Biochemical and structural insights into dehairing proteases for efficient leather processing

Abstract

Dehairing proteases are eco-friendly alternatives to chemical dehairing process. Yet, their widespread industrial adoption requires focused research on key factors influencing their efficacy. Earlier research on two microbial proteases, SP01 (from Bacillus cereus VITSP01) and SP02 (from Brevibacterium luteolum VITSP02), revealed a correlation between substrate specificity for proteoglycans and dehairing efficacy. Herein, a comprehensive comparative analysis of their biochemical, thermodynamic, and molecular characteristics was performed to elucidate the determinants of their performance. In comparison to SP01, SP02 could cleave skin proteoglycans and dehair goat skins efficiently at lower temperatures (15 °C and 37 °C). Additionally, it exhibited thermolability, lower activation energy (Ea = 27.003 kJ mol⁻¹) and tolerance to 10% SDS, non-ionic detergents, and 20% NaCl, while Ca²⁺ had a stabilizing effect on its structure. The protease genes of SP01 and SP02 were identified to be trypsin-like peptidase domain-containing protein and S8 family peptidase, respectively. The docking and molecular dynamics simulation revealed stabler interactions between proteoglycans and SP02 compared to SP01. The penetration of SP02 was faster than that of SP01 through skin matrix, probably due to its lower molecular weight (around 30 kDa), pI (about 4.6), proteoglycan degrading property, and smaller Rg and Rh. In silico structural analyses suggested some salient structural features responsible for the cold tolerance of SP02, and the resultant structural flexibility along with a larger catalytic pocket and longer multiple substrate-interacting tunnels could enable better substrate accommodation. In conclusion, SP02 was demonstrated as a potential dehairing enzyme with cold tolerant structural features. This study revealed some key determinants influencing its efficacy and stability, thereby implying improvements for process optimization.

Keywords

Dehairing / Leather / Protease / Enzyme activity / Molecular simulation / Cold adaptation

References

[1] Leather goods market size & share analysis report, 2030. May 2023. Accessed 18 July 2025. https://www.grandviewresearch.com/industry-analysis/leather-goods-market

[2] Novozymes, proteases for biocatalysis, Novoenzymes. 2016; 1–6. Accessed 14 Feb 2022. https://www.novozymes.com/en/solutions/fine-chemicals/biocatalysis/proteases

[3] Shakilanishi S, Shanthi C. Specificity studies on proteases for dehairing in leather processing using decorin as model conjugated protein. Int J Biol Macromol, 2017, 103: 1069-1076

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

[5] Shakilanishi S, Mrudula P, Shanthi C. Production of dehairing protease by Bacillus cereus VITSN04: a model cradle-to-cradle approach for sustainable greener production of leathers. Environ Technol, 2024, 45: 180-191

[6] Zhang X, Gao M, Zhang C, Peng B. Enzymatic processes for animal hide/skin collagen fiber purification processing: recent progress, challenges and recommendations. Bioresour Technol, 2025, 418: 131955

[7] Isaksen GV, Åqvist J, Brandsdal BO. Enzyme surface rigidity tunes the temperature dependence of catalytic rates. Proc Natl Acad Sci U S A, 2016, 113: 7822-7827

[8] Sivasubramanian S. Mechanism of enzymatic dehairing of skins using a bacterial alkaline protease. Chemosphere, 2008, 70: 1025-1034

[9] Sujitha P, Shanthi C. Insights into substrate specificity of proteases for screening efficient dehairing enzymes. Int J Biol Macromol, 2021, 172: 360-370

[10] Hollingsworth SA, Dror RO. Molecular dynamics simulation for all. Neuron, 2018, 99: 1129-1143

[11] Cantera C, Garro M, Goya L, Babeito C, Galarza B. Hair shaving unhairing process. Part 6. Stratum corneum as a diffusion barrier: chemical–mechanical injury of epidermis. J Soc Leath Technol Chem, 2004, 88: 121-130

[12] Yates BJR. Studies in depilation. Aust J Biol Sci, 1968, 21: 1249-1262

[13] Luo F, Zhong X, Gao M, Peng B, Long Z. Progress and mechanism of breaking glycoconjugates by glycosidases in skin for promoting unhairing and fiber opening-up in leather manufacture. A review. J Leather Sci Eng, 2020

[14] Chauhan JV, Mathukiya RP, Singh SP, Gohel SD. Two steps purification, biochemical characterization, thermodynamics and structure elucidation of thermostable alkaline serine protease from Nocardiopsis alba strain OM-5. Int J Biol Macromol, 2021, 169: 39-50

[15] da Silva OS, Silva JdeC, de Almeida EM, Sousa F, Gonçalves OSL, Sarmento B, et al.. Biophysical, photochemical and biochemical characterization of a protease from URM4634. Int J Biol Macromol, 2018, 118: 1655-1666

