Bioresource Technology 98 (2007) 1238–1245

Production and partial characterization of dehairing protease

from Bacillus cereus MCM B-326
S.S. Nilegaonkar, V.P. Zambare, P.P. Kanekar ¤, P.K. Dhakephalkar, S.S. Sarnaik
Microbial Sciences Division, Agharkar Research Institute, G.G. Agarkar Road, Pune, Maharashtra State 411 004, India

Received 12 January 2006; received in revised form 4 May 2006; accepted 7 May 2006
Available online 19 June 2006

Abstract

Bacillus cereus MCM B-326, isolated from buValo hide, produced an extracellular protease. Maximum protease production occurred
(126.87 § 1.32 U ml¡1) in starch soybean meal medium of pH 9.0, at 30 °C, under shake culture condition, with 2.8 £ 108 cells ml¡1 as
initial inoculum density, at 36 h. Ammonium sulphate precipitate of the enzyme was stable over a temperature range of 25–65 °C and pH
6–12, with maximum activity at 55 °C and pH 9.0. The enzyme required Ca2+ ions for its production but not for activity and/or stability.
The partially puriWed enzyme exhibited multiple proteases of molecular weight 45 kDa and 36 kDa. The enzyme could be eVectively used
to remove hair from buValo hide indicating its potential in leather processing industry.
© 2006 Elsevier Ltd. All rights reserved.

Keywords: Bacillus cereus; BuValo hide; Dehairing; Protease; Starch–soybean meal

1. Introduction reported by many researchers (Huang et al., 2003; RiZe

In leather processing, the most constrained operation
from environmental point of view is the dehairing of skin/
hide. The conventional method of dehairing involves the
use of lime and sodium sulphide. Presence of these chemi-
cals in tannery waste is responsible for tremendous pollu-
tion, causing health hazards to the tannery workers. Lime
produces a poisonous sludge while sodium sulphide is
highly toxic and has obnoxious odor. Although enzyme
assisted dehairing process reduces the pollution load to
some extent, a technology based on enzyme alone, without
the use of sulphide and other chemical inputs, has yet to be
explored (Thanikaivelan et al., 2004).
Proteases Wnd applications at various steps of leather
processing, e.g., neutral proteases in soaking (Deshpande
et al., 2004), alkaline proteases in dehairing (Dayanandan
et al., 2003), and acid proteases in bating (Padmavathi et al.,
1995). Dehairing enzymes from Bacillus sp. have been
*
Corresponding author. Tel.: +91 20 25653680; fax: +91 20 25651542.
E-mail address: PPKanekar@aripune.org (P.P. Kanekar).
et al., 2003; Alexandre et al., 2005). The concrete mixture of
dehairing enzymes from Bacillus subtilis and Bacillus cereus
with sodium carbonate, caustic soda and thioglycolic acid,
is described in a patent (Monsheimer and PXeiderer, 1976).
The aim of the present work was to study the production,
optimization and properties of extracellular proteolytic
enzyme of B. cereus B-326 having application in dehairing
of buValo hide.
2. Methods
2.1. Microorganism and taxonomic study
B. cereus BSA-26 producing protease was isolated from
buValo hide obtained from Local Municipal Corporation
Slaughterhouse (Pune, India). The strain was identiWed
according to the methods described in Bergey’s Manual of
Systematic Bacteriology (Sneath, 1984) and on the basis of
16 s ribosomal DNA sequence The stock culture was main-
tained on nutrient agar at 4 °C and as a glycerol stock at
¡20 °C.

