Then, several typical industrial protease preparations were chosen to measure collagenolytic activities at different temperatures and pH values, whose change trends were quite distinct from those of proteolytic activity assay method based on casein or dye-labeled hide powder substrate. Especially, in the pH 5–7, casein hydrolytic activities of these proteases showed sharper peaks with relative activity from 6% to 100%, whereas, their collagen hydrolytic activities based on native hide powder exhibited 30–100% with broader peaks. And collagen hydrolytic activities resulted from using dye-labeled substrate reached a lower optimum pH value than that of other methods. Besides, the results of these measurements displayed a moderate degree of reproducibility. This method is more reasonable than the protease assay method using casein or labeled hide powder as the substrate in many fields.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  1. Hong H, Fan H, Chalamaiah M, Wu J (2019) Preparation of low-molecular-weight, collagen hydrolysates (peptides): current progress, challenges, and future perspectives. Food Chem 301:125222

    Article  CAS  PubMed  Google Scholar 

  2. Asaduzzaman AKM, Getachew AT, Cho Y, Park J, Haq M, Chun B (2020) Characterization of pepsin-solubilised collagen recovered from mackerel (Scomber japonicus) bone and skin using subcritical water hydrolysis. Int J Biol Macromol 148:1290–1297

    Article  CAS  PubMed  Google Scholar 

  3. Feng M, Betti M (2017) Transepithelial transport efficiency of bovine collagen hydrolysates in a human Caco-2 cell line model. Food Chem 224:242–250

    Article  CAS  PubMed  Google Scholar 

  4. Li J, Wang M, Qiao Y, Tian Y, Liu J, Qin S, Wu W (2018) Extraction and characterization of type I collagen from skin of tilapia (Oreochromis niloticus) and its potential application in biomedical scaffold material for tissue engineering. Process Biochem 74:156–163

    Article  CAS  Google Scholar 

  5. Liu X, Zheng C, Luo X, Wang X, Jiang H (2019) Recent advances of collagen-based biomaterials: Multi-hierarchical structure, modification and biomedical applications. Mater Sci Eng C 99:1509–1522

    Article  CAS  Google Scholar 

  6. Alemán A, Martínez-Alvarez O (2013) Marine collagen as a source of bioactive molecules. A review. Nat Prod J 3:105–114

    Google Scholar 

  7. Sugihara F, Inoue N, Kuwamori M, Taniguchi M (2012) Quantification of hydroxyprolyl-glycine (Hyp-Gly) in human blood after ingestion of collagen hydrolysate. J Biosci Bioeng 113:202–203

    Article  CAS  PubMed  Google Scholar 

  8. Sibilla S, Godfrey M, Brewer S, Budh-Raja A, Genovese L (2015) An Overview of the beneficial effects of hydrolysed collagen as a nutraceutical on skin properties: scientific background and clinical studies. T O Nutra J 8:29–42

    CAS  Google Scholar 

  9. Prow TW, Grice JE, Lin LL, Faye R, Butler M, Becker W, Wurm EM, Yoong C, Robertson TA, Soyer HP, Roberts MS (2011) Nanoparticles and microparticles for skin drug delivery. Adv Drug Deliv Rev 63:470–491

    Article  CAS  PubMed  Google Scholar 

  10. Anzani C, Álvarez C, Mullen AM (2020) Assessing the effect of Maillard reaction with dextran on the technofunctional properties of collagen-based peptides obtained from bovine hides. LWT Food Sci Technol 118:1–7

    Article  CAS  Google Scholar 

  11. Rýglová BM, Suchý T (2017) Collagen and its modifications—crucial aspects with concern to its processing and analysis. Macromol Mater Eng 302:1600460

    Article  CAS  Google Scholar 

  12. Paul T, Jana A, Mandal AK, Mandal A, Das Mohpatra PK, Mondal KC (2016) Bacterial keratinolytic protease, imminent starter for NextGen leather and detergent industries. Sustain Chem Pharm 3:8–22

    Article  CAS  Google Scholar 

  13. Sujitha P, Kavitha S, Shakilanishi S, Babu NKC, Shanthi C (2018) Enzymatic dehairing: a comprehensive review on the mechanistic aspects with emphasis on enzyme specificity. Int J Biol Macromol 118:168–179

    Article  CAS  PubMed  Google Scholar 

  14. Bhagwat PK, Dandge PB (2018) Collagen and collagenolytic proteases: a review. Biocatal Agric Biotechnol 15:43–55

    Article  Google Scholar 

  15. Wusigale LL, Luo Y (2020) Casein and pectin: structures, interactions, and applications. Trends Food Sci Tech 97:391–403

    Article  CAS  Google Scholar 

  16. Errasti ME, Caffini NO, López LMI (2018) Proteolytic extracts of three Bromeliaceae species as eco-compatible tools for leather industry. Environ Sci Pollut R 25:21459–21466

