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Contents lists available at SciVerse ScienceDirect

Process Biochemistry

journa l h o me page: www.elsev ier .com/ locate /procbio

ron containing keratinolytic metallo-protease producedy Chryseobacterium gleum

riti N. Chaudhari, Bhushan L. Chaudhari ∗, Sudhir B. Chincholkarepartment of Microbiology, School of Life Sciences, North Maharashtra University, P B 80, Jalgaon 425 001, India

r t i c l e i n f o

rticle history:eceived 19 June 2012eceived in revised form 19 October 2012ccepted 8 November 2012

eywords:hryseobacterium gleum

a b s t r a c t

Chryseobacterium gleum exhibited complete dissolution of whole chicken-feathers (10 g l−1, pH 8) after72 h at 30 ◦C through synthesis of keratinolytic protease when inoculated at 1% (v/v). This enzyme waspurified to 67-fold with yield of 2.25% having a specific activity of 1670 U mg−1 and ∼36 kDa Mw. MALDI-TOF MS of this keratinase showed some similarity with the keratinase peptides of Bacillus subtilis (BOFXJ2).The keratinase action was inhibited by EDTA, iodoacetamide and metal ions like mercury, copper andzinc (1 mM each), while it was enhanced by iron and calcium. Keratinase showed presence of 3 mM of

−1

ronetalloprotease

oultry waste management

Fe M as tested by atomic absorption spectroscopy and addition of Fe in its apoenzyme retained about79% of original residual feather degradation activity which portrayed it to be metalloprotease. Purifiedkeratinase revealed significant degradation (85%) of feather concentrate (20 g l−1) to 3.9 �M ml−1 of freeamino groups in 24 h at an initial pH of 8.0, 30 ◦C and 120 rpm shaking. This keratinase activity can becontrolled precisely by presence of chemical or metal ions which could be of use in biotechnology industry

sed i

. Introduction

Proteases are industrially important enzymes bearing tremen-ous interests in the enzyme-market toward recycling oferatin-wastes because of their multipurpose applications in theeed, fertilizer, detergent, leather, and pharmaceutical industries1]. Keratin has peptide chains packed as tight secondary structureor instance �-helices (hair and wool) or in �-sheet arrangementsfeathers) making it difficult to hydrolyze by common proteolyticnzymes like trypsin, pepsin and papain. Additionally, disulphideinkages also contribute its toughness. Although, complex structure

akes keratin more resistant to chemical and enzymatic destruc-ion, it is a rich source of amino acids [2]. Therefore, an efficientpproach to degrade keratin restoring its high biological value isesired.

Enzymatic hydrolysis allows the formation of hydrolysates withhe high amount of labile amino acids conserved due to the rela-ively mild treatment conditions. Hence, it is the most attractiveption in view of ecological safety and the preservation of func-

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ional properties of the hydrolysis products. Generally, alkaline andubtilisin-like proteases are used for the hydrolysis of keratin. Aarge amount of keratin waste is generated by poultry industry

hose utilization for preparation of keratin hydrolysates throughnzymatic route is a lucrative and eco-friendly option.

∗ Corresponding author. Tel.: +91 2572257421; fax: +91 2572258403.E-mail addresses: [email protected], [email protected]

B.L. Chaudhari).

359-5113/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.procbio.2012.11.009

n poultry waste management.© 2012 Elsevier Ltd. All rights reserved.

Other objectives of such keratinolytic proteases involve themodification of fibers such as silk and wool [3] for shrink proofingand dyeing of wool in the textile industry [4] while in medical andpharmaceutical fields to eliminate acne, psoriasis or human callusand for making vaccines of dermatophytosis [5]. It is also reportedto degrade prion protein leading to the prevention/cure of mad cowdisease [6].

Keratinolytic activity has been reported in several bacteriaincluding Bacillus spp. [7,8], Microbacterium sp. [9], Chryseobac-terium sp. [10,11]. The genus Chryseobacterium was built onthe ruins of the genus Flavobacterium followed by extensivephylogenetic investigations [12]. The culture under study; Chry-seobacterium gleum (LMG P-22264) was a non motile, nonspore-forming, Gram negative, non-hemolytic, aerobic small rodsof soil origin. It was characterized taxonomically and molecularlyand reported for the production of sulfobacin A [13] as well ascholesterol biotransformation [14]. Iron containing proteases aremerely reported in bacteria [15,16] and fungi [17]. Metallopro-teases reported in Chryseobacteria were generally zinc containingkeratinases [18,33], however, through current investigations, wereport iron containing keratinolytic protease of C. gleum.

