Leather manufacturing still leans heavily on chemistry that is hard on the environment. Traditional dehairing soaks hides and skins in lime and sodium sulphide, a step that generates polluted effluent and consumes large amounts of energy. Enzymes that strip hair without harsh chemicals have long been proposed as a greener alternative, but few have made it to industry because their performance varies unpredictably from batch to batch and skin to skin. A new comparative study of two microbial proteases now maps out, in unusual detail, why one of them works so much better than the other, and what structural features make it a promising candidate for cold, low-energy dehairing.
The research, published in the journal Collagen and Leather, focused on two purified proteases isolated at Vellore Institute of Technology in India: SP01, produced by Bacillus cereus VITSP01, and SP02, produced by Brevibacterium luteolum VITSP02. Earlier work had already shown that both enzymes could remove hair from goat skins, but with very different efficiency, and that their ability to attack proteoglycans, the sugar-protein complexes that cement hair into the skin matrix, seemed to track with how well they dehaired. The new study set out to explain that difference by combining biochemical and thermodynamic measurements with molecular docking, molecular dynamics simulations and structural comparisons.
The temperature experiments revealed a striking contrast. Both enzymes peaked in proteolytic activity at 37 degrees Celsius, with SP01 reaching 1785.33 plus or minus 49.65 U/mg protein/min and SP02 reaching 1856.16 plus or minus 58.03 U/mg protein/min. But SP02 stayed active across a much broader low-temperature window, from 5 to 40 degrees Celsius for up to 90 minutes of incubation, retaining 29.18 plus or minus 1.8 percent of its maximum activity at 5 degrees and 40.91 plus or minus 2.93 percent at 10 degrees. SP01, by comparison, showed limited dehairing activity at 15 degrees. SP02, in contrast, dehaired goat skins efficiently at both 15 and 37 degrees, a property that could allow tanneries to run enzymatic dehairing without heating large volumes of water.
Thermodynamic measurements helped explain this cold tolerance. The activation energy of SP02 was 27.003 kJ/mol, far lower than the 78.99 kJ/mol measured for SP01, indicating a faster reaction rate and greater structural flexibility that likely eases substrate binding and speeds the conformational changes needed for catalysis. SP02 also proved thermolabile: it remained stable up to 45 degrees, with a half-life of 203.86 minutes, but denatured rapidly above 50 degrees, where its half-life dropped to 86.64 minutes. Its higher deactivation energy of 85.160 kJ/mol, compared with 49.35 kJ/mol for SP01, along with higher deactivation enthalpy and entropy, pointed to a less rigid structure with more disordered regions, a hallmark of enzymes adapted to work in cold environments.
The enzyme also tolerated the harsh chemicals found in the beamhouse. SP02 retained 71 percent of its activity in 10 percent SDS and 73 to 78 percent in 10 percent non-ionic surfactants such as Triton X-100 and Tween 20, and it withstood up to 20 percent NaCl. That halotolerance could spare tanneries the extensive soaking needed to rehydrate salted hides before enzymatic treatment. Only the cationic surfactant CTAB, which likely binds the acidic groups on SP02, shut the enzyme down. When the metal chelator EDTA inactivated SP02, adding calcium ions restored 94.22 plus or minus 2.47 percent of its activity at 100 mM, and gelatin zymography and ANS fluorescence confirmed that calcium stabilizes the protein’s folded structure. Because lime dehairing already uses roughly 150 mM of calcium hydroxide in the float, the enzyme’s tolerance of calcium concentrations up to 200 mM suggests it could work alongside existing chemical processes rather than replacing them outright.
Molecular identification showed the two proteases belong to different enzyme families. The gene encoding SP01 matched trypsin-like peptidase domain-containing proteins from the S1 family, while SP02 matched S8 family peptidases, or subtilases, from Brevibacterium species. Their physical properties also differed sharply: SP01 has a pI of about 7.2 and a molecular weight near 40 kDa, whereas SP02 is smaller, around 30 kDa, and more acidic, with a pI of about 4.6. Peptide mass fingerprinting by LC-MS/MS identified 17 peptides from SP02 that matched S8 family serine peptidases from Brevibacterium luteolum, covering 37 percent of the protein sequence, and the predicted catalytic triads, His-Asp-Ser for SP01 and Asp-His-Ser for SP02, fit their respective families.