    Article  CAS  Google Scholar 

  17. Lopez L, Viana CA, Errasti ME, Garro ML, Martegani JE, Mazzilli GA, Freitas C, Araujo I, Da SR, Ramos MV (2017) Latex peptidases of Calotropis procera for dehairing of leather as an alternative to environmentally toxic sodium sulfide treatment. Bioprocess Biosyst Eng 40:1391–1398

    Article  CAS  PubMed  Google Scholar 

  18. Chavira R, Burnett TJ, Hageman JH (1984) Assaying proteinases with azocoll. Anal Biochem 136:446–450

    Article  CAS  PubMed  Google Scholar 

  19. Komsa-Penkova RS, Rashap RK, Yomtova VM (1997) Advantages of orange-labelled collagen and gelatine as substrates for rapid collagenase activity measurement. J Biochem Bioph Meth 34:237–249

    Article  CAS  Google Scholar 

  20. Foroughi F, Keshavarz T, Evans CS (2006) Specificities of proteases for use in leather manufacture. J Chem Technol Biot 81:257–261

    Article  CAS  Google Scholar 

  21. Wanderley MCDA, Neto JMWD, Filho JLDL, Lima CDA, Teixeira JAC, Porto ALF (2017) Collagenolytic enzymes produced by fungi: a systematic review. Braz J Microbiol 48:13–24

    Article  CAS  PubMed  Google Scholar 

  22. Zhang Y, Fu Y, Zhou S, Kang L, Li C (2013) A straightforward ninhydrin-based method for collagenase activity and inhibitor screening of collagenase using spectrophotometry. Anal Biochem 437:46–48

    Article  CAS  PubMed  Google Scholar 

  23. Hattori S, Fujisaki H, Kiriyama T, Yokoyama T, Irie S (2002) Real-time zymography and reverse zymography: a method for detecting activities of matrix metalloproteinases and their inhibitors using FITC-labeled collagen and casein as substrates. Anal Biochem 301:27–34

    Article  CAS  PubMed  Google Scholar 

  24. Krizkova S, Zitka O, Masarik M, Adam V, Stiborova M, Eckschlager T, Chavis GJ, Kizek R (2011) Assays for determination of matrix metalloproteinases and their activity. Trac Trend Anal Chem 30:1819–1832

    Article  CAS  Google Scholar 

  25. Pellegrini D, Corsi M, Bonanni M, Bianchini R, D’Ulivo A, Bramanti E (2015) Study of the interaction between collagen and naturalized and commercial dyes by Fourier transform infrared spectroscopy and thermogravimetric analysis. Dyes Pigments 116:65–73

    Article  CAS  Google Scholar 

  26. Singha NR, Chattopadhyay PK, Dutta A, Mahapatra M, Deb M (2019) Review on additives-based structure-property alterations in dyeing of collagenic matrices. J Mol Liq 293:111470

    Article  CAS  Google Scholar 

  27. Hoffmann C, Leroy-Dudal J, Patel S, Gallet O, Pauthe E (2008) Fluorescein isothiocyanate-labeled human plasma fibronectin in extracellular matrix remodeling. Anal Biochem 372:62–71

    Article  CAS  PubMed  Google Scholar 

  28. Dean DD Jr, Woessner JF (1985) A sensitive, specific assay for tissue collagenase using telopeptide-free [3 H]acetylated collagen. Anal Biochem 148:174–181

    Article  CAS  PubMed  Google Scholar 

  29. Cheng X, Wang Q, Fang H, Tang W, Xu W (2008) Synthesis of new sulfonyl pyrrolidine derivatives as matrix metalloproteinase inhibitors. Bioorgan Med Chem 16:7932–7938

    Article  CAS  Google Scholar 

  30. Tran LH, Nagano H (2002) Isolation and characteristics of Bacillus subtilis CN2 and its collagenase production. J Food Sci 67:1184–1187

    Article  CAS  Google Scholar 

  31. Ignat’Eva NY, Danilov NA, Averkiev SV, Obrezkova MV, Lunin VV, Sobol EN (2007) Determination of hydroxyproline in tissues and the evaluation of the collagen content of the tissues. J Anal Chem 62:51–57

    Article  CAS  Google Scholar 

  32. Li Y (2016) Effects of protease hydrolysis of elastin and collagen on enzymatic dehairing and the degree of action of hide, vol Doctor. Sichuan University Chengdu, China

  33. Reddy GK, Enwemeka CS (1996) A simplified method for the analysis of hydroxyproline in biological tissues. Clin Biochem 29:225–229

    Article  CAS  PubMed  Google Scholar 

  34. Da Silva CML, Spinelli E, Rodrigues SV (2015) Fast and sensitive collagen quantification by alkaline hydrolysis/hydroxyproline assay. Food Chem 173:619–623

