APPLIED AND ENVIRONMENTAL MICROBIOLOGY, OCt. 1992, p. 3271-32750099-2240/92/103271-05$02.00/0Copyright © 1992, American Society for Microbiology

Vol. 58, No. 10

Purification and Characterization of a Keratinase from a

Feather-Degrading Bacillus licheniformis StrainXIANG LIN,t CHUNG-GINN LEE,4 ELLEN S. CASALE, AND JASON C. H. SHIH*

Department ofPoultry Science and University Biotechnology Program, North Carolina State University,Raleigh, North Carolina 27695-7608

Received 27 April 1992/Accepted 21 July 1992

A keratinase was isolated from the culture medium of feather-degrading Bacilus licheniformis PWD-1 by use

of an assay of the hydrolysis of azokeratin. Membrane ultrafiltration and carboxymethyl cellulose ion-exchangeand Sephadex G-75 gel chromatographies were used to purify the enzyme. The specific activity of the purifiedkeratinase relative to that in the original medium was approximately 70-fold. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and Sephadex G-75 chromatography indicated that the purifiedkeratinase is monomeric and has a molecular mass of 33 kDa. The optimum pH and the pI were determinedto be 7.5 and 7.25, respectively. Under standard assay conditions, the apparent temperature optimum was

50°C. The enzyme is stable when stored at -20°C. The purified keratinase hydrolyzes a broad range ofsubstrates and displays higher proteolytic activity than most proteases. In practical applications, keratinase isa useful enzyme for promoting the hydrolysis of feather keratin and improving the digestibility of feather meal.

The major component of feathers is keratin. Because of ahigh degree of cross-linking by cystine disulfide bonds,hydrogen bonding, and hydrophobic interactions, keratin isinsoluble and not degradable by proteolytic enzymes, suchas trypsin, pepsin, and papain (7, 8, 14). Despite the unusualstability of keratin, feathers do not accumulate in nature.Our laboratory has reported the isolation and characteriza-tion of a feather-degrading bacterium, Bacillus licheniformisPWD-1 (23, 24). The bacterium grows on feathers as theprimary organic substrate for supplying carbon, sulfur, andenergy. Biodegradation of feathers by this bacterium repre-sents a method for improving the utilization of feathers as,for example, a feed protein. Recently, feeding experimentswith chickens demonstrated a significantly better growthresponse when the bacterial fermentation product featherlysate (18) was added to the diet than when untreatedfeathers or commercial feather meal was added (18, 22). Thisstudy reports the purification and characterization of thekeratinase secreted by feather-degrading B. lichenifornisPWD-1.

MATERIALS AND METHODS

Organism and growth conditions. The bacterium used inthis study was a patented strain of B. licheniformis PWD-1isolated in our laboratory (19, 23). All culture conditions andthe feather culture medium were as previously described.The medium contained, per liter, the following: 0.5 g ofNH4Cl, 0.5 g of NaCl, 0.3 g of K2HPO4, 0.4 g of KH2PO4,0.1 g of MgCl2 6H20, 0.1 g of yeast extract, and 10 g ofhammer-milled chicken feathers. The pH was adjusted to7.5. Feathers were washed, dried, and hammer milled priorto being added to the medium. The medium was sterilized byautoclaving.The bacterium was cultured in a test tube (20 by 1.5 cm)

* Corresponding author.t Permanent address: Shenyang Agricultural University, Shen-

yang, Liaoning, People's Republic of China.t Present address: Department of Animal Production, Council of

Agriculture, Taipei, Taiwan, Republic of China.

containing 10 ml of culture medium. After 4 days of incuba-tion, 10 ml of the culture medium was transferred to a 3-literFernbach flask containing 1.0 liter of medium. After 4 daysof incubation, the medium was collected for keratinasepurification. All incubations were done at 50°C with shakingat 120 rpm in a controlled-environment shaker (New Bruns-wick Scientific Co., New Brunswick, N.J.).Enzyme purification. The culture medium was prefiltered

