Microbial keratinases: industrial enzymes with waste management potential

Proteases are ubiquitous enzymes that occur in various biological systems ranging from microorganisms to higher organisms. Microbial proteases are largely utilized in various established industrial processes. Despite their numerous industrial applications, they are not efficient in hydrolysis of recalcitrant, protein-rich keratinous wastes which result in environmental pollution and health hazards. This paved the way for the search of keratinolytic microorganisms having the ability to hydrolyze “hard to degrade” keratinous wastes. This new class of proteases is known as “keratinases”. Due to their specificity, keratinases have an advantage over normal proteases and have replaced them in many industrial applications, such as nematicidal agents, nitrogenous fertilizer production from keratinous waste, animal feed and biofuel production. Keratinases have also replaced the normal proteases in the leather industry and detergent additive application due to their better performance. They have also been proved efficient in prion protein degradation. Above all, one of the major hurdles of enzyme industrial applications (cost effective production) can be achieved by using keratinous waste biomass, such as chicken feathers and hairs as fermentation substrate. Use of these low cost waste materials serves dual purposes: to reduce the fermentation cost for enzyme production as well as reducing the environmental waste load. The advent of keratinases has given new direction for waste management with industrial applications giving rise to green technology for sustainable development.     

Microbial keratinases and their prospective applications: an overview

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.     

Similarities and specificities of fungal keratinolytic proteases: comparison of keratinases of Paecilomyces marquandii and Doratomyces microsporus to some known proteases

Based on previous screening for keratinolytic nonpathogenic fungi, Paecilomyces marquandii and Doratomyces microsporus were selected for production of potent keratinases. The enzymes were purified and their main biochemical characteristics were determined (molecular masses, optimal temperature and pH for keratinolytic activity, N-terminal amino acid sequences). Studies of substrate specificity revealed that skin constituents, such as the stratum corneum, and appendages such as nail but not hair, feather, and wool were efficiently hydrolyzed by the P. marquandii keratinase and about 40% less by the D. microsporus keratinase. Hydrolysis of keratin could be increased by the presence of reducing agents. The catalytic properties of the keratinases were studied and compared to those of some known commercial proteases. The profile of the oxidized insulin B-chain digestion revealed that both keratinases, like proteinase K but not subtilisin, trypsin, or elastase, possess broad cleavage specificity with a preference for aromatic and nonpolar amino acid residues at the P-1 position. Kinetic studies were performed on a synthetic substrate, succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. The keratinase of P. marquandii exhibited the lowest Km among microbial keratinases reported in the literature, and its catalytic efficiency was high in comparison to that of D. microsporus keratinase and proteinase K. All three keratinolytic enzymes, the keratinases of P. marquandii and D. microsporus as well as proteinase K, were significantly more active on keratin than subtilisin, trypsin, elastase, chymotrypsin, or collagenase.     

Biochemical features of microbial keratinases and their production and applications

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.     

Similarities and Specificities of Fungal Keratinolytic Proteases: Comparison of Keratinases of Paecilomyces marquandii and Doratomyces microsporus to Some Known Proteases

Based on previous screening for keratinolytic nonpathogenic fungi, Paecilomyces marquandii and Doratomyces microsporus were selected for production of potent keratinases. The enzymes were purified and their main biochemical characteristics were determined (molecular masses, optimal temperature and pH for keratinolytic activity, N-terminal amino acid sequences). Studies of substrate specificity revealed that skin constituents, such as the stratum corneum, and appendages such as nail but not hair, feather, and wool were efficiently hydrolyzed by the P. marquandii keratinase and about 40% less by the D. microsporus keratinase. Hydrolysis of keratin could be increased by the presence of reducing agents. The catalytic properties of the keratinases were studied and compared to those of some known commercial proteases. The profile of the oxidized insulin B-chain digestion revealed that both keratinases, like proteinase K but not subtilisin, trypsin, or elastase, possess broad cleavage specificity with a preference for aromatic and nonpolar amino acid residues at the P-1 position. Kinetic studies were performed on a synthetic substrate, succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. The keratinase of P. marquandii exhibited the lowest K_m among microbial keratinases reported in the literature, and its catalytic efficiency was high in comparison to that of D. microsporus keratinase and proteinase K. All three keratinolytic enzymes, the keratinases of P. marquandii and D. microsporus as well as proteinase K, were significantly more active on keratin than subtilisin, trypsin, elastase, chymotrypsin, or collagenase.     