0960-8524/$ - see front matter © 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2006.05.003
 S.S. Nilegaonkar et al. / Bioresource Technology 98 (2007) 1238–1245
2.2. Inoculum preparation and production
Seed inoculum was prepared by growing the isolate on
nutrient agar slants/Roux bottle at 30 °C for 24 h. The cells
were suspended in saline and cell density was measured
spectrophotometrically (Shimatzu UV-2501 PC, Japan) at
600 nm. Following media were studied for protease produc-
tion: synthetic medium casein (SMC: 0.7% K2HPO4, 0.3%
KH2PO4, 0.01% MgSO4, 1% casein), nutrient broth supple-
mented with 1% casein (NBC), starch soybean meal (SS:
2% starch, 1% soybean meal, 0.3% CaCO3) and soybean
meal-tryptone (ST: 1% soybean meal, 1% tryptone). The
Xasks were inoculated with 24 h old inoculum and incu-
bated on orbital shaker (150 rpm) at 30 °C for 60 h. The
medium yielding maximum protease activity was selected
for further investigation.
2.3. Protease assay
The protease activity was determined by caseinolytic
assay method of (Kanekar et al., 2002). The cell free super-
natant (1 ml) was mixed with 4 ml of casein (0.625% w/v)
and incubated at 37 °C for 30 min. The reaction was
stopped by addition of 5 ml of 5% trichloroacetic acid.
Enzymatically hydrolyzed casein was measured by modi-
Wed Folin Ciocalteu method (Jayaraman, 2003), against
casein treated with inactive enzyme as blank. A standard
graph was generated using standard tyrosine solutions of
5–50 g ml¡1. One unit of protease activity was deWned as
the amount of enzyme which liberated 1 g tyrosine per min
at 37 °C.
2.4. EVect of diVerent environmental conditions on protease
production
For optimization, production of protease by isolate
BSA-26 was studied using SS medium, under the following
conditions: shake and static culture, age of inoculum-21 to
24 h, initial inoculum density: 0.5–5.0% (v/v) of cell density
2.8 £ 108 cells ml¡1, temperature: 25–40 °C, with increments
of 5 °C, pH: 6–12, with increments of one unit. The Xasks
were incubated for 36 h and cells were removed by cold cen-
trifugation at 13,000 £ g for 10 min. The cell free superna-
tant was analyzed for protease activity.
2.5. EVect of diVerent nitrogen and carbon sources
To test the eVect of diVerent nitrogen source on the pro-
tease production, the liquid medium of starch (2%) and cal-
cium carbonate (0.3%) was supplemented with various
complex nitrogen source such as yeast extract, beef extract,
casein, tryptone, peptone, soybean meal, corn steep liquor
and inorganic nitrogen sources viz. ammonium chloride,
ammonium sulphate, ammonium phosphate, ammonium
nitrate and sodium nitrite all at 1% concentration. Likewise
the eVect of diVerent carbon sources on protease produc-
tion was studied by supplementing; a liquid medium of soy-
1239
bean meal (1%) and calcium carbonate (0.3%), with starch,
glucose, sucrose at 1% concentration. The Xasks were incu-
bated for 36 h and cell free supernatant was analyzed for
protease activity.
2.6. EVect of metal ions
To determine the eVect of metal ions on the protease
production, diVerent metal ions viz. CaCO3, CaCl2,
K2HPO4, KH2PO4, FeSO4, MgSO4, NaCl and MnSO4 were
individually added with 0.3% strength in starch (2%) and
soybean meal (1%) liquid medium. The Xasks were incu-
bated for 36 h and cell free supernatant was analyzed for
protease activity.
2.7. Time course of protease production
The growth of the organism and production of protease
was studied under all optimum conditions viz SS medium,
pH 9.0, 30 °C, shake culture condition, 1% inoculum size at
