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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 2005, p. 3420–3426 Vol. 71, No. 7 0099-2240/05/$08.00 0 doi:10.1128/AEM.71.7.3420–3426.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved. Similarities and Speciﬁcities of Fungal Keratinolytic Proteases: Comparison of Keratinases of Paecilomyces marquandii and Doratomyces microsporus to Some Known Proteases Helena Gradisar,1* Jozica Friedrich,1 Igor Krizaj,2 and Roman Jerala1 ˇ ˇ ˇ Laboratory of Biotechnology, National Institute of Chemistry, Hajdrihova 19, Ljubljana 1000, Slovenia,1 and ˇ Department of Biochemistry and Molecular Biology, Jozef Stefan Institute, Jamova 39, Ljubljana 1000, Slovenia2 Received 3 January 2005/Accepted 6 January 2005 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. Keratins are the most abundant proteins in epithelial cells of mostly active in alkaline environments, with optimal activity at vertebrates and represent the major constituents of skin and its temperatures up to 50°C. Thermostable keratinases with opti- appendages such as nail, hair, feather, and wool. The protein mal temperatures of around 85°C and a higher molecular mass chains are packed tightly either in -chain ( -keratins) or in have been reported (5, 8, 31). The potential use of keratinases -sheet ( -keratins) structures. Keratins belong to the super- is in different applications where keratins should be hydro- family of intermediate ﬁlament proteins. Their high degree of lyzed, such as the leather and detergent industries, textiles, cross-linking by disulﬁde bonds, hydrophobic interactions, and waste bioconversion, medicine, and cosmetics for drug delivery hydrogen bonds stabilizes keratin ﬁlament structure (11). through nails and degradation of keratinized skin. Therefore, keratinous material is water insoluble and ex- In our previous studies potent keratinase producers, among tremely resistant to degradation by proteolytic enzymes such as them Aspergillus ﬂavus (9) and Doratomyces microsporus (12), trypsin, pepsin, and papain. were selected out of 300 nonpathogenic fungi which are pre- A group of proteolytic enzymes which are able to hydrolyze ferred for biotechnological applications. The keratinases were insoluble keratins more efﬁciently than other proteases are produced by cultivating strains in optimized media under sub- called keratinases (29). They are produced by some insects and merged aerobic conditions. The enzymes were isolated and mostly by microorganisms. The best studied are keratinases puriﬁed and their main biochemical characteristics were deter- from the dermatophytic genera Microsporum (26, 37) and mined. A keratinase produced by the fungus Paecilomyces mar- Trichophyton (30, 38) as well as from bacteria of the genera quandii promised to be still more active than the previously Bacillus (4, 14, 20, 24, 34, 35) and Streptomyces (2, 3, 27). There studied keratinolytic enzymes. In the present report, we inves- are relatively few reports on characterization of the keratinases tigated its characteristics. We examined in detail the catalytic from nondermatophytic fungi (5, 7, 12, 25, 32, 33). properties as well as the speciﬁcity of the P. marquandii and D. Most keratinases have some common characteristics despite microsporus keratinases for both natural and synthetic sub- their different origins. They belong mainly to the extracellular strates. A comparison of the enzyme properties with those of serine proteases, with the exception of keratinases from yeasts, some commercial proteases is presented. Our aim was to elu- which belong to the aspartic proteases (23, 28). The molecular cidate speciﬁc characteristics which distinguish keratinases masses of the enzymes range from 20 kDa to 60 kDa. They are from nonkeratinolytic proteases. MATERIALS AND METHODS * Corresponding author. Mailing address: Laboratory of Biotech- nology, National Institute of Chemistry, Hajdrihova 19, Ljubljana Production and puriﬁcation of keratinases. Fungal strains Paecilomyces mar- 1000, Slovenia. Phone: 386 1 4760 331. Fax: 386 1 