azokeratin protocol

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Abstract A novel feather-degrading microorganism wasisolated from poultry waste, producing a high keratinolyticactivity when cultured on broth containing native feather.Complete feather degradation was achieved during culti-vation. The bacterium presents potential use for biotech-nological processes involving keratin hydrolysis.Chryseo-bacterium sp. strain kr6 was identified based on morpho-logical and biochemical tests and 16S rRNA sequencing.The bacterium presented optimum growth at pH8.0 and30 C; under these conditions, maximum feather-degrad-ing activity was also achieved. Maximum keratinase pro-duction was reached at 25 C, while concentration of sol-uble protein was similar at both 25 and 30C. Reductionof disulfide bridges was also observed, increasing withcultivation time. The keratinase of strain kr6 was activeon azokeratin and azocasein as substrates, and presentedoptimum pH and temperature of 7.5 and 55 C, respec-tively. The keratinase activity was inhibited by 1,10-phen-anthroline, EDTA, Hg2+, and Cu2+ and stimulated by Ca2+.

Keywords Keratin Proteolysis Poultry waste Chicken feather Bacteria

Introduction

Feathers are produced in large amounts as a waste by-product at poultry processing plants, reaching millions oftons per year worldwide (Williams et al. 1991). Sincefeathers are almost pure keratin protein, feather wastesrepresent a potential alternative to more expensive dietary

ingredients for animal feedstuffs (Shih 1993). However,feathers are currently utilized on a limited basis as a di-etary protein supplement for animal feed because feathermeal production is an expensive process, requiring signif-icant amounts of energy. In addition this process destroyscertain amino acids, yielding a product with poor digest-ibility and variable nutrient quality (Papadopoulos et al.1986; Wang and Parsons 1997).

Keratin is the insoluble structural protein of feathersand wool and is known for its high stability (Bradbury1973). The composition and molecular configurations ofits constituent amino acids warrant structural rigidity. Thekeratin chain is tightly packed in the -helix (-keratin)or -sheet (-keratin) into a supercoiled polypeptide chain(Parry and North 1998), resulting in mechanical stabilityand resistance to common proteolytic enzymes such aspepsin, trypsin, and papain. In addition, cross-linking ofprotein chains by cysteine bridges confers high mechani-cal stability and resistance to proteolytic degradation ofkeratins. Nevertheless, feathers do not accumulate in na-ture, since structural keratin can be degraded by some mi-croorganisms (Onifade et al. 1998). The current investiga-tion has been focused on proteolytic microorganisms;however, reduction of cysteine bridges may significantlyinfluence keratin degradation (Noval and Nickerson 1959;Kunert and Stransky 1988).

Keratinolytic enzymes may have important uses inbiotechnological processes involving keratin-containingwastes from poultry and leather industries, through thedevelopment of non-polluting processes (Shih 1993; Oni-fade et al. 1998). After hydrolysis, the feathers can beconverted to feedstuffs, fertilizers, glues, and films orused for the production of the rare amino acids serine,cysteine, and proline (Papadopolous et al. 1986; Yamauchiet al 1996). Known keratinases are mainly produced bymesophilic fungi and actinomycetes (Noval and Nicker-son 1959; Kushwaha 1983; Bckle et al. 1995; Santos etal. 1996), but some thermophilic species ofBacillus pro-duce feather-degrading enzymes (Williams et al. 1990;Kim et al. 2001). The use of keratinase to upgrade the nu-tritional value of feather meal has been described. Com-

Alessandro Riffel Franoise Lucas Philipp Heeb Adriano Brandelli

Characterization of a new keratinolytic bacterium

that completely degrades native feather keratin

Arch Microbiol (2003) 179: 258265DOI 10.1007/s00203-003-0525-8

Received: 15 October 2002 / Revised: 26 December 2002 / Accepted: 22 January 2003 / Published online: 28 February 2003