6 h interval. The cell growth was measured at 600 nm. Cell
free supernatants were analyzed for enzyme activity, pro-
tein and residual starch, at each interval, up to 72 h. Protein
content was measured by Buret method (Jayaraman, 2003).
The residual starch was estimated by iodometric method
(Jayaraman, 2003).
2.8. Scale-up studies
Production of the enzyme was carried out in a 2 l glass
bottle with a working volume of 800 ml of SS medium.
Parallel experiment was conducted at Xask level with the
same medium. The culture was inoculated in nutrient
broth. After an incubation period of 21 h, cell growth of
2.8 £ 108 cells ml¡1 was inoculated in 800 ml SS medium
(pH 9.0) and incubated at 30 °C with constant agitation
speed of 150 rpm. Enzyme samples were removed at 12 h
intervals for measuring cell growth by recording OD at
600 nm. Biomass was separated by centrifugation. The cell
free culture broth was assayed for extracellular protease
activity and protein content as described above. The
enzyme was partially puriWed using ammonium sulphate
(60% saturation). The precipitated enzyme was used as a
crude enzyme for further studies.
2.9. Substrate speciWcity
Protease was produced in SS medium (pH 9.0) at 30 °C
for 36 h, and cell free supernatant was assayed using diVer-
ent substrates such as casein, Bovine serum albumin (BSA),
haemoglobin, collagen, gelatin and keratin. The enzymatic
hydrolysis of the casein and BSA was studied by assay as
described above. The proteolytic activity of the enzyme
with 2% haemoglobin as substrate was carried out accord-
ing to Sarath et al. (1996). The enzymatic hydrolysis of col-
lagen, gelatin was studied as described by Woessner (1961).
Keratinase activity was determined using keratin azure
1240 S.S. Nilegaonkar et al. / Bioresource Technology 98 (2007) 1238–1245
substrate (Takami et al., 1990). Enzyme units were deWned
according to the respective assay.
2.10. EVect of pH and temperature
The pH stability of precipitated protease was determined
with casein (0.625% w/v) as a substrate dissolved in diVer-
ent buVers [sodium acetate, pH 4–6, sodium phosphate, pH
7–8, Tris HCl, pH 9–10.6 and Glycine-NaOH, pH 11–12].
The pH stability of the protease was determined by pre-
incubating the enzyme in diVerent buVers for 30 min at
37 °C. Likewise, thermal stability of the precipitated prote-
ase was determined by pre-incubating the enzyme at diVer-
ent temperatures from 25–80 °C for 30 min. All experiments
were carried out in duplicate and each analysis was also
performed in duplicate.
2.11. EVects of diVerent inhibitors, metals, detergents and
oxidant on enzyme activity
To study inhibition of the protease, enzyme was pre-
incubated with inhibitors such as phenylmethylsulphonyl
Xuoride (PMSF, 1 mM), ethylenediamine tetraacetic acid
(EDTA, 2 & 5 mM), dithiothretol (DTT, 2 & 5 mM), iodo-
acetamide (2 mM), trypsin inhibitor (100 g), metal ions
such as Hg+ (1 & 5 mM), Na+, Fe2+, Cu2+, Ca2+, Mg2+,
Mn2+, Zn2+ (5 mM); detergents such as sodium dodecyl
sulfate (SDS, 1%), sodium tripolyphosphate (1%), sodium
tetraborate (1%) and oxidizing agent H2O2 (5%, 10% and
15%) for 30 min at 37 °C. Subsequently the enzyme assay
was performed as described above. The percent residual
enzyme activity was calculated with reference to the activity
of the enzyme without these supplements.
2.12. Gel electrophoresis and zymogram
Proteins were analyzed by tube gel electrophoresis using
comassive brilliant blue R250. The proteins were precipi-
tated with ammonium sulphate (60% saturation) followed
