Biochemical and Molecular Characterization of a Serine Keratinase from Brevibacillus brevis US575 with Promising Keratin-Biodegradation and Hide-Dehairing Activities

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.

Purification and biochemical characterization of two detergent-stable serine alkaline proteases from Streptomyces sp. strain AH4

“…These observations strongly suggested that the SASP-P1 and SAPS-P2 proteases were monomeric proteins comparable to those previously reported for other Streptomyces proteases (Tatineni et al 2008;Yum et al 1994). The N-terminal amino acid sequences of the enzymes were identified and noted to display high homologies with those of The activity of each substrate was determined by measuring absorbance at specified wavelengths according to the relative method reported by Zaraî Jaouadi et al (2013) Streptomyces proteases, thus strongly suggesting that SAPS-P1 and SAPS-P2 were novel proteases. The two alkaline proteases SAPS-P1 and SAPS-P2 showed high alkaline pH optima, which are compatible with the alkaline environment encountered under harsh washing conditions (pH 9.0-11.0, temperature of 20-60°C, and high salt, bleach, and surfactant concentrations).…”

“…Peptides were dissolved in 10 % (v/v) N,N-dimethylformamide prior to dilution in buffer and prepared immediately before the experiment since nitroanilide substrates show varying degrees of autolysis during storage. Enzymatic activities were determined on each substrate according to standard conditions previously described by Zaraî Jaouadi et al (2013).…”

A novel organic solvent- and detergent-stable serine alkaline protease from Trametes cingulata strain CTM10101

“…The pH was kept constant throughout hydrolysis by adding NaOH 5 N. An amount of 4 g of casein was dissolved in 100 ml of 50 mM glycine-NaOH buffer containing 2 mM CaCl2 at pH 9 and then treated with 500 U of the purified enzymes, namely SPTC, Flavourzyme® 500 L, and Thermolsyin. The degree of hydrolysis (DH) was calculated for each case from the amount of base (NaOH) added to keep the pH constant during hydrolysis [34] as previously described by Zaraî Jaouadi et al [33].…”

Thermotolerant bacteria of biotechnological potential from hot springs in Eritrea

“…Thermotolerant bacteria have attracted industrial and biotechnological attention as their enzymes are well suited for harsh industrial processes (Abdel-Rahman et al, 2016;Archna et al, 2015;Verma et al, 2015). Proteases obtained from thermophilic or thermotolerant (Adhikari et al, 2015;Bozoglu et al, 2015;Lele and Deshmukh, 2016;Panda et al, 2013;Remigio et al, 2012;Verma et al, 2014) bacteria, for example, have found applications such as hide dehairing in the leather industry and stain removal in laundry detergents (Chandrashekhar and Narayan, 2015;Dudhgara et al, 2015;Jaouadi et al, 2013). Cellulases have showed great potential in the production of bioethanol and other speciality chemicals from renewable agricultural residues (Hardiman et al, 2010).…”