[16] Basu B, Apte SK. A novel serralysin metalloprotease from *Deinococcus radiodurans*. Biochim Biophys Acta Proteins Proteom, 2008, 1784: 1256-1264

[17] Xu X, Chen J, Zhang L, Wang S, Shen D, Liu Q. Calcium ion-induced stabilization and refolding of agkisacutacin from *Agkistrodon acutus* venom studied by fluorescent spectroscopy. J Fluoresc, 2007, 17: 215-221

[18] Friedman DB, Hoving S, Westermeier R. Isoelectric focusing and two-dimensional gel electrophoresis. In: Burgess RR, Deutscher MP, editors. Methods in enzymology. 1st ed. Amsterdam: Elsevier Inc; 2009. p. 515–40.

[19] Shevchenko A, Tomas H, Havliš J, Olsen J, Mann M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc, 2007, 1: 2856-2860

[20] Kumari R, Kumar R, Lynn A. G-mmpbsa -a GROMACS tool for high-throughput MM-PBSA calculations. J Chem Inf Model, 2014, 54: 1951-1962

[21] Shah D, Guo Y, Ocando J, Shao J. FITC labeling of human insulin and transport of FITC-insulin conjugates through MDCK cell monolayer. J Pharm Anal, 2019, 9: 400-405

[22] Tomoda K, Yabuki N, Terada H, Makino K. Application of polymeric nanoparticles prepared by an antisolvent diffusion with preferential solvation for iontophoretic transdermal drug delivery. Colloid Polym Sci, 2014, 292: 3195-3203

[23] Stourac J, Vavra O, Kokkonen P, Filipovic J, Pinto G, Brezovsky J, et al.. Caver Web 1.0: identification of tunnels and channels in proteins and analysis of ligand transport. Nucleic Acids Res, 2019, 47: W414-W422

[24] Pravda L, Sehnal D, Toušek D, Navrátilová V, Bazgier V, Berka K, et al.. MOLEonline: a web-based tool for analyzing channels, tunnels and pores. Nucleic Acids Res, 2018, 46: W368-W373

[25] Zamudio-Prieto O, Benítez-Cardoza C, Arroyo R, Ortega-López J. Conformational changes induced by detergents during the refolding of chemically denatured cysteine protease ppEhCP-B9 from *Entamoeba histolytica*. Biochim Biophys Acta Proteins Proteom, 2014, 1844: 1299-1306

[26] Monera OD, Kay CM, Hodges RS. Protein denaturation with guanidine hydrochloride or urea provides a different estimate of stability depending on the contributions of electrostatic interactions. Protein Sci. 1994;3:1984-91. https://doi.org/10.1002/pro.5560031110

[27] Wauters G, Avesani V, Laffineur K, Charlier J, Janssens M, Van Bosterhaut B, et al.. sp. nov., from human and environmental samples. Int J Syst Evol Microbiol, 2003, 53: 1321-1325

[28] Li X, Zhang S, Zhang Q, Gan L, Jiang G, Tian Y, et al.. Characterization and application of a novel halotolerant protease with no collagenase activity for cleaner dehairing of goatskin. Process Biochem, 2022, 113: 203-215

[29] Gohel SD, Singh SP. Thermodynamics of a Ca²⁺ dependent, highly thermostable and detergent compatible purified alkaline serine protease from *Nocardiopsis xinjiangensis* strain OM-6. Int J Biol Macromol, 2018, 113: 565-574

[30] Ertan H, Cassel C, Verma A, Poljak A, Charlton T, Aldrich-Wright J, et al.. A new broad specificity alkaline metalloprotease from a . isolated from refrigerated milk: role of calcium in improving enzyme productivity. J Mol Catal B Enzym, 2015, 113: 1-8

[31] Covington ADTanning chemistry: the science of leather, 2009LondonRoyal Society of Chemistry

[32] Wilkins DK, Grimshaw SB, Receveur V, Dobson CM, Jones JA, Smith LJ. Hydrodynamic radii of native and denatured proteins measured by pulse field gradient NMR techniques. Biochemistry, 1999, 38: 16424-16431

[33] Mitragotri S. Modeling skin permeability to hydrophilic and hydrophobic solutes based on four permeation pathways. J Control Release, 2003, 86: 69-92

[34] Gao M, Song J, Zhang X, Zhang C, Peng B, Chattha SA. Key mechanism of enzymatic dehairing technology for leather - making: permeation behaviors of protease into animal hide and the mechanism of charge regulation. Collagen Leather, 2023

[35] Stanton H, Melrose J, Little CB, Fosang AJ. Proteoglycan degradation by the ADAMTS family of proteinases. Biochim Biophys Acta Mol Basis Dis, 2011, 1812: 1616-1629