through glass wool to remove residual undegraded feathers.The medium was then filtered through a 0.45-,um-pore-sizemembrane with a Pellicon cassette system (Millipore Corp.,Bedford, Mass.) to remove bacterial cells and other parti-cles. The filtrate was concentrated by membrane ultrafiltra-tion (molecular weight cutoff, >10,000) with the same (Pel-licon) system. The crude concentrated keratinase solutionwas applied to a column of carboxymethyl cellulose (CM-cellulose) (2.5 by 60 cm) at 4°C. The column was equilibratedwith buffer A (25 mM potassium phosphate buffer [pH 5.8]).Approximately 50 mg of protein was loaded on the column.Elution was begun with 500 ml of buffer A. The elutedproteins were monitored by measuring the A280 of thefractions. When the 280-nm reading was a continuous base-line, elution with buffer B (25 mM potassium phosphatebuffer [pH 6.2], 20mM NaCl) was begun. Approximately 300ml was used for this elution. Finally, buffer C (25 mMpotassium phosphate buffer [pH 6.8], 20 mM NaCl) wasused. The elution flow rate was 0.4 ml/min, and 5-mlfractions were collected. Fractions were screened with themilk-agarose plate assay. Fractions exhibiting proteolyticactivity were assayed for keratinase activity with the azo-keratin hydrolysis test. Keratinase active fractions werepooled and concentrated with an Amicon stirred cell filtra-tion system with Diaflo ultrafilters (molecular weight cutoff,>10,000) (Amicon Div., W. R. Grace and Co., Beverly,Mass.) at 4°C. Purification of keratinase was continued witha Sephadex G-75 column (1.5 by 90 cm) at 4°C. Equilibrationand elution were carried out at 4°C with 50 mM potassiumphosphate buffer (pH 7.0) at a flow rate of 0.3 ml/min.Fraction volumes of 3 ml were collected. Protein elution wasfollowed by monitoring of the A280 of each fraction. Frac-tions were screened with the milk-agarose plate assay, and

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then the active fractions were assayed for keratinase activitywith the azokeratin hydrolysis test. Keratinase active frac-tions were pooled and concentrated with the Amicon stirredcell unit.Enzyme hydrolysis of azokeratin. Azokeratin was prepared

by reacting ball-milled feather powder (24) with sulfanilicacid and NaNO2 by use of a method similar to that describedby Tomarelli et al. for azoalbumin (21). For a standard assay,5 mg of azokeratin was added to a 1.5-ml centrifuge tubealong with 0.8 ml of 50 mM potassium phosphate buffer (pH7.5). This mixture was agitated until the azokeratin was

completely suspended. A 0.2-ml aliquot of an appropriatelydiluted enzyme solution was mixed with the azokeratin, andthe mixture was incubated for 15 min in a 50°C water bath.The reaction was terminated by the addition of 0.2 ml of 10%trichloroacetic acid, and the mixture was filtered. The A450of the filtrate was measured with a UV-160 spectrophotom-eter (Shimadzu, Columbia, Md.). A control was prepared byadding trichloroacetic acid to a reaction mixture beforeadding the enzyme solution. Without knowing the molarextinction coefficient of azopeptides, we defined 1 U ofkeratinase activity as an increase in the A450 of 0.01 after 15min in the test reaction compared with the control reaction.

Milk-agarose plate assay. A method that was an alternativeto the azokeratin hydrolysis assay was used to screen largenumbers of fractions collected from CM-cellulose and Seph-adex G-75 columns. One gram of agarose was dissolved in 98ml of 50 mM potassium phosphate buffer (pH 7.5). After theagarose solution was cooled to 60 to 70°C, 2 ml of evaporatedwhole milk was added with gentle agitation. The mixture waspoured rapidly onto a clean glass plate (20 by 20 cm) andspread evenly. After the agarose had solidified, 81 (9 by 9)small wells (5 mm in diameter) were punched in the agarose.A 25-,l aliquot of a sample was applied to each well. Theplate was incubated in a moist atmosphere either overnightat 37°C or for 2 h at 50°C. Clear zones around wells wereindicative of proteolytic activity. A similar method waspreviously published (17). Keratinase activity was furtheridentified by azokeratin hydrolysis as described above.