    Article  PubMed  CAS  Google Scholar 

  35. Zhang X, Bu D, Tian Y, Xian J, Zhang C, Li Y, Li B (2019) Optimization of method for determination of hydroxyproline in enzymatic hydrolysate of collagen fibers. Leather Sci Eng 29:25–30

    Article  CAS  Google Scholar 

  36. Stegemann H, Stalder K (1967) Determination of hydroxyproline in tissue. Clin Chim Acta 18:267–273

    Article  CAS  PubMed  Google Scholar 

  37. Nethery A, Lyons JG, O’Grady RL (1986) A spectrophotometric collagenase assay. Anal Biochem 159:390–395

    Article  CAS  PubMed  Google Scholar 

  38. Pal GK, Pv S (2016) Microbial collagenases: challenges and prospects in production and potential applications in food and nutrition. Rsc Adv 6:33763–33780

    Article  CAS  Google Scholar 

  39. Lima CA, Rodrigues PMB, Porto TS, Viana DA, Lima Filho JL, Porto ALF, Carneiro Da Cunha MG (2009) Production of a collagenase from Candida albicans URM3622. Biochem Eng J 43:315–320

    Article  CAS  Google Scholar 

  40. Lima CA, Viana Marques DA, Neto BB, Lima Filho JL, Carneiro-da-Cunha MG, Porto ALF (2011) Fermentation medium for collagenase production by Penicillium aurantiogriseum URM4622. Biotechnol Progr 27:1470–1477

    Article  CAS  Google Scholar 

  41. Cawston TE, Barrett AJ (1979) A rapid and reproducible assay for collagenase using [1-14C] acetylated collagen. Anal Biochem 99:340–345

    Article  CAS  PubMed  Google Scholar 

  42. Gisslow MT, McBride BC (1975) A rapid sensitive collagenase assay. Anal Biochem 68:70–78

    Article  CAS  PubMed  Google Scholar 

  43. Ishikawa T, Nimni ME (1979) A modified collagenase assay method based on the use of p-dioxane. Anal Biochem 92:136–143

    Article  CAS  PubMed  Google Scholar 

  44. Dean DD, Woessner JF (1985) A sensitive, specific assay for tissue collagenase using telopeptide-free [3H] acetylated collagen. Anal Biochem 148:174–181

    Article  CAS  PubMed  Google Scholar 

  45. Ratnikov B, Deryugina E, Leng J, Marchenko G, Dembrow D, Strongin A (2000) Determination of matrix metalloproteinase activity using biotinylated gelatin. Anal Biochem 286:149–155

    Article  CAS  PubMed  Google Scholar 

  46. Peterkofsky B (1982) Bacterial collagenase. In: Cunningham LW, Frederiksen DW (eds) Methods in enzymology, vol 82. Academic Press, New York, pp 456–461

    Google Scholar 

  47. Petrova DH, Shishkov SA, Vlahov SS (2006) Novel thermostable serine collagenase from Thermoactinomyces sp. 21E: purification and some properties. J Basic Microb 46:275–285

    Article  CAS  Google Scholar 

  48. Petrova D, Derekova A, Vlahov S (2006) Purification and properties of individual collagenases from Streptomyces sp. strain 3B. Folia Microbiol 51:93–98

    Article  CAS  Google Scholar 

  49. Jackson RJ, Lien Dao M, Lim DV (1995) Modified FALGPA assay for cell-associated collagenolytic activity. J Microbiol Meth 21:209–215

    Article  CAS  Google Scholar 

  50. Wart HEV, Steinbrink DR (1981) A continuous spectrophotometric assay for Clostridium histolyticum collagenase. Anal Biochem 113:356–365

    Article  PubMed  Google Scholar 

  51. Rosen H (1957) A modified ninhydrin colorimetric analysis for amino acids. Arch Biochem Biophys 67:10–15

    Article  CAS  PubMed  Google Scholar 

  52. Mandl I, Zipper H, Ferguson LT (1958) Clostridium histolyticum collagenase: Its purification and properties. Arch Biochem Biophys 74:465–475

    Article  CAS  PubMed  Google Scholar 

  53. Bleeg HS (1991) Collagenolytic enzymes assayed by spectrophotometry with suspensions of reconstituted collagen fibrils. Connect Tissue Res 26:247–257

    Article  CAS  PubMed  Google Scholar 

  54. Pissarenko A, Ruestes CJ, Meyers MA (2020) Constitutive description of skin dermis: Through analytical continuum and coarse-grained approaches for multi-scale understanding. Acta Biomater 106:208–224

    Article  CAS  PubMed  Google Scholar 

  55. Yu X, Ma S, Xu Y, Fu C, Jiang C, Zhou C (2017) Construction and application of a novel genetically engineered Aspergillus oryzae for expressing proteases. Electron J Biotechn 29:32–38

