Applied Microbiology and Biotechnology

, Volume 102, Issue 21, pp 9159–9170 | Cite as

A novel “trifunctional protease” with reducibility, hydrolysis, and localization used for wool anti-felting treatment

  • Jingxia Mei
  • Nan Zhang
  • Yuanyuan Yu
  • Qiang WangEmail author
  • Jiugang Yuan
  • Ping Wang
  • Li Cui
  • Xuerong Fan
Biotechnologically relevant enzymes and proteins


Proteases can cause unacceptable fiber damage when they are singly applied to wool anti-felting treatment which can make wool textiles machine-washable. Even if protease is attached by synthetic polymers, the modified protease plays a limited role in the degradation of keratin with dense structure consisting of disulfide bonds in the scales. Here, to obtain “machine-washable” wool textiles, a novel “trifunctional protease” with reducibility, hydrolysis, and localization is developed by means of covalent bonding of protease molecules with poly (ethylene glycol) bis (carboxymethyl) ether (HOOC-PEG-COOH) and l-cysteine using carbodiimide/N-hydroxysuccinimide (EDC/NHS) coupling, aiming at selectively degrading the scales on the surface of wool. The formation of polymer is confirmed with size exclusion chromatography (SEC) and Fourier transform infrared spectroscopy (FT-IR). Ellman’s test and fluorescence microscopy reveal that the modified protease can reduce disulfide bonds and restrict hydrolysis of peptide bonds on the wool scales. Furthermore, when applied to wool fabrics, the modified protease reach better treatment effects considering dimensional stability to felting (6.12%), strength loss (11.7%) and scale dislodgement proved by scanning electron microscopy (SEM), alkali solubility, wettability, and dyeability. This multifunctional enzyme is well-designed according to the requirement of the modification of wool surface, showing great potential for eco-friendly functionalization of keratin fibers rich in disulfide linkage.


Protease modification PEG wool anti-felting 


Funding information

This study was funded by the National Key R&D Program of China (2017YFB0309200), the National Natural Science Foundation of China (51673087, 31771039), the Program for Changjiang Scholars and Innovative Research Teams in Universities (IRT_15R26), Fundamental Research Funds for the Central Universities (JUSRP51717A), and national first-class discipline program of Light Industry Technology and Engineering (LITE2018-21).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Informed consent

Informed consent was obtained from all individual participants included in the study.

Supplementary material

253_2018_9276_MOESM1_ESM.pdf (298 kb)
ESM 1 (PDF 297 kb)


