Novel Keratinase from Bacillus subtilis S14 Exhibiting Remarkable Dehairing Capabilities

Abstract

We report the isolation of a keratinolytic-producing Bacillus subtilis strain and the characterization of the exceptional dehairing properties of its subtilisin-like keratinase. This enzyme can be an alternative to sodium sulfide, the major pollutant from tanneries, and may completely replace it. Its unique nonactivity upon collagen enhances its industrial potential.Enzymatic dehairing in tanneries has been envisaged as an alternative to sulfides (4,6,9,22,23). Tanneries are constantly concerned about the obnoxious odor and pollution caused by the extremely toxic sodium sulfide used in the dehairing process step (24). Deaths due to this toxic chemical process have even been reported (2,8). Worldwide, it is estimated that 315 million bovine leathers are produced per year. Considering a waste treatment cost of $0.30 per m 2 of leather produced (A. Klein, personal communication), more than $1 million is spent per day to treat the waste from tanneries around the world. We report here a novel keratinase from Bacillus subtilis that has the potential to replace sodium sulfide in the dehairing process.Microorganism isolation. Bovine hair, skins wastes, and soil samples were suspended and cultivated in a feather-broth medium (composition in grams per liter: delipidated feather meal [the sole carbon and nitrogen source], 10.0; NaCl, 0.5; K 2 HPO 4 , 0.3; and KH 2 PO 4 , 0.4 [pH 7.5]).The best keratinase-producing organism was identified as a B. subtilis strain (named strain S14) after classification based on homology (99%) of its 16S fragment with sequences from the NCBI databank by use of BLASTN 2.2.6 (1) (accession number AY345856).Keratinase production. The microorganism was cultivated in a 14-liter bioreactor in a culture medium composed of whey milk (a dairy byproduct containing 94.0% water, 5% lactose, 0.9% protein, and 0.1% fat), pH 8.5, and bovine hair (40 g/liter). A crude extract (supernatant) was obtained after centrifugation of the culture. Keratinolytic, subtilisin, and collagenase activities were assayed by using azokeratin, as described elsewhere (15). One unit of enzyme activity was defined as the amount of enzyme that increases absorbance by 0.1 per hour in the conditions described above (15).Dehairing assay. A fresh fleshed bovine hide was washed with a commercial detergent solution and cut into 15-by 5-cm pieces. Two hundred grams of skin (usually two pieces) was processed in a drum flask at 4 rpm with crude extract or water (control) in a proportion of 1.0 ml of liquid per g of skin. When necessary, pH was adjusted with lime. At the end of the process, the skin pieces were gently scraped with fingers to remove loose hairs. This procedure was necessary because rubbing in this laboratory-scale process was not as vigorous as in industrial drums. Total skin depilation was observed in the pH range from 7 to 10, with 4.8 U/g of skin. A complete depilation was reached in 9 h at pH 9.0, 24°C, with 4.8 U/g of skin.Samples of bovine skin were kept in contact with the crude extract, and the skin fragments were fi...

Purification and characterization of keratinase from a new Bacillus subtilis strain

“…Accompanied by feather degradation and keratinase production, the pH value of the culture increased to about 8.5. B. subtilis strains had been widely utilized for enzyme production, including the keratinases (Lal et al, 1999;Suh and Lee, 2001;Kim et al, 2001;Macedo et al, 2005). Other Bacillus species including B. licheniformis, B. pumilis, B. cereus, B. halodurans and B. pseudofirmus (Williams et al, 1990;Takami et al, 1999;Rozs et al, 2001;Kim et al, 2001;Gessesse et al, 2003;El-Refai et al, 2005) had also been reported for their keratinolytic activity.…”

“…Among these strains, B. subtilis KD-N2 degraded feathers more quickly than any other reported until now, except for B. licheniformis RG1, which degraded feathers completely in 24 h (Ramnani and Gupta, 2004). Among the keratinases reported, only two of them had been purified (Suh and Lee, 2001;Macedo et al, 2005).…”

“…Also, the keratinase can degrade the infectious form of prion, PrP sc , in the presence of detergents and heat treatment (Langeveld et al, 2003), which could be important for the utilization of animal meal as feed. As for leather industry, the keratinase from Bacillus subtilis S14 exhibits remarkable dehairing capabilities (Macedo et al, 2005) without the degradation of collagen and this ecofriendly dehairing approach shows great utilization potential. We had screened a new feather-degrading Bacillus subtilis KD-N2 strain which degraded feathers completely within 30 h in submerged cultivation (Cai et al, 2008), and the crude keratinase from the isolate showed capabilities of removing calf hair and sheep wool from the skins.…”

Lime and Sulphide-Free Dehairing of Animal Skin Using Collagenase-Free Alkaline Protease from Vibrio metschnikovii NG155

“…As a positive control, collagen was incubated with collagenase from Clostridium histolyticum. Samples were collected at 0 and 24 h of incubation and analyzed on SDS PAGE [11].…”

Hide unhairing and characterization of commercial enzymes used in leather manufacture

“…Enzymes can be applied in different steps of the process, such as in soaking, liming, unhairing, bating, dyeing, and degreasing, as well as for effluent treatment, both solid and liquid (Kanth et al, 2009;Kanth et al, 2008;Macedo et al, 2005;Dayanandan et al, 2003;Lutckmeier et al, 2008;Kumar et al, 2008). Despite being consolidated in other industrial applications, the use of enzymes in the leather industry needs more research to be effectively used in large scale operations and at competitive costs.…”