Microbial keratinases have become biotechnologically important since they target the hydrolysis of highly rigid, strongly cross-linked structural polypeptide “keratin” recalcitrant to the commonly known proteolytic enzymes trypsin, pepsin and papain. These enzymes are largely produced in the presence of keratinous substrates in the form of hair, feather, wool, nail, horn etc. during their degradation. The complex mechanism of keratinolysis involves cooperative action of sulfitolytic and proteolytic systems. Keratinases are robust enzymes with a wide temperature and pH activity range and are largely serine or metallo proteases. Sequence homologies of keratinases indicate their relatedness to subtilisin family of serine proteases. They stand out among proteases since they attack the keratin residues and hence find application in developing cost-effective feather by-products for feed and fertilizers. Their application can also be extended to detergent and leather industries where they serve as specialty enzymes. Besides, they also find application in wool and silk cleaning; in the leather industry, better dehairing potential of these enzymes has led to the development of greener hair-saving dehairing technology and personal care products. Further, their prospective application in the challenging field of prion degradation would revolutionize the protease world in the near future.

Microbial keratinases and their prospective applications: an overview.

Fungi are an understudied, biotechnologically valuable group of organisms. Due to 
the immense range of habitats that fungi inhabit, and the consequent need to compete against a diverse array of other fungi, bacteria, and animals, fungi have developed numerous survival mechanisms. The unique attributes of fungi thus herald great promise for their application in biotechnology and industry. Moreover, fungi can be grown with relative ease, making production at scale viable. The search for fungal biodiversity, and the construction of a living fungi collection, both have incredible economic potential in locating organisms with novel industrial uses that will lead to novel products. This manuscript reviews fifty ways in which fungi can potentially be utilized as biotechnology. We provide notes and examples for each potential exploitation and give examples from our own work and the work of other notable researchers. We also provide a flow chart that can be used to convince funding bodies of the importance of fungi for biotechnological research and as potential products. Fungi have provided the world with penicillin, lovastatin, and other globally significant medicines, and they remain an untapped resource with enormous industrial potential.

The amazing potential of fungi: 50 ways we can exploit fungi industrially

Keratinases are exciting proteolytic enzymes that display the capability to degrade the insoluble protein keratin. These enzymes are produced by diverse microorganisms belonging to the Eucarya, Bacteria, and Archea domains. Keratinases display a great diversity in their biochemical and biophysical properties. Most keratinases are optimally active at neutral to alkaline pH and 40–60°C, but examples of microbial keratinolysis at alkalophilic and thermophilic conditions have been well documented. Several keratinases have been associated to the subtilisin family of serine-type proteases by analysis of their protein sequences. Studies with specific substrates and inhibitors indicated that keratinases are often serine or metalloproteases with preference for hydrophobic and aromatic residues at the P1 position. Keratinolytic enzymes have several current and potential applications in agroindustrial, pharmaceutical, and biomedical fields. Their use in biomass conversion into biofuels may address the increasing concern on energy conservation and recycling.

Biochemical features of microbial keratinases and their production and applications.

Biodegradation of keratin waste: Theory and practical aspects

Keratinous materials such as wool, feathers and hooves are tough unique biological co-products that usually have high sulfur and protein contents A high cystine content (7–13%) differentiates keratins from other structural proteins, such as collagen and elastin Dissolution and extraction of keratin is a difficult process compared to other natural polymers, such as chitosan, starch, collagen, and a large-scale use of keratin depends on employing a relatively fast, cost-effective and time efficient extraction method Keratin has some inherent ability to facilitate cell adhesion, proliferation, and regeneration of the tissue, therefore keratin biomaterials can provide a biocompatible matrix for regrowth and regeneration of the defective tissue Additionally, due to its amino acid constituents, keratin can be tailored and finely tuned to meet the exact requirement of degradation, drug release or incorporation of different hydrophobic or hydrophilic tails This review discusses the various methods available for the dissolution and extraction of keratin with emphasis on their advantages and limitations The impacts of various methods and chemicals used on the structure and the properties of keratin are discussed with the aim of highlighting options available toward commercial keratin production This review also reports the properties of various keratin-based biomaterials and critically examines how these materials are influenced by the keratin extraction procedure, discussing the features that make them effective as biomedical applications, as well as some of the mechanisms of action and physiological roles of keratin Particular attention is given to the practical application of keratin biomaterials, namely addressing the advantages and limitations on the use of keratin films, 3D composite scaffolds and keratin hydrogels for tissue engineering, wound healing, hemostatic and controlled drug release

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Keratin: dissolution, extraction and biomedical application

The fungus Doratomyces microsporus produced an extracellular keratinase during submerged aerobic cultivation in a medium containing a protein inducer for enzyme synthesis. The keratinase was purified to homogeneity using hydrophobic interaction chromatography followed by gel chromatography. The molecular weight was estimated to be 33 kDa (from SDS-PAGE analysis) or 30 kDa (by gel chromatography), suggesting a monomeric structure. The isoelectric point of the enzyme was determined to be around 9. The optimal pH and temperature for keratinolytic activity were pH 8-9 and 50 degrees C, respectively. The serine protease inhibitor PMSF totally inhibited the keratinase. The enzyme was not glycosylated. It was capable of hydrolysing different keratinous materials as well as some non-keratinous proteins. Hydrolysis of some synthetic substrates, specific for known proteinases, suggested that the keratinase of D. microsporus is close to proteinase K.

Keratinase of Doratomyces microsporus.


 Crossbred pigs (Fast Genetics 276 × PIC 800; n = 2,388; initial BW = 74.1 ± 2.0 kg) were used in two, 63-d growth trials to evaluate the addition of a water source (fixed double nipple waterer) to wet/dry feeders on finishing pig performance. Pigs were assigned randomly to 1 of 2 treatments with 27 to 30 pigs per pen and 40 pens per treatment. Pens were equipped with a wet/dry feeder (SDI, drop shelf wet/dry feeder) with no supplemental water or a wet/dry feeder with a supplemental water source available from 74 kg BW until marketing. Growth data were analyzed using the GLIMMIX procedure with repeated measures over time. The model included fixed effects of water treatment, trial, and time with all possible two-way interactions. Removals, mortalities, and total removals were analyzed using a binomial distribution. No differences (P > 0.05) in body weight were observed initially or at the last marketing event between water source treatments; however, a supplemental water source tended (P = 0.061) to decrease average market weight compared to pigs not given a supplemental waterer (Table). Overall average daily gain (ADG) was lower (P = 0.011) and average daily feed intake (ADFI) was decreased (P = 0.002) for pigs given a supplemental waterer compared to pigs only drinking from a wet/dry feeder. No differences were observed for the percentage of mortalities, pigs removed, or total removals (P > 0.05) regardless of treatment. Finally, daily water disappearance was 2.5 liters greater for pigs with a supplemental waterer compared to pigs with no supplemental waterer. In conclusion, the addition of a supplemental water source to wet/dry feeders decreased growth performance.


S. Kern

Papers

Keratinase of Doratomyces microsporus.

PSII-12 Effect of a Supplemental Water Source on Growth Performance of Finishing Pigs