Biochemical characterization of an extreme alkaline and surfactant-stable keratinase derived from a newly isolated actinomycete Streptomyces aureofaciens K13

Abstract

Keratinase has attracted increasing attention in the field of biocatalysis in recent years because of its critical role in keratin resource exploitation and keratin waste degradation. However, conventional studies focused on keratinases from bacterial and fungal strains, especially those of the Bacillus genus, keratinase resources from actinomycetes are far from being fully explored. In this study, a novel keratinase-producing strain was isolated with wool as the sole carbon and nitrogen source and identified as Streptomyces aureofaciens K13. The keratinase was purified to electrophoretic homogeneity with a molecular mass of 46 kDa. The purified enzyme exhibited optimum activity at 75 C and pH of 12.0. It remained extremely stable at alkaline pH values between 6 and 12 and at a high reaction temperature of 65 C. The keratinase displayed significant activity toward casein, keratin, BSA and wool. It could be activated in the presence of K + , Cu 2+ , Mn 2+ , Ca 2+ , Li + , and Sr 2+ . The keratinase was completely inhibited by PMSF and moderately inhibited by EDTA, indicating that this keratinase is a metallo-serine keratinase. This enzyme could remain stable and even be promoted in the presence of surfactants, including SDS, Tween, and Triton; especially, 1% of Tween 80 and Triton X-100 could substantially enhance the activity by 46% and 38%, respectively. These results indicated certain advantages over conventional keratinases. The keratinase can completely remove blood stains when combined with detergents. The improvement effect of S. aureofaciens K13 keratinase by various surfactants and the favourable washing performance might indicate its significant application potential in the detergent industry. There are rare reports on keratinase production from S. aureofaciens.

Microbial Keratinase: Next Generation Green Catalyst and Prospective Applications

“…Majority of keratinolytic proteases are catalytically active within neutral to alkaline pH with optima values between pH 7.0 and 9.0 (Gradišar et al, 2000;Bach et al, 2011;Kuo et al, 2012;Jaouadi et al, 2013;Bose et al, 2014;Akhter et al, 2020). However, a few extremely alkalophilic keratinases with optimum pH between 10 and 13 were previously reported (Friedrich and Antranikian, 1996;Letourneau et al, 1998;Mitsuiki et al, 2006;Jaouadi et al, 2008Jaouadi et al, , 2010Tatineni et al, 2008;Benkiar et al, 2013;Paul et al, 2014b;Gong et al, 2015;Tian et al, 2019). The optimum temperature reported for the activity of several microbial keratinases was between 37 and 65 • C (Hossain et al, 2007;Bach et al, 2011;Jaouadi et al, 2013;Wu et al, 2017;Hassan et al, 2020b), whereas thermophilic keratinolytic proteases already characterized function optimally between 70 and 100 • C (Nam et al, 2002;Jaouadi et al, 2010;Kuo et al, 2012;Benkiar et al, 2013;Paul et al, 2014b;Gong et al, 2015).…”

“…However, a few extremely alkalophilic keratinases with optimum pH between 10 and 13 were previously reported (Friedrich and Antranikian, 1996;Letourneau et al, 1998;Mitsuiki et al, 2006;Jaouadi et al, 2008Jaouadi et al, , 2010Tatineni et al, 2008;Benkiar et al, 2013;Paul et al, 2014b;Gong et al, 2015;Tian et al, 2019). The optimum temperature reported for the activity of several microbial keratinases was between 37 and 65 • C (Hossain et al, 2007;Bach et al, 2011;Jaouadi et al, 2013;Wu et al, 2017;Hassan et al, 2020b), whereas thermophilic keratinolytic proteases already characterized function optimally between 70 and 100 • C (Nam et al, 2002;Jaouadi et al, 2010;Kuo et al, 2012;Benkiar et al, 2013;Paul et al, 2014b;Gong et al, 2015). Many keratinases, irrespective of the sources, have been found to be catalytically active and stable over a temperature and pH range of 20 to 100 • C, and 4 to 13, respectively (Jaouadi et al, 2008(Jaouadi et al, , 2010Paul et al, 2014b;Gong et al, 2015;Jin et al, 2019).…”

Improved catalytic efficiency, thermophilicity, anti-salt and detergent tolerance of keratinase KerSMD by partially truncation of PPC domain

“…However, the variant V355 obtained significant tolerability to chaotropic compounds, and reserved more than 40% activity in 4% (w/v) urea or SDS. Remarkably, the addition of 1% (w/v) SDS to variant V355 improved its enzymatic activity, similar to a surfactant-stable keratinase from Streptomyces aureofaciens K13 32 . Though site-saturation mutagenesis and recombination of subtilisin E obtained excellent mutants that had 40–60% activity in 4% (w/v) SDS 31 33 , it might be more time-consuming than our C-terminal truncation strategy.…”

In vitro degradation of β‐amyloid fibrils by microbial keratinase

“…Microbial keratinases are proteases that effectively degrade sturdy proteins such as keratin [20]. Keratinases are produced by several microbial species including fungi [21,22], actinomycetes [23] especially genus Streptomyces [24,25], Bacillus [26,27], and archaea [28]. A keratinase purified from Bacillus licheniformis PWD‐1 degraded prions efficiently [29].…”