Free amino group assay. For the native feather keratin andother protein substrates, including casein, elastin, collagen,and bovine serum albumin (BSA) (all from Sigma ChemicalCo., St. Louis, Mo.), the proteolytic activities of keratinasewere determined on the basis of the production of free aminogroups. A modified ninhydrin method was used for detectingfree amino groups (16). Five milligrams of a protein substratewas placed in a 1.5-ml tube with 0.8 ml of buffer and 0.2 mlof enzyme solution containing 8 ,ug of purified keratinase.After incubation at 50°C for 30 min, the reaction was stoppedby the addition of trichloroacetic acid. The reaction mixturewas filtered with a 5.0-,um-pore-size syringe filter (MicronSeparations, Inc., Westboro, Mass.) or centrifuged to re-move insoluble protein, and a 0.5-ml aliquot was removedfor a reaction with ninhydrin reagent to determine theproduction of free amino groups. The standard curve wasdetermined with leucine at 0.1 to 0.5 ,umol.Both the free amino group assay and the azokeratin

hydrolysis assay were used to compare the keratinolyticactivities of a variety of proteases (Sigma). The enzymestested were papain (EC 3.4.22.2), bovine trypsin (EC3.4.4.4), porcine elastase (EC 3.4.21.36), Clostridium his-tolyticum collagenase (EC 3.4.24.3), Tntirachium proteinaseK (EC 3.4.21.14), and keratinase.

Protein determination. Protein concentrations were deter-mined by the Bio-Rad (Richmond, Calif.) protein assaymethod as described by Bradford (3). The microassay pro-

cedure was performed, and BSA was used as the standardprotein.Enzyme properties. The native molecular weight of kera-

tinase was estimated by Sephadex G-75 column chromatog-raphy; calibration was done with standard proteins from a kitobtained from Pharmacia Fine Chemicals (Piscataway,N.J.). To examine the purity and determine the subunitmolecular weight, we performed discontinuous sodium do-decyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described by Laemmlli (9). The protein bandswere stained with a solution of Coomassie blue R-250.Isoelectric focusing, as described in Bio-Rad InstructionManual 161-0310, was used to determine the isoelectric pointof keratinase. The experiment was conducted with an LKBhorizontal electrophoresis unit (2117 Multiphor) and LKBAmpholines. The protein bands were stained with a Bio-Radsilver stain kit by use of the method developed by Mehta andPatrick (11) and Confavreux et al. (4). The pH and apparenttemperature optimum for keratinase activity were assayedby the azokeratin hydrolysis assay. To determine whether aprosthetic group is an integral part of keratinase, we scannedthe enzyme solution with a Shimadzu UV-160 UV-visiblerecording spectrophotometer. A BSA solution of the sameprotein concentration was used for comparison.

Determination of the production of sulfhydryl groups. Re-duction of disulfides to sulfhydryls in keratin may be part ofthe mechanism of enzymatic keratinolysis. To test thishypothesis, we determined the production of sulfhydrylgroups by use of the Ellman reaction (5) during the enzy-matic reaction, while hydrolysis was monitored as the in-crease in the production of free amino groups as describedabove. One hundred milligrams of feather keratin was incu-bated at 50°C with 50 ,ug of keratinase in 15 ml of 50 mMphosphate buffer (pH 7.5). Every 10 min, 1.6 ml of reactionmixture was removed for analysis, 1.0 ml for sulfhydryls and0.6 ml for free amino groups.

RESULTS

Enzymatic hydrolysis of azokeratin. Insoluble azokeratinas the chromogenic substrate was incubated with the kera-tinase solution to produce a soluble colored product. Thesupernatant demonstrated an increase in the A450 in a linearfashion in the first 30 min because of the release of azopep-tide derivatives into the solution. This assay, in conjunctionwith prescreening by the milk-agarose plate assay, was usedfor the purification of keratinase.

Purification of keratinase. A summary of the purification ofkeratinase from the culture medium of B. licheniformisPWD-1 is presented in Table 1. Membrane ultrafiltration andCM-cellulose ion-exchange and Sephadex G-75 gel chro-matographics yielded a purified keratinase fraction having anoverall purification factor of 70-fold. The final product had aspecific activity of about 6,000 U/mg. The ultrafiltration stepremoved approximately 60% of the total protein while main-taining 70% of the enzyme activity. CM-cellulose columnchromatography purified the enzyme 43-fold and removed98% of the original total protein. Elution from the SephadexG-75 column yielded a homogeneous protein, as shown by asingle protein band in SDS-PAGE (Fig. 1A).Enzyme properties. The purified PWD-1 keratinase is a

monomeric protein with a molecular mass of 33 kDa, asestimated by both Sephadex G-75 chromatography (data notshown) and SDS-PAGE (Fig. 1). The pH optimum andapparent temperature optimum of keratinase activity, asdetermined by the azokeratin hydrolysis assay, are 7.5 and