  1. Araújo, R., Silva, C., Machado, R., Casal, M., Cunha, A. M (2009) Proteolytic enzyme engineering: a tool for wool. Biomacromolecules 10(6):1655–1661 doi: CrossRefGoogle Scholar
  2. Chen R, Yuan J, Yu Y, Fan X, Wang Q, Zhu Y (2012) Preparation of chitosan conjugated protease used for shrink resistance of wool and its properties. J food Sci Biotechnolo 31(5):63–68. CrossRefGoogle Scholar
  3. Fu J, Su J, Wang P, Yu Y, Wang Q, Cavaco-Paulo A (2015) Enzymatic processing of protein-based fibers. Appl Microbiol Biotechnol 99(24):10387–10397. CrossRefPubMedGoogle Scholar
  4. Gaffar Hossain KM, Juan AR, Tzanov T (2009) Simultaneous protease and transglutaminase treatment for shrink resistance of wool. Biocatal Biotransform 26(5):405–411. CrossRefGoogle Scholar
  5. Hosseinkhani S, Ranjbar B, Naderi-Manesh H, Nemat-Gorgani M (2004) Chemical modification of glucose oxidase: possible formation of molten globule-like intermediate structure. FEBS Lett 561(1–3):213–216. CrossRefPubMedGoogle Scholar
  6. Ibrahim NA, El-Shafei HA, Abdel-Aziz MS, Ghaly MF, Eid BM, Hamed AA (2012) The potential use of alkaline protease from Streptomyces albidoflavus as an eco-friendly wool modifier. J Text Inst 103(5):490–498. CrossRefGoogle Scholar
  7. Jus S, Schroeder M, Guebitz GM, Heine E, Kokol V (2007) The influence of enzymatic treatment on wool fibre properties using PEG-modified proteases. Enzyme Microb Technol 40(7):1705–1711. CrossRefGoogle Scholar
  8. Mojsov K (2017) Enzymatic treatment of wool fabrics - opportunity of the improvement on some physical and chemical properties of the fabrics. J Text Inst 108(7):1136–1143. CrossRefGoogle Scholar
  9. Kaisersberger-Vincek M, Štrancar J, Kokol V (2016) Antibacterial activity of chemically versus enzymatic functionalized wool with ɛ-poly-L-lysine. Textile Res J 87(13):1604–1619. CrossRefGoogle Scholar
  10. Kaur A, Chakraborty JN, Dubey KK (2016) Enzymatic functionalization of wool for felting shrink-resistance. J Nat Fibers 13(4):437–450. CrossRefGoogle Scholar
  11. Kelly SM, Jess TJ, Price NC (2005) How to study proteins by circular dichroism. Biochim Biophys Acta 1751(2):119–139. CrossRefPubMedGoogle Scholar
  12. Kynclova E, Elsner E, Kopf A, Hawa G, Schalkhammer T, Fritz P (1996) Novel method for coupling of poly(ethyleneglycol) to carboxylic acid moieties of proteins. J Mol Recognit 9(5–6):644–651.<644::AID-JMR314>3.0.CO;2-7 CrossRefPubMedGoogle Scholar
  13. Kantouch A, Bendak A, Sadek M (1978) Studies on the shrink resist treatment of wool with potassium permanganate. Text Res J 48(11):619–624. CrossRefGoogle Scholar
  14. Liu B, Zhang J, Li B, Liao X, Du G, Chen J (2013) Expression and characterization of extreme alkaline, oxidation-resistant keratinase from Bacillus licheniformis in recombinant Bacillus subtilis WB600 expression system and its application in wool fiber processing. World J Microb Biot 29(5):825–832. CrossRefGoogle Scholar
  15. Moser M, Behnke T, Hamers-Allin C, Klein-Hartwig K, Falkenhagen J, Resch-Genger U (2015) Quantification of PEG-maleimide ligands and coupling efficiencies on nanoparticles with Ellman's reagent. Anal Chem 87(18):9376–9383. CrossRefPubMedGoogle Scholar
  16. Roberts MJ, Bentley MD, Harris JM (2012) Chemistry for peptide and protein PEGylation. Adv Drug Deliver Rev 64:116–127. CrossRefGoogle Scholar
  17. Schroeder M, Lenting HB, Kandelbauer A, Silva CJ, Cavaco-Paulo A, Gubitz GM (2006) Restricting detergent protease action to surface of protein fibres by chemical modification. Appl Microbiol Biotechnol 72(4):738–744. CrossRefPubMedGoogle Scholar
  18. Schroeder M, Schweitzer M, Lenting HBM, Guebitz GM (2009) Chemical modification of proteases for wool cuticle scale removal. Biocatal Biotransfor 22(5–6):299–305. CrossRefGoogle Scholar
  19. Shen J, Rushforth M, Cavaco-Paulo A, Guebitz G, Lenting H (2007) Development and industrialisation of enzymatic shrink-resist process based on modified proteases for wool machine washability. Enzyme Microb Tech 40(7):1656–1661. CrossRefGoogle Scholar
  20. Shen J, Smith E, Chizyuka M, Prajapati C (2017) Development of durable shrink-resist coating of wool with sol-gel polymer processing. Fiber Polym 18(9):1769–1779. CrossRefGoogle Scholar
  21. Silva CJSM, Gübitz G, Cavaco-Paulo A (2006a) Optimisation of a serine protease coupling to Eudragit S-100 by experimental design techniques. J Chem Technol Biot 81(1):8–16. CrossRefGoogle Scholar
  22. Silva CJSM, Prabaharan M, Gübitz G, Cavaco-Paulo A (2005) Treatment of wool fibres with subtilisin and subtilisin-PEG. Enzyme Microb Tech 36(7):917–922. CrossRefGoogle Scholar
  23. Silva CJSM, Zhang Q, Shen J, Cavaco-Paulo A (2006b) Immobilization of proteases with a water soluble–insoluble reversible polymer for treatment of wool. Enzyme Microb Tech 39(4):634–640. CrossRefGoogle Scholar
  24. Smith E, Farrand B, Shen J (2010a) The removal of lipid from the surface of wool to promote the subsequent enzymatic process with modified protease for wool shrink resistance. Biocatal Biotransfor 28(5–6):329–338. CrossRefGoogle Scholar
  25. Smith E, Schroeder M, Guebitz G, Shen J (2010b) Covalent bonding of protease to different sized enteric polymers and their potential use in wool processing. Enzyme Microb Tech 47(3):105–111. CrossRefGoogle Scholar
  26. Smith E, Shen J (2011) Surface modification of wool with protease extracted polypeptides. J Biotechnol 156(2):134–140. CrossRefPubMedGoogle Scholar
  27. Smith E, Shen J (2012) Enzymatic treatment of wool pre-treated with cetyltrimethylammonium bromide to achieve machine washability. Biocatal Biotransfor 30(1):38–47. CrossRefGoogle Scholar
  28. Smith E, Zhang Q, Shen J, Schroeder M, Silva C (2009) Modification of Esperase® by covalent bonding to Eudragit® polymers L 100 and S 100 for wool fibre surface treatment. Biocatal Biotransfor 26(5):391–398. CrossRefGoogle Scholar
  29. Vílchez S, Jovančić P, Erra P (2010) Influence of chitosan on the effects of proteases on wool fibers. Fiber Polym 11(1):28–35. CrossRefGoogle Scholar
  30. Wang K, Li R, Ma JH, Jian YK, Che JN (2016) Extracting keratin from wool by using L-cysteine. Green Chem 18(2):476–481. CrossRefGoogle Scholar
  31. Wang P, Yuan J, Ren X, Cui L, Wang Q, Fan X (2013) Bio-antifelting of wool based on mild methanolic potassium hydroxide pretreatment. Eng Life Sci 13(1):102–108. CrossRefGoogle Scholar
  32. Yu F, Du P, Lei X, Zhang S (2009) Investigation of voltammetric enzyme-linked immunoassay system based on N-heterocyclic substrate of 2,3-diaminopyridine. Talanta 78(4–5):1395–1400. CrossRefPubMedGoogle Scholar
  33. Zhu Y, Fan X, Wang Q, Chen R, Yu Y, Yuan J (2011) Chemical modification of Savinase by dextran and its properties. China Biotechnol 31(10):45–49 doi:

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Key Laboratory of Science and Technology of Eco-Textile, Ministry of EducationJiangnan UniversityWuxiChina

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