ORIGINAL PAPER

A. Riffel A. Brandelli ()Departamento de Cincia de Alimentos, ICTA,Universidade Federal do Rio Grande do Sul,Av. Bento Gonalves 9500, 91501970 Porto Alegre, Brazile-mail: [email protected]

F. Lucas P. HeebInstitut dEcologie, University of Lausanne,1015 Lausanne, Switzerland

Springer-Verlag 2003


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parable growth rates were observed between chickens fedwith isolated soybean and those fed with feather meal fer-mented withStreptomyces fradiae plus methionine sup-plementation (Elmayergi and Smith 1971). The utilizationof aBacillus licheniformis feather-lysate with amino acidsupplementation in test diets fed to growing broilers pro-duced a weight gain identical to that achieved with soy-bean meal (Williams et al. 1991). The use of crude kera-

tinase significantly increased the amino acid digestibilityof raw feathers and commercial feather meal (Lee et al.1991).

The aim of this study was to identify new keratinolyticbacteria showing high feather degradation at room tem-perature, with potential application in biotechnological pro-cesses. Such microorganisms will be less energy-consum-ing than the thermophilic strains usually used in featherprocessing. We have isolated mesophilic feather-degrad-ing bacteria from industrial poultry waste. In a previouswork, we described the characterization of the proteolyticVibrio sp. strain kr2, which may be potentially useful forprocesses involving keratin hydrolysis (Sangali and Bran-

delli 2000). This report describes the identification, growth,and keratinase production by another novel feather-de-grading isolate.

Materials and methods

Isolation of keratinolytic micro-organism

Feathers were collected from several sites at a local poultry indus-try. Feathers were flooded in peptone broth (5g l1)and incubatedfor 24 h at 30 C. The suspension was used to streak feather-mealagar plates (10 g feather-meal l1 , 0.5 g NaCl l1 , 0.3g K2HPO4l

1,0.4g KH2PO4l

1 , and 15g agar l1 ) which were incubated at 30Cfor 3 days. Single colonies were isolated and screened for theirability to hydrolyze keratin in feather-meal agar plates. Coloniesproducing clear zones in this medium were selected for furtheranalysis.

Growth determination

The isolate from a 106 colony forming units (CFU) ml1 culturewas cultivated for 72 h in whole-feather medium (10g wholefeathers l1 , 0.5g NaCl l1 , 0.3 g K2HPO4 l

1 , 0.4g KH2PO4l1).

Initial pH was set at 5.0, 6.0, 7.0 or 8.0. Cultures were grown in500-ml flasks containing 100ml of medium and incubated at either25, 30 and 37 C in an orbital shaker at 180rpm. Bacterial growthwas monitored by measuring the CFU ml1, as described elsewhere(Sangali and Brandelli 2000). The bacterial suspension was diluted

to 10

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in phosphate-buffered saline. The samples were then ho-mogenized and loaded (20l) in triplicate onto nutrient agar plates,which were incubated for 24 h at 30C. Thirty to 100 colonies weresubsequently counted.

Taxonomic studies

Bacteria were identified based on morphological and biochemicaltests (MacFadin 2000), comparing the data with standard speciesand using an API 20E kit (Bio-Mrieux). Morphological and phys-iological characteristics of the isolated bacterium were comparedwith data fromBergeys manual of systematic bacteriology (Palle-roni 1984).