2. Materials and methods

2.1. Reagents and materials

eratinolytic metallo-protease produced by Chryseobacterium gleum.

The chemicals and reagents were purchased from Hi-Media and Qualigens(Mumbai, India) while phenylmethylsulphonyl fluoride (PMSF) was from Sigma(USA). Azokeratin was synthesized in laboratory as described previously [10]. Wholefeathers (WF) of poults were obtained from a local industry, washed extensively with

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ater, sun dried for 3 days, chopped and ball milled to yield uniform ≤100 mesh sizeeather powder (FP). Initially, whole feathers were used for its dissolution. Later, forest of the studies, FP was used to bring uniformity in feather particle size.

.2. Keratinolytic activity of C. gleum and growth in keratinaceous medium

C. gleum was grown for 72 h in whole-feather (WF) medium containing g l−1 ofhole feathers (10), NaCl (0.5), K2HPO4 (0.3), KH2PO4 (0.4) in water added with its

noculum 1% (v/v) from a 106 colony forming units (CFU) ml−1. Its initial pH was setn the range of 4–11 at difference of pH 1 using 0.1 mM each citrate (pH 4–5), phos-hate (pH 6–7), Tris (pH 7.5–9) and carbonate (pH 10–11) buffers so as to determineptimum pH for growth and enzyme production. In each case, culture was grownn 500 ml capacity Erlenmeyer flasks containing 100 ml of medium and cultivatedt various temperatures ranging from 25 to 50 ◦C in an orbital shaking incubator at20 rpm up to 144 h. Bacterial growth was monitored by measuring the CFU ml−1.amples withdrawn after each 24 h were centrifuged at 10,000 × g for 10 min at 4 ◦Cnd the supernatant was used as a crude enzyme. A culture supernatant was usedor the determination of its protein content with Folin phenol reagent [19] whereSA was used as a standard. Amino acid content was determined by photometricinhydrin method [20] while free thiol groups were determined by DTNB (5,5′-ithio-bis-2-nitrobenzoic acid) assay [21] using N-acetyl-l-cysteine as standard.he A412 was measured and the thiol concentration was calculated from the cali-ration curve plotted using N-acetyl-l-cysteine as a standard. Enzyme activity waslso observed on plates containing keratin as sole substrate. Culture was spot inoc-lated on medium in Petri plate containing g l−1 of feather powder (10), NaCl (0.5),2HPO4 (0.3), KH2PO4 (0.4) having pH 8.0 and agar (15). The plate was incubated at0 ◦C for 72 h and observed for zone of clearance.

.3. Enzyme assay

Enzyme activity was assayed using azokeratin as a substrate according to Riffelt al. [10]. Since, azokeratin has uniform particulate size; it was used as substratelthough insoluble in water. In brief, the reaction mixture contained 100 �l of crudenzyme and 400 �l of 10 g l−1 azokeratin in 50 mM Tris buffer, pH 8.0 to a total vol-me of 1 ml. The reaction mixture was well dispersed and incubated for 60 min at0 ◦C in water bath and later the reaction was stopped by the addition of TCA 10%w/v). In the blank, enzyme was replaced by Tris buffer (50 mM, pH 8.0). After cen-rifugation at 10,000 × g for 5 min, spectrophotometrically the A440 of supernatantas determined. One unit of enzyme activity was the amount of enzyme that caused

change of A440 by 0.01 in 60 min at 30 ◦C.

.4. Enzyme production

The cell culture 1% (v/v) of 18 h age having 106 CFU ml−1 was inoculated intohe FP medium containing g l−1 of feather powder (10), NaCl (0.5), K2HPO4 (0.3),H2PO4 (0.4) in water (pH 8.0) and cultivated for 72 h at 30 ◦C and 120 rpm. After

ncubation, the cell free extract was subjected for enzyme purification.

