Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Purification and properties of a keratinolytic

metalloprotease from Microbacterium sp.
R.C.S. Thys and A. Brandelli
Laboratório de Bioquı́mica e Microbiologia Aplicada, Departamento de Ciência de Alimentos, ICTA, Universidade Federal do Rio Grande do Sul,
Porto Alegre, Brazil

Keywords
actinomycetes, enzyme, feather degradation,
keratin, protease.
Correspondence
Dr A. Brandelli, ICTA-UFRGS, Av. Bento
Gonçalves 9500, 91501-970 Porto Alegre,
Brazil.
E-mail: abrand@ufrgs.br
2005/1059: received 15 September 2005,
revised 15 March 2006 and accepted 22
March 2006
doi:10.1111/j.1365-2672.2006.03050.x
Abstract
Aims: This study was developed to purify and to characterize a keratinolytic
protease from the bacterium Microbacterium sp. strain kr10.
Methods and Results: Enzyme purification was carried out by sequential liquid
chromatography on Sephadex G-100 and Q-Sepharose columns. The purifica-
tion was about 255-fold, with a yield of 34%, as determined with azocasein as
substrate. The molecular weight of the enzyme was estimated as 42 000 Da by
SDS-PAGE. The enzyme had pH and temperature optima of 7Æ5 and 50C
respectively. This keratinase was inhibited by EDTA and 1,10-phenanthroline,
and analysis of metal content indicates that Zn2+ and Mg2+ are present. A 22
factorial design was developed to investigate the effect of keratinase and merca-
ptoacetate concentration on feather keratinolysis. Statistical analysis showed
that both variables have a significant effect on hydrolysis of keratin.
Conclusions: A new keratinase produced by Microbacterium sp. was purified
and characterized.
Significance and Impact of the Study: This keratinolytic enzyme offers an
interesting potential for the hydrolysis of keratin wastes to be used as feed sup-
plement or bioconversion to added-value products.

upgrade of feather meal and the use of microbial feather

Introduction
Keratins are insoluble fibrous proteins highly cross-linked
with disulfide bonds, which in addition to a tightly
packed supercoiled polypeptide chain results in high
mechanical stability and resistance to proteolytic hydroly-
sis (Jones et al. 1999). Keratin sources such as feather,
horn, nails and hair, are abundantly available in nature as
wastes. Worldwide, commercial poultry processing gener-
ate an excess of million tons of feathers per year (Shih
1993; Schrooyen et al. 2001), which are currently conver-
ted to feather meal through steam pressure and chemical
treatments. However, these methods destroy amino acids
and require significant energy input (Papadopoulos et al.
1986). Alternatively, keratin can be converted to useful
biomass, protein concentrate or amino acids using pro-
teases derived from certain micro-organisms. The use of
keratinolytic micro-organisms or keratinases has been
investigated (Onifade et al. 1998). The nutritional
lysate in feed trials showed that the treatment with kera-
tinase might significantly increase amino acid digestibility
of feather keratin (Williams et al. 1991; Odetallah et al.
2003).
Keratinases may have other important uses in biotech-
nological processes. The use of keratinolytic enzymes as
an alternative to de-hairing pelts and skins has been
investigated (Raju et al. 1996; Riffel et al. 2003a). The
potential for commercial use of enzymes in leather indus-
try is considerable because of their properties as highly
efficient and selective catalysts, avoiding the problem
caused by sulfide in tanneries (Wiegant et al. 1999). In
addition, keratin wastes have the potential for conversion
to products with high added value such as slow-release
nitrogen fertilizers, cosmetics and biodegradable films
(Choi and Nelson 1996; Schrooyen et al. 2001).
Keratinases are produced by fungi, particularly dermat-
ophytes and bacteria (Onifade et al. 1998). Concerning