Biotechnological applications and prospective market of microbial keratinases

Keratinases are well-recognized enzymes with the unique ability to attack highly cross-linked, recalcitrant structural proteins such as keratin. Their potential in environmental clean-up of huge amount of feather waste has been well established since long. Today, they have gained importance in various other biotechnological and pharmaceutical applications. However, commercial availability of keratinases is still limited. Hence, to attract entrepreneurs, investors and enzyme industries it is utmost important to explicitly present the market potential of keratinases through detailed account of its application sectors. Here, the application areas have been divided into three parts: the first one is dealing with the area of exclusive applications, the second emphasizes protease dominated sectors where keratinases would prove better substitutes, and the third deals with upcoming newer areas which still await practical documentation. An account of benefits of keratinase usage, existing market size, and available commercial sources and products has also been presented.     

Versatility and commercial status of microbial keratinases: a review

The world’s increasing population and shortage of food and feed is creating an urgently for us to look for new protein sources from waste products like keratinous waste. Poor management of these wastes has made them one of the major recalcitrant pollutants in nature. Microbial keratinases offers an economic and eco-friendly alternative for degrading and recycling keratinous waste into valuable byproducts. Diverse groups of microorganisms viz., bacteria, fungi and actinomycetes have the ability to degrade recalcitrant keratin by producing keratinase enzyme. Microbial keratinases exhibits great diversity in its biochemical properties with respect to activity and stability in various pH and temperature ranges as well as in the range of recalcitrant proteins it degrades like those present in feathers, hairs, nails, hooves etc. Owing to diverse properties and multifarious biotechnological implications, keratinases can be considered as promising biocatalysts for preparation of animal nutrients, protein supplements, leather processing, fiber modification, detergent formulation, feather meal processing for feed and fertilizer, the pharmaceutical, cosmetic and biomedical industries, and waste management. This review article presents an overview of keratin structure and composition, mechanism of microbial keratinolysis, diversity of keratinolytic microorganisms, and their potential applications in various fields.     

Exocellular proteases of Malbranchea gypsea and their role in keratin deterioration

Malbranchea gypsea IMI 338168 isolated from the soils of Keoladeo National Park, Bharatpur was studied for its ability to produce exocellular proteases on glucose – gelatin medium at pH 7; 28°C. The fungus was observed to be a potent producer of such enzymes. Protease production was optimal at 15 days of incubation. Asparagine was repressive to protease expression. No relationship existed between the amount of enzyme production and increase in biomass. Exogenous sugars suppressed enzyme production in descending order as follows: glucose > mannose > maltose > arabinose > fructose. The enzymes expressed showed the ability to degrade three keratinous substrate tested. Buffalo skin was the most actively degraded substrate when exogenous glucose was present, and was also the most resistant to degradation in the absence of glucose. The rate of keratin deterioration was independent of enzyme activity. Production of protease enzymes especially keratinases is ecologically important in a place like a National Park because such enzymes degrade keratinous detritus derived from mammals and birds. Accumulation of such materials can be a cause of pollution and can provide a breeding spot for various types of pathogens. This revised version was published online in June 2006 with corrections to the Cover Date.     

