Characterization of a keratinolytic protease produced by the feather-degrading Amazonian bacterium Bacillus sp. P45

Abstract

An extracellular keratinolytic protease produced by Bacillus sp. P45 was purified and characterized. The keratinase had a molecular weight of approximately 26 kDa and was active over wide pH and temperature ranges, with optimal activity at 55°C and pH 8.0. However, this enzyme displayed low thermostability, being completely inactivated after 10 min at 50°C. Keratinase activity increased with Ca²⁺, Mg²⁺, Triton X-100, ethanol and DMSO, was stable in the presence of the reducing agent 2-mercaptoethanol, and was inactivated by SDS. PMSF (phenylmethylsulfonyl fluoride) completely inactivated and EDTA strongly inhibited the enzyme, indicating that the keratinase is a serine protease depending on metal ions for optimal activity and/or stability. Accordingly, analysis of tryptic peptides revealed sequence homologies which characterize the keratinase as a subtilisin-like serine protease. The purified enzyme was able to hydrolyze azokeratin and keratin azure. Casein was hydrolyzed at higher rates than keratinous substrates, and 2-mercaptoethanol tended to enhance keratin hydrolysis. With synthetic substrates, the keratinase showed a preference for aromatic and hydrophobic residues at the P1 position of tetrapeptides; the enzyme was not active, or the activity was drastically diminished, towards shorter peptides. Keratinase from Bacillus sp. P45 might potentially be employed in the production of protein hydrolysates at moderate temperatures, being suitable for the bioconversion of protein-rich wastes through an environmentally friendly process requiring low energy inputs.     

Hydrolysis of Feather Keratin by Immobilized Keratinase

Keratinase isolated from Bacillus licheniformis PWD-1 was immobilized on controlled-pore glass beads. The immobilized keratinase demonstrated proteolytic activities against both insoluble feather keratin and soluble casein. It also displayed a higher level of heat stability and an increased tolerance toward acidic pHs compared with the free keratinase. During a continuous reaction at 50(deg)C, the immobilized keratinase retained 40% of the original enzyme activity after 7 days. The immobilized keratinase exhibits improved stability, thereby increasing its potential for use in numerous applications.     

角蛋白酶的研究与应用前景

角蛋白酶(keratinase) 是一种可以特异性降解角蛋白的酶类,其来源广泛,多种微生物在羽毛降解过程中均可产生角蛋白酶。不同菌种来源的角蛋白酶,其结构、理化性质、活性和底物也不同。其在饲料行业、制革工业和环境废弃物处理等多个方面具有广泛的应用前景,能够产生巨大的社会效益和经济效益。本文系统总结了角蛋白酶的来源、分类、理化性质、作用机理及其在基因工程研究等方面的一些最新进展,简要介绍了其应用研究现状,并展望了角蛋白酶的应用前景。     

Multifarious potential applications of keratinase of Bacillus subtilis K-5

Abstract

Bacillus subtilis K-5, an isolate from compost, utilized a wide range of keratinous wastes viz. diverse feather types, nails, hair, scales, etc. for growth and produced a thermostable alkaline protease (keratinase) with broad proteolytic activity. Optimization of cultural and environmental variables using a Plackett–Burman design and response surface methodology resulted in enhanced keratinase production (89%). Keratinase was partially purified (15-fold) by ammonium sulfate precipitation and carboxymethyl cellulose chromatography. The optimum pH and temperature for keratinase activity were 9.0 and 60°C, however, considerable activity and stability was observed over broad pH (5–10) and temperature range (50–90°C). B. subtilis K-5 keratinase exhibited excellent stability toward detergents (cetyl trimethylammonium bromide, Tween 80, and sodium dodecyl sulfate) and organic solvents (benzene, acetonitrile, phenylmethylsulfonyl fluoride); however, metal ions like Mn²⁺, Cu²⁺, Na⁺, Hg²⁺, K⁺, Ca²⁺, and Zn²⁺ inhibited the activity. B. subtilis K-5 protease showed remarkable potential for diverse applications like blood stain removal, gelatin hydrolysis from waste X-ray films and dehairing of animal hide.     