.5. Enzyme purification

All purification procedures were performed at 4 ◦C. The crude enzyme was sepa-ated by ultrafiltration using hollow fiber cartridge (Amicon, Ireland) having 10 kDaut-off. The concentrate was precipitated by addition of ammonium sulfate (70%)nd the precipitate was dissolved in Tris buffer (50 mM) of pH 8.0. The proteaseample was dialyzed against the same buffer and subjected to further purification.

.5.1. Gel filtration (size exclusion) chromatographyThe dialyzed protease was loaded onto a Seralose CL-6B gel (Sisco Research Lab,

umbai) column (2.0 cm × 10 cm; diameter × length) having separation range of04–106 Da and particle size of 40–190 �m equilibrated with Tris buffer (50 mM,H 8.0). The protein was eluted from the column with same buffer at flow ratef 0.2 ml min−1 and collected in fractions. The protein concentration of the frac-ions collected was determined as A280 and assayed for enzyme activity. The activeractions were pooled together.

.5.2. DEAE cellulose-52 (ion exchange) chromatographyThe protein obtained was applied to a DEAE cellulose-52 (Hi-Media, Mumbai)

olumn (2 cm × 10 cm; diameter × length) pre-equilibrated with 50 mM Tris–HCluffer; pH 8.0. It was eluted with a step gradient of NaCl ranging from 0.0 to 0.5 Mrepared in the same buffer with a flow rate of 0.2 ml min−1. Total of 30 fractionsach of 2 ml were collected for each NaCl pitch and subsequently enzyme activityf each fraction was determined. All the fractions were dialyzed overnight against0 mM Tris–HCl buffer; pH 8.0. The active fractions were pooled, vacuum driedsing a lyophilizer (VirTis 3.3 L, USA) and the purity of protein was checked by gellectrophoresis.

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.6. Polyacrylamide gel electrophoresis

The SDS-PAGE method (sodium dodecyl sulfate-polyacrylamide gel elec-rophoresis) of Laemmli [22] was followed to run protein on gel electrophoresis,

PRESSemistry xxx (2012) xxx– xxx

using a 10% polyacrylamide gel and compared with standard molecular weightmarker (range: 97.4–14.3 kDa; PMWM, Genei, Bangalore). Protein was run on nativePAGE also to obtain zymogram using 1% casein substrate and detected by coomassiebrilliant blue R250 (CBB) [23].

2.7. Peptide sequencing

The obtained protein band in CBB stained gel was trypsin digested and peptideswere extracted according to standard techniques [24]. Peptides were analyzed byMALDI TOF/TOF mass spectrometer using a 5800 Proteomics Analyzer [AB Sciex].Spectra were analyzed to identify protein of interest using Mascot sequence match-ing software [Matrix Science] with Ludwig NR Database and taxonomy set toBacteria (http://www.matrixscience.com).

2.8. Enzyme kinetics using different substrates

The purified enzyme (10 �g ml−1) was assayed for its proteolytic activity againstdifferent substrates like BSA, casein, feather powder, gelatin, soy meal and azok-eratin having working concentration of 10 mg ml−1. The most suitable substrateamongst these was determined from the kinetic studies of the enzyme-substratereaction based on Lineweaver–Burk plot.

2.9. Effect of pH and temperature on enzyme activity

Enzyme (10 �g ml−1) was assayed at different pH values (4–11) of reaction mix-tures prepared in different buffers mentioned earlier at 30 ◦C. The temperature rangeof 4–100 ◦C was used to determine the optimum temperature for the enzyme actionin Tris buffer (0.1 mM, pH 8.0). The enzyme activity (%) and stability was determinedat various conditions of pH and temperature.

2.10. Effect of chemicals and metal ions on enzyme activity

The enzyme (10 �g ml−1) was mixed with each chemical of specific concentra-tion separately. The test metal ions were added to reach a working concentrationof 1 mM. A control was set up where the enzyme was mixed with distilled waterinstead of chemical/metal solution and its activity was considered as 100%.

2.11. Analysis of metal content in enzyme

The purified enzyme was dialyzed extensively against pure water at 4 ◦C. It wasthen hydrolyzed completely using 15.6 M HNO3 and subjected for metal analysison atomic absorption spectrophotometer (S2-Thermo Electron Corporation, USA).The amount of metals per enzyme molecule was estimated from the protein con-tent, assuming a molecular weight observed on SDS-PAGE analysis. Absorbance wasmeasured at 422.7, 324.8, 279.5, 253.6, 248.3 and 213.8 nm for Ca, Cu, Mn, Hg, Feand Zn, respectively by comparing with known standards.