Profiling serine protease substrate specificity with solution phase fluorogenic peptide microarrays

A novel microarray-based proteolytic profiling assay enabled the rapid determination of protease substrate specificities with minimal sample and enzyme usage. A 722-member library of fluorogenic protease substrates of the general format Ac-Ala-X-X-(Arg/Lys)-coumarin was synthesized and microarrayed, along with fluorescent calibration standards, in glycerol nanodroplets on microscope slides. The arrays were then activated by deposition of an aerosolized enzyme solution, followed by incubation and fluorometric scanning. The specificities of human blood serine proteases (human thrombin, factor Xa, plasmin, and urokinase plasminogen activator) were examined. The arrays provided complete maps of protease specificity for all of the substrates tested and allowed for detection of cooperative interactions between substrate subsites. The arrays were further utilized to explore the conservation of thrombin specificity across species by comparing the proteolytic fingerprints of human, bovine, and salmon thrombin. These enzymes share nearly identical specificity profiles despite approximately 390 million years of divergent evolution. Fluorogenic substrate microarrays provide a rapid way to determine protease substrate specificity information that can be used for the design of selective inhibitors and substrates, the study of evolutionary divergence, and potentially, for diagnostic applications.     

微生物角蛋白酶的特性及其应用研究进展

角蛋白作为家禽加工和农业废弃物的主要成分,因其结构中富含能抵抗普通蛋白酶和化学催化剂降解的稳定交联二硫键而难以被利用,因此每年都在环境中大量积累,造成了严重的环境污染。微生物角蛋白酶可将角蛋白废弃物转化为可再次利用的产物,带来了经济的可行性及环境的可持续发展。本文主要综述了角蛋白酶的生物化学特性、角蛋白酶的基本结构及其表达特性,总结了其应用价值及角蛋白降解机制,最后展望了微生物角蛋白酶的进一步研究方向。     

PROSPER: An Integrated Feature-Based Tool for Predicting Protease Substrate Cleavage Sites

The ability to catalytically cleave protein substrates after synthesis is fundamental for all forms of life. Accordingly, site-specific proteolysis is one of the most important post-translational modifications. The key to understanding the physiological role of a protease is to identify its natural substrate(s). Knowledge of the substrate specificity of a protease can dramatically improve our ability to predict its target protein substrates, but this information must be utilized in an effective manner in order to efficiently identify protein substrates by in silico approaches. To address this problem, we present PROSPER, an integrated feature-based server for in silico identification of protease substrates and their cleavage sites for twenty-four different proteases. PROSPER utilizes established specificity information for these proteases (derived from the MEROPS database) with a machine learning approach to predict protease cleavage sites by using different, but complementary sequence and structure characteristics. Features used by PROSPER include local amino acid sequence profile, predicted secondary structure, solvent accessibility and predicted native disorder. Thus, for proteases with known amino acid specificity, PROSPER provides a convenient, pre-prepared tool for use in identifying protein substrates for the enzymes. Systematic prediction analysis for the twenty-four proteases thus far included in the database revealed that the features we have included in the tool strongly improve performance in terms of cleavage site prediction, as evidenced by their contribution to performance improvement in terms of identifying known cleavage sites in substrates for these enzymes. In comparison with two state-of-the-art prediction tools, PoPS and SitePrediction, PROSPER achieves greater accuracy and coverage. To our knowledge, PROSPER is the first comprehensive server capable of predicting cleavage sites of multiple proteases within a single substrate sequence using machine learning techniques. It is freely available at http://lightning.med.monash.edu.au/PROSPER/.     

Cloning,heterologous expression and characterization of two keratinases from Stenotrophomonas maltophilia BBE11-1

The keratin-degrading strain Stenotrophomonas maltophilia BBE11-1 secretes two keratinolytic proteases, KerSMD and KerSMF. However, the genes encoding these proteases remain unknown. Here, we have isolated these two genes with a modified TAIL-PCR (thermal asymmetric interlaced PCR) method based on the N-terminal amino acid sequences of mature keratinases. These two keratinase genes encode serine proteases with PPC (bacterial pre-peptidase C-terminal) domain, which are successfully expressed with the help of pelB leader in Escherichia coli cells. Recombinant KerSMD (48 kDa) shows a better activity in feather degradation, higher thermostability and substrate specificity than KerSMF (40 kDa). KerSMD has a t_1/2 of 90 min at 50 °C and 64 min at 60 °C, and a better tolerance to surfactants SDS and triton X-100. The predicted model of KerSMD helps to explain the phenomenon of auto-catalytic C-terminal propeptide truncation, the special function of PPC domain, and the molecular weight of the C-terminal-processed mature keratinase KerSMD. This work not only provides a new way to overproduce keratinases but also helps to explore keratinases folding mechanism.     