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Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 101 (2006) 1259–1268 1259
Keratinase of Microbacterium sp.
bacterial keratinases, research has been focused on Bacillus
spp. and Streptomyces spp., whose proteolytic activities
are often associated to serine-type proteases (Böckle et al.
1995; Lin et al. 1995; Bressollier et al. 1999). Keratinolytic
activity has been recently described for Gram-negative
bacteria (Sangali and Brandelli 2000; Riffel and Brandelli
2002; Lucas et al. 2003). We identified a novel feather-
degrading strain, the first Microbacterium species capable
of completely disintegrate chicken feathers (Thys et al.
2004). In this report, the purification and characterization
of a metalloprotease produced by this feather-degrading
micro-organism is described.
Materials and methods
Reagents
Azocasein, azoalbumin, phenylmethylsulfonyl fluoride
(PMSF), N-benzoyl-l-arginine p-nitroanilide (BAPNA),
Ala-Ala p-nitroanilide, Ala-Ala-Pro-Met p-nitroanilide,
N-carboxybenzoyl-l-phenylalanine p-nitroanilide, were
from Sigma (St Louis, MO, USA). Sephadex G-100 and
Q-Sepharose were from Pharmacia (Uppsala, Sweden).
Other reagents were from Merck (Darmstadt, Germany).
Azokeratin and azogelatin were synthesized as described
elsewhere (Riffel et al. 2003a).
Micro-organism
The Microbacterium sp. strain kr10, previously isolated
from chicken feathers in decomposition (Thys et al.
2004), was used for protease production. The organism
was stored at )20C in BHI broth containing 20% (v/v)
glycerol. The bacterium was propagated twice in fresh
BHI medium before it was used for protease production.
Enzyme activity
Enzyme activity was measured as described elsewhere
(Thys et al. 2004). The enzyme solution (120 ll) was
added to 480 ll of azocasein solution (10 mg ml)1) in
reaction buffer 100 mmol l)1 phosphate pH 7Æ0. The
mixture was incubated at 50C for 30 min and the reac-
tion was stopped by adding 600 ll of 10% (w/v) trichlo-
roacetic acid (TCA) and leaving the preparation on ice
for 30 min. The mixture was then centrifuged at
10 000 g for 10 min at 4C and 800 ll of the superna-
tant was added to 200 ll of 1Æ8 mol l)1 NaOH. Absorb-
ance at 420 nm was measured with a Hitachi U-1100
spectrophotometer (Hitachi, Tokyo, Japan). One unit of
enzyme activity was defined as the amount of protein
that resulted in an increase of absorbance at 420 nm of
0Æ01, under the assay conditions used. The data points
1260 Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 101 (2006) 1259–1268
R.C.S. Thys and A. Brandelli
given in tables and figures are averages of three separate
experiments.
Enzyme production
Microbacterium sp. kr10 was grown in feather meal broth
(15 g l)1 feather meal, 0Æ3 g l)1 Na2HPO4, 0Æ4 g l)1
NaH2PO4, 0Æ5 g l)1 NaCl) for 24 h at 30C in an orbital
shaker at 125 cycles min)1. An inoculum of 2% was used.
After 72 h at 30C under shaking the culture supernatant
was obtained by centrifugation at 10 000 g for 10 min at
4C. The supernatant was taken as crude enzyme prepar-
ation.
Enzyme purification
The crude supernatant was freeze-dried, resuspended in
10 mmol l)1 phosphate buffer pH 7Æ0, and the applied to
a column of Sephadex G-100 (0Æ8 · 30 cm) pre-equili-
brated and eluted with the same buffer. Fractions positive
for keratinase activity were pooled and applied to a col-
umn of Q-Sepharose (2Æ0 · 5Æ0 cm), equilibrated with
10 mmol l)1 phosphate buffer pH 7Æ0. The column
was eluted with a gradient of 0–1 mol l)1 NaCl in
10 mmol l)1 phosphate buffer pH 7Æ0. The active frac-
tions were pooled and used for enzyme characterization.
Chromatographic procedures were performed using an
Econo System low-pressure chromatography system (Bio-
Rad Laboratories, Hercules, CA, USA) at 20C. The puri-
fied enzyme was freeze-dried and stored at )20C.
Polyacrylamide gel electrophoresis (SDS-PAGE)
The lyophilized protease samples were solubilized in
65 mmol l)1 Tris buffer pH 6Æ8, containing 2% (w/v)
SDS, 10% (v/v) glycerol and 5% (v/v) b-mercaptoethanol,
boiled for 5 min at 100C and applied to a 10% poly-
acrylamide gel. Molecular weight standards (range: 14Æ2–
205 kDa; Sigma) were run simultaneously for calculation
of molecular mass. After running at 20 mA, the gel was
fixed overnight in a solution of 100 g l)1 TCA and
stained with Coomassie brilliant blue G-250 using the
ultrasensitive method (Neuhoff et al. 1994). Alternatively
the gels were submitted to silver staining (Switzer et al.
1979).
Substrate specificity
The enzyme activity was tested on different natural and
synthetic substrates. Activity on azoproteins was carried
out as described above. Activity on native proteins
(casein, gelatin, keratin, bovine serum albumin and
haemoglobin) was essentially as follows: the enzyme
ª 2006 The Authors
R.C.S. Thys and A. Brandelli
solution (120 ll; 22 U; 1Æ4 lg) was added to 480 ll of
substrate solution (10 mg protein ml)1) in reaction buf-