2.12. Preparation of apoenzyme and effect of metals on its activity

Apoenzyme was prepared by removing the metal ions present in enzyme bytreating the purified enzyme (0.1 mg ml−1) with EDTA.2Na (10 mM) at 30 ◦C for 1 h.The EDTA.2Na was removed by dialyzing it with Tris buffer (50 mM, pH 8.0) at 4 ◦Cfor 6 h. The prepared apoenzyme was again modified as enzyme by dialyzing itagainst the buffer containing 1 mM of each metal ion separately and later tested forits activity.

2.13. Feather degradation by purified enzyme

The ability of purified enzyme to degrade feather was checked. The lyophilizedenzyme (2 mg ml−1) was added to 100 ml of FP medium containing substrate1–10 g l−1 feather powder at pH 8 in 500 ml capacity of Erlenmeyer flask and incu-bated on rotary incubator shaker with 120 rpm at 30 ◦C for 24 h. Sodium azide (1 mM)was added to avoid any microbial contamination in flask. Separate controls withoutenzyme were prepared for each substrate concentration. The hydrolysis was ter-minated by heating mixture at 85 ◦C for 15 min to inactivate the enzyme. Sampleswere removed at 6, 12, 18, and 24 h of incubation time and % feather hydrolysis wasestimated by comparing amount of soluble protein with residue. Insoluble matterwas determined by passing hydrolysates through filter paper and then drying toa constant weight at 50 ◦C for 24 h. The weight of residual matter was determinedby subtracting the weight of filter paper. The soluble protein and free thiol groupswere estimated as stated earlier [19,21]. The free amino groups were determinedby a modified ninhydrin method [25] using leucine as a standard.

3. Results

eratinolytic metallo-protease produced by Chryseobacterium gleum.

3.1. Keratinolytic activity of C. gleum

C. gleum demonstrated pronounced growth in whole-feathermedium after 72 h of incubation at 30 ◦C at 120 rpm resulting in

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Fig. 2. (a) Purification of keratinolytic protease from C. gleum: fractions eluted after

TP

ig. 1. (a) Growth of C. gleum in WF medium with the protease activity and (b)oluble protein, amino acids and thiol content in the medium.

issolution of feathers and produced zone of clearance exhibit-ng keratinolytic activity on FP agar in Petri plate. C. gleum grewn the range of pH 7.0–9.0 and temperature of 25–42 ◦C whileH 8.0 and 30 ◦C were optimum for feather degradation and 72 hf incubation was found to be appropriate when run for 144 h.ater on, complete dissolution of feather powder was observed,ut as cells began to lyse soon after reaching stationary phase,light increase in pH of the medium was observed (data nothown). Maximum enzyme activity was observed during cultiva-ion at 30 ◦C (44.77 U ml−1), followed by 37 ◦C (37.2 U ml−1) andhen at 42 ◦C (33.34 U ml−1) at 72 h. Keratinolytic activity androwth of C. gleum in the FP medium is shown in Fig. 1a whilets soluble protein, amino acid and thiol contents are shown inig. 1b.

.2. Enzyme purification

Ultrafiltration and ammonium sulfate precipitation lead to par-ial purification of enzyme. Further, on gel filtration; some ofhe fractions bearing high protein content (A280) coincided withigher enzyme activities (Fig. 2a). Subsequently, ion exchange

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hromatography of pooled fractions exhibited peaks with var-ed protein content and keratinolytic activities (Fig. 2b). Thisurification could increase enzyme activity to several foldsTable 1).

able 1urification of C. gleum keratinolytic protease.

Purification step Total activity (U) Total protein (mg)

Culture filtrate 93500.0 3740.00

Post-ultrafiltration 47166.5 579.64

(NH4)2SO4 dialysate 22392.5 72.50

Seralose CL-6B 17567.0 13.16

DEAE cellulose-52 2106.0 1.26

Seralose CL-6B gel filtration chromatography and (b) the elution profile of kerati-nolytic protease on DEAE cellulose-52 ion exchange chromatography using NaClgradient in 50 mM Tris–HCl buffer, pH 8.0.