To probe why SP02 targets proteoglycans so effectively, the researchers docked both enzymes against bovine decorin and biglycan, two model proteoglycans, and then ran 50-nanosecond molecular dynamics simulations at 37 degrees. The SP02-biglycan complex showed minimal deviation, with RMSD values of 0.2 to 0.3 nm, and remained compact and poorly solvent-accessible throughout. SP01-decorin, by contrast, destabilized after 40 nanoseconds. At 20 degrees, the SP02-biglycan complex formed fewer interactions than at 37 degrees but still showed stable contacts and moderate binding energy, suggesting the enzyme keeps working on proteoglycans even at lower temperatures. The simulations support the idea that SP02’s substrate specificity, not just its raw catalytic power, drives its dehairing performance.
Penetration through the skin matrix turned out to be another decisive advantage. When the enzymes were fluorescently labeled and applied to the corium side of fresh goat skin, SP02 reached the opposite side within 6 hours, while SP01 needed 12 hours. Because roughly 70 percent of total dehairing time is spent waiting for the enzyme to travel from the skin surface to the hair roots, this difference matters enormously. The authors attribute SP02’s speed to its lower molecular weight, its acidic pI, which creates electrostatic repulsion with the negatively charged proteoglycans and reduces surface retention, and its compact shape, reflected in a smaller radius of gyration of 1.85 nm and hydrodynamic radius of 2.47 nm compared with 2.67 nm for SP01. The enzyme likely moves through natural pores and through gaps it creates itself by digesting the cementing proteoglycans around each hair.
Structural comparisons with a psychrophilic homolog and the mesophilic subtilisin E revealed the molecular basis of SP02’s flexibility. SP02 carries a higher proportion of glycine-rich loops, which can reposition active site residues to accommodate large substrates, and a surplus of acidic over basic residues that explains its low pI and negatively charged surface, a feature common in cold-adapted proteins. DynaMine analysis flagged a flexible region spanning residues 161 to 170, rich in beta-turns and coiled structure, that participates in substrate interaction. SP02 also possesses three tunnels connecting its buried active site to the protein surface, measuring 9, 9.3 and 14 angstroms, compared with a single tunnel in SP01, and a larger catalytic pocket of 461 cubic angstroms. Together, these features likely allow SP02 to process bulky, hydrated proteoglycans more readily than the rigid fibrillar collagen that it leaves largely untouched, preserving leather quality.
The study stops short of claiming industrial readiness, noting that further work is needed to fully elucidate the molecular mechanisms governing protease-substrate interactions within the skin matrix. But the message for enzyme screening is clear: dehairing efficiency depends on a constellation of properties, including substrate specificity for proteoglycans, low activation energy, structural flexibility, small size, acidic pI, and tolerance to surfactants, salt and calcium, rather than on any single parameter. By identifying these determinants in SP02, the researchers provide a template for finding or engineering similar enzymes that could make enzymatic dehairing reliable enough to displace chemical dehairing in commercial tanneries, cutting greenhouse gas emissions and energy use in a sector that is projected to grow substantially over the coming decade.
Subject of Research: Comparative biochemical and structural analysis of two microbial dehairing proteases for leather processing
Article Title: Biochemical and structural insights into dehairing proteases for efficient leather processing
Article References: Sujitha, P., Poornima, K. G., & Shanthi, C. (2026). Biochemical and structural insights into dehairing proteases for efficient leather processing. Collagen and Leather, 8(1), Article 20. https://doi.org/10.1186/s42825-026-00250-z
Image Credits: AI Generated
DOI: 10.1186/s42825-026-00250-z
Keywords: dehairing proteases, SP02, Brevibacterium luteolum, leather processing, proteoglycans, cold tolerance, molecular docking, molecular dynamics, enzyme penetration, goat skin, activation energy, sustainable tanning
News Source: Morgan Morrow. (November 5, 2026). Microbial Protease SP02 Shows Faster, Cooler Route to Greener Leather Dehairing. Scienmag.