The sequence of the 16S rRNA gene was determined after ge-nomic DNA extraction, PCR amplification, and sequencing. Ge-nomic DNA was extracted from one colony of isolate kr6, whichwas resuspended in 50 l of distilled water and boiled for 10min.Samples were centrifuged and DNA was amplified by PCR using5l of supernatant, according to the method of Osborn et al.(1999). The bacterial 16S rRNA primers were 63f (5 CAGGCC-TAACACATGCAAGTC 3), 907r (5 CCGTCAATTCCTTTG-AGTTT 3) and 1389r (5 ACGGGCGGTGTGTACAAG 3), cor-responding to Escherichia coli 16S rRNA gene position. PCR

products were purified using the columns from Wizard PCR prepsDNA purification systems (Promega). Sequencing reactions werecarried out in a 7.5-l reaction volume with 15ng of purifiedDNA, 1.5l of 1-M primer 63f, 3 l of BigDye Terminator 3.0(ABI Prism, PE Applied Biosystems), and 0.5l of distilled water.PCR was run for 25 cycles under the following conditions: 96 Cfor 20 s, 55C for 10 s, and 60 C for 4min. An ABI PRISM373 XL (PE Applied Biosystems) was used for sequencing. The1,237-bp sequence was submitted to Genbank (accession numberAY157745). The BLAST algorithm was used to search for homol-ogous sequences in Genbank. The sequence was reversed, aligned,and compared to similar database sequences using the GeneticsComputer Group package (Madison, Wis.). The phylogenetic treewas inferred from Jukes-Cantor distances using the neighbor-join-ing method (software PHYLIP 3.6 a2, Felsenstein 1989). Thebranching pattern was checked by 1,000 bootstrap replicates.

Enzyme production

The organism was cultivated for 72h in whole-feather medium,from a 106 CFU ml1 culture. Samples were centrifuged at10,000g for 5 min, and the supernatant was used as a crude en-zyme preparation.

Synthesis of azokeratin

Azokeratin was synthesized based on the methodology describedfor azoalbumin (Tomarelli et al. 1949). Keratin was coupled witha diazotized aryl amine to produce a chromophoric derivative, sul-fanilic acid-azokeratin. Fifteen grams of feather keratin were sus-

pended in 1 l distilled water, and 100 ml of a 100 g NaHCO3 l1

so-lution were added with stirring. Simultaneously, 8.65 g of sul-fanilic acid were dissolved in 200 ml of 0.12M NaOH; then 1.7 gNaNO2 were added. The solution was stirred and 10ml of 5.0 MHCl added. The solution was stirred again for 2 min and then 10mlof 5.0 M NaOH were added with stirring. This solution was mixedwith the keratin suspension. The reaction mixture was stirred for5 min and dialyzed against distilled water at 4 C. The dialysed so-lution was freeze-dried. Azokeratin is a deep red-orange com-pound with an absorption maximum at 440450 nm.

Enzyme assays

Keratinase activity was assayed with azokeratin as a substrate bythe following method. The reaction mixture contained 100 l of

enzyme preparation and 800l of 10 g azokeratin l1

in 50mM trisbuffer, pH 8.0. The mixture was incubated for 15 min at 50 C; thereaction was stopped by the addition of trichloroacetic acid to a fi-nal concentration of 100 g l1. After centrifugation at 10,000g for5 min, the absorbance of supernatant was determined at 440 nm.One unit of enzyme activity was the amount of enzyme that causeda change of absorbance of 0.01 at 440nm for 15min at 50 C. Asimilar protocol was used to determine enzyme activity on azoca-sein. Activity on benzoyl-arginine-p-nitroanilide (BAPNA) wascarried out as described previously (Sangali and Brandelli 2000).

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Determination of protein concentration

A cell-free supernatant of each feather culture was used for the de-termination of soluble protein by the Folin phenol reagent method(Lowry et al. 1951), with bovine serum albumin as standard.

Determination of thiol formation

Free thiol groups were determined essentially as described else-

where (Sullivan et al. 1942). To 1ml of sample were added 0.2 mlof NH4OH, 1ml of 0.5 g NaCN l1 , and 1 ml of water. The mixture

was incubated for 20 min at 25C, and then 0.2 ml of 0.5g sodiumnitroprusside l1 were added. Absorbance at 530 nm was measuredwithin 2 min.