fer 100 mmol l)1 phosphate pH 7Æ0. The mixture was
incubated at 50C for 30 min and the reaction was
stopped by adding 600 ll of 100 g l)1 TCA and leaving
the preparation on ice for 30 min. The mixture was
then centrifuged at 10 000 g for 10 min at 4C and
absorbance at 280 nm was measured with a spectropho-
tometer (Hitachi U-1100). One unit of enzyme activity
was defined as the amount of protein that resulted in
an increase of absorbance at 280 nm of 0Æ01, under the
assay conditions used. Activity on p-nitroanilide deriva-
tives was determined as described previously (Sangali
and Brandelli 2000).
Effect of inhibitors
Chemicals were added to the enzyme preparations and
incubated for 10 min at 25C before being tested for
proteolytic activity. The protease inhibitors phenyl-
methylsulfonyl fluoride (PMSF), ethylenediaminetetraace-
tic acid (EDTA), 1,10-phenanthroline, p-chloromercury
benzoate (pCMB), and pepstatin, the detergents SDS
and Triton X-100, and the organic solvents dimethyl
sulfoxide (DMSO) and isopropanol, and the reducing
agent b-mercaptoethanol were used at the working concen-
trations listed in Table 2.
Effect of metal ions
The effect of metal ions on protease activity was investi-
gated using a concentration range of 0Æ05–5 mmol l)1
(working concentration). The metal ion stock solutions
were prepared in distilled water and diluted to the appro-
priate concentrations (Venter et al. 1999). The enzyme
solution (22 U; 1Æ4 lg) was mixed with the different
metal solutions and pre-incubated for 10 min at 25C
before assayed for activity. A control was also included
where the enzyme was mixed with distilled water instead
of metal solution. The activity of control was taken as
100%.
Determination of metal content
The enzyme from Q-Sepharose chromatography was
extensively dialyzed (cellulose membrane, cut off 12 kDa)
against distilled, deionized water at 4C. Ca2+, Zn2+,
Mg2+ were determined by atomic emission spectroscopy
with an Optima 2000DV ICP-atomic emission spectro-
photometer (Perkin-Elmer, Shelton, CT, USA). Absorb-
ance was measured at 422Æ7, 213Æ9 and 285Æ2 nm for
Ca2+, Zn2+ and Mg2+ respectively. The absorbance of dif-
ferent standard solutions were used as standards.
ª 2006 The Authors
Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 101 (2006) 1259–1268
Keratinase of Microbacterium sp.
Feather keratin hydrolysis
Feathers were reduced to powder in a mortar with liquid
nitrogen, and sieved through a 0Æ20 mm mesh. The sub-
strate powder (20 mg) was added to 1 ml sodium merca-
ptoacetate solutions (0Æ3–1Æ7 mg ml)1) and immediately
the keratinase (76–104 U) was mixed to give the desired
final activity units. The mixtures were incubated in a
water bath at 50C with agitation (125 rev min)1) for
60 min. To separate the insoluble powder from the super-
natant, samples were centrifuged at 10 000 g for 10 min.
Then, soluble proteins released in the solutions were
determined by the Folin phenol reagent method (Lowry
et al. 1951).
Chemometric methodology
To study the hydrolysis of feather keratin, a chemometric
approach was based on the use of a matrix of experi-
ments by which the simultaneous variations of the factors
can be studied (Box 1954; Myers and Montgomery 2002).
A full 22 factorial design was developed to study the
effect of keratinase and mercaptoacetate concentration on
the formation of soluble protein. For the two factors, this
design was made up with its four points augmented with
three replications of the center points (all factors at level
0) and the four star points, that is, points having for one
factor an axial distance to the center of ±a, whereas the
other two factors are at level 0. The axial distance a was
chosen to be 1Æ41 to make this design orthogonal. A set
of 11 experiments was carried out. The central values
(zero level) chosen for experimental design were: kera-
tinase activity at 90 U and mercaptoacetate at 1 mg ml)1.
For two factors the equation model is:
Y ¼ bo þ b1 x1 þ b2 x2 þ b12 x1 x2 þ b11 x12 þ b22 x22 ð1Þ
where Y is the response (soluble protein, lg ml)1); x1, the
enzyme concentration (U) and x2 is the mercaptoacetate
concentration (mg ml)1). The bi,bii and bij terms repre-
sent the parameters of the model.
The results were analysed by the Experimental Design
Module of the Statistica 5.0 software (Statsoft, Tulsa, OK,
USA). The model permitted evaluation of the effects of
linear, quadratic and interactive terms of the independent
variables on the chosen dependent variables. Three-
dimensional surface plots were drawn to illustrate the
main and interactive effects of the independent variables
on keratin hydrolysis.
The quality of the fit of the polynomial model equation
was expressed by the coefficient of determination R2 and
its statistical significance was checked by an F-test. The
significance of the regression coefficient was tested by a
t-test. The level of significance was given as P < 0Æ05.
1261
Keratinase of Microbacterium sp. R.C.S. Thys and A. Brandelli