3.3. SDS-PAGE

The major protein obtained was near to homogeneity as checkedby SDS-PAGE that showed a single band of enzyme having appar-ent molecular mass of ∼36 kDa when compared with standardprotein markers (Fig. 3). Native PAGE showed a single band of non-denatured enzyme and zymogram staining of protein exhibited itsproteolytic activity (Fig. 3).

3.4. Peptide sequencing by MS

Peptides of enzyme on fragmentation in the mass spectrometerproduced ions that gave amino acid sequence information and themass spectrum showed several protonated ions [M+H]+ of the pep-tide fragments (Fig. 4a). Out of many fragments, six major peptides(Table 2) matched with the sequences of keratinolytic proteases ofB. licheniformis, B. subtilis and B. mojavensis in Uniprot. One of themwas keratinase (fragment) (BOFXJ2-Uniprot accession number) ofB. subtilis with 35.9 kDa mass and 410 score (p < 0.05) (Fig. 4b). Thesequencing could reveal 102 amino acids of keratinase of C. gleum.

eratinolytic metallo-protease produced by Chryseobacterium gleum.

3.5. Enzyme kinetics using different substrates

Enzyme was active against all the substrates tested as observedthrough Lineweaver–Burk plot (Fig. 5). The Vmax was 1.56 U min−1

Specific activity (U mg−1) Yield (%) Purification fold

25.0 100.00 1.081.4 50.40 3.3

308.9 24.00 12.41334.9 18.80 53.41671.4 2.25 66.9

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Fig. 3. Electrophoretic mobility of keratinolytic protease of C. gleum: SDS-PAGE ofsamples at various purification stages i.e. culture extract (lane 1), precipitate onaddition of ammonium sulfate (lane 2), and the Seralose CL-6B column fraction (lane3); lane M, marker proteins (10 �l) (molecular weight, kDa), 97.4 (phosphorylase b),66 (bovine serum albumin), 43 (ovalbumin), 29 (carbonic anhydrase), 20 (soybeantrypsin inhibitor), 14.3 (lysozyme); the DEAE cellulose-52 column fraction (lane 4),Native PAGE of keratinolytic protease (lane 5) and zymogram staining of proteinon native PAGE (lane 6); The amount of protein deposited in each lane was ∼50 �gexcept marker protein.

Fig. 5. Lineweaver–Burk plot for the enzymatic hydrolysis of different natural pro-tein substrates viz. azokeratin (Az), feather powder (FP), casein (Csn), bovine serumalbumin (BSA), soy meal (SM) and gelatin (Glt).

Fig. 4. (a) MALDI-TOF MS of trypsin digested peptides of keratinolytic protease of C. gleum and (b) peptides of keratinolytic protease of C. gleum matching with keratinase(fragment) of B. subtilis (BOFXJ2).

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Table 2Peptide mass profile of trypsin digested keratinase of C. gleum having similarity with that of B. subtilis (KPT).a

KPTs of C. gleum Amino acid sequence Peptide sequence [M+H]+ mass (m/z) in Da

From To Observed Calculated

KPT1 216 224 K.QAVDNAYAR.G 1007.52 1006.48KPT2 93 102 K.ADKVQAQGFK.G 1091.61 1090.58KPT3 317 326 K.HPNLSASQVR.N 1108.62 1107.58KPT4 250 265 K.YDSVIAVGAVDSNSNR.A 1666.84 1665.80

GK.G 1744.86 1743.84SSGNTNTIG YPAKYDSVIAVGAVDSNSNR.A 3938.94 3937.94

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Chemical (working concentration) Residual activity (%) a

Control 100 ± 1EDTA (1.0 mM) 32 ± 1.5PMSF (1.0 mM) 90 ± 1.2IAA (1.0 mM) 88 ± 2SDS (0.1%, w/v) 64 ± 1.5Triton X-100 (0.1%, v/v) 194 ± 1.7Tween 80 (0.1%, v/v) 146 ± 1.2Acetonitrile (0.1%, v/v) 54 ± 2.3DMSO (0.1%, v/v) 62 ± 1.2Isopropanol (0.1%, v/v) 58 ± 1�-Mercaptoethanol (0.1%, v/v) 219 ± 2.5

a Values are the mean ± standard deviation of three independent determinations.

Table 4Effect of different metal ions on the keratinase activity.