4760 300. E-mail: quandii (MZKI B639) and Doratomyces microsporus (MZKI B399) were used as firstname.lastname@example.org. producers of the keratinolytic enzymes. Both strains have been isolated and 3420 VOL. 71, 2005 FUNGAL KERATINOLYTIC PROTEASES 3421 identiﬁed in the Laboratory of Botany, Faculty of Medicine and Pharmacy, TABLE 1. Effect of protease inhibitors and additives on activity of University of Franche-Comte, Besancon, France, and are deposited in the Cul- ´ ¸ P. marquandii and D. microsporus keratinases ture Collection of the National Institute of Chemistry (MZKI), Ljubljana, Slo- venia. The fungi are maintained on potato dextrose agar (Fluka) slants. Residual activity (%) For keratinase production, strains of P. marquandii and D. microsporus were Compound Concn Keratinase of Keratinase of cultivated in shaken ﬂasks. Each strain was grown in a liquid medium containing P. marquandii D. microsporus soy ﬂour as an enzyme inducer and (g/liter): KH2PO4, 1.5; K2HPO4, 1.0; MgSO4 · 7H2O, 0.2; CaCl2 · 2H2O, 0.2; NaCl, 0.2; ZnSO4 · 7H2O, 0.002; peptone, 0.4; Control 100 100 malt extract, 1.0; glucose, 1.0; soy ﬂour, 5.0; and glycerol, 2 ml/liter. The pH of PMSF 1 mM 0 0 the medium was adjusted to 6, and aliquots of 100 ml were distributed into EDTA 5 mM 70 75 500-ml Erlenmayer ﬂasks and autoclaved. After inoculation with a spore sus- Iodoacetamide 0.05 mM 98 99 pension (106 spores/ml medium), the fermentation was carried out at 30°C and DTT 0.5 mM 143 175 100 rpm on a rotary shaker until the keratinolytic activity reached its maximum, 1 mM 230 322 i.e., for 5 days with P. marquandii and for 4 days with D. microsporus. 5 mM 197 266 Crude enzymes were prepared by concentration of the broth ﬁltrate (PLCC 5K -ME 5 mM 108 160 membrane, Minitan system, Millipore, Bedford, MA) followed by overnight 25 mM 115 215 dialysis against deionized water and subsequent lyophilization. For enzyme pu- SDS 0.1% 29 23 riﬁcation, the lyophilized powder was dissolved in 50 mM phosphate buffer (pH 0.5% 14 16 7.0) containing 1 M ammonium sulfate and the enzyme solution was applied onto DMSO 1% 103 100 a preparative column for hydrophobic interaction chromatography (HiPrep 5% 105 104 16/10 Phenyl FF, Pharmacia), previously equilibrated with the same buffer. The 10% 88 90 enzyme was eluted by stepwise decreasing salt concentrations from 1 M to 0 M Isopropanol 1% 88 85 at a ﬂow rate of 3.0 ml/min. The keratinolytic active fraction was pooled and concentrated by ultraﬁltration (YM10 membrane, Amicon). Subsequently the enzyme solution was applied on a gel ﬁltration column (Superose 12 HR 10/30, Pharmacia) equilibrated with a 50 mM phosphate buffer (pH 7.5) containing 0.2 was determined spectrophotometrically at 280 nm by measuring of TCA-soluble M NaCl. The puriﬁed enzyme was stored in aliquots at 20°C. peptides released from the substrate during the incubation. Soluble protein determination. The concentration of soluble protein was mea- In addition, the keratinase activity assay was used to compare the activities of sured at 562 nm by the bicinchoninic acid assay according to the manufacturer’s selected proteases (Sigma). The enzymes tested were keratinases from P. mar- instructions (Sigma). The protein concentration was determined by using a quandii and D. microsporus, chymotrypsin, collagenase, elastase, proteinase K, calibration curve that was established with known concentrations of bovine subtilisin, and trypsin. Keratin of the stratum corneum and casein were chosen as serum albumin. model substrates for enzyme activity determination. Stock solutions in concen- The purity of the keratinases and their molecular masses were determined by tration of 1 mg/ml were prepared in 28 mM Tris-HCl buffer (pH 8.0) for each sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) accord- enzyme. They were diluted adequately with the same buffer and then the sub- ing to the method of Laemmli (19). The MiniProtean II system was used ac- strate was added. Incubation and determination conditions were the same as described above. cording to the manufacturer’s instructions (Bio-Rad). The proteins were sepa- Effects of pH, temperature, proteinase inhibitors, organic solvents, detergents, rated on a 12% gel and stained with a 0.1% (wt/vol) solution of Coomassie and reducing agents on keratinolytic activity. The effect of pH on the keratino- brilliant blue R (Sigma). Low-molecular-mass markers (Dalton mark VII-L, lytic activity of the keratinases was assayed at 45°C using 28 mM buffers of Sigma) were used as protein standards for determination of molecular masses. various pH values: citrate buffer (pH 4 to 6), phosphate buffer (pH 6 to 8), N-terminal amino acid sequencing of keratinases and sequence analysis. The Tris-HCl (pH 7 to 9), and glycine-NaOH (pH 9 to 11). The optimum tempera- N-terminal sequences of the puriﬁed keratinases were determined by automated ture for the enzyme activity was determined by performing the enzyme reaction Edman degradation using a pulsed-liquid sequence analyzer (Procise 492A pro- at various temperatures between 20°C and 80°C at pH 8.0 (28 mM Tris-HCl). All tein sequencing system, Applied Biosystems). Samples were puriﬁed by reversed- other conditions were the same as described in the keratinolytic activity assay. phase high-performance liquid chromatography (RP-HPLC), dried on a Speed- The effects of protease inhibitors (EDTA, phenylmethylsulfonyl ﬂuoride Vac concentrator (Savant), dissolved in 20% (vol/vol) acetonitrile/water [PMSF], and iodoacetamide) and other chemicals, dithiothreitol (DTT), -mer- containing 0.001% (vol/vol) triﬂuoroacetic acid (TFA), and sequenced. The captoethanol ( -ME), sodium dodecyl sulfate (SDS), dimethyl sulfoxide sequences obtained were compared to sequences in the TrEMBL/Swissprot (DMSO), and isopropanol, on keratinolytic activity were determined (for con- database using BLASTp algorithm (http://www.expasy.org/tools/BLAST/). centrations, see Table 1). The puriﬁed keratinases were preincubated with each Determination of keratinase activity. Keratinolytic activity was examined using compound in 28 mM Tris-HCl buffer (pH 8.0) at 30°C for 10 min. The control keratin from the stratum corneum of the human sole as a substrate. The prep- was preincubated without the compound. Keratin of the stratum corneum was aration of keratin powder was described previously (12). Brieﬂy, scrapings of then added to each preparation, and the residual keratinolytic activity was mea- human sole were defatted, well rinsed, dried, ground to a powder, and sifted sured at 45°C as described above for determination of keratinase activity. through a 0.2-mm screen. The previously described activity assay (12) was slightly Proteolytic speciﬁcity of keratinases. The proteolytic speciﬁcity was deter- modiﬁed and performed as follows: the reaction mixture comprised 4.0 ml of 28 mined by measuring the ability of the keratinases to hydrolyze selected synthetic mM Tris-HCl buffer (pH 8.0), 1.0 ml of the enzyme solution, and 20 mg of the substrates and the oxidized B-chain of insulin. keratin powder. Incubation was carried out in a water bath at 45°C for 30 min The synthetic substrates used in this study (all purchased from Bachem) were with constant agitation of 160 rpm. The enzyme reaction was terminated by the prepared as stock solutions in DMSO: 200 mM N-succinyl-Ala-Ala-Ala-pNA addition of 2.0 ml 10% (wt/vol) trichloroacetic acid (TCA) and then allowed to (AAA), Ac-Tyr-OEt (ATEE), Bz-Arg-pNA · HCl (L-BAPA), 100 mM N-succi- stand at 4°C for 30 min. After centrifugation (10,000 rpm, 15 min) in a cooled nyl-Ala-Ala-Pro-Phe-pNA (AAPF), FA-Leu-Gly-Pro-Ala-OH (FALGPA), or in centrifuge (Sorvall), the absorbance of the supernatant was measured spectro- isopropanol (100 mM Bz-Tyr-pNA). The enzymatic reactions were carried out in photometrically at 280 nm (spectrophotometer from Beckman). The control was 28 mM Tris-HCl buffer (pH 8.0) at 45°C. The substrate concentration was 1 mM, treated the same way except that TCA was added before the incubation. One while the keratinase was added at an amount adequate to be able to follow the unit of keratinolytic activity was deﬁned as an increase of