by membrane dialysis. The protein pattern of crude enzyme
was carried out with native PAGE. A zymogram of dia-
lyzed enzyme was obtained using polyacrylamide gel elec-
trophoresis followed by treatment with 1% (w/v) casein as
substrate in Tris buVer (pH 9.0). The molecular weight of
the protease enzyme was determined by compairing with
mobility of standard molecular weight marker proteins
(bovine albumin, 66 kDa, chicken ovalbumin, 45 kDa, gly-
ceroldehyde-3-phosphate dehydrogenase 36 kDa, trypsino-
gen 24 kDa, cytochrome C 12.4 kDa).
2.13. Dehairing of buValo hide
Ammonium sulfate precipitate of enzyme was applied at
1% concentration of to piece buValo hide (2.5 cm £ 2.5 cm),
presoaked in tap water from Xesh side, and kept at ambient
temperature (28 § 2 °C) in a dry place. Loosening of hair
and epidermis were observed by mechanical means at an
hourly interval.
2.14. Statistical analysis
Analysis of variance with repeated measures was carried
out using GLM command of software SPSS (SPSS version
10, Windows 98 version). Activity means and standard
deviations were calculated. Univariate analysis of variance
(ANOVA) was employed on the data for protease activity
at diVerent % inoculum, pH, temperature, culture condi-
tion-incubation period, complex and inorganic nitrogen
sources, carbon supplement and metal ions supplement and
tested for their signiWcance.
3. Results and discussion
The culture B. cereus BSA-26 was deposited in MACS
collection of microorganisms (WFCC code 561) and desig-
nated as MCM B-326.The isolate was identiWed as on the
basis of morphological and physiological characteristics,
biochemical tests and 16 S rRNA sequencing. A compari-
son of the DNA sequence with sequences in the National
Center for Biotechnology Information (NCBI) database
with BLAST software (Altschul et al., 1997) showed 98.5%
sequence identity with the published 16 s rRNA sequences
of B. cereus. The 16 s rDNA sequence of the isolate has
been deposited in GenBank database with accession num-
ber DQ479314. Strains of Bacillus species producing pro-
tease activity have been widely described in literature
(Mehrotra et al., 1999; Kanekar et al., 2002; He et al., 2006).
Their applications, especially those of alkaline protease, are
mainly directed towards detergent industry (Anwar and
Saleemuddin, 1998).
3.1. Optimization of protease production
Maximum production of protease by B. cereus MCM B-
326 was obtained in SS medium at 36 h of incubation, and
was signiWcantly (p < 0.001) higher than that observed in
other media tested. The protease activity was highest with
2% starch, 1% soybean meal and 0.3% CaCO3 with 0.5–
1.0% inoculum of cell density 2.8 £ 108 cells/ml of 21 h age.
The results showed close resemblance with the alkaline
protease by thermophilic B. licheniformis (Sinha and Satya-
narayana, 1991). The results on the eVect of initial pH and
temperature on protease production showed maximum
activity at pH 9.0 (p < 0.001) and 30 °C (p < 0.001)
(113.33 § 14.43 and 112 § 14.83 U ml¡1, respectively, results
not shown). Comparable results were obtained for B. alcal-
ophilus, isolated from Lonar lake, India (Kanekar et al.,
2002).
It was observed that, in presence of sucrose, starch
or glucose (1%), the protease activity (116.27 § 5.72–
122.45 § 20.59 U ml¡1, results not shown) was almost con-
stant; however it decreased signiWcantly in the absence of
carbon sources (29.35 § 9.36 U ml¡1) at 36 h. The activity
 S.S. Nilegaonkar et al. / Bioresource Technology 98 (2007) 1238–1245 1241