ized (Fig. 2). Denatured enzyme in SDS-PAGE showed a

Results
Proteolytic activity from Microbacterium sp.
The proteolytic activity was detected in supernatant cul-
tures of the actinomycetes Microbacterium sp. kr10. Kera-
tinolytic protease activity increased with cultivation time,
reaching maximum values after 36 h when stationary
phase took place. Activity on both azokeratin and azoca-
sein substrates was observed. The initial pH was 7Æ0,
increasing to 8Æ5 at 48 h and remaining at the last value
until the end of incubation.
Enzyme purification
molecular weight of about 42 kDa, when compared with
molecular weight standards. Omission of the reducing
agent b-mercaptoethanol did not affect the apparent size
of the protein (Fig. 2).
The samples of each purification step were assayed for
proteolytic activity and compared to control experiments
(without enzyme addition) to demonstrate that the
(a) 4 300
250
3

Keratinase activity (U ml–1)

The enzyme purification was performed by liquid chro-

Absorbance 280 nm
200
matography using gel filtration and ion exchange columns
sequentially. A representative example is presented in
2 150
Table 1. The overall purification was about 255-fold, with
a recovery of 34% as measured by assay with azocasein.
The maximum enzymatic activity was 15 125 U m)1 of 100
protein. The enzyme freeze-dried culture supernatant was
submitted to gel filtration chromatography on a Sephadex 1
G-100 column. The enzyme activity eluted as a symmetri- 50
cal peak, coinciding with a small absorbance at 280 nm
(Fig. 1a). The active fractions were pooled and submitted
to ion exchange chromatography. Enzymatic activity elut- 0 0
0 10 20 30 40
ed as a single peak (Fig. 1b).
Elution volume (ml)
The purified enzyme was analysed by polyacrylamide
gel electrophoresis, and a unique protein band was visual- (b)

Table 1 Purification of keratinase of Microbacterium sp. kr10 0·10

60
Total Total Specific

Keratinse activity (U ml–1)

activity protein activity Yield Purification
Absorbance 280 nm

Step (U) (mg) (U mg)1) (%) fold

Supernatant 2650Æ0 44Æ7 59Æ3 100 1Æ0 40

Freeze-dried 1878Æ7 28Æ4 66Æ1 70Æ9 1Æ1
Sephadex G-100 1400Æ0 0Æ98 1428Æ6 52Æ8 24Æ1
0·05
Q-Sepharose 907Æ5 0Æ06 15125Æ0 34Æ2 255Æ0

20

Table 2 Effect of different inhibitors on the activity of protease kr10

Concentration Remaining
Inhibitor (mmol l)1) activity (%)
0·00 0
None – 100 0 10 20 30 40 50 60
EDTA 5 0 Elution volume (ml)
PMSF 5 93
Figure 1 Purification of keratinase from Microbacterium sp. kr10.
1,10-phenanthroline 1 0
Freeze-dried culture filtrate was submitted to sequential chromato-
Benzamidine 1 100
graphy on Sephadex G-100 (a) and Q-Sepharose (b). Keratinase activ-
Pepstatin 0Æ1 102
ity (circles) and protein (solid line) were monitored. Arrow indicates
p-CMB 5 15
the start of the linear gradient from 0 to 1Æ5 mol l)1 NaCl.