corrected A280 for 0.100 initial reaction velocity. The hydrolysis of peptides was monitored spectropho- under the conditions described. The data presented are mean values of three tometrically at 237 nm for ATEE, at 324 nm for FALGPA, and at 405 nm for parallel determinations. p-NA peptides. Then the best substrate was chosen for kinetic studies of kera- Further, more native keratins (keratin from human nail and hair, porcine nail, tinase activity. At least ﬁve concentrations of the chosen synthetic peptide were chicken feather, sheep wool, and commercial bovine keratin [ICN Biomedicals assayed under the same conditions. The ﬁnal concentration of organic solvent in Inc.]) and nonkeratinous ﬁbrillar proteins (collagen and elastin) as well as bovine the reaction mixture never exceeded 5% (vol/vol). The hydrolysis was followed serum albumin and casein were used as substrates to examine keratinase activity continuously and the initial velocities were determined. The values of Michaelis- on different native proteins. The preparation of keratinous materials was de- Menten constant (Km), maximal velocity (Vmax), and catalytic constant (kcat) scribed in our previous publication (12). The incubation procedure was the same were calculated by nonlinear regression using Michaelis-Menten equation. as described for keratinolytic activity determination. The extent of proteolysis To determine the hydrolysis sites, each keratinase (0.2 g) was incubated with 3422 ˇ GRADISAR ET AL. APPL. ENVIRON. MICROBIOL. the oxidized insulin B-chain (0.1% [wt/vol]) in 28 mM Tris-HCl buffer (pH 8.0), and the ﬁnal volume was 100 l. The reaction mixture was incubated at 30°C for 20 min and 12 h. The reaction was stopped by adding 1 volume of 5% (vol/vol) acetonitrile in 0.05% (vol/vol) TFA. An aliquot of 100 l of the sample was applied on RP-HPLC column C18 (Chromospher, HPLC system; Knauer). The peptides were eluted with an increasing gradient of acetonitrile from 5% to 95% (vol/vol) in 0.05% (vol/vol) TFA. The detection was performed at 215 nm. The molecular masses of the peptide products were determined by electrospray ionization mass spectroscopy (ESI-MS). The cleavage sites were deﬁned by using a computer program, FindPept (http://ca.expasy.org/tools/ﬁndpept/), which en- ables peptide identiﬁcation after protein cleavage on the basis of the experimen- tally determined size of the products. RESULTS AND DISCUSSION Keratinases which are able to degrade extremely resistant keratins are mostly isolated from pathogenic dermatophytes or bacteria. We searched for prospective keratinase producers among the nonpathogenic ﬁlamentous fungi. The most out- standing keratinolytic activity was found in keratinases from Paecilomyces marquandii, Doratomyces microsporus, and As- pergillus ﬂavus strains. Due to the reputation of A. ﬂavus as the potential producer of aﬂatoxins, we have focused our research on the other two strains. Production and puriﬁcation of keratinases. Fungal strains FIG. 1. SDS-PAGE of the puriﬁed keratinases of P. marquandii of D. microsporus and P. marquandii were cultivated in a sub- (P.m.) and D. microsporus (D.m.). The positions of low-molecular- mass markers (st.) from Sigma are shown. The gel (12%) was stained merged fermentation. Keratinolytic activity of the culture ﬁl- with Coomassie brilliant blue. trate appeared after 40 h for both strains and increased until reached its maximum, i.e., 49.5 U/ml after 95 h for D. micros- porus culture ﬁltrate and 230.6 U/ml after 110 h for that of P. marquandii. The culture ﬁltrates were collected and then con- perature was examined. The enzyme solution of each kera- centrated, dialyzed, and lyophilized; 0.25 g of the crude kera- tinase (0.06 mg/ml) was incubated at 45°C. Every 5 min the tinase from D. microsporus and 0.4 g of that of P. marquandii residual activity was determined spectrophotometrically by fol- were obtained from 1 liter of fermentation broth. The kera- lowing hydrolysis of 1 mM AAPF under the conditions de- tinases were puriﬁed, as described in the Materials and Meth- scribed for activity measurement