Homogenous photocatalytic decontamination of prion infected stainless steel and titanium surfaces

Prions are notorious for their extraordinary resistance to traditional methods of decontamination, rendering their transmission a public health risk. Iatrogenic Creutzfeldt–Jakob disease (iCJD) via contaminated surgical instruments and medical devices has been verified both experimentally and clinically. Standard methods for prion inactivation by sodium hydroxide or sodium hypochlorite have failed, in some cases, to fully remove prion infectivity, while they are often impractical for routine applications. Prion accumulation in peripheral tissues and indications of human-to-human bloodborne prion transmission, highlight the need for novel, efficient, yet user-friendly methods of prion inactivation. Here we show both in vitro and in vivo that homogenous photocatalytic oxidation, mediated by the photo-Fenton reagent, has the potential to inactivate the pathological prion isoform adsorbed on metal substrates. Photocatalytic oxidation with 224 μg mL⁻¹ Fe³⁺, 500 μg mL⁻¹ h⁻¹ H₂O₂, UV-A for 480 min lead to 100% survival in golden Syrian hamsters after intracranial implantation of stainless steel wires infected with the 263K prion strain. Interestingly, photocatalytic treatment of 263K infected titanium wires, under the same experimental conditions, prolonged the survival interval significantly, but failed to eliminate infectivity, a result that we correlate with the increased adsorption of PrP^Sc on titanium, in comparison to stainless steel. Our findings strongly indicate that our, user- and environmentally friendly protocol can be safely applied to the decontamination of prion infected stainless steel surfaces.     

Optimization of an extracellular protease of Chrysosporium keratinophilum and its potential in bioremediation of keratinic wastes

Chrysosporium keratinophilum IMI 338142 isolated from a waste site containing organopollutants was studied for its ability to produce extracellular proteases on glucose-gelatin medium. Fungus was observed to be a potent producer of such enzymes. Enzyme secretion was best at 15 days of incubation period at pH 8 and temperature 40 degrees C. Asparagine was repressive to protease expression. No relationship existed between the enzyme yield and increase in biomass. Exogenous sugars suppressed enzyme production in the descending order as follows: glucose > arabinose > maltose > mannose > fructose. The enzyme released showed the ability to decompose two keratin substrates tested. Buffalo skin was the most actively degraded substrate when exogenous glucose was absent. Presence of glucose suppressed both enzyme production and degradation of keratin. However, the rate of keratin degradation was independent of enzyme production.     

Substrate profiling of cysteine proteases using a combinatorial peptide library identifies functionally unique specificities

The substrate specificities of papain-like cysteine proteases (clan CA, family C1) papain, bromelain, and human cathepsins L, V, K, S, F, B, and five proteases of parasitic origin were studied using a completely diversified positional scanning synthetic combinatorial library. A bifunctional coumarin fluorophore was used that facilitated synthesis of the library and individual peptide substrates. The library has a total of 160,000 tetrapeptide substrate sequences completely randomizing each of the P1, P2, P3, and P4 positions with 20 amino acids. A microtiter plate assay format permitted a rapid determination of the specificity profile of each enzyme. Individual peptide substrates were then synthesized and tested for a quantitative determination of the specificity of the human cathepsins. Despite the conserved three-dimensional structure and similar substrate specificity of the enzymes studied, distinct amino acid preferences that differentiate each enzyme were identified. The specificities of cathepsins K and S partially match the cleavage site sequences in their physiological substrates. Capitalizing on its unique preference for proline and glycine at the P2 and P3 positions, respectively, selective substrates and a substrate-based inhibitor were developed for cathepsin K. A cluster analysis of the proteases based on the complete specificity profile provided a functional characterization distinct from standard sequence analysis. This approach provides useful information for developing selective chemical probes to study protease-related pathologies and physiologies.     