Effects of chemicals on keratinase activity

Chemicals were added to the enzyme preparations and incubatedfor 15 min at 25C before being tested for keratinase activity. Theprotease inhibitors phenylmethylsulfonyl fluoride (PMSF), EDTA,and pepstatin, the detergents SDS and Triton X-100, the organicsolvents dimethyl sulfoxide (DMSO) and isopropanol, and the re-ducing agent 2-mercaptoethanol were used at the working concen-trations listed in Table 2. The metal ions tested were added to reacha working concentration of 10 mM.

Chemicals

Azocasein, BAPNA, PMSF, pepstatin, and Folin-Ciocalteaus re-agent were from Sigma (St. Louis, Mo., USA). Other reagentswere from Merck (Darmstadt, Germany) unless otherwise stated.

Results

Characterization of keratinase-producing strain

The feather-degrading microorganism kr6 isolated fromfeathers in decomposition demonstrated pronounced growth

in whole-feather medium. This isolate was selected foridentification and for its adaptation to feather degradation.Cells of the isolate were grown on whole feathers andtransferred at frequent intervals to basal medium contain-ing whole feathers. Feather barbules were completely de-graded and rachis was also attacked by the bacterium(Fig. 1). The isolate was able to complete the disintegra-tion of whole feathers.

The results of taxonomic studies on the isolated strainkr6 are summarized in Table1. The identification of thisbacterial isolate was based on cell and colony morphol-ogy, growth characteristics, several biochemical tests,API20E, and 16S rRNA sequence data. Microscopic ob-

servation of the isolate showed a gram-negative straightrod; the bacterium grew aerobically and formed typicalyellow colonies. Together with physiological and API20E profiling, these characteristics suggest theFlavobac-teriaceae family, genusFlavobacterium (Palleroni 1984)and genusChryseobacterium (Vandamme et al. 1994). Inthe genusChryseobacterium, the new isolate was similartoC. gleum andC. indologenes.

The genus determination based on physiological traitswas confirmed by phylogenetic analysis of the 16S rRNAgene. The 16S rRNA sequence of strain kr6 showed highsimilarities to those of a group consisting of severalChry-seobacterium strains (similarity 95.699.2%). The isolate

kr6 shared 99.2% sequence similarity with Chryseobac-terium gleum ATCC 35910 and 98.6% similarity withChryseobacterium indologenes ATCC 29897 (Fig. 2). Boot-strap analysis resulted in relatively high values for thebranching of kr6 within the Chryseobacterium cluster.Other related members of the Flavobacteriaceae (Berge-

yella, Empedobacter, Capnocytophaga, Coenonia, Areni-bacter, Aequorivita, Flavobacterium species) shared 81.294.4% sequence similarity with strain kr6 (Fig.2).

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Fig. 1A, B Feather degradation by keratinolytic bacteria. Nativefeathers were autoclaved and incubated at 30 C for 3 days inA theabsence orB the presence of strain kr6.Bar10m

Table 1 Morphological and physiological characteristics of ker-atinase-producing bacterial strain kr6

Morphological characteristics

Form Rods

Size 0.51-2 m

Gram stain Negative

Spore Non-sporulating

Cultural characteristics

Feather meal agar colonies Yellow color, circular, smooth,convex, undulate, moist

Physiological characteristics

Catalase Positive

Oxidase Positive

Oxidation-fermentation test Oxidative

Voges-Proskauer test Negative

Citrate Negative

Nitrate reduction Positive

Gelatin liquefaction Positive

Starch hydrolysis Negative

Lipase Positive

DNAse Negative

Lysine decarboxylase NegativeOrnithine decarboxylase Negative

Arginine dihydrolase Negative

Triple sugar iron agar Acid slant/alkaline butt/no H2Sproduction

Motility Negative


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Optimal growth conditions

The newly isolated strain grew at pH 5.08.0 and 2237C,with an optimum at pH8.0 and 30 C.Chryseobacte-rium sp. strain kr6 grew at approximately the same rate at

2537 C and from a starting pH of 58, although at neu-tral to alkaline pH, cells began to lyse soon after reachingstationary phase (data not shown).

