Biodegradation of α and β-keratins by Gram-negative bacteria
Introduction
An increasing amount of fibrous insoluble protein comprised by feathers, hair, nails, horns, hoofs and wool has been generated as byproducts of the agroindustrial processing. These hard-to-degrade animal proteins are mostly disposed by land filling or incineration, which is ecologically disadvantageous owing to the apparent energy loss and the production of large amounts of carbon dioxide (Suzuki et al., 2006). Thus, there is a demand for an environmentally friendly alternative to the management of these recalcitrant keratinous wastes.
Keratin is difficult to degrade since the polypeptide is densely packed and strongly stabilized by several hydrogen bonds, hydrophobic interactions, and disulfide bonds (Brandelli et al., 2010). Keratins can be divided into soft and hard keratin according to their content of sulfur amino acids. Skin keratin, called soft keratin, possesses up to 10% of cysteine residues. Hard keratin, present in skin appendages, has more than 15% of cysteine residues and is more resistant to chemical factors (Gradisar et al., 2005). Besides that, keratin can also be classified as α-keratin (present in hair and wool) or β-keratin (present in feathers) due to its secondary structure (Korniłłowicz-Kowalska and Bohacz, 2011).
Keratinases are microbial enzymes capable of degrading keratin. This enzyme shows importance in the hydrolysis of feather, hair and wool to clear obstructions in the sewage system during wastewater treatment and exhibits many other remarkable biotechnological applications (Brandelli et al., 2015). Specifically, keratinases that degrade α-keratin may selectively dehair hides in the leather industry without affecting the tensile strength of leather and reducing the pollution caused by the chemical counterpart (Chaturvedi et al., 2014). Furthermore, keratinases can also be added to detergents for wool cleaning (Saleem et al., 2012) and used for degumming of silk and finishing textile fibers, which increases its smoothening, shining, dyeing and shrink-proof capacities (Souza et al., 2007, Lv et al., 2010).
However, the utility of keratinases is underexploited due to limited availability of efficient enzymes with versatile substrate specificity. For instance, a limited number of studies have described the isolation of wool and hair-degrading bacteria (Mazotto et al., 2009). Bacterial keratinases have been widely documented for Gram-positive strains, mostly capable of β-keratin degradation, including substrates like feathers (Daroit et al., 2009, Bach et al., 2011a, Tork et al., 2013). The Gram-negative bacteria Aeromonas hydrophila K12, Chryseobacterium indologenes A22 and Serratia marcescens P3 showed potential for feather degradation and a preliminary study indicates that a major metalloprotease is produced by each strain during growth in feather meal (Bach et al., 2011b). This work describes the degradation of α- and β-keratins by these Gram-negative bacteria and also, the effect of pH and feather meal concentration on the enzyme production by C. indologenes A22.
Section snippets
Microorganisms
The strains A. hydrophila K12, C. indologenes A22 and S. marcescens P3 (Bach et al., 2011b) were used for degrading keratinous substrates. These bacteria were maintained as stock cultures in BHI broth containing 20% (v/v) glycerol at −20 °C, and replicated twice in fresh BHI broth before utilization in degradation experiments.
Media and culture conditions
All cultivation media contained 0.5 g l−1 NaCl, 0.3 g l−1 K2HPO4, 0.4 g l−1 KH2PO4 and 10 g l−1 of the keratinous substrate as the sole carbon and nitrogen source. Chicken
Degradation of keratinous wastes
Degradation of feathers and feather meal by the Gram-negative bacteria was initially investigated. As expected, the isolates K12 and P3 produced more soluble protein in FMB than in FB (Table 1). Feather meal is a thermal and mechanically processed waste and therefore should be more susceptible to degradation than feather. Interestingly, the isolate A22 showed higher production of soluble protein in FB than in FMB. Some indirect measurements are useful indicators of microbial keratinolytic
Conclusions
In this work, the degradation of β-keratin and most importantly, the degradation of α-keratin wastes (hair and wool) by the Gram-negative bacteria C. indologenes A22 and A. hydrophila K12 was described. The strains A22 and K12 degraded feathers easier than hair and wool, confirming that the α-keratin conformation is more difficult to be hydrolyzed by microbial keratinases. Moreover, C. indologenes A22 presented an optimal keratinase production at 30 °C, neutral pH and 15 g L−1 of feather meal
Acknowledgments
Authors thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, grant 305653/2009-3), Brazil for financial support.
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