obtained with 1% starch was similar to that with 2% starch meal (122.24 § 2.06 U ml¡1, p < 0.001). Soybean meal was
in SS medium. The protease activity enhanced 4 fold in used as inducer for protease production from Conidiobolus
presence of starch. Such enhancement of alkaline protease coronatus (Deshpande et al., 2004). The protease activity
has been reported in alkaliphilic B. licheniformis (Sinha and was increased when production medium was supplemented
Satyanarayana, 1991). EVect of diVerent complex and inor- with potassium, magnesium, iron and calcium ions.
ganic nitrogen sources on protease activity are presented in Enhancement of 7.4 fold in activity was achieved by supple-
Table 1. The protease activity was highest with soybean mentation of 0.3% CaCO3 (p < 0.001) (Table 1). This indi-
cated that the calcium ion was necessary for enzyme
induction (Ghorbel et al., 2005).
Table 1 Thus, optimization studies resulted in the following
EVect of nitrogen sources and metal ions on protease production by Wndings: the most suitable nutrient medium starch (1%),
B. cereus MCM B-326 soybean meal (1%) and CaCO3 (0.3%) of initial pH 9.0,
Substrates supplied Mean enzyme activity (U ml¡1) temperature 30 °C, 1% inoculum and period of incubation
Complex nitrogen sources (1%)
36 h. (Tayler et al., 1987) have reported the best production
Soybean meal 122.24 § 2.06 of dehairing protease using nutrient medium containing
Yeast extract 61.26 § 7.63 corn steep liquor, lactose, sodium sulfate and potassium
Peptone 39.97 § 10.03 acid phosphate at initial pH 6.8–7.2 and 37 °C for 96 h.
Tryptone 30.37 § 5.40
Corn steep liquor 22.31 § 4.43
Beef extract 9.04 § 2.53 3.2. Time course of protease production
Casein 1.36 § 0.72
Inorganic nitrogen sources (1%) Under the optimum conditions, the protease activity
Ammonium sulphate 58.91 § 1.41 reached to 126.87 § 1.32 U ml¡1 within 36 h of the fermenta-
Ammonium nitrate 58.38 § 0.19
tion when the cell growth reached late log phase or early
Ammonium chloride 100.88 § 8.03
Ammonium phosphate 0.42 § 0.80 stationary phase (Fig. 1). An alkaline protease from marine
Sodium nitrite 112.13 § 6.99 yeast produced protease activity within 30 h when the cell
Metal ions (0.3%)
growth reached mid-log phase (Chi et al., 2006). The reduc-
CaCO3 125.99 § 1.91 tion in starch level indicated that B. cereus MCM B-326
CaCl2 62.63 § 3.65 had strong amylolytic activity in addition to protease activ-
K2HPO4 43.30 § 5.47 ity. The starch level was almost negligible after 6 h. The pH
KH2PO4 41.51 § 2.75 of the medium decreased towards acidic side because of
FeSO4 30.76 § 1.61
MgSO4 21.20 § 1.72
hydrolysis of starch to acids via glucose (data not shown).
NaCl 17.66 § 1.73 The protein content was decreased because of the utiliza-
MnSO4 13.90 § 3.19 tion of protein by the organism. The decline in protease
Without metal ions 17.06 § 0.86 activity upon prolonged incubation may be due to autolysis
Data represent the mean values and standard deviation (n D 9). of the enzyme.