Production of keratinase and soluble protein

The effects of pH and temperature on the production ofkeratinase and soluble protein were investigated. Maxi-mum enzyme activity was observed during cultivation at25C (96U ml1 at 48h), followed by 30 C (64 U ml1 at36 h) and then at 37C (40 U ml-1 at 48h). Keratinase pro-

duction was similar at starting pH values of 5.08.0, andsoluble protein concentration was higher with increasingstarting pH (data not shown). More efficient feather degra-dation was observed at 30C and initial pH 8.0.

Keratinase activity was followed during cultivation of

strain kr6 in whole-feather medium at 30C and initialpH 8.0. Keratinase reached a maximum activity at 48 h,coinciding with the end of the exponential phase, then de-creased, and increased again at 100h (Fig.3A). The kinet-ics of production of soluble protein and free amino acidsare shown in Fig. 3B. Maximum values were reached at36 h, at which the concentration of soluble protein was ap-proximately five times higher than the amino acid con-centration.

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Fig. 2 Phylogenetic position of strainkr6 within the genusChryseobacte-rium and allied bacteria. The branch-ing pattern was generated by theneighbor-joining method. The Genbankaccession number of the 16S rRNAnucleotide sequences used are indi-cated inparentheses. The number ofeach branch indicates the bootstrapvalues.BarJukes-Cantor distance of

0.05.Cytophaga fermentas was usedas an outgroup


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Reduction of disulfide bonds

The high stability of keratin proteins against mechanicaland enzymatic attack is due, at least in part, by the occur-rence of cysteine bridges. Reduction of disulfide bonds bystrain kr6 was investigated. Thiol formation increasedwith cultivation time (Fig. 4), reaching a maximum at 40 h.The rate of thiol formation was maximum during the ex-ponential phase.

Characterization of keratinase

The effect of pH on keratinase activity was determined.The enzyme was active in the range of pH68 with amaximum at pH7.5 (Fig. 5). The effect of temperature onkeratinase activity is shown in Fig. 6. Activity was ob-served in the range of 4585C with a maximum at 75C(Fig. 6A). The activity was stable for 30 min at 37C,with 80% of the initial activity remaining after 1h and60% after 2 h. At 55 C and 70C, the enzyme maintained50% its initial activity after 2h of incubation (Fig. 6B).

The keratinase was active on azocasein but not onBAPNA as substrate. Keratinase activity was investigatedafter preincubation of the enzyme with several chemicalsfor 15 min. The effects of these chemicals on keratinaseactivity are shown in Tables 2 and 3. The enzyme was in-hibited by 1,10 phenanthroline and EDTA (Table 2), al-though other protease inhibitors also had minor effects(1424% inhibition). The enzyme maintained at least 50%of its activity after incubation with SDS or organic sol-vents. The use of the non-ionic detergent Triton X-100and reducing agent caused important increases in enzymeactivity (Table 2). The enzyme was totally inhibited byHg2+ and Cu2+ (Table 3). Activity increased in the pres-ence of Ca2+ and was partially inhibited by Zn2+ (Table3).

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Fig. 3A, B Proteolytic activity during growth of Chryseobac-terium strain kr6 in whole-feather medium at 30 C and initialpH 8.0.A Keratinase activity measured using azokeratin as sub-strate() and viable cell counts ().B Concentration of soluble

protein (

) and free amino acids (

). Each point represent themeanSEM of three independent experiments

Fig.4 Formation of extracellular thiol groups during growth ofChryseobacterium strain kr6 in whole-feather medium at 30 Cand initial pH 8.0. Each point represent the meanSEM of three in-dependent experiments

Fig.5 Effect of pH on the activity of Chryseobacterium sp. kr6keratinase. Keratinolytic activity was measured at different pHvalues. Each point represent the meanSEM of three independentdeterminations


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Discussion

A feather-degrading bacterium was isolated from a poul-try-processing industry. Based on phenotypical and phylo-genetical characteristics, strain kr6 belongs to the Flavo-bacteriaceae of theCytophaga-Flavobacterium group and

was identified as Chryseobacterium sp. strain kr6. The16S rRNA sequence of strain kr6 showed 99% similarityto that ofChryseobacterium gleum ATCC 35910.