[36] Nowak JS, Otzen DE. Helping proteins come in from the cold: 5 burning questions about cold-active enzymes. BBA Adv, 2024, 5: 100104

[37] Davail S, Feller G, Narinx E, Gerday C. Cold adaptation of proteins. purification, characterization, and sequence of the heat-labile subtilisin from the Antarctic psychrophile Bacillus TA41. J Biol Chem, 1994, 269: 17448-17453

[38] Almog O, González A, Godin N, de Leeuw M, Mekel MJ, Klein D, et al.. The crystal structures of the psychrophilic subtilisin S41 and the mesophilic subtilisin Sph reveal the same calcium-loaded state. Proteins, 2009, 74: 489-496

[39] Liu H, Ding S, Nardin C, Zhang Y. A new cold-active transglutaminase: discovery, computational insights, and recombinant expression. Enzyme Microb Technol, 2025, 191: 110712

[40] Thomas T, Cavicchioli R. Archaeal cold-adapted proteins: structural and evolutionary analysis of the elongation factor 2 proteins from psychrophilic, mesophilic and thermophilic methanogens. FEBS Lett, 1998, 439: 281-286

[41] Liu Y, Jia K, Chen H, Wang Z, Zhao W, Zhu L. Cold-adapted enzymes: mechanisms, engineering and biotechnological application. Bioprocess Biosyst Eng, 2023, 46: 1399-1410

[42] Mavromatis K, Tsigos I, Tzanodaskalaki M, Kokkinidis M, Bouriotis V. Exploring the role of a glycine cluster in cold adaptation of an alkaline phosphatase. Eur J Biochem, 2002, 269: 2330-2335

[43] Arnórsdóttir J, Kristjánsson MM, Ficner R. Crystal structure of a subtilisin-like serine proteinase from a psychrotrophic Vibrio species reveals structural aspects of cold adaptation. FEBS J, 2005, 272: 832-845

[44] Óskarsson KR, Kristjánsson MM. Improved expression, purification and characterization of VPR, a cold active subtilisin-like serine proteinase and the effects of calcium on expression and stability. Biochim Biophys Acta Proteins Proteom, 2019, 1867: 152-162

[45] Smith CA, Toogood HS, Baker HM, Daniel RM, Baker EN. Calcium-mediated thermostability in the subtilisin superfamily: the crystal structure of Bacillus Ak.1 protease at 1.8 Å resolution. J Mol Biol, 1999, 294: 1027-1040

[46] Eijsink VGH, Matthews BW, Vriend G. The role of calcium ions in the stability and instability of a thermolysin-like protease. Protein Sci, 2011, 20: 1346-1355

[47] Almog O, González A, Klein D, Greenblatt HM, Braun S, Shoham G. The 0.93 Å crystal structure of sphericase: a calcium-loaded serine protease from Bacillus sphaericus. J Mol Biol, 2003, 332: 1071-1082

[48] Siddiqui KS, Poljak A, Guilhaus M, Feller G, D’Amico S, Gerday C, et al.. Role of disulfide bridges in the activity and stability of a cold-active α-amylase. J Bacteriol, 2005, 187: 6206

[49] Feller G, Gerday C. Psychrophilic enzymes: hot topics in cold adaptation. Nat Rev Microbiol, 2003, 1: 200-208

[50] Kokkonen P, Bednar D, Pinto G, Prokop Z, Damborsky J. Engineering enzyme access tunnels. Biotechnol Adv, 2019, 37: 107386

[51] Kingsley LJ, Lill MA. Substrate tunnels in enzymes: structure-function relationships and computational methodology. Proteins, 2015, 83: 599

[52] Alberts B, Johnson A, Lewis JRobertson M. The extracellular matrix of animals the extracellular matrix is made and oriented by the cells within its glycosaminoglycan (GAG) chains occupy large amounts of space and form hydrated gels. Molecular biology of the cell, 20024New YorkGarland1-24

[53] Jian S, Wenyi T, Wuyong C. Kinetics of enzymatic unhairing by protease in leather industry. J Clean Prod, 2011, 19: 325-331

[54] Paredes DI, Watters K, Pitman DJ, Bystroff C, Dordick JS. Comparative void-volume analysis of psychrophilic and mesophilic enzymes: structural bioinformatics of psychrophilic enzymes reveals sources of core flexibility. BMC Struct Biol, 2011, 20: 11-42

[55] Struvay C, Feller G. Optimization to low temperature activity in psychrophilic enzymes. Int J Mol Sci, 2012, 13: 11643-11665

Funding

Vellore Institute of technology(VIT/SG/2020-21/46)

RIGHTS & PERMISSIONS

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