2.5 5 140 10

120
2.0 4 8
Cell density (x10 cells ml )
-1

100
Enz yme ac tivity (U m l

Residual starch (mg ml )

Protein (mg ml )
-1

1.5 3 6
9

80

1.0 60
2 4

40
-1
)

-1

0.5 1 2
20

0.0 0 0 0
0 10 20 30 40 50 60 70 80
Tim e ( h)
Cell density Protein Enzyme activity Residual starch

Fig. 1. Time course of protease production by B. cereus MCM B-326. Standard deviations are represented by error bars.
1242 S.S. Nilegaonkar et al. / Bioresource Technology 98 (2007) 1238–1245

3.3. Scale-up studies 160

140
Protease activity was 150.52 § 11.24 U ml¡1 in a 2 l glass

% Enzyme activity
bottle, (Fig. 2) and 126.87 § 1.32 U ml¡1 in Xask level at the 120
Temperature
end of 36 h, showing 1.13 fold increase. (Wang and Shih, 100
1999) reported that keratinase activity from B. licheniformis
80
and a recombinant B. subtilis was increased 1.04 and 1.01
fold respectively in 15 l fermentor as compared to Xask 60
level. 40 pH

20
3.4. Characteristics of protease
0
2 4 6 8 10 12 14
The protease from B. cereus MCM B-326 hydrolyzed
pH
casein, haemoglobin, BSA with activity of 128.64 U ml¡1,
14.25 U ml¡1 and 50.89 U ml¡1 respectively (data not 20 30 40 50 60 70 80 90
shown). But it was not able to hydrolyze collagen, gelatin, Temperature (ºC)
and keratin. Protease from Alkaligenes faecalis could not
Fig. 3. EVect of pH and temperature on activity of B. cereus MCM B-326.
hydrolyze the Wbrous proteins such as collagen and kera-
Protease symbols and bars represent the mean values and the standard
tin (Thangam and Rajkumar, 2002). The enzyme was deviations. The 100 ml¡1 for pH and 1721.38 U ml¡1 for temperature.
active in the pH range of 6–12, with optimum activity at
pH 9.0, although, a small peak at pH 10.6 was also
observed, suggesting a presence of two alkaline proteases After 30 min, activity decreased to 53% and 85% at 50 °C
(Fig. 3). The preliminary studies on the extracellular and 60 °C, respectively and completely inactivated at 70 °C
dehairing protease secreted by the Bacillus sp. showed that (Huang et al., 2003). (Annapurna et al., 1996) reported that
it has dual pH maxima at 7.5 and 9.0 (Annapurna et al., the enzyme was active in temperature range of 20–50 °C,
1996). with optimum activity at 37 °C. Thus the protease of B.
The enzyme was active in the temperature range of 30– cereus under study is more thermo tolerant than the pro-
65 °C with maximum activity at 55 °C, although, a small teases reported above.
peak at 30 °C was also observed, suggesting presence of two The eVect of inhibitors, metal ions, detergents and oxi-
proteases. The activity increased from 134% to 149% with dant on the ammonium sulphate precipitated enzyme is
increase in temperature from 40–55 °C. The enzyme was detailed in Table 2. The protease was completely inhibited
completely inactivated at 80 °C (Fig. 3). In an earlier report, by EDTA indicating that it might be a metalloprotease.
the dehairing enzyme was active between the temperature Similar results for B. cereus KCTC 3674 were observed by
range of 30–60 °C, and showed optimum activity at 55 °C. (Kim et al., 2001). The protease activity was 97% inhibited

180 5 25

160
4 20
140
Cell density (x109 cells ml-1)
Enzyme activity (U ml-1)

120
Protein (mg ml-1)

3 15
100

80
2 10
60

40 1 5
20

0 0 0
0 10 20 30 40 50 60 70 80
Time (h)
Enzyme activity Protein (mg ml-1) Cell density
Fig. 2. Scale-up of protease by B. cereus MCM B-326. Standard deviations are represented by error bars.
 S.S. Nilegaonkar et al. / Bioresource Technology 98 (2007) 1238–1245 1243

Table 2
EVect of inhibitors and activators on protease activity of B. cereus MCM B-326
Compound Concentration % residual activity Compound Concentration % residual activity
Control – 100 § 1.96 CuSO4 5 mM 17.03 § 1.16
Inhibitors CaCl2 5 mM 101.03 § 5.07
PMSF 1 mM 84.63 § 3.93 MnSO4 5 mM 49.6 3 § 1.86
EDTA 2 mM 7.68 § 1.55 MgSO4 5 mM 7.81 § 2.69
5 mM 0.00 § 0.00 ZnSO4 5 mM 17.96 § 1.08
DTT 2 mM 11.52 § 1.60 Detergents
5 mM 2.80 § 1.11 Tween 80 1% 79.64 § 1.78
Iodoacetamide 2 mM 78.81 § 1.92 SDS 1% 0.72 § 0.92
Trypsin inhibitor 100 g 98.13 § 0.81 Sodium tripolyphosphate 1% 11.00 § 0.77
Metal ions Sodium tetraborate 1% 62.30 § 2.94
NaCl 5 mM 69.78 § 3.55 Oxidant
HgCl2 1 mM 15.78 § 2.31 H 2O 2 5% 121.91 § 1.88
5 mM 0.00 § 0.53 10% 113.60 § 3.04
FeSO4 5 mM 36.24 § 2.99 15% 104.77 § 2.80
Data represent the mean values and standard deviation (n D 3).
The 100% activity correspond to 1393.22 U ml¡1.
Abbreviation: PMSF– phenymethylsulphonyl Xuoride, EDTA– Ethylenediamine tetraaceticacid, DTT– Dithiothreitol, SDS– Sodium dodecyl sulphate.