Chryseobacterium strains have been isolated from var-ious ecosystems such as water, soil, fish, marine environ-ments, and clinical specimens. SeveralChryseobacteriumstrains produce highly proteolytic activities (Jooste andBritz, 1986; Yamaguchi and Yokoe 2000). However, theisolate kr6 is the firstChryseobacterium strain describedas a feather degrader. In two earlier studies, two Cytopha-gales isolates were discovered that solubilized autoclavedfeathers and wool, the organisms resembled Cytophaga

johnsoniae but were never clearly classified (Reichem-

back 1992). Until recently, feather degradation by bacteriahas been described only in gram-positive bacteria such as

Bacillus andStreptomyces species (Williams et al., 1990;Bockle et al., 1995; Onifade et al. 1998; Zaghloul 1998;Kim et al. 2001). We described a Vibrio sp. presentinghigh feather-degrading activity (Sangali and Brandelli2000). Lucas et al. (2003) also isolated feather-degradingstrains from soils, belonging to theCytophaga-Flavobac-terium group. The isolate presented optimum growth atmesophilic temperatures, as expected from its environ-mental origin. Other previously described keratinolyticbacteria generally have optimum growth and featherdegradation activity at highest temperatures (Williams et

al. 1990; Nam et al. 2002).Strain kr6 caused a significant increase in the pH of themedium during cultivation on raw feathers and was ableto complete feather degradation, indicating its strong kera-tinolytic character. Organisms with higher keratinolyticactivity alkalinize the media to a greater extent than thoseexhibiting lower keratinolytic activity (Kaul and Sumbali1997). This tendency to alkalinize the medium resultsfrom the production of ammonia by means of the deami-nation of peptides and amino acids originating from ker-atin degradation. The resulting increase of pH is typical ofmicroorganisms growing on protein substrates.

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Table 2 Effect of chemicals on keratinase activity of Chryseo-bacterium sp. strain kr6. Values are representative of three inde-pendent determinations

Chemical Concentration Residualactivity (%)

Control 100

PMSF 1 mM 83

E-64 5 M 86

Pepstatin 1 mM 76

EDTA 5 mM 43

EGTA 5 mM 601,10 phenanthroline 1 mM 12

SDS 0.1% (w/v) 50

SDS 0.5% (w/v) 88

Triton X-100 0.1% (v/v) 231

0.5% (v/v) 77

DMSO 1% (v/v) 75

5% (v/v) 68

Isopropanol 1% (v/v) 56

5% (v/v) 56

2-Mercaptoethanol 0.1% (v/v) 238

0.5% (v/v) 172

Table 3 Effect of ions on ker-atinase activity ofChryseobac-terium sp. strain kr6. Valuesare representative of three in-dependent determinations.Compounds were tested ata working concentration of10mM

Ion Residualactivity (%)

Control 100

CaCl2 116

BaCl2 96

Fe2Cl3 102

HgCl2 0

MgCl2 81

CuSO4 0

MnCl2 81

ZnCl2 62

Fig. 6A, B Effect of temperature on the activity and stability ofChryseobacterium sp. kr6 keratinase.A Enzyme activity was de-termined at various temperatures.B Enzyme was incubated for in-dicated times at 37C (), 55C (), or 70 C () and residual ac-tivity was measured


