Abstract
Dehairing is one of the highly polluting operations in the leather industry. The conventional lime-sulfide process used for dehairing produces large amounts of sulfide, which poses serious toxicity and disposal problems. This operation also involves hair destruction, a process that leads to increased chemical oxygen demand (COD), biological oxygen demand (BOD), and total suspended solid (TSS) loads in the effluent. With these concerns in mind, enzyme-assisted dehairing has often been proposed as an alternative method. The main enzyme preparations so far used involved keratinases. The present paper reports on the purification of an extracellular keratinase (KERUS) newly isolated from Brevibacillus brevis strain US575. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis revealed that the purified enzyme was a monomer with a molecular mass of 29121.11 Da. The sequence of the 27 N-terminal residues of KERUS showed high homology with those of Bacillus keratinases. Optimal activity was achieved at pH 8 and 40°C. Its thermoactivity and thermostability were upgraded in the presence of 5 mM Ca2+. The enzyme was completely inhibited by phenylmethanesulfonyl fluoride (PMSF) and diiodopropyl fluorophosphates (DFP), which suggests that it belongs to the serine protease family. KERUS displayed higher levels of hydrolysis, substrate specificity, and catalytic efficiency than NUE 12 MG and KOROPON® MK EG keratinases. The enzyme also exhibited powerful keratinolytic activity that made it able to accomplish the entire feather-biodegradation process on its own. The kerUS gene encoding KERUS was cloned, sequenced, and expressed in Escherichia coli. The biochemical properties of the extracellular purified recombinant enzyme (rKERUS) were similar to those of native KERUS. Overall, the findings provide strong support for the potential candidacy of this enzyme as an effective and eco-friendly alternative to the conventional chemicals used for the dehairing of rabbit, goat, sheep and bovine hides in the leather processing industry.
Highlights
Leather making is an important socio-economic activity for several countries throughout the world
Mono Q Sepharose aThe experiments were conducted three times and 6 standard errors are reported. bOne keratin unit is defined as an increase of 0.1 absorbance at 440 nm per minute, using keratin azure as a substrate under the experimental conditions used. cAmounts of protein were estimated by the method of Bradford [21]. doi:10.1371/journal.pone.0076722.t001
The effects of phenylmethanesulfonyl fluoride (PMSF), diiodopropyl fluorophosphates (DFP), soybean trypsin inhibitor (SBTI), benzamidine hydrochloride hydrate, Na-p-tosyl L-phenylalanine chloromethyl ketone (TPCK), Na-p-tosyl L-lysine chloromethyl ketone (TLCK), dithio-bis-2-nitro benzoic acid (DTNB), monoiodoacetic acid (MIA), LD-dithiothreitol (DTT), 2-mercaptoethanol (2-ME), N-ethylmalemide (NEM), leupeptin hemisulfate salt, pepstatin A, 1,2-epoxy-3-(pnitrophenoloxy) propane (EPNP), EDTA, EGTA, and various monovalent and divalent metal ions (5 mM) on keratinase stability were investigated by pre-incubating the purified KERUS enzyme for 1 h at room temperature with each inhibitor, and for 1 h at 40uC in the presence of metal ions
Summary
Leather making is an important socio-economic activity for several countries throughout the world. Leather processing involves a complex set of steps, from skin to finished product, including soaking, dehairing, bating, and tanning. Namely Aquaderm, NUE (Novozymes A/S, Danemark), and KOROPON (MK Michael Kors leathers, Brazil), are currently manufactured for use in soaking, dehairing, and bating, respectively Due to their attractive properties and attributes, keratinases have been isolated from various microorganisms and introduced into a wide range of biotechnological applications, including those in the feed, fertilizer, detergent, leather and pharmaceutical industries [3]. The isolation and screening of new keratinolytically active Bacillus strains from natural habitats or neutral/alkaline wastewater could, open new opportunities for the discovery and use of novel keratinases for application in poultry and leather processing industries. The nucleotide and amino acid sequences, cloning, and expression of the encoding gene (kerUS) were determined
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