STUDYING THE DYNAMICS OF KERATINASES IN IONIC LIQUIDS FOR SUSTAINABLE HUMAN HAIR KERATIN EXTRACTION

Abstract

The increasing concern regarding degradation of keratinaceous wastes, specifically human hair, because of its exceptional durability, has currently made it an emerging environmental problem. Keratin is a fibrous structural protein found abundantly in hair, nails, feathers, and other animal tissues; it owes its recalcitrant nature to tightly packed polypeptide chains stabilized by strong disulfide linkages. This physicochemical stability causes keratin-rich wastes to persist in the environment, contributing to their accumulation in landfills and drainage systems, hence causing problems in sanitation, blocking urban drainage, and ecological imbalance. Decomposition of human hair waste, which is mainly generated from barber shops and salons, is very slow under natural conditions and may release fine particulates that pose respiratory health risks upon inhalation. Other significant sources of keratin-rich wastes come from slaughterhouses, poultry farms, and wool processing units, improper waste disposal from which may facilitate the proliferation of pathogens, thereby jeopardizing environmental and public health. Amongst all, biological approaches to keratinous waste management have come out to be of great significance due to their eco-friendly potential compared to conventional chemical and physical treatments. Certain microorganisms have developed special enzymatic mechanisms for the degradation of keratin. These organisms secrete a class of serine- or metalloproteases, keratinases (EC 3.4.21/24/99), which are able to hydrolyze the resistant structure of keratin into simpler peptides and amino acids. Unlike regular proteases, which cannot degrade keratin, the activity of keratinases involves disruption of disulfide bonds, thereby allowing bioconversion of keratinous materials. Besides playing a significant role in the natural recycling of keratin, keratinases provide potential for various industrial applications such as organic fertilizer, animal feed additives, cosmetics ingredients, and biomedical materials. Current research interest is generally focused on studies of their catalytic mechanism, screening of novel keratinases with high activity, and the application of protein engineering to modify their functionality and stability for industrial applications. Human hair, containing about 65–95% keratin depending on geography and individual genetics, has recently become a promising biomaterial. The excellent biocompatibility, low immunogenicity, and spontaneous self-assembly driven by its high cysteine content make human hair keratin an attractive biomaterial for regenerative medicine, drug delivery, and tissue engineering applications. In this context, various functional materials, such as hydrogels, scaffolds, and films, have been successfully prepared with human hair keratin, illustrating the great value of human hair keratin as a renewable biopolymer. The extraction of keratin remains difficult due to the characteristics of insolubility and strong 5 structural organization, so environmentally benign methods are under exploration to assist the recovery of keratin. Ionic Liquids (ILs), sometimes referred to as "green" or "designer solvents," are usually defined as salts that are liquid below 100 °C and are composed of a wide range of combinations of organic cations and inorganic or organic anions. Tunable ILs allow millions of possible variants, each with a specific solvating capability. They are characterized by non-volatility, non-flammability, low vapor pressure, and excellent thermal stability, presenting themselves as potentially viable alternatives to conventional organic solvents. Their multifunctional interactions, such as electrostatic, hydrogen bonding, van der Waals, and π-π interactions, allow the disruption of covalent and non covalent bonds present in complex biomolecules, therefore allowing the dissolution of recalcitrant materials like keratin. Various ILs have been used for the dissolution of human hair, including tetramethylammonium hydroxide (TMAOH), tetrabutylammonium hydroxide (TBAH), choline hydroxide (Ch-OH), and several imidazolium-based ILs, such as [BMIM]Cl, [BMIM]Br, and [AMIM]Cl. Due to their higher extraction efficiency, reusability, and mild operating conditions when compared to water and other organic solvents, this class of solvent now represents a sustainable tool in biopolymer recovery. Given that both keratinases and ILs degrade human hair, an integrated approach using enzymatic hydrolysis together with IL-mediated dissolution might offer an eco-friendly route for extracting keratin. The present study investigates the stability and possible compatibility of keratinases in a range of ILs to find the best match for combined use. Computational and experimental investigations so far have indicated that ILs can both stabilize and destabilize protein conformation depending on their ionic structure and thus have to be analyzed systematically for enzyme performance. In this context, our study focuses on three bacterial keratinases-ker6 from Bacillus cereus, kerC from Bacillus subtilis, and kerF from Stenotrophomonas maltophilia-to investigate their conformational stability in IL systems previously reported to dissolve hair. Protein sequences were retrieved from the UniProt database. Since no experimentally determined structures existed in the Protein Data Bank for these enzymes, high-confidence tertiary structure predictions were generated using AlphaFold2, a state-of-the-art artificial intelligence system capable of resolving protein structures from amino acid sequences with near-atomic accuracy. Subsequent stereochemical validation was performed using the PDBSum server to verify structural integrity and active site analysis via PyMOL. Six ionic liquids-Tetrabutylammonium hydroxide, Tetramethylammonium hydroxide, Choline hydroxide, 1-butyl-3-methylimidazolium bromide, 1-butyl-3-methylimidazolium chloride, and 1-allyl-3-methylimidazolium chloride-were shortlisted based on their documented ability to disrupt the keratin matrix. Their three-dimensional structures were collected from the PubChem database, and system preparation was carried out using the CHARMM-GUI web interface, ensuring correct atom typing and force field 6 compatibility for all components. Molecular dynamics simulations were then performed to analyze the stability of these keratinases in water and in six selected human hair-degrading ILs. The simulation parameters, including root-mean-square deviation (RMSD), root mean-square fluctuation (RMSF), radius of gyration (Rg), solvent-accessible surface area (SASA), and intra-protein hydrogen bonding, were evaluated to assess conformational stability and solvent interactions. The results showed that molecular stability and residue-level flexibility of keratinases were strongly dependent on the solvent used. Results from molecular dynamics simulations of three keratinases-ker6, kerC, and kerF-in water and different ionic liquids have shown that this solvent dependency affects enzyme structural stability that is important for sustainable extraction of keratin. ker6 exhibited the highest stability in tetramethylammonium hydroxide (TMAOH), characterized by reduced backbone fluctuations, increased intra protein hydrogen bonding, and compact structure as reflected by the low radius of gyration and solvent-accessible surface area. In contrast, kerC showed optimal stability in 1-allyl-3 methylimidazolium chloride (AMIMCl), maintaining low structural deviations and compactness, while kerF remained stable in both AMIMCl and 1-butyl-3 methylimidazolium bromide (BMIMBr), showing uniform maintenance of structural integrity. Other tested ionic liquids resulted in increased fluctuations and destabilization of these keratinases, underlining the specificity in enzyme-solvent interactions. Understanding how keratinases behave in various ILs will help to shed light on the feasibility of combining these biocatalysts with IL extraction systems for sustainable and efficient recovery of human hair keratin. The outcome of such integrated biocatalytic and green solvent systems may pioneer new methodologies for waste valorization, reduce environmental contamination, and facilitate circular bioeconomy strategies by converting underutilized keratinaceous wastes into high-value biomaterials. Collectively, this thesis provides an advanced understanding of microbial keratinase behavior in designer solvents regarding biowaste valorization and offers a scientific basis for sustainable, scalable keratin extraction. The molecular insights that have been gained are promising directions for the rational design of enzyme-IL combinations, hence potentiating the upcycling of human hair into next-generation biomaterials for applications such as wound healing, drug delivery, tissue engineering, and environmental remediation.