Production and characterization of a novel thermo- and detergent stable keratinase from Bacillus sp. NKSP-7 with perceptible applications in leather processing and laundry industries

https://doi.org/10.1016/j.ijbiomac.2020.07.146Get rights and content

Highlights

  • Extracellular keratinase from Bacillus sp. NKSP-7 is purified and characterized.

  • Keratinase is a serine and monomeric protein with a molecular mass of 25 kDa.

  • Purified keratinase exhibited optimal activity at 65 °C and pH 7.5.

  • It showed stability over a range of pH (5.5–9.5) and temperature (20–60 °C) for 8 h.

  • Crude enzyme displayed potential application in leather and laundry industry.

Abstract

Keratinase has the ability to degrade the recalcitrant keratinous wastes that cannot be degraded by conventional proteases. The present study describes a novel hyperstable keratinolytic enzyme from Bacillus sp. NKSP-7, which has excellent efficiency of keratin-feather biodegradation, washing and dehairing. The production of extracellular keratinase was improved by 3.02-fold through optimization of various parameters. Purified keratinase (25 kDa) showed optimal activity at 65 °C and pH 7.5, and displayed stability over a range of pH (5.5–9.5) and temperature (20–60 °C) for 8 h. No conspicuous effect was perceived with various chemicals and organic solvents, however, the catalytic efficiency was enhanced in the presence of Ca2+, Cd2+, Na+, Mn2+, sodium sulfite, and β-mercaptoethanol. The enzyme was completely inhibited by phenylmethanesulfonyl fluoride (PMSF), suggesting that this keratinase belongs to serine protease family. It displayed prodigious stability and compatibility to salinity and commercial detergents. Enzyme exhibited great substrate specificity but high affinity was observed with keratin-rich substrates. Crude and purified keratinase revealed perceptible potential for destaining of blood-stained fabric (10 min), and dehairing of hide (8 h) without any damage. All these auspicious features make this enzyme a promising candidate for various industrial applications, especially in keratin-waste management, detergent formulations and leather processing.

Introduction

With the advent of modern industrial era, where the mankind has technologically revolutionized, many environmental and health problems have also been arisen due to various chemicals that are being used for carrying out various industrial processes, and this detriment has called for safer and eco-friendly alternatives. Replacing the chemical catalysis with bio-catalysis is one of the effective solution to cope with this problem. Several microbial biocatalysts can be effectively employed in various biotechnological and industrial processes. Keratinase is one of the imperative biocatalyst, which has a unique ability to degrade highly recalcitrant, tough and abundant proteins known as keratins, which constitute various epidermal structures of vertebrates like feathers, hair, nails, beaks, horns, hooves, scales and wool [1]; and are found abundantly in different dumping sites. Annually, millions of tons of keratinous wastes are generated from poultry, husbandry, herding and related processing industries that highlight a potential use of keratinolytic enzymes for solid waste management [2].

Keratins are fibrous, insoluble and highly stable proteins that are copiously present in nature and are divided into two groups based on their secondary structure viz. α-helix that is present in hair and wool, and β-sheet conformation which is largely found in feathers [3]. On the basis of sulfur content, they are classified as: (i) soft keratins (in skin and callus), which have less disulphide bonds and hence are less tough, and (ii) hard keratins (in horns, hoofs and feathers), which have larger quantity of disulphide bonds in their structures and resultantly are tougher and more complex. The highly complex structure of keratin consists of supercoiled polypeptide chains, cross-linked hydrogen bonds, hydrophobic interactions and disulphide bridges that provide them mechanical stability and make them resistant to various conventional proteolytic enzymes (pepsin, trypsin and papain) as well as to chemical agents [4]. This recalcitrant behavior of these proteins makes them difficult to be degraded by conventional proteases, but fortunately, nature has given a great solution for this problem i.e. keratinolytic enzymes.

Keratinases (E.C. 3.4.21/24/99.11), a group of proteolytic enzymes (serine and/or metallo-proteases), are extracellular inducible biocatalysts specific for keratin-based substrates. They have gained much attention not only because of their stability over a range of temperature and pH, but also due to their implausible affinity towards various insoluble and complex keratin-based substrates as compared to other proteases [5,6]. The mechanism of enzyme adsorption is still not clearly understood; however, it is known that the adsorption capacity of enzyme is directly proportional to the degree of keratin hydrolysis [7]. They have excellent ability to completely degrade recalcitrant keratinous wastes which makes them prospective candidates for solid waste management and several other imperative industrial bio-processes.

Keratinases are ubiquitous in nature and produced by several bacteria, actinomyces, and fungi especially dermatophytes. Keratinolytic bacterial enzymes have acquired far more attention from industries and researchers as compared to the enzymes from other sources, because of their specific properties and ease of large-scale production [8]. Due to their effectiveness on feather degradation, keratinolytic enzymes from the genus Bacillus have been studied extensively [5,9]. Generally, keratin-feathers are converted to animal feedstuff through chemical and hydrothermal treatment [10], which result in the great loss of essential amino acids residues (histidine, lysine, tryptophan and methionine). Chemical treatment of keratin-waste has caused negative impact on environment, and is considered as an eco-destructive process. However, keratinolytic enzymes represent a promising environmentally sound and cost-effective alternative technology for recycling keratinous byproducts (wastes), especially poultry feathers, leading to the production of products with upgraded nutritional value [11].