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B. LICHENIFORMIS KERATINASE 3273

TABLE 1. Purification of keratinase from the medium ofB. licheniforinis PWD-1

Total Total Sp act Purifi-Step protein ua (U/mg of % of U cation

(mg) protein) (fold)

Medium 142 12,200 86 100 1.0Membrane concen- 54.5 8,450 150 69.3 1.8

trationCM-cellulose chro- 3.3 12,080 3,720 99 43matography

Sephadex G-75 1.5 8,910 5,990 73.1 70chromatography

a One unit is defined as an increase in the A450 of 0.01 after reaction withazokeratin for 15 min.

M.W(KDa) 1

97.4

66.2

2 3 4 54-

3 1 -m -_

50°C, respectively. Isoelectric focusing determined that theisoelectric point is 7.25. The keratinase is fairly stable uponstorage at low temperatures. A solution sample (30 ,ug/ml)was found to lose only 7% of its activity at -20°C and 20%of its activity at 4°C after 19 days of storage. However, atroom temperatures (20 to 25°C), the half-life of keratinaseactivity is 4 to 5 days. The loss of enzyme activity is largelydue to enzymatic autolysis, which was detected by SDS-PAGE (data not shown). The UV-visible absorption spec-trum of the keratinase was found to be identical to that ofBSA. No absorption peak besides the typical absorptionpeaks at 280 and <220 nm for a protein molecule wasdetected.Enzyme specificity. A free amino group assay was used to

compare the substrate specificities of a variety of proteases.The keratinase was capable of hydrolyzing all the proteinstested, including BSA, casein, collagen, elastin, and featherkeratin. As demonstrated in Table 2, both the native featherkeratin and synthetic azokeratin were specific substrates forkeratinase. They were degraded less by other proteases.

Sulfhydryl analysis. During the enzymatic hydrolysis offeather keratin, sulfhydryl groups in the reaction mixturewere monitored (Fig. 2). No increase in the production offree sulfhydryl groups was detected during the reaction. Onthe other hand, the production of free amino groups in-creased as a result of peptide bond cleavage in a linearfashion over a 1-h period.

DISCUSSION

The combination of the milk-agarose plate assay and theazokeratin hydrolysis assay was demonstrated to be effec-tive for the purification of the keratinase from the culturemedium of B. licheniformis PWD-1. The milk-agarose plateassay is a simple method for screening large numbers offractions for proteolytic activity. For the identification ofkeratinase activity, the azokeratin hydrolysis assay is simpleand specific. The two methods correlated well in that theactive fractions showing proteolytic activity on the milk-agarose plate also showed keratinase activity. Therefore,only one major protease, the keratinase, was present in thebacterial medium. To further confirm the keratinolytic activ-ity, we also used a third method, measuring the increase inthe production of free amino groups upon hydrolysis of thenative feather keratin. Both the native feather keratin andsynthetic azokeratin have been shown to be more specifi-cally hydrolyzed by keratinase than by other proteases(Table 2).The purification of keratinase was effective and efficient.

21.5

1 4.4 m

A

1 0 5

0

4 n 4

phosphorylase b

BSA

ovalbumin

keratinase w. M carbonic anhydrase

trypsin inhibitor

lysozyme

Iv T

0.0 0.2 0.4 0.6 0.8 1.0

Relative mobilityB

FIG. 1. SDS-PAGE of purified keratinase. (A) SDS-polyacryl-amide gel. Lanes: 1 and 3, purified keratinase; 2, molecular massstandards; 4, CM-cellulose-treated preparation; 5, crude enzymepreparation. (B) Molecular mass standard curve. Standards: phos-phorylase b, 97.4 kDa; BSA, 67 kDa; ovalbumin, 43 kDa; carbonicanhydrase, 31 kDa; soybean trypsin inhibitor, 21.5 kDa; lysozyme,14.4 kDa.