(A) Holoenzyme (B) Apoenzyme

Ions (1 mM) Residualactivity (%)a

Ions (1 mM) Residualactivity (%)a

None 100 ± 0.6 None 0 ± 0.6Ba2+ 89 ± 1.2 Ca2+ 16 ± 1.2Ca2+ 92 ± 2.3 Cu2+ 0 ± 0.6Cu2+ 0 ± 0 Fe2+ 79 ± 0.6Fe2+ 141 ± 1 Fe3+ 67 ± 1Fe3+ 132 ± 0.6 Hg2+ 0 ± 0Hg2+ 0 ± 0 Mn2+ 14 ± 1.7Mg2+ 86 ± 2.5 Zn2+ 11 ± 1

KPT5 329 344 R.LSSTATYLGSSFYYKPT6 225 265 R.GVVVVAAAGNSG

a Keratinase peptide.

hile Kcat was 0.52 min−1 with low Km value of 1.70 mg ml−1 forzokeratin. Among the substrates tested, azokeratin an artificiallyrepared semi-synthetic substrate was the best substrate. How-ver, keratin in feather powder was the best natural substrate forhe enzyme while it showed moderate activity against soy mealollowed by casein, BSA and gelatin.

.6. Effect of pH and temperature on keratinase activity

The enzyme was stable in the pH range of 6–10 and up to 50 ◦C.nzyme remained active in the pH range of 8–9 and temperatureange of 30–50 ◦C. However, the enzyme activity was highest at pH.0 (Fig. 6a) and at 30 ◦C temperature (Fig. 6b). But, it was almost

nactivated at and above pH 11, 75 ◦C.

.7. Effect of chemicals and metal ions on keratinase activity

The enzyme activity was strongly inhibited by EDTA whileMSF and IAA (iodoacetamide) had minor effects. The enzymeaintained about 50–60% of its activity after incubation with SDS

nd organic solvents. The use of Tween 80, Triton X-100 and �-ercaptoethanol caused significant increase in enzyme activity

Table 3). Among the metal ions tested, Fe2+ and Fe3+ had a sti-ulatory effect on the enzyme activity. The activity was not much

eviated in presence of Ca2+, Ba2+, Mg2+, Mn2+ while inhibitedtrongly by Hg2+, Cu2+ (Table 4A).

Please cite this article in press as: Chaudhari PN, et al. Iron containing keratinolytic metallo-protease produced by Chryseobacterium gleum.Process Biochem (2012), http://dx.doi.org/10.1016/j.procbio.2012.11.009

.8. Evidence for the presence of metals in the keratinase

Atomic absorption spectroscopy showed Fe as a major con-tituent of enzyme (3 mM) followed by Ca (130 �M), Zn (71.43 �M)

Fig. 6. Effect of (a) pH and (b) temperature on the purified C. gleum keratinase activity.

Mn2+ 65 ± 1.5Zn2+ 48 ± 2

a Values are the mean ± standard deviation of three independent determinations.

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s minor constituent while other metals (Cu, Hg and Mn) were inrace amounts per mole of enzyme.

.9. Effect of metals on apoenzyme of keratinase

The activity of the apoenzyme was completely lost, but recov-red significantly by the addition of iron. Remarkable increase inhe activity of enzyme was observed in presence of Fe2+ (79%) fol-owed by Fe3+ (67%) while other metal ions did not show muchncrease (Table 4B).

.10. Feather degradation by the purified keratinase

The ability of the purified enzyme to hydrolyze feather powderrotein was studied under the predetermined optimal conditions.he % feather hydrolysis for each FP concentration in specific dura-ion is shown in Fig. 7. A maximum of 85% hydrolysis was achievedhen 20 g l−1 FP was used in the medium. This hydrolysis efficacy

f enzyme was decreased slightly up to 30 g l−1 substrate concen-ration where 81% feather hydrolysis was achieved. At 50 g l−1 FPoncentration, the rate of feather hydrolysis decreased to 72% on4 h of incubation, but beyond this concentration, the enzyme was

neffective. During feather degradation, the amount of thiol groupsroduced was monitored (Fig. 7) where free thiol groups did not

ncrease during the reaction. Conversely, the production of freemino groups increased proving the 20 g l−1 FP as the best sub-trate concentration. At this concentration, the highest amount ofree amino groups (3.9 �M ml−1) was produced (Fig. 7).