in Materials and Methods. ods section, 3.8-fold to a speciﬁc activity of 1,005 U/mg and The reaction rate decreased for 50% in 15 min with D. micros- 4.9-fold to a speciﬁc activity of 326 U/mg, respectively. By porus keratinase and in 150 min with P. marquandii keratinase, SDS-PAGE, a single protein band was obtained in each case so the P. marquandii keratinase was stable 10 times longer than (Fig. 1). that of D. microsporus. SDS-PAGE showed complete degra- Characterization of keratinases. The basic biochemical dation of the enzyme and appearance of smaller peptides due characteristics of D. microsporus keratinase were described in to enzyme autolysis (data not shown). our previous report (12). In this study, the molecular mass of The addition of the inhibitor PMSF prevents autodegrada- the keratinase of P. marquandii was estimated to be 33 kDa by tion of keratinases. Since stabilization of the native conforma- both SDS-PAGE and gel ﬁltration on Superose 12. The en- tion with calcium ions is commonly observed with extracellular zyme seemed to be a monomer. The molecular masses of the enzymes, the effect of Ca2 ions on the stability of D. micros- puriﬁed keratinases, 33 kDa for P. marquandii, 30 kDa for D. porus keratinase was studied in our previous work. It was microsporus (12), and 22 kDa for A. ﬂavus keratinase (not shown that Ca2 ions at 1 mM concentration did not signiﬁ- published), ﬁt into the range between 20 kDa and 60 kDa cantly inﬂuence enzyme stability (15). However, autolysis did reported for other keratinases. All three keratinases are serine not represent a problem when other proteins were present as proteases, as are the majority of keratinases. The keratinolytic a substrate. If the stability of the P. marquandii keratinase was activity of the keratinase of P. marquandii on stratum corneum compared to the data for keratinases from the literature, only keratin was detected in a broad range of pH values (pH 6 to a keratinase from Streptomyces pactum (2) and protease D-1 11), with the maximum activity at pH 8 (Fig. 2A). The optimal from Stenotrophomonas sp. (39) proved to be similarly stable, pH of keratinolytic activity for both the P. marquandii and D. in addition to keratinolytic enzymes of thermophilic microor- microsporus enzymes is around pH 8, while the keratinase of A. ganisms (5, 31). ﬂavus has its optimum at pH 11, which is very high among the We have analyzed the effect of protease inhibitors and re- keratinases reported before. The P. marquandii keratinase is ducing agents on enzyme activity (Table 1). Both puriﬁed kera- special for its temperature optimum, which was determined to tinases were totally inhibited by the serine protease inhibitor be 60 to 65°C (Fig. 2B). Other reported keratinases, except PMSF and partially by EDTA (around 30%), while iodoacet- thermostable ones, were mostly active up to 50°C. amide did not cause any inhibition. The maximal increase of Since enzyme assays were performed at 45°C, a slightly high enzyme activity on stratum corneum keratin was observed by temperature, the enzyme stability of keratinases at that tem- the presence of the reducing agent DTT. The activity was VOL. 71, 2005 FUNGAL KERATINOLYTIC PROTEASES 3423 FIG. 2. pH (A) and temperature (B) optima for the keratinase of P. marquandii. Œ, citrate buffer; F, phosphate buffer; Œ, Tris-HCl; , glycine-NaOH buffer. increased twofold for the P. marquandii keratinase and three- D. microsporus keratinase vary by ﬁve amino acid residues. The fold for the D. microsporus keratinase. The reducing agent highest similarity with the sequence of the P. marquandii en- -ME only slightly increased the activity of the P. marquandii zyme, 12 out of 13 residues identical, was found in the se- keratinase but signiﬁcantly increased the D. microsporus kera- quence of a serine protease from another species of the same tinase activity. genus, Paecilomyces lilacinus, while the sequence most homol- Since DTT and -ME are known to cleave disulﬁde bridges, ogous to the D. microsporus enzyme, 10 out of 13 residues an inﬂuence either on enzyme or on