About 73% of the total activity was retained, while only 1%of the original total protein remained. However, concentra-tion of the medium by membrane ultrafiltration resulted in adecrease in the total enzyme units. The total enzyme unitsreturned to the original level after ion-exchange chromatog-raphy (Table 1). This phenomenon was repeatedly observed.The concentration of the proteins may be such that the

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TABLE 2. Relative keratinolytic activities of proteases in two different assays

Keratinolytic activity of:Substrate Temp (°C)

Papain Trypsin Collagenase Elastase Proteinase K Keratinasea

Feather keratin 37 0 13 0 17 36 4150 0 16 0 27 51 100

Azokeratin 37 8 23 2 35 39 6450 7 27 1 47 52 100

a For the feather keratin substrate, the specific activity of purified keratinase is 118 pmol of leucine equivalents released per h per mg of enzyme protein; forthe azokeratin substrate, the specific activity is 4,700 U, and 1 U is defined as an increase in the A450 of 0.01 after reaction for 15 min under standard assayconditions.

keratinase is inhibited by an unknown but coconcentratedfactor.The keratinase molecule is monomeric. Molecular weights

were found to be identical when they were determined bytwo different methods, SDS-PAGE and Sephadex G-75 gelchromatography. The enzyme is relatively thermostable,with an apparent optimum temperature of 50'C. Cold storageat -20°C is stable. At room temperatures, autolysis resultsin a 50% loss of activity in 4 to 5 days.On the basis of its pl of 7.25, determined by isoelectric

focusing, keratinase is a nearly neutral protein. This conclu-sion is verified by the fact that keratinase binds to CM-cellulose at an acidic pH (5.8) and is not released until pH 6.8is reached. The optimum pH (7.5) for keratinase is close toits pI, indicating that keratinase is most active when itcarries the least net.The keratinase not only can hydrolyze keratin but also has

the ability to hydrolyze a wide variety of soluble andinsoluble protein substrates. The soluble substrates BSA andcasein are readily degradable, while the insoluble substratescollagen, elastin, and keratin are less so. The keratinase is

0.4 - 0.2

E 0.3-[-NH 2]0~~~~~~~~~E ID

=10.2 0.2 0.1Lo

Time(min)~~~~~~~~~~~m-

0

U)0.1 CD

[-HJ0.0 ~ * I 0.0

0 2 0 4 0 6 0 8 0

Time (min)FIG. 2. Analysis of free amino and sulfhydryl groups during

enzymatic keratinolysis.

more hydrolytic for elastin and collagen than some elastasesand collagenases (data not shown). Elastin and collagen aresimilar to keratin in that they are insoluble, highly struc-tured, and recalcitrant to many proteases. However, exam-ination of the individual structures of these proteins revealsquite different assemblies. Collagen, like keratin, is a fibrousprotein and consists of three a chains assembled in a triplehelix conformation, resulting in a rod-like shape. Elastin isnot a fibrous protein but rather is a polymer of globularproteins assembled in a fibrous arrangement. Both elastinand collagen are stabilized by covalent intermolecular cross-linkages involving lysine and lysine derivatives (1, 2, 13, 15,20). These properties provide some degree of resistance topeptide hydrolysis.The unique structure of keratin makes it very resistant to

proteolytic digestion. The resistance is due not only to thesupercoiled helical structure of polypeptide chains but alsoto the strength of intermolecular disulfide bonds and othermolecular interactions (6, 12). One of the possible mecha-nisms in breaking down keratin is the reduction of disulfidebonds. For instance, pretreatment of keratin with a reducingagent, such as thioglycolate, disrupts the disulfide bonds andincreases digestibility by trypsin (7). However, such a mech-anism is not likely in the case of the keratinase. First, itdegrades keratin without a reducing agent or coenzyme, andsecond, enzymatic keratinolysis is not coupled with anincrease in the production of detectable sulfhydryls (Fig. 2).The molecular mechanism of the enzymatic action remainsto be determined.

In summary, a simple colorimetric assay for keratinasewas developed. With this method, a keratinase was purifiedfrom newly discovered B. licheniformis PWD-1. The kerati-nase was characterized in terms of its biochemical propertiesand found to have a broad substrate specificity. The reduc-tion of disulfide bonds to sulfhydryls was not detected in thehydrolysis process. The use of keratinase as a feed additiveto improve the digestibility of feather meal in chickens hasbeen demonstrated in our laboratory (10). The purificationand characterization studies of the keratinase have providedthe basis to develop further the production and uses of thisenzyme.

ACKNOWLEDGMENT

We appreciate the grant support from Southeastern Poultry andEgg Association.

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