Characterization of a novel <Emphasis Type="Italic">Stenotrophomonas</Emphasis> isolate with high keratinase activity and purification of the enzyme

A feather-degrading bacterium was isolated from poultry decomposition feathers in China. The strain, named L1, showed significant feather-degrading activity because it grew and reproduced quickly on basal medium containing 10 g/L of native feather as the source of energy, carbon, and nitrogen. According to the phenotypic characteristics and 16S rRNA profile, the isolate belongs to Stenotrophomonas maltophilia. Keratinase activity of the isolate was determined during cultivation on raw feathers at different temperatures and initial pH. Maximum growth and feather-degrading activity of the bacterium were observed at 40°C and initial pH ranging from 7.5 to 8.0. The crude enzyme was purified by ammonium sulphate precipitation, Sephadex G-100 chromatographic and ceramic hydroxyapatite (CHT) chromatographic. Its molecular mass estimated as 35.2 kDa in SDS-PAGE. The enzyme had an optimum activity at the pH was 7.8 and the temperature was 40°C. The keratinase was wholly inhibited by a serine protease inhibitor, PMSF. Its activity was activated or inhibited by different metal ions. The keratinase activity of enzyme from strain L1 functioned on different keratins, such as feather, hair, wool, horn, and so on.     

微生物角蛋白酶的分子生物学研究进展

角蛋白酶具有分解角蛋白的活性,在饲料,食品加工,环境废物处理等方面,具有广泛的应用前景。本文综述了微生物角蛋白酶的来源,角蛋白酶的理化性质,作用机理,基因分离和表达等分子生物学和基因工程研究进展,并对其应用前景进行了展望,对其今后的发展趋势进行了讨论。     

Exocellular enzyme activity of dermatophytes and other fungi isolated from ruminants in Southern Iraq

Sixteen fungal species were isolated from 182 specimens collected from four ruminants (buffalo, camel, cattle and sheep) in Southern Iraq. Fungi represented by five species of dermatophytes and eleven species of other fungi were screened for the activity of four enzymes; keratinase, proteinase, lipase and amylase. Keratinase was found to be produced by all of the dermatophytes and non-dermatophytes, except for Paecillomyces variottii and Scytalidium lignicola. However, high keratinase activity was expressed by the dermatophytic species particularly by Trichophyton mentagrophytes var. erinacei and Microsporum gypseum. Three dermatophytes viz. M. gypseum, T. verrucosum and T. mentagrophytes var. nodulare were capable of producing protease, lipase and amylase. Although, T. mentagrophytes var. erinacei showed high protease activity, it did not produce lipase and amylase. On the contrary most of the non-dermatophytic species revealed protease and lipase activities higher than the dermatophytes. The Curvularia spp. isolates showed the highest protease and amylase activity, while Aspergillus parasiticus revealed the highest activity of lipase and amylase. No correlation was observed between enzyme activity and the growth rate of the examined fungi. This revised version was published online in June 2006 with corrections to the Cover Date.     

Iron containing keratinolytic metallo-protease produced by Chryseobacterium gleum

Chryseobacterium gleum exhibited complete dissolution of whole chicken-feathers (10 g l^?1, pH 8) after 72 h at 30 °C through synthesis of keratinolytic protease when inoculated at 1% (v/v). This enzyme was purified to 67-fold with yield of 2.25% having a specific activity of 1670 U mg^?1 and ～36 kDa Mw. MALDI-TOF MS of this keratinase showed some similarity with the keratinase peptides of Bacillus subtilis (BOFXJ2). The keratinase action was inhibited by EDTA, iodoacetamide and metal ions like mercury, copper and zinc (1 mM each), while it was enhanced by iron and calcium. Keratinase showed presence of 3 mM of Fe M^?1 as tested by atomic absorption spectroscopy and addition of Fe in its apoenzyme retained about 79% of original residual feather degradation activity which portrayed it to be metalloprotease. Purified keratinase revealed significant degradation (85%) of feather concentrate (20 g l^?1) to 3.9 μM ml^?1 of free amino groups in 24 h at an initial pH of 8.0, 30 °C and 120 rpm shaking. This keratinase activity can be controlled precisely by presence of chemical or metal ions which could be of use in biotechnology industry while the culture can be used in poultry waste management.     