    Article  CAS  Google Scholar 

  56. Sánchez Blanco A, Palacios Durive O, Batista Pérez S, Díaz Montes Z, Pérez Guerra N (2016) Simultaneous production of amylases and proteases by Bacillus subtilis in brewery wastes. Braz J Microbiol 47:665–674

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  57. Xu F, Ding H (2007) A new kinetic model for heterogeneous (or spatially confined) enzymatic catalysis: Contributions from the fractal and jamming (overcrowding) effects. Appl Catal A-Gen 317:70–81

    Article  CAS  Google Scholar 

  58. Kari J, Olsen JP, Jensen K, Badino SF, Krogh KBRM, Borch K, Westh AP (2018) Sabatier principle for interfacial (Heterogeneous) enzyme catalysis. Acs Catal 8:11966–11972

    Article  CAS  Google Scholar 

  59. Zhang X (2019) Characterization and Application of protease on the functional properties of skin collagen fibers, vol Master. Sichuan University Chengdu, China

  60. Nilegaonkar SS, Zambare VP, Kanekar PP, Dhakephalkar PK, Sarnaik SS (2007) Production and partial characterization of dehairing protease from Bacillus cereus MCM B-326. Bioresour Technol 98:1238–1245

    Article  CAS  PubMed  Google Scholar 

  61. Prakash P, Jayalakshmi SK, Sreeramulu K (2010) Production of keratinase by free and immobilized cells of Bacillus halodurans strain PPKS-2: partial characterization and its application in feather degradation and dehairing of the goat skin. Appl Biochem Biotechnol 160:1909–1920

    Article  CAS  PubMed  Google Scholar 

  62. Mortuza MF, Rahman MH, Rahman MH, Nahar A, Islam Khan MR, Hasan AKMM, Rahman M (2017) Isolation, biochemical and genetic characterization of extracellular protease producing cattle hide dehairing bacterium—a potential alternative to chemical dehairing. Eco Genet Genomics 2:3–12

    Google Scholar 

  63. Shrinivas D, Naik GR (2011) Characterization of alkaline thermostable keratinolytic protease from thermoalkalophilic Bacillus halodurans JB 99 exhibiting dehairing activity. Int Biodeter Biodegr 65:29–35

    Article  CAS  Google Scholar 

  64. Contesini FJ, Melo RR, Sato HH (2018) An overview of Bacillus proteases: from production to application. Crit Rev Biotechnol 38:321–334

    Article  CAS  PubMed  Google Scholar 

  65. Chao L, Zhaoyang L, Aimin L, Wei L, Zhenmao J, Jinlong C, Quanxing Z (2005) Adsorption of reactive dyes onto polymeric adsorbents: effect of pore structure and surface chemistry group of adsorbent on adsorptive properties. Sep Purif Technol 44:115–120

    Article  CAS  Google Scholar 

  66. Khandelwal HB, Khandelwal HB, More SV, More SV, Kalal KM, Kalal KM, Laxman RS, Laxman RS (2015) Eco-friendly enzymatic dehairing of skins and hides by C. brefeldianus protease. Clean Technol Envir 17:393–405

    Article  CAS  Google Scholar 

  67. Lyu B, Cheng K, Ma J, Hou X, Gao D, Gao H, Zhang J, Qi Y (2017) A cleaning and efficient approach to improve wet-blue sheepleather quality by enzymatic degreasing. J Clean Prod 148:701–708

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was financially supported by National Key R&D Program of China (2017YFB0308402).

Author information

Authors and Affiliations

  1. Key Laboratory of Leather Chemistry and Engineering of Ministry of Education, Sichuan University, Chengdu, 610065, China

    Mengchu Gao, Xu Zhang, Yongxin Tian, Chunxiao Zhang & Biyu Peng

  2. National Engineering Research Center of Clean Technology in Leather Industry, Sichuan University, Chengdu, 610065, China

    Chunxiao Zhang & Biyu Peng

Authors
  1. Mengchu Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xu Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yongxin Tian
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Chunxiao Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Biyu Peng
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

MG Investigation, formal analysis, resources, data curation, writing—original draft. XZ: Investigation, validation. YT: Investigation, validation. CZ: Writing—review & editing. BP: Conceptualization, methodology, supervision, project administration.

Corresponding author

Correspondence to Biyu Peng.

Ethics declarations

Conflict of Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Gao, M., Zhang, X., Tian, Y. et al. Development and validation of a label-free method for measuring the collagen hydrolytic activity of protease. Bioprocess Biosyst Eng 44, 2525–2539 (2021). https://doi.org/10.1007/s00449-021-02624-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00449-021-02624-5

Keywords

Search

Navigation