Similarities and Specificities of Fungal Keratinolytic Protease - PDF

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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
helena.gradisar@ki.si.                                                       producers of the keratinolytic enzymes. Both strains have been isolated and

                                                                      3420
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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. MICROBIOL.

                          ˇ
thank B. Kralj from the Jozef Stefan Institute in Ljubljana, Slovenia,               21. Letourneau, F., V. Soussotte, P. Bressollier, P. Branland, and B. Verneuil.
for ESI-MS analyses.                                                                     1998. Keratinolytic activity of Streptomyces sp. S.K1-02: a new isolated strain.
                                                                                         Lett. Appl. Microbiol. 26:77–80.
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