Degradation of chicken feathers by Chrysosporium georgiae

Using a baiting technique, Chrysosporium georgiae was isolated from chicken feathers. Twenty-eight different fungal isolates were evaluated for their ability to produce keratinase enzymes using a keratin–salt agar medium containing either white chicken feathers or a prepared feather keratin suspension (KS). The Chrysosporium species were able to use keratin and grow at different rates. Chrysosporium georgiae completely degraded the added keratin after 9 days of incubation. Degradation of feathers by C. georgiae was affected by several cultural factors. Highest keratinolytic activity occurred after 3 weeks of incubation at 6 and 8~pH at 30 °C. Chrysosporium georgiae was able to degrade white chicken feathers, whereas bovine and human hair and sheep wool were not degraded and did not support fungal growth. Addition of 1% glucose to the medium containing keratin improved fungal growth and increased enzyme production. Higher keratin degradation resulted in high SH accumulation and the utilization of the carbohydrate carbon in the medium resulted in high keto-acid accumulation but decreased ammonia accumulation. Supplementation of the keratin–salt medium with minerals such as NH4Cl and MgSO4 slightly increased mycelial growth, but decreased production of extracelluar keratinase. Keratinase enzymes were very poorly produced in the absence of keratin, indicating its inducible nature. Analysis of endocellular keratinases in the mycelial homogenate indicated higher activity of intracellular keratinase as compared to the extracellular enzyme in culture filtrates. Chrysosporium georgiae was the most superior for keratinase production among the Chrysosporium species tested in the presence or absence of glucose. It produced more of the intracellular enzymes than the exocellular ones. This revised version was published online in June 2006 with corrections to the Cover Date.     

Characterization of an alkaline active-thiol forming extracellular serine keratinase by the newly isolated Bacillus pumilus

Aims: The aim of the study was to optimize microbial degradation of keratinous waste and to characterize the alkaline active keratinase showing its biotechnological importance. Method and Results: An extracellular keratinase enzyme was purified from the culture medium of a bacterial isolate and the conditions were optimized. The molecular weight of DEAE‐Sepharose‐purified keratinase was determined by SDS‐PAGE. Instrumental analyses were investigated to study the mechanism of bovine hair hydrolysis. Isolate was identified as Bacillus pumilus based on phenotypic characteristics and 16S rDNA sequence. The optimized condition for its growth was pH 8 and 35°C. The molecular weight of the keratinase was estimated as 65 kDa. Activity inhibition by phenyl methyl sulphonyl fluoride confirmed keratinase as serine protease type. Instrumental analysis revealed the sulphitolysis and proteolysis involved mechanism in bovine hair hydrolysis. Conclusion: This study indicates that the isolated keratinase is an alkaline active serine protease with a high degree of activity towards bovine hair. Significance and Impact of the Study: This study examines a serine protease with high keratinolytic activity and degradation mechanism for bovine hair. The keratinolytic activity of the isolated strain and its reaction mechanism on bovine hair could show biotechnological potential in the leather industry.     

Stability and Stabilisation of Doratomyces microsporus Keratinase

Thermal and pH stabilities of a new crude keratinase ( Doratomyces microsporus ) were investigated in the ranges of 20-40°C and pH 4-10, respectively. The stability test was followed by activity measurement on two different substrates: human stratum corneum and haemoglobin. Activity measurement lasted more than 100 h. The effect of calcium ions on enzyme stability was also studied. Crude keratinase was stabilised by crosslinking with glutaraldehyde (GA). The same characteristics were determined for Proteinase K, the commercial enzyme, for comparative purposes. Crude keratinase was most stable at pH 8 in Tris/HCl and borate buffers. The type of buffer used proved to have higher effect on crude keratinase stability than on Proteinase K. Both enzymes were most stable at 20°C. Keratinase stability rapidly decreased at 40°C while Proteinase K showed higher thermal stability. A 1 mM solution of Ca 2+ ions did not significantly influence enzyme stability, but 2.5% GA solution stabilised crude keratinase at 40°C reducing the k d value by about 50%. Crude and crosslinked crude keratinase were used for crude calf skin degradation. A mathematical model, based on Michaelis-Menten kinetics, was developed to describe the crude calf skin degradation in a batch reactor. Validation of the model showed that it could describe the process over a defined range of its conditions.     

Stability and Stabilisation of Doratomyces microsporus Keratinase

Thermal and pH stabilities of a new crude keratinase ( Doratomyces microsporus ) were investigated in the ranges of 20-40°C and pH 4-10, respectively. The stability test was followed by activity measurement on two different substrates: human stratum corneum and haemoglobin. Activity measurement lasted more than 100 h. The effect of calcium ions on enzyme stability was also studied. Crude keratinase was stabilised by crosslinking with glutaraldehyde (GA). The same characteristics were determined for Proteinase K, the commercial enzyme, for comparative purposes. Crude keratinase was most stable at pH 8 in Tris/HCl and borate buffers. The type of buffer used proved to have higher effect on crude keratinase stability than on Proteinase K. Both enzymes were most stable at 20°C. Keratinase stability rapidly decreased at 40°C while Proteinase K showed higher thermal stability. A 1 mM solution of Ca 2+ ions did not significantly influence enzyme stability, but 2.5% GA solution stabilised crude keratinase at 40°C reducing the k d value by about 50%. Crude and crosslinked crude keratinase were used for crude calf skin degradation. A mathematical model, based on Michaelis-Menten kinetics, was developed to describe the crude calf skin degradation in a batch reactor. Validation of the model showed that it could describe the process over a defined range of its conditions.     