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Strain kr6 produced an extracellular keratinase, thelevels of which varied during cultivation on whole feath-ers. This pattern suggests that the enzyme is inducible andthat substrate and metabolite levels in the extracellularmilieu regulate its secretion. These results resemble thoseobtained with cultures ofStreptomyces pactum (Bckle etal. 1995),Trichophyton species (Grzywnowicz et al. 1989),

Bacillus sp. (Atalo and Gashe 1993) andVibrio sp. (San-

gali and Brandelli 2000) growing on keratin substrates.Thiol formation by strain kr6 suggests the presence of

disulfide reductase activity. Reduction of disulfide bridgeswas also observed for Streptomyces pactum grown onfeathers (Bckle and Mller 1997), andStreptomyces fra-diae on wool (Kunert and Stransky 1988). Kunert (1989)reported that keratin degradation by S. fradiae andMi-crosporum gypseum extracellular proteases follows sulfi-tolysis of cystine. In addition, the use of reducing agentsto enhance keratin degradation by keratinases has beendescribed (Bckle et al. 1995; Letourneau et al. 1998).The stimulatory effect of 2-mercaptoethanol on kerati-nolytic activity of strain kr6 may be explained by the re-

duction of disulfide bridges of azokeratin, allowing amore accessible substrate. Thus, screening for kerati-nolytic micro-organisms should also lead to the isolationof organisms possessing disulfide reductases.

The main proteolytic activity of keratinases is nor-mally associated with serine proteinase activity (Bckle etal. 1995; Lin et al. 1995), The keratinases produced by

B. licheniformis andB. subtilis are serine proteases (Lin etal. 1995; Suh and Lee 2001), and their genes (kerA andaprAk) show significant similarity with subtilisins, whichare typical members of the serine-protease family (Lin etal. 1995; Zaghloul 1998). However, the keratinase pro-duced by strain kr6 appears to belong to the metallopro-

tease type since it was inhibited by EDTA and 1,10-phen-anthroline (Zn2+-specific chelator) and did not hydro-lyze the substrate BAPNA. These features resemble thoseofFlavobacterium/Chryseobacterium proteases. The twomajor proteases secreted by Flavobacterium meningo-septicum are zinc metalloendopeptidases, one of thempresenting unusualO-glycosylation (Tarentino et al. 1995).A protease purified from Chryseobacterium indologeneswas inhibited by EDTA and 1,10-phenanthroline, andatomic absorption analysis showed that the enzyme con-tained Ca2+ and Zn2+ (Venter et al. 1999). The partial in-hibition by EDTA and EGTA could be explained by theincreased strength of zinc binding to the active site at neu-

tral to alkaline pH (Auld 1995).The inhibition by 10 mMZn2+ is also in agreement with the fact that several zincpeptidases are inhibited by excess zinc, particularly atneutral to alkaline pH (Auld 1995). Activation by Ca2+

and inhibition by Zn2+ is also a feature of keratinase ofVibrio kr2 (Sangali and Brandelli 2000) and of some cal-pains (Sorimachi et al. 1997). The results of inhibitorstudies also suggest that gram-negative bacteria possesskeratinases that differ from those previously isolated fromgram-positive bacteria.

The new Cytophagale strain described here has highkeratinolytic activity and is very effective in feather degra-

dation, suggesting its potential use in biotechnologicalprocesses involving keratin hydrolysis. In addition, thekeratinase produced by strain kr6 was active over a widerange of pH values and temperatures, and was relativelyheat stable. These are interesting properties for regardingindustrial use of the enzyme.

Acknowledgements A.R. is the recipient of a M.Sc. fellowshipfrom CNPq. A.B. is Research Fellow of CNPq, Brazil. F.L. P.H.

acknowledges the Fondation de Famille Sandoz for financing hisresearch (subsidy to P. Heeb). We thank Jitka Lipka for runningour samples at the sequencing facility of the Institute of Ecology(University of Lausanne).

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azokeratin protocol

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