. Discussion

Various proteolytic activities like degradation of skim milk,asein, and gelatin on agar media and H2S production were foundn C. gleum strains isolated from Cape marine fish [26]. Kerati-olytic activity was also detected in Chryseobacterium sp. kr6 whichas close to C. gleum. Many other neighboring bacteria belonging

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o the Cytophaga-Flavobacterium group and other Chryseobacteriaave been described for feather degradation [11,18,27]. The isolatexhibited optimum growth and proteolytic activity at mesophilicemperatures corresponding to its environment.

ons of feather powder (FP) with the production of free amino and thiol groups in

C. gleum produced an extracellular enzyme bearing an abilityto degrade whole feathers and grew where the levels of expressedenzyme varied during incubation period of growth. The soluble pro-tein increased up to 48 h possibly because of the rapid degradationof hairy feather barbules and utilization of degraded products by theorganism resulting in rapid growth (Fig. 1a) while remaining thickfeather rachises were degraded slowly by C. gleum. Perhaps thiscould be the reason for sharp decrease of soluble protein (32%) after48–72 h which remained constant up to 144 h (Fig. 1b). This patternof protein degradation suggested inducible nature of enzyme whichwas regulated by substrate and metabolite levels in the extra-cellular medium. Keratin degradation by C. gleum resulted in theproduction of amino acids, thiol groups indicating the reduction ofdisulfides to sulfhydryls in keratin which may be due to disulfidereductase activity of the organism as like in Bacillus sp. [28], Vib-rio sp. [29] and Chryseobacterium sp. kr6 [10] growing on keratinsubstrates.

The keratin degrading enzyme of C. gleum was purified from anutritionally poor medium containing feather protein in majority.The medium contained only one type of complex protein keratinas a substrate which directed the production of keratinase withnegligible amount of other contaminating proteins. Feather ker-atin as the sole carbon and nitrogen source in the culture mediumresulted in preferential expression of the keratinase by C. gleum asobserved by Lin et al. [30] in B. licheniformis. Keratin degradationby C. gleum could probably be multiple proteases as evident fromadditional proteolytic activity peaks of proteins obtained on col-umn chromatography; hence it can be called as keratinase complex.Riffel et al. [18] and Wang et al. [33] also showed that Chryseobac-terium strains produce three proteolytic enzymes, one of themshowing major keratinolytic activity. However, the major enzymewas having relative molecular mass of (∼36 kDa) and here called askeratinase was similar to proteases of Cytophaga-Flavobacteriumgroup, ranging from 20 to 40 kDa [31,32]. Zymogram stainingrevealed monomeric nature and protease activity of this protein.The keratinase of C. gleum resembled the previously investigatedkeratinolytic proteases from Chryseobacterium sp. strain kr6 [10]

eratinolytic metallo-protease produced by Chryseobacterium gleum.

and C. indologenes [33]. The keratinase of C. gleum showed thehighest activity against semisynthetic substrate azokeratin fol-lowed by feather protein keratin comparable to keratinases of B.licheniformis PWD-1 [28], Chryseobacterium sp. [11,32] and other

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eratinolytic bacteria [10,34]. Practically, keratin being insoluble inater bearing uneven size is difficult to use as substrate in studying

nzyme-substrate kinetics, while azokeratin; although insolubleaving evenly dispersible uniform particle size is suitable, never-heless giving apparent values for kinetic parameters under study.

Keratin is very resistant to proteolytic digestion due to its uniqueupercoiled �-helical structure of polypeptide chains as well ashe strength of intermolecular disulfide bonds and other molecularnteractions [29]. Reduction of disulfide bonds is one of the possible

echanisms in breaking down keratin such as the pretreatment oferatin with a reducing agent (thioglycolate) which disrupts theisulfide bonds and makes it easy to digest by certain proteolyticnzymes like trypsin [2]. �-Mercaptoethanol stimulated the activ-ty of the keratinase may be because of the reduction of disulfideridges of keratin, generating more accessible substrate. However,eratinase did not show such mechanism as it degrades keratinithout a reducing agent or coenzyme. The enzymatic keratinoly-

is was not coupled with an increase in the production of detectablehiol groups, but with an increase in the free amino groups due toeptide bond cleavage (Fig. 7).