Phosphate-Mediated Alteration of the Microsporum gypseum Germination Protease Specificity for Substrate: Enhanced Keratinase Activity

Inorganic phosphate was found to decrease the caseinolytic and ethyl-esterase activities of the Microsporum gypseum germination protease. The germination protease possessed exokeratinase (beta-keratinase) activity immediately after release from the fungal spore. After phosphate treatment of the enzyme, the germination protease also possessed endo-keratinase (alpha-keratinase) activity. Phosphate altered the protease's pH optimum from 9.0 to 7.0 and decreased the molecular weight from 33,000 to 16,000. These values were identical to those found for the keratinase. Alpha- and beta-keratinase activities were stimulated in excess of 200-fold by disulfide reducing agents. Natural and suspected keratin degradation products also enhanced keratinase activity. Cell fractionation and in vitro conversion of the alkaline germination protease into a functional keratinase suggested that the subunits comprising the germination protease and the keratinase were of a common origin.     

Optimizing the fermentation conditions and enhanced production of keratinase from Bacillus cereus isolated from halophilic environment

Keratinase degrading Bacillus cereus was isolated from the halophilic environment in Tamilnadu, India and keratinase production was optimized using wheat bran substrate. Of the screened bacterial isolates, four were found to have the ability to produce keratinolytic enzyme. The process parameters were optimized using one-variable-at-a-time approach and response surface methodology. Supplementation of 1% lactose supported more keratinase production (120?U/g). Among the selected nitrogen sources, addition of casein significantly enhanced maximum keratinase production (132.5?U/g). Among the ions, manganese chloride significantly enhanced keratinsase production (102.6?U/g), however addition of zinc sulphate and copper sulphate decreased keratinase production. The maximum keratinase production was obtained in the wheat bran medium containing 1% lactose, 0.5% manganese with 80% moisture (292?U/g). Statistics based contour plots were generated to explore the variations in the response surface and to find the relationship between the keratinase yield and the bioprocess conditions.     

Immobilization of a keratinolytic protease from Purpureocillium lilacinum on genipin activated-chitosan beads

Keratinase from Purpureocillium lilacinum LPSC # 876 was immobilized on chitosan beads using two different cross-linking agents: glutaraldehyde and genipin. For its immobilization certain parameters were optimized such as cross-linker concentration, activation time and activation temperature. Under optimum conditions, enzyme immobilization resulted to be 96 and 92.8% for glutaraldehyde and genipin, respectively, with an activity recovery reaching up to 81% when genipin was used. The immobilized keratinase showed better thermal and pH stabilities compared to the soluble form, retaining more than 85% of its activity at pH 11 and 74% at 50 °C after 1 h of incubation. The residual activity of immobilized keratinase remained more than 60% of its initial value after five hydrolytic cycles. The results in this study support that glutaraldehyde could be replaced by genipin as an alternative cross-linking eco-friendly agent for enzyme immobilization.     

Biodegradation of feather waste by keratinase produced from newly isolated Bacillus licheniformis ALW1

Keratinase are proteolytic enzymes which have gained much attention to convert keratinous wastes that cause huge environmental pollution problems. Ten microbial isolates were screened for their keratinase production. The most potent isolate produce 25.2?U/ml under static condition and was primarily identified by partial 16s rRNA gene sequence as Bacillus licheniformis ALW1. Optimization studies for the fermentation conditions increased the keratinase biosynthesis to 72.2?U/ml (2.9-fold). The crude extracellular keratinase was optimally active at pH 8.0 and temperature 65?°C with 0.7% soluble keratin as substrate. The produced B. licheniformis ALW1 keratinase exhibited a good stability over pH range from 7 to 9 and over a temperature range 50–60?°C for almost 90?min. The crude enzyme solution was able to degrade native feather up to 63% in redox free system.     

