Structure, Application, and Biochemistry of Microbial Keratinases

Abstract

Keratinases belong to a class of proteases that are able to degrade keratins into amino acids. Microbial keratinases play important roles in turning keratin-containing wastes into value-added products by participating in the degradation of keratin. Keratin is found in human and animal hard tissues, and its complicated structures make it resistant to degradation by common proteases. Although breaking disulfide bonds are involved in keratin degradation, keratinase is responsible for the cleavage of peptides, making it attractive in pharmaceutical and feather industries. Keratinase can serve as an important tool to convert keratin-rich wastes such as feathers from poultry industry into diverse products applicable to many fields. Despite of some progress made in isolating keratinase-producing microorganisms, structural studies of keratinases, and biochemical characterization of these enzymes, effort is still required to expand the biotechnological application of keratinase in diverse fields by identifying more keratinases, understanding the mechanism of action and constructing more active enzymes through molecular biology and protein engineering. Herein, this review covers structures, applications, biochemistry of microbial keratinases, and strategies to improve its efficiency in keratin degradation.

Keywords: keratin; keratinase; microorganisms; protease; structure; waste treatment.

FIGURE 1

A strategy to screen keratinase-producing microorganisms. In this strategy, a method to identify keratinase-producing strain is critical. Molecular biology, bioinformatics, biochemistry, and sensitive analytical methods such as MS are critical in the screening. In addition, as keratin degradation is a complicated step, sample isolation and enriching steps are important to make sure that the desired strains are sustained. Many keratinase-producing strains have been isolated and identified from the environment (Nnolim and Nwodo, 2021).

FIGURE 2

A simplified diagram showing the degradation of keratin by proteases. Two-step disulfide bond breakage and polypeptide degradation are usually included in keratin degradation. Keratins are simplified as helices. The degradation includes the steps such as releasing of keratin and degradation of keratin by multiple enzymes, which has been described in several reviews (Korniłłowicz-Kowalska and Bohacz, 2011; Sharma and Devi, 2018; Vidmar and Vodovnik, 2018; Qiu et al., 2020).

FIGURE 3

Crystal structures of microbial keratinases. The crystal structures of five keratinases were shown to understand their mechanism of action. The PDB access codes of the structures are indicated. The amino acids in active sites are highlighted in sticks and labeled with sequence numbers. Ca²⁺ and Co²⁺ atoms in the structures are shown as yellow and green spheres, respectively. In the crystal structure of fervidolyis (PDB:1R6V) (Kim et al., 2004), His208 was mutated into Ala. Ala 208 was labeled as His208 in this figure to show the active site. The β-sheet structures in FisCP (PDB: 5E3X) are highlighted in blue (Lee et al., 2015). All the figures were made using PyMOL (www.pymol.org). The details of the structures can be found in the reports (Betzel et al., 2001; Kim et al., 2004; Teruo et al., 2015; Wu et al., 2017).

FIGURE 4

Structure of rMtaKer and its insights into protease and substrate interactions. Surface presentation of one keratinase in the absence and presence of a peptide sequence binding to the active site. The crystal structure of the protease (PDB ID 5WSL) is shown (Wu et al., 2017). The orientation of the figure is similar to those in Figure 3. The residues forming the catalytic triad are shown in green, and the peptide from the adjacent molecule in the crystal structure is shown in sticks. The peptide sequence is shown as sticks in the figure.

FIGURE 5

Application of microbial keratinases in different fields. Applications of keratinases and their products are highlighted in green, which has been introduced in multiple reports (Onifade et al., 1998; Gupta and Ramnani, 2006; Brandelli et al., 2010; Chaturvedi et al., 2014; Sahni et al., 2015; Sharma and Gupta, 2016; Vidmar and Vodovnik, 2018).