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1262 Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 101 (2006) 1259–1268
R.C.S. Thys and A. Brandelli Keratinase of Microbacterium sp.

A B C D E

1·2
205 –

Absorbance 440 nm
116 – 0·8
97 –

84 –

66 – 0·4

45 –
0·0
29 – 0 2 4 6 8 10 12
Keratinase (µg ml–1)
20 –
Figure 3 Linear dependence of keratinase activity on protein concen-
14·2 – tration. Enzyme activity was measured at 50C for 30 min using
azocasein as substrate.

Figure 2 SDS-PAGE of Microbacterium sp. kr10 protease. The puri-

fied enzyme was applied to 10% polyacrylamide gel and stained with
Coomassie blue (lanes B,D,E) or AgNO3 (lane C). Samples were loaded 100
for SDS-PAGE under reducing (lane D) or non-reducing conditions
(lane E). Lane A, molecular weight standards (kDa) were miosin (205),
b-galactosidase (116), phosphorilase b (97), fructose-6-phosphate 80
Relative activity (%)

kinase (84), bovine serum albumin (66), ovalbumin (45), carbonic

anhydrase (29), trypsin inhibitor (20), a-lactoalbumin (14Æ2).
60

observed values are actual velocities of keratinase. Enzyme 40

activity of purified keratinase was linearly dependent on
the enzyme amount added to the reaction mixture
20
(Fig. 3), thereby showing that the initial velocity is pro-
portional to total enzyme concentration and that true ini-
tial velocities are being measured. 0
2 4 6 8 10
pH
Effect of pH
Figure 4 Effect of pH on keratinase activity. The activity was meas-
Enzyme assays were carried out at different pH values ured at 50C for 30 min using azokeratin as substrate.
using the following buffers: 0Æ1 mol l)1 citrate (pH 3–5),
0Æ1 mol l)1 phosphate (pH 6–7), 0Æ1 mol l)1 tris (pH 7Æ5–
9) and 0Æ1 mol l)1 carbonate (pH 9–11). The optimum pre-incubation in 0Æ1 mol l)1 phosphate buffer pH 7Æ0 in
pH of enzyme was measured as 7Æ5 (Fig. 4). Relative the range of 4–100C for 60 min. The results are showed
activity decrease to 40% and 55% at pH 6Æ0 and 9Æ0 in Fig. 5b. Keratinase was stable up to 55C and com-
respectively. pletely inactivated at 75C and higher temperatures.

Effect of temperature Substrate specificity

The temperature range of 4–100C was used to determine
the optimum temperature of the enzyme in 0Æ1 mol l)1
phosphate buffer pH 7Æ0. Under these conditions the tem-
perature optima of the enzyme was 50C (Fig. 5a).
Enzyme activity strongly decreased out of the range of
40–55C. The heat stability of the enzyme was tested after
Proteolytic activity was tested on several substrates. Incu-
bation were carried out using the substrates at the con-
centration of 10 mg ml)1 for diazoted and natural
proteins and 1 mmol l)1 for synthetic substrates. Among
the azoproteins tested, the enzyme was active on casein,
keratin and albumin, but not on gelatin (Fig. 6). The

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Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 101 (2006) 1259–1268 1263
Keratinase of Microbacterium sp. R.C.S. Thys and A. Brandelli

(a) 120
100
100
80

Enzyme activity (U ml–1)

Relative activity (%)

80
60

60
40

40
20

20
0
0 20 40 60 80 100
Temperature (oC) 0

in

og in

ok n

BA n

AL A

PM A
A
Az lati

i
PN

AA N

pN
Az ase

um

at

Ap

p
er
e

BZ
(b)

lb
oc
oa

C
Az

Az
100

Figure 6 Hydrolysis of substrates by Microbacterium sp. keratinase.