Purification and characterization of extreme alkaline, thermostable keratinase, and keratin disulfide reductase produced by Bacillus halodurans PPKS-2

Two alkaline keratinases-I and II secreted by Bacillus halodurans PPKS-2 were purified and characterized. Both the keratinases were purified using ammonium sulfate, DEAE-Sephadex followed by Sephadex G-200 column chromatography. The purification was 21.5-fold and 11.17% yield for keratinase-I and 23.7-fold with yield 18.46 for keratinase-II and its molecular weights 30 and 66 kDa. Both purified enzymes were relatively stable over a broad pH range 7.0–13.0 and optimally active at pH 11.0 and 60–70 °C. Keratinase-II was found to be more stable at 70 °C for 3 h and retained 100% of its activity, whereas keratinase-I lost 10% activity. Keratinase-I had high keratin disulfide reductase activity with low keratinase activity whereas keratinase-II had high keratinase activity with low keratin disulfide reductase activity. Keratinase activities of both the enzymes were completely inhibited by PMSF at 1 mM, whereas keratin disulfide reductase activity of keratinase-I was not affected. Enzymes were active and stable in the presence of the surfactants, bleaching agents (20% H₂O₂), commercial detergents (1%), and SDS (20%). Both the enzymes were partially sequenced and found that keratinase-I and II had a homology with disulfide reductases and serine type of proteases, respectively.     

Subtilisins of Bacillus spp. hydrolyze keratin and allow growth on feathers

Keratinase is a serine protease produced by Bacillus licheniformis PWD-1 that effectively degrades keratin and confers the ability to grow on feathers to a protease-deficient B. subtilis strain. Studies presented herein demonstrate that B. licheniformis Carlsberg strain NCIMB 6816, which produces the well-characterized serine protease subtilisin Carlsberg, also degrades and grows on feathers. The PWD-1 and Carlsberg strains showed a similar time-course of enzyme production, and the purified serine proteases have similar enzymatic properties on insoluble azokeratin and soluble FITC-casein. Kinetic analysis of both enzymes demonstrated that they have high specificity for aromatic and hydrophobic amino acids in the P1 substrate position, although keratinase discriminates more than subtilisin Carlsberg against charged residues at this site. Nucleotide sequence analysis of the serine protease genes from B. licheniformis strains PWD-1, Carlsberg NCIMB 6816, ATCC 12759, and NCIMB 10689 showed that the kerA-encoded protease of PWD-1 differs from the others only by having V222, rather than A222, near the active site serine S220. Further, high-level expression of subE-encoded subtilisin from B. subtilis (78% similar to subtilisin Carlsberg) also confers growth on feathers on a protease-deficient B. subtilis strain. While strain PWD-1 and the kerA protease efficiently degrade keratin, keratin hydrolysis and growth on feathers is a property that can be conferred by appropriate expression of the major subtilisins, including the industrially produced enzymes.     

Purification and characterization of a keratinolytic serine protease from Purpureocillium lilacinum LPS # 876

A keratinolytic serine protease secreted by Purpureocillium lilacinum (formerly Paecilomyces lilacinus) upon culture in a basal medium containing 1% (w/v) hair waste as carbon and nitrogen source was purified and characterized. After purification the keratinase was resolved by SDS-PAGE as a homogeneus protein band of molecular mass 37.0 kDa. The extracellular keratinase of P. lilacinum was characterized by its appreciable stability over a broad pH range (from 4.0 to 9.0), and up to 65 °C, along with its strong inhibition by phenylmethylsulphonyl fluoride among the protease inhibitors tested (98.2% of inhibition), thus suggesting its nature as a serine protease. The enzyme was active and stable in the presence of organic solvents such as dimethylsulfoxide, methanol, and isopropanol; certain surfactants such as Triton X-100, sodium dodecylsulfate, and Tween 85; and bleaching agents such as hydrogen peroxide. These biochemical characteristics suggest the potential use of this enzyme in numerous industrial applications.     

Heterologous production of Aspergillus fumigatus keratinase in Pichia pastoris

Aspergillus fumigatus Fresenius was previously shown to grow in mineral medium containing chicken feather flour as carbon and nitrogen source. Substantial proteolytic keratin-degrading activity was present in the culture supernatant after 24–72 h of growth at 42 °C. The keratinase was successfully purified by a single ion exchange chromatographic procedure and had a molecular mass of 31 kDa as determined by SDS–PAGE. The keratinase cDNA was expressed in Pichia pastoris cells and the recombinant clones were shown to be able to produce substantial caseinolytic, azo-keratinolytic and keratinolytic activities. SDS–PAGE and Western-blotting analysis using antibody against keratinase of A. fumigatus showed the presence of a single protein in the culture supernatants of several recombinant P. pastoris cells. This protein had a molecular mass corresponding to that of the A. fumigatus keratinase. The enzyme production profile showed that theP. pastoris recombinant cells produced an increasing amount of proteolytic and azo-keratinolytic activities over a 72 h growth period. Dry weight determination analysis indicated that 10% of the keratin flour was hydrolysed over a 24 h incubation period with 510 U (caseinolytic activity) of the recombinant keratinase.