Biodegradation of feathers by keratinase is found to be an efficient method to attain useful feather-lysate (contains free amino acids and peptides) that can be employed as a nitrogenous fertilizers for plants, and as a protein-rich meal for animals [2]. Bacterial keratinases have been recognized for various potential biotechnological and industrial applications such as bioprocessing of agro-industrial wastes, detergent formulation, cosmetic formulations (hair-removing creams and lotions, etc.), food, textile, bio-fertilizers (nitrogen and minerals rich) production, feedstuff, fiber modification, manufacturing of biodegradable films, wastes management, and manufacturing of glues by hydrolyzing the keratin substrates [2,7,12,13]. Keratinases are being used for the manufacturing of protein supplements and biomedical products [6,13]; and for the processing of edible bird's nests, pearl bleaching, and for the production of biogas and bioenergy [7]. They also have pharmaceutical and medical applications as enhancing the drug delivery system, prion degradation, earwax removal, personal hygiene products, transfer accelerators, treatment of skin ailments (psoriasis and acne), for fungal infections especially nail disorder (onychomycosis), removal of human callus and as vaccines against dermatophytosis [7,12]. Furthermore, they have been studied as an active component of pesticide against root-knot nematodes [14].

Among active industries, leather and tanneries have recognized the usage of keratinase as an effective enzyme for dehairing/depilation process that can be an efficient alternative to the detrimental chemicals [8,9,15]. Conventionally, different harmful chemicals including soda lime, sulfur substances, salts, chromate, emulsified fatty matter and waste liquid lime are used for dehairing in leather industries, which are not only corrosive and hazardous for health but are also toxic for the environment and cause enough pollution [16]. Moreover, the usage of conventional proteases (alkaline proteases) for dehairing process mars the quality of leather because of low substrate specificity of these enzymes [17]. Additionally, the dehairing efficiency declines because the keratin in the hair resists the common proteases [15]. Due to these issues, many leather industries are now focusing on non-toxic and sustainable alternatives for dehairing processes and one of these is the microbial keratinolytic enzymes.

Industrially significant biocatalyst must have the ability to withstand harsh processing conditions. Therefore, it has become a priority of the research world to discover new enzymes that exhibit astounding stability at elevated temperature and variant pH. Previously, we had isolated an efficient keratinolytic bacterial strain Bacillus sp. NKSP-7 from the soil of a poultry dumping site (Punjab, Pakistan). Bacillus sp. NKSP-7 exhibited great potential to degrade whole chicken feathers within 24 h and produced extracellular keratinase under submerged fermentation [18]. The present study is focused to increase the production of keratinase from Bacillus sp. NKSP-7 through optimization of various cultivation parameters that have played essential role to enhance the growth of bacterium and revamp the targeted protein. Keratinase enzyme was completely characterized after purification; and the biocatalytic applications of crude and purified enzyme for cleaning of blood-stained cloth and dehairing of goat skin are also discussed.

Section snippets

Substrates and chemicals

All the required chemicals, reagents, components of media, salts, substrates, surfactants, and organic solvents were obtained from Merck (Darmstadt, Germany), Fluka (Switzerland) and Sigma-Aldrich Co. (St Louis, USA), unless otherwise specified. Protein marker was acquired from Novogen (Madison, WI, USA).

Keratinolytic bacterial strain

In this study, a newly isolated keratinolytic bacterium Bacillus sp. NKSP-7 was used to produce extracellular keratinase enzyme as described in earlier study [18]. Glycerol stocks of Bacillus

Optimal growth medium for keratinase production

Keratinolytic bacterium Bacillus sp. NKSP-7 was cultivated in sixteen different media supplemented with 1% (w/v) feathers to screen the best growth medium for enzyme production and feathers degradation. Maximum extracellular keratinolytic activity (51.50 U mL−1) and biodegradation of feathers (89.51%) were observed in a modified medium 2 × NB, which seemed to be due to higher cell density. So, high-yield of bacterial density (14.78) and DCW (8.91 g L−1) were achieved in 2 × NB, which was

Conclusion

As hazardous chemical methods in various industrial processes including leather processing are causing some serious problems, they are required to be replaced with the non-toxic and eco-friendly alternatives. Therefore, in recent times, microbial biocatalyst has gained immense attention for numerous biotechnological and industrial applications. The present study describes the optimization of various cultivation parameters for optimal keratinase production, and purification and characterization

Compliance with ethical standards/ethical statement

The authors declare that they have no competing interests. We assure the integrity and quality of our research work. It is also stated that there is no plagiarism in this work and all points taken from other authors are well cited in the text. This study is completely independent and impartial.

Research involving human participants and/or animals

N/A. This research did not involve human participants and/or animals.

Informed consent

N/A. This research did not involve human participants.

Contribution of authors

F Akram wrote the manuscript, carried out experiments, analyzed the data and performed statistical analysis.

IU Haq: designed and conceptualized the study and supervised all work.

Z Jabbar: as a research assistant carried out some experiments work.

Declaration of competing interest

The authors declare that they have no conflict of interest.

Acknowledgements

This work was supported by a grant No. 5-9/PAS/727 from Pakistan Academy of Science, Islamabad, Pakistan.

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