keratin substrate was identical, was found at the N terminus of a serine protease possible. Thus, the enzyme activity was measured with 1 mM from Acremonium chrysogenum. Among the proteases from AAPF, which does not contain disulﬁde bridges. The activity Table 2, only proteinase K from Tritirachium album is de- on the synthetic substrate remained unchanged in the presence scribed as keratinolytic. of DTT and -ME for both keratinases (data not shown). The Proteolytic speciﬁcity of keratinases. The peptide cleavage result conﬁrmed that the reducing agents acted on the keratin speciﬁcity of the enzymes was tested using synthetic substrates substrate and not on the enzymes. The inﬂuence of some and oxidized insulin B-chain. A variety of synthetic oligopep- additives which were present in some assays was also measured tides were examined as a substrate for keratinases. The most (Table 1). DMSO did not inﬂuence the activity of keratinases favored substrate for both enzymes was AAPF; cleavage of the up to a concentration of 5%, while isopropanol decreased the substrate AAA was 100-fold lower than cleavage of AAPF. activity about 13% at a concentration of 1%. SDS inhibited The keratinases also possessed an esterase activity, as they both enzymes at a concentration of just 0.1%. were able to hydrolyze ATEE. The other substrates, FALGPA, Thirteen-amino-acid-residue sequences of the N termini of L-BAPA, and Bz-Tyr-pNA, were not hydrolyzed (not shown). the puriﬁed keratinases from P. marquandii and D. microsporus This experiment demonstrated that keratinases preferentially were determined, and a sequence similarity search was per- cleave the peptide bond at hydrophobic aromatic (AAPF) and formed in the SwissProt/TrEMBL database. The comparisons aliphatic (AAA) amino acids at the P-1 site of synthetic oli- between determined sequences and 13-residue sequences of gopeptides. Several researchers also found AAPF to be the the N termini of various proteases are presented in Table 2. best substrate for their keratinase (6, 17, 26, 32, 38). Therefore, The sequence of the P. marquandii keratinase and that of the the tetrapeptide AAPF was chosen to determine kinetic pa- rameters. Besides the two keratinases, proteinase K was also TABLE 2. Alignment of the N-terminal sequences of P. marquandii tested for comparison. and D. microsporus keratinases with N-terminal sequences of The initial velocities of hydrolysis for different AAPF con- similar proteasesa centrations in the range from 0.1 mM to 5.0 mM were deter- Enzymeb Sequence mined. Then, kinetic parameters were calculated using the molar absorption coefﬁcient for p-nitroaniline determined un- Keratinase of P. marquandii .......................................ALTQQPGAPWGLG Serine protease of P. lilacinus (Q01471)...................AYTQQPGAPWGLG der our experimental conditions (ε 8,800 l mol 1 cm 1). The Keratinase of D. microsporus......................................ATVTQNNAPWGLG results are summarized in Table 3. The highest afﬁnity towards Alkaline proteinase of A. chrysogenum the substrate AAPF was exhibited by the keratinase of P. (P29118) ....................................................................ALVTQNGA–WGLG marquandii. Its catalytic efﬁciency (kcat/Km) of 241 mM 1 s 1 Proteinase K (P06873).................................................––AAQTNAPWGLA Subtilisin (SUBSCL)....................................................ALA–QTV–PYGIP was higher than those of the D. microsporus keratinase and of a proteinase K, 9 mM 1 s 1 and 95 mM 1 s 1, respectively. Our Underlined amino acid residues are identical to corresponding residues in the P. marquandii sequence. measurements on AAPF showed that the kinetic constants for b The SwissProt/TrEMBL data base number is in parentheses. the P. marquandii keratinase are comparable to the constants 3424 ˇ GRADISAR ET AL. APPL. ENVIRON. MICROBIOL. TABLE 3. Kinetic parameters for hydrolysis of AAPF with P. marquandii and D. microsporus keratinases and with proteinase K Enzyme Km Vmax kcat kcat/Km (mM) (107 mmol/s) (s 1) (mM 1s 1) Keratinase of P. marquandii 