Biology 2022, 11, 244. https://doi.org/10.3390/biology11020244 www.mdpi.com/journal/biology Article Isolation of Bacillus sp. A5.3 Strain with Keratinolytic Activity Saniya Aktayeva 1,2 , Kairat Baltin 1 , Assel Kiribayeva 1 , Zhiger Akishev 1 , Dmitriy Silayev 1 , Yerlan Ramankulov 1 and Bekbolat Khassenov 1, * 1 Laboratory of Genetics and Biochemistry of Microorganisms, National Center for Biotechnology, Nur‐Sultan 010000, Kazakhstan; aktayevasa@gmail.com (S.A.); Baltin@biocenter.kz (K.B.); kiribayeva@biocenter.kz (A.K.); akishev@biocenter.kz (Z.A.); silayev@biocenter.kz (D.S.); ramanculov@biocenter.kz (Y.R.) 2 Department of General Biology and Genomics, L.N. Gumilyov Eurasian National University, Nur‐Sultan 010000, Kazakhstan * Correspondence: khassenov@biocenter.kz Simple Summary: In this study, we described keratinolytic properties of a strain of Bacillus (sp. A5.3) isolated from sites of feather waste accumulation. The proteolytic enzymes secreted by Ba‐ cillus sp. A5.3 are serine proteases, are alkaline enzymes, have a wide substrate specificity, and have high thermal stability. Bacillus sp. A5.3 effectively hydrolyzes feathers and can be used in feather‐processing technologies and as a source of alkaline and thermostable proteases and keratinases. Abstract: Environmental safety and economic factors necessitate a search for new ways of pro‐ cessing poultry farm feathers, which are 90% β‐keratin and can be used as a cheap source of amino acids and peptones. In this study, feather‐decomposing bacteria were isolated from a site of ac‐ cumulation of rotten feathers and identified as Bacillus. Among them, the Bacillus sp. A5.3 isolate showed the best keratinolytic properties. Scanning electron microscopy indicated that Bacillus sp. A5.3 cells closely adhere to the feather surface while degrading the feather. It was found that Ba‐ cillus sp. A5.3 secretes thermostable alkaline proteolytic and keratinolytic enzymes. Zymographic analysis of the enzymatic extract toward bovine serum albumin, casein, gelatin, and β‐keratin revealed the presence of proteases and keratinases with molecular weights 20–250 kDa. The pro‐ teolytic and keratinolytic enzymes predominantly belong to the serine protease family. Proteome analysis of the secreted proteins by nano‐HPLC coupled with Q‐TOF mass spectrometry identified 154 proteins, 13 of which are proteases and peptidases. Thus, strain Bacillus sp. A5.3 holds great promise for use in feather‐processing technologies and as a source of proteases and keratinases. Keywords: feathers; protease; keratinase; Bacillus; proteomics 1. Introduction Feather biomass consists of 90% of protein [1], which is a valuable biological prod‐ uct, and feather hydrolysates can be a source of peptones [2,3]. The feather is rich in es‐ sential amino acids leucine, valine, arginine, isoleucine, phenylalanine, and threonine, with smaller proportions of lysine, methionine, histidine, and tryptophan [4,5]. Sul‐ fur‐containing cysteine and methionine, together with threonine, tyrosine, and phenyl‐ alanine, are important for the biosynthesis of hair and feather keratin [6]. Unfortunately, feather proteins consist of insoluble protein extensively cross‐linked by disulfide bonds, e.g., β‐keratin [7]. Due to a sharp reduction in biological resources, the use of feather hydrolysates as a source of amino acids and peptones is relevant for biotechnological industries [8]. On the other hand, feather keratin is insoluble in water and has low digestibility by enzymes of the pepsin family due to disulfide bonds, hydrogen bonds, and hydrophobic interactions be‐ Citation: Aktayeva, S.; Baltin, K.; Kiribayeva, A.; Akishev, Z.; Silayev, D.; Ramankulov, Y.; Khassenov, B. Isolation of Bacillus sp. A5.3 Strain with Keratinolytic Activity. Biology 2022, 11, 244. https://doi.org/10.3390/ biology11020244 Academic Editor: Ansgar Poetsch Received: 15 December 2021 Accepted: 2 February 2022 Published: 4 February 2022 Publisher’s Note: MDPI stays neu‐ tral with regard to jurisdictional claims in published maps and insti‐ tutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses /by/4.0/).