80 Aliquots were incubated and assayed with proteinaceous and p-nitro-
Residual activity (%)

anilide derivatives as substrates. Bars give the means and standard

errors of three replicate experiments. BAPNA, N-benzoyl-l-arginine
60
p-nitroanilide; ALApNA, Ala-Ala p-nitroanilide; CBZpNA, N-carbo-
xybenzoyl-l-phenylalanine p-nitroanilide; AAPMpNA, Ala-Ala-Pro-Met
40 p-nitroanilide.

20
Table 3 Effect of ions on the activity of protease kr10

Ion* Remaining activity (%)

0
0 20 40 60 80 100 None 100
Temperature (oC) CaCl2 90
MgCl2 119
Figure 5 Effect of temperature on keratinase activity (a) and stability
BaCl2 100
(b).
LiCl 99
SnCl2 57
HgCl2 8
enzyme presented a KM value for azokeratin of CuCl2 2
2Æ1 mg ml)1. The enzyme showed no activity on the sev- MnCl2 62
eral p-nitroanilide derivatives, excepting a marked activity ZnCl2 20
on CBZ-Phe-p-NA. ZnCl2 (0Æ5 mmol l)1) 107
ZnCl2 (0Æ05 mmol l)1) 97

Effect of chemicals *Ions were tested at 5 mmol l)1 unless otherwise stated.

The protease activity was investigated after pre-incubation

of the enzyme with several chemicals for 15 min. The
activity was inhibited by specific metalloprotease inhibi-
tors such as EDTA and 1,10-phenanthroline (Table 2).
Other protease inhibitors such as PMSF and pepstatin
had no effect. The thiol-reactive agent p-CMB also inhib-
ited the enzyme activity (Table 2). Among the metal ions
tested, Mg2+ had a stimulatory effect, but the activity was
strongly inhibited by Hg2+ and Cu2+, and partially inhib-
ited by Sn2+ (Table 3). The inhibitory effect of zinc was
observed at 5 mmol l)1, but not at 0Æ5 or 0Æ05 mmol l)1
(Table 3).
Determination of metal content
The metal content was determined by atomic emission
spectroscopy, indicating that the enzyme have Zn2+ and
Mg2+. The number of metal atoms per enzyme molecule
was estimated from the protein content, assuming a

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1264 Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 101 (2006) 1259–1268
R.C.S. Thys and A. Brandelli Keratinase of Microbacterium sp.

Table 4 Experimental design and results of the 22 factorial design

Coded values Variables

Enzyme Mercaptoacetate Soluble protein 280

Run x1 x2 (U) (mg ml)1) [Y (lg ml)1)] 240

n
Soluble protei
1 )1 )1 80 0Æ5 165 200
2 )1 +1 80 1Æ5 112 160
3 +1 )1 100 0Æ5 233 120
4 +1 +1 100 1Æ5 208 80
5 0 )1Æ41 90 0Æ3 185 40
6 )1Æ41 0 76 1Æ0 82 0
7 0 +1Æ41 90 1Æ7 180
8 +1Æ41 0 104 1Æ0 250 98 1·8
En
9 0 0 90 1Æ0 218 zy 90 1·4
m e
e 1·0 tat
10 0 0 90 1Æ0 220 un 82 ace
11 0 0 90 1Æ0 217
its 0·6 pto
74 0·2 e rca
M

molecular weight of 42 000. One mol of zinc was found Figure 7 Response surface plot of soluble protein release from
per mol of keratinase. The ratio of Zn2+ to Mg2+ was cal- feather keratin as a function of keratinase and mercaptoacetate
culated as 1 : 2Æ3. concentration.