0.17 0.02 7.3 0.1 41.0 241 Keratinase of D. microsporus 1.03 0.17 11.7 0.4 8.8 9 Proteinase K 1.49 0.30 8.2 0.4 142.0 95 given in the literature for other microbial keratinases (2, 3, 6, 26, 32). Among them, the Michaelis-Menten constant Km for AAPF was the lowest for the keratinase of P. marquandii. Further, the proteolytic speciﬁcity of both keratinases was examined by hydrolysis of the oxidized insulin B-chain. The cleavage sites were compared with the cleavage sites of some other proteases (Fig. 3). The two fungal keratinases exhibited very broad speciﬁcity, with preferential selectivity for hydro- phobic and aromatic amino acids on the P-1 site for Leu (L), Val (V), Tyr (Y), and Phe (F). The proﬁle of cleavage sites for the keratinase of D. microsporus was more similar to that of proteinase K, while the keratinase of P. marquandii revealed a FIG. 4. Hydrolysis of different keratinous and nonkeratinous sub- smaller number of cleavage sites. However, a large number of strates by keratinases of P. marquandii and D. microsporus after 30 min of incubation. The activity of D. microsporus keratinase on stratum cleavage sites for the keratinases of P. marquandii and D. corneum was deﬁned as 100%. microsporus as well as for proteinase K in the oxidized B-chain of insulin distinguished these enzymes from subtilisin and es- pecially from trypsin and elastase. Substrate speciﬁcity of keratinases. To investigate the kera- keratinases are able to hydrolyze -keratins from skin, nail, tinase substrate speciﬁcity, we prepared different keratinous and hair but not -keratins from chicken feathers. substrates from natural human and animal keratinous materi- It was shown that the presence of reducing agents stimulated als. Skin keratins, called soft keratins, possess up to 10% of enzyme hydrolysis of keratin. At a 1 mM concentration of cysteine residues. Hard keratins, which are constituents of skin DTT, the activity of the D. microsporus keratinase was three appendages, possess more than 15% of cysteine residues and times higher and that of the P. marquandii keratinase was two are more resistant to proteolysis than soft keratins. The activity times higher than the activity of the enzymes without DTT of the P. marquandii and D. microsporus keratinases on kera- addition. The stimulating effect of reducing agents has been tins and on other proteins is presented in Fig. 4. reported by many authors (16, 18, 20, 22, 36). They agreed that Among keratins, the soft keratins of the stratum corneum reducing agents reduced disulﬁde bonds of keratinous ﬁla- were preferably hydrolyzed. The keratinases also showed the ments and allowed access of the enzymes to the substrate for ability to degrade commercial bovine keratin and human and proteolytic attack. The amounts of soluble products formed porcine nail, but to a much lesser degree. Human hair, sheep during casein hydrolysis were comparable to the product wool, and chicken feathers were not hydrolyzed in short incu- amount after stratum corneum hydrolysis, whereas the globu- bation times of 30 min. With prolonged incubation of up to lar protein bovine serum albumin was not hydrolyzed to such a 24 h, the D. microsporus (10) and P. marquandii keratinases high level. also hydrolyzed hair and wool keratin (data not shown). Thus, The keratinases of P. marquandii and D. microsporus as well FIG. 3. Cleavage sites on oxidized insulin B-chain for the keratinases of P. marquandii and D. microsporus. For comparison, the cleavage sites for proteinase K (14) as well as subtilisin, trypsin, and elastase (1) are presented. Thick arrow, major cleavage site, 20 min of hydrolysis; thin arrow, minor cleavage site, 12 h of hydrolysis. VOL. 71, 2005 FUNGAL KERATINOLYTIC PROTEASES 3425 FIG. 5. Amount of soluble products during hydrolysis of (A) keratin and (B) casein by keratinases and selected proteases. as keratinases reported by other authors, with one single ex- As expected, the P. marquandii and D. microsporus kera- ception (5), in addition to keratin hydrolyzed the nonkerati- tinases, as well as proteinase K, hydrolyzed