The production of proteases in chemically defined growthedium often promotes vigorous growth and high enzyme yields

35,36], but being expensive, it is unsuitable for a large-scale pro-uction. Hence, it is beneficial to use raw materials like industryastes or by-products to reduce the cost of culture media. C.

leum exhibited its potential by utilizing feather protein or raweathers that have been used as a substrate for preparation ofther keratinolytic enzymes [10,29]. Purified enzyme showed goodegradation of feather protein up to 50 g l−1 concentration, beyondhich it decreased. This might be due to competitive inhibition

xerted by the increased substrate concentration interfering theatalytic efficiency of the enzyme. Similar results were observed byoo et al. [36] in case of protease production by Bacillus horikoshiising soy meal as substrate. This indicates that appropriate amountf substrate is needed for higher enzyme yields. Riffel et al. [10]as also observed that substrate and metabolite levels both in thextracellular medium can regulate enzyme secretion.

Although, the existence of protease was also reported inhryseobacterium taeanense TKU001 [37], Fe containing proteasenhancing the feather degradation has not been reported in Chry-eobacterium spp. Setyorini et al. [15] reported the halotolerantxtracellular protease activated by Fe in B. subtilis strain FP-133ontaining high concentration of Fe in protein. An alkaline �-eratinase produced by B. subtilis RM-01 using chicken-feather asubstrate was reported which required Fe2+ for enzyme activityaving application in laundry detergents [16]. Protease from thehermophilic fungus was also enhanced by the presence of Fe2+

17]. The Fe2+ leads to augmentation of keratinolytic activity forhrysosporium keratinophilum [38] and B. subtilis RM-01 [16].

Keratinolytic proteases generally belong to the class of seriner metallo proteases irrespective of the microorganism. Most ofhe keratinolytic enzymes are serine-type proteases. The genesf serine proteases exhibited sequence homology with subtilisinsroduced by Bacillus spp. [8,30]. MALDI-TOF MS analysis of kerati-olytic protease of C. gleum revealed some amino acid sequencesatching with those of keratinase of B. subtilis. At a glance, it

ave impression that the keratinase of C. gleum may belong tohe serine-type proteases, but; its activity was not strongly inhib-ted by PMSF. There is no solid evidence on production of serineroteases by Chryseobacterium spp. except a moderately charac-erized protease from Chryseobacterium sp. L99 was inhibited byMSF which could be serine protease reported by Lv et al. [39].

Please cite this article in press as: Chaudhari PN, et al. Iron containing kProcess Biochem (2012), http://dx.doi.org/10.1016/j.procbio.2012.11.009

owever, it may need additional substantiation to establish typef that enzyme preferentially based on gene or protein sequence(s)hile its catalytic triad and mechanism of action if known would

scertain it.

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PRESSemistry xxx (2012) xxx– xxx 7

Nevertheless, the presence of metal; especially iron and stronginhibition by EDTA indicated keratinase of C. gleum to be iron con-taining metallo-protease. Some of the keratinolytic enzymes arereported to be metalloproteases [9,18,33]. The divalent metal ionslike Mg2+ and Ca2+ enhanced activity of keratinases in Microbac-terium sp. kr10 [9] and Chryseobacterium sp. kr6 [18], respectively.However, keratinase activity was inhibited by transition and heavymetal ions Cu2+, Hg2+, Ag2+, Pb2+, Zn2+, Ba2+ and Co2+ [38,40,41].The presence of metal in the enzyme can be effectively used forimmobilization by chelation which increases its stability because ofa reduced autolysis [42]. Among the metal ions tested, iron exertedremarkable increase in keratinase activity of C. gleum. This wasconfirmed on the observation of decreased activity of apoenzymewhich regained on addition of iron. Hence, iron was essential forthe enhancement in keratin degradation by the enzymatic actionof C. gleum as observed by Rai et al. [16] and Nam et al. [43] in otherorganisms.

5. Conclusions

C. gleum exhibited efficient keratin degradation through enzymekeratinase, hence; it can be used for whole feather degradation inpoultry waste management. Its keratinase was of an inducible typebearing ∼36 kDa Mw. Presence of iron and its inhibition by EDTAsuggested it to be an iron containing metallo-protease. This enzymecan also be of great interest; since its activity can be controlledcategorically by metals or chemicals reported that can be useful forbiotechnology industry.

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.procbio.2012.11.009.

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