S1 pocket of a bacterially derived subtilisin-like protease underpins effective tissue destruction

The ovine footrot pathogen, Dichelobacter nodosus, secretes three subtilisin-like proteases that play an important role in the pathogenesis of footrot through their ability to mediate tissue destruction. Virulent and benign strains of D. nodosus secrete the basic proteases BprV and BprB, respectively, with the catalytic domain of these enzymes having 96% sequence identity. At present, it is not known how sequence variation between these two putative virulence factors influences their respective biological activity. We have determined the high resolution crystal structures of BprV and BprB. These data reveal that that the S1 pocket of BprV is more hydrophobic but smaller than that of BprB. We show that BprV is more effective than BprB in degrading extracellular matrix components of the host tissue. Mutation of two residues around the S1 pocket of BprB to the equivalent residues in BprV dramatically enhanced its proteolytic activity against elastin substrates. Application of a novel approach for profiling substrate specificity, the Rapid Endopeptidase Profiling Library (REPLi) method, revealed that both enzymes prefer cleaving after hydrophobic residues (and in particular P1 leucine) but that BprV has more restricted primary substrate specificity than BprB. Furthermore, for P1 Leu-containing substrates we found that BprV is a significantly more efficient enzyme than BprB. Collectively, these data illuminate how subtle changes in D. nodosus proteases may significantly influence tissue destruction as part of the ovine footrot pathogenesis process.     

Current and prospective applications of non-proteinogenic amino acids in profiling of proteases substrate specificity

Abstract Proteases recognize their endogenous substrates based largely on a sequence of proteinogenic amino acids that surrounds the cleavage site. Currently, several methods are available to determine protease substrate specificity based on approaches employing proteinogenic amino acids. The knowledge about the specificity of proteases can be significantly extended by application of structurally diverse families of non-proteinogenic amino acids. From a chemical point of view, this information may be used to design specific substrates, inhibitors, or activity-based probes, while biological functions of proteases, such as posttranslational modifications can also be investigated. In this review, we discuss current and prospective technologies for application of non-proteinogenic amino acids in protease substrate specificity profiling.     

Purification and characterization of keratinase from recombinant Pichia and Bacillus strains

The keratinase gene from Bacillus licheniformis MKU3 was cloned and successfully expressed in Bacillus megaterium MS941 as well as in Pichia pastoris X33. Compared with parent strain, the recombinant B. megaterium produced 3-fold increased level of keratinase while the recombinant P. pastoris strain had produced 2.9-fold increased level of keratinase. The keratinases from recombinant P. pastoris (pPZK3) and B. megaterium MS941 (pWAK3) were purified to 67.7- and 85.1-folds, respectively, through affinity chromatography. The purified keratinases had the specific activity of 365.7 and 1277.7 U/mg, respectively. Recombinant keratinase from B. megaterium was a monomeric protein with an apparent molecular mass of 30 kDa which was appropriately glycosylated in P. pastoris to have a molecular mass of 39 kDa. The keratinases from both recombinant strains had similar properties such as temperature and pH optimum for activity, and sensitivity to various metal ions, additives and inhibitors. There was considerable enzyme stability due to its glycosylation in yeast system. At pH 11 the glycosylated keratinase retained 95% of activity and 75% of its activity at 80 degrees C. The purified keratinase hydrolyzed a broad range of substrates and displayed effective degradation of keratin substrates. The K(m) and V(max) of the keratinase for the substrate N-succinyl-Ala-Ala-Pro-Phe-pNA was found to be 0.201 mM and 61.09 U/s, respectively. Stability in the presence of detergents, surfactants, metal ions and solvents make this keratinase suitable for industrial processes.