keratin more ex- nous substrates casein and bovine serum albumin. Interest- tensively than the other proteases. The keratinase of P. mar- ingly, elastin and collagen are also ﬁbrillar constituents of skin, quandii was about 20% and the keratinase of D. microsporus but only slight activity of the tested keratinases on these sub- was about 50% less active on stratum corneum keratin than strates was detected. The same observations were reported for was proteinase K. However, all the other enzymes tested were the Streptomyces albidoﬂavus (3) and Stenotrophomonas sp. only slightly active or not active at all, as was the case with the keratinases (39). However, on the majority of substrates tested, collagenase. When caseinolytic activity was measured, the the keratinase of P. marquandii proved to be about twice as keratinases and proteinase K were closely followed by elastase, active as the D. microsporus keratinase. A quantitative com- chymotrypsin, and subtilisin. parison to other keratinases reported in the literature is prac- The ratio between the velocity of keratin hydrolysis and the tically impossible because keratinous substrates were not iden- velocity of casein hydrolysis for each enzyme was a criterion for tical and methods of activity measurements and deﬁnitions of the enzyme speciﬁcity for keratinous substrates (Table 4). The keratinolytic units are not uniﬁed. keratinase of P. marquandii and proteinase K revealed the Hydrolysis of model substrates with keratinases and other highest speciﬁcity, with a ratio of around 0.7, and the speciﬁcity proteases. We have chosen two model substrates (keratin of of the D. microsporus keratinase was lower, with a ratio of 0.52. the stratum corneum and casein) and compared the keratinase However, with the other enzymes the values were lower than activities on these substrates with the activities of some pro- 0.3. The value for trypsin was 0.42 due to the weak hydrolysis teases of fungal (proteinase K), bacterial (collagenase and of casein and not due to the signiﬁcant degradation of keratin. subtilisin), and animal (chymotrypsin, elastase, and trypsin) A ratio higher than 0.5 for the keratinases and proteinase K origin. Proteinase K is a keratinolytic protease with a well- conﬁrms that these enzymes degrade keratins more efﬁciently known broad speciﬁcity. Except for the collagenase, which is a than other proteases do. Similar results were reported by metalloprotease, all other enzymes are serine-type proteases, Cheng et al. (4) for the bacterial keratinase of Bacillus licheni- as are the two keratinases studied. The keratinolytic and case- formis. Generally, the keratinases reported in the literature inolytic activities of the proteolytic enzymes are shown in Fig. were 5- to 20-fold more active on keratinous substrates than 5. other proteases (2, 3, 21, 39). In summary, the keratinases are enzymes which catalyze the degradation of keratins. All of them are proteases, but not all TABLE 4. Ratio of velocities of keratin to casein hydrolysis as a proteases hydrolyze keratins. Taking into account that in kera- criterion of enzyme speciﬁcity for keratinous substrates tins a high percentage of the molecule represents hydrophobic and aromatic amino acids (approximately 50%) (13), it could Velocity (U/mg/min) Ratio, Enzyme be concluded that keratinases are successful in hydrolysis of Keratin Casein keratin/casein keratinous materials due to the speciﬁc amino acid composi- Proteinase K 35.1 50.4 0.70 tion of keratins as well as to their broad speciﬁcity. Keratinase of P. marquandii 24.8 36.3 0.69 Keratinase of D. microsporus 14.6 27.9 0.52 Elastase 8.2 32.0 0.25 ACKNOWLEDGMENTS Subtilisin 6.6 21.3 0.30 Trypsin 4.2 10.0 0.42 This work was partially supported by the Ministry of Higher Edu- Chymotrypsin 3.0 25.8 0.12 cation, Science and Technology of Slovenia. Collagenase 0 4.8 0 We thank J. P. Chamount from the Faculty of Medicine and Phar- ¸ macy in Besancon, France, for the donation of fungal strains. We also 3426 ˇ GRADISAR ET AL. APPL. ENVIRON. 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