Keratin hydrolysis
The results of the central composite design experiments
for studying the effects of enzyme and mercaptoacetate
concentration on feather keratin hydrolysis are presented
in Table 4.
Statistical analysis of results showed that, in the range
studied, the two variables, as well as their interactions,
have a significant effect on keratin hydrolysis. Analysis of
variance (anova) and Fischer’s F-test showed the value
F(9Æ7) ¼ 18Æ31, which is 3Æ6 times higher than the F tabu-
lated (Ft(9Æ7) ¼ 5Æ05), and that demonstrates significance
for the regression model. The following regression equa-
tion was obtained, presenting the R2 value of 0Æ948
(a value of R2 > 0Æ75 indicates the aptness of the model):
Y ¼ 2183115 þ 502595x1  10663x2 þ 700x1 x2
 249256x12  166262x22
with Y, soluble protein (response); x1, enzyme units; and
x2, mercaptoacetate concentration (coded values). The
significance of each coefficient was determined by Stu-
dent’s t-test and P-values, which were all significant at
95% of confidence level.
The three dimensional response surface curve was plot-
ted (Fig. 7). Maximum keratin hydrolysis was achieved at
mercaptoacetate between 0Æ6 and 1Æ4 mg ml)1 and 100 U
of keratinolytic protease enzyme.
Discussion
A keratinolytic metalloprotease produced by Microbacte-
rium sp. kr10 was purified through a liquid chromato-
graphy protocol. The enzyme was purified from a poor
medium, containing only keratin protein and therefore
the protease could be purified through a relatively simple
procedure. Some described purification protocols for
Streptomyces and Bacillus keratinases often involve similar
steps although resulting lower purification fold (Lin et al.
1992; Böckle et al. 1995; Bressollier et al. 1999; Suh and
Lee 2001). The gene kerA, which encodes a B. lichenifor-
mis keratinase, is expressed specifically for feather hydro-
lysis (Lin et al. 1995). Therefore, the presence of feather
keratin as the sole carbon and nitrogen source in the cul-
ture medium may result in preferential expression of the
keratinolytic protease.
The molecular weight for keratinases of mesophilic
micro-organisms, including Bacillus spp. and Streptomyces
spp. is around 20–50 kDa (Friedrich and Antranik-
ð2Þ ian 1996). The keratinase kr10 showed a similar MW of
B. licheniformis K-503 (Rozs et al. 2001) and Chryseobac-
terium sp. kr6 (Brandelli 2005) keratinases, whose MW
are 42 and 38 kDa respectively.
Mostly of the keratinases are classified as serine-type
proteases. The keratinases produced by Bacillus lichenifor-
mis, Bacillus subtilis, Microsporum canis are serine protea-
ses, and their genes shown important sequence homology
with subtilisins, which are typical serine-type proteases
(Lin et al. 1995; Zaghloul 1998; Deschamps et al. 2003).
Among actinomycetes, several keratinases described for
Streptomyces spp. are also classified as serine proteases
(Böckle et al. 1995; Bressollier et al. 1999). However, the
enzyme produced by the strain kr10 seems to belong to
the metalloprotease type since it was completely inhibited
by EDTA and 1,10-phenanthroline, but not by PMSF,

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Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 101 (2006) 1259–1268 1265
Keratinase of Microbacterium sp.
benzamidine or pepstatin. Metalloproteases have not been
frequently associated with keratinolytic activity, being
only recently associated with Lysobacter (Allpress et al.
2002) and Chryseobacterium (Riffel et al. 2003b). In this
way, some properties of Microbacterium sp. kr10 kera-
tinase resemble those of Chryseobacterium proteases (Ven-
ter et al. 1999; Riffel et al. 2003b).
The activity on synthetic substrates give some indica-
tion of enzyme specificity. The keratinase kr10 was only
active on CBZ-Phe-pNA, a substrate for chymotrypsin-
like proteases. In agreement with our results, Allpress
et al. (2002) reported a keratinolytic metalloprotease
from Lysobacter that was strongly active towards CBZ-
Phe-pNA. S. albidoflavus produces a chymotrypsin-like
keratinase that exhibited specificity with aromatic and
hydrophobic amino acid residues, as demonstrated by
using synthetic peptides (Bressollier et al. 1999). The
keratinolytic B. licheniformis K-508 secreted a mixture
trypsin-like and chymotrypsin-like proteases that are not
inhibited by PMSF (Rozs et al. 2001). That enzyme was
inhibited by Hg2+ and characterized as a novel trypsin-
like thiol protease. In this concern, keratinase kr10 inhibi-
tion by pCMB and Hg2+ may suggest that a free cysteine
is present at or near the active site. It has been suggested
that inhibition by Hg2+ is not just related to binding of
the thiol groups but may be a result of an interaction
with tryptophan residues or with the carbonyl group of
amino acids in the enzyme (Lusterio et al. 1992).
The soluble substrates azoalbumin and azocasein were
readily degradable by the keratinase, while the insoluble
azokeratin was less so. Similar data was reported for kera-
tinases of B. licheniformis PWD-1 (Lin et al. 1992) and
Chryseobacterium sp. (Brandelli 2005). The unique struc-
ture of keratin makes it very resistant to proteolytic diges-
tion, and therefore this protein is a more resistant
substrate to many proteases.
The inhibition by 5 mmol l)1 Zn2+ agrees with the fact
that several zinc peptidases are inhibited by excess zinc,
particularly at neutral to alkaline pH (Auld 1995). The
inhibition is effected by zinc monohydroxide (ZnOH+),
through a bridge between the inhibitory zinc and catalytic
zinc ions at the active site (Larsen and Auld 1991). Sim-
ilar effect of zinc ions was demonstrated for Chryseobacte-
rium sp. keratinase (Riffel et al. 2003b) and for a recently
described keratinolytic metalloprotease of Bacillus cereus
(Werlang and Brandelli 2005).
Thermolysin, a characteristic bacterial metalloprotease,
contains one atom of zinc located in the active site and
four atoms of calcium, which are bound to different
sites of the enzyme. The calcium on thermolysin contri-
butes to heat stability, prevention of autolysis, and
maintenance of structural integrity (Feder et al. 1971).
The liability of the keratinase kr10 against 1,10-phen-
1266 Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 101 (2006) 1259–1268
R.C.S. Thys and A. Brandelli
anthroline also indicates that it contains zinc in the act-
ive site, which was confirmed by atomic emission
spectroscopy. The presence and the stimulatory effect of
Mg2+ may be related to thermal stability of the enzyme.
The extracellular protease of Pseudomonas fluorescens
T20 contained Mg2+, which plays an important role on
enzyme stability (Patel et al. 1986).
The use of reducing agents to enhance keratin degrada-
tion by keratinases has been described (Böckle et al. 1995;
Riffel et al. 2003b). In this study, the interaction between
keratinase and mercaptoacetate on hydrolysis of feather
keratin was demonstrated by response surface methodo-
logy. The two variables have significant effects on keratin
hydrolysis, indicating that the release of soluble protein
enhances around the center point. The optimization
of experimental conditions for keratin hydrolysis by
Doratomyces microsporum keratinase was studied using
two-order experimental design. An increased quantity of
soluble protein was achieved when thioglycolated nails or
hooves were treated with keratinase (Vignardet et al.
2001). The optimum concentration for mercaptoacetate
was between 0Æ6 and 1Æ4 mg ml)1, and keratin hydrolysis
decreases above this value. Despite the reducing agent
may help the keratin hydrolysis, a deleterious effect on
enzyme may occur when it was used at more elevated
concentrations. In fact, pCMB has an inhibitory effect on
keratinase suggesting that cysteine residues are associated
to enzyme activity.
It has been suggested that keratinolytic metalloprotea-
ses may have great biotechnological promise (Allpress
et al. 2002). As secondary keratinases, they may overcome
the limited keratinolysis on the surface of keratin particles
because of restricted enzyme-substrate interaction (Böckle
et al. 1995). In addition, they may be temporarily inacti-
vated by chelating agents during storage, reducing auto-
lysis. The metalloenzyme nature presents a potential
method of enzyme immobilization, which has been repor-
ted to increase stability because of a reduced autolysis
(Wang et al. 2003).
This enzyme has potential for utilization in several
important processes where keratin hydrolysis must be
achieved. The enzyme did not hydrolyze gelatin and syn-
thetic substrate for collagenase, and its use for de-hairing
bovine pelts caused no collagen damage (Riffel et al.
2003a), indicating desirable property for cosmetic and
pharmaceutical purposes, or leather processing where col-
lagen should not be attacked. The recent finding that
B. licheniformis PWD-1 keratinase cause enzymatic break-
down of prion protein PrPSc (Langeveld et al. 2003) leave
open a novel relevant application for keratinases. Also,
hydrolysis of feather keratin is a potential use as poultry
feathers are generated in large amounts as potential pol-
lutants.
ª 2006 The Authors
R.C.S. Thys and A. Brandelli
Acknowledgments
This work was supported by CNPq (Brazil).
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