by DTT, thus the enzyme might contain S–S bond as a

part of its monomeric structure. The eVect was consistent
with its above-described thermal stability, which had been
shown primarily to be the result of disulWde bond (Kara-
dzic et al., 2004). Protease activity was inhibited by 15%
by PMSF and trypsin inhibitor had no eVect on activity.
This suggested that this protease was not of serine type. In
an earlier report, dehairing protease was completely inhib-
ited by PMSF and partially inhibited by EDTA (Huang
et al., 2003). Similarly, the dehairing protease of Bacillus
sp. kr10 was completely inhibited by PMSF, whereas 84%
inhibited by EDTA (RiZe et al., 2003).Thus, both the
reported dehairing proteases of Bacillus sp. were serine
proteases.
The protease was markedly inhibited (80–93%) by Cu2+,
Mg2+, Zn2+, and sodium tripolyphosphate; while partially
inhibited (30–60%) by Na+, Fe2+, Mn2+. Sodium tetra-
borate and iodoacetamide inhibited the activity by about
20%. The protease enzyme was stable and active in the pres- Fig. 4. Enzyme proWle of protease from B. cereus MCM B-326.
ence of oxidizing agent H2O2 (5% and 10%) and, therefore,
could be used in bleach-based detergent formulations. Sim-
ilar results were obtained for alkaline protease from alkali (Kim et al., 2001). A calcium dependent protease from
tolerant B. patagoniensis (Olivera et al., 2006). Results also B. cereus BG1 was reported to have molecular weight of
showed that (data not shown) Ca2+ was required for the 34 kDa (Ghorbel et al., 2005). According to (Huang et al.,
production only and not for activity and/or stability of the 2003) the molecular weight of the puriWed dehairing prote-
enzyme. However, protease from B. cereus BG1 required ase from B. pumilus was 32 kDa. Thus, molecular weight of
calcium ion for its activity as well as stability (Ghorbel Cereus 2 was similar to the molecular weight reported
et al., 2005). Thus, the present enzyme diVered from the for other proteases from B. cereus while molecular weight
previous reports on B. cereus. of Cereus 1 was diVerent. The enzyme showed two optima
The crude enzyme preparation showed Wve protein for both pH and temperatures and two enzyme bands on
bands. After dialysis the ammonium sulphate precipitated PAGE-zymogram, suggesting the extracellular secretion of
enzyme showed two bands, viz., Cereus 1 and Cereus 2 on two proteases from B. cereus MCM B-326.
zymogram. The non-denaturing PAGE showed two pro-
teases with approximate molecular weights 45 kDa and 3.5. Dehairing of buValo hide
36 kDa of band Cereus 1 and Cereus 2 respectively (Fig. 4).
It has been reported that B. cereus has two proteases with The enzyme dehaired the buValo hide within 21 h at pH
molecular masses of approximately 38 kDa and 36 kDa 7.0 and ambient temperature 28 § 2°C, with 1% enzyme
1244 S.S. Nilegaonkar et al. / Bioresource Technology 98 (2007) 1238–1245
used for a 2.5 cm £ 2.5 cm piece of buValo hide (results not
shown). The enzyme used in dehairing process should not
be collagenolytic in nature, so that hide matrix remains
intact. Earlier studies with the dehairing protease from
B. subtilis S14 have reported elastase, keratinase and colla-
genase activities (Alexandre et al., 2005) while the present
enzyme had non collagenolytic and non keratinolytic prop-
erties. In dehairing process, pH tolerance for the enzyme
is an important factor. In earlier report, an alkaline prote-
ase from Aspergillus tamarri, dehaired the goat skin at pH
9–11, temperature 30–37 °C with 1% enzyme concentration
and incubation period of 18–24 h (Dayanandan et al.,
2003). Similarly, an enzyme isolated from Bacillus sp. was
used for dehairing of goat skin with 2–3% concentration
and was active in pH 7.5 and 9.0 at 37 °C (Annapurna et al.,
1996). In general, the dehairing process required activity of
enzyme under alkaline condition; this criterion was satisWed
by protease from B. cereus MCM-B326 and thus suitable
for dehairing.
4. Conclusion
The production of extracellular protease from B. cereus
MCM B-326 was optimum in starch- soybean meal-CaCO3
medium of pH 9.0 at 30 °C under shake culture condition
with 2.8 £ 108 cells ml¡1 as an initial inoculum density and
incubation of 36 h. The protease exhibited important prop-
erties such as activity under alkaline pH, at broad tempera-
ture range, in presence of oxidizing agent, and dehairing
activity for animal hide without chemical assistance and
without hydrolyzing Wbrous proteins. Due to these proper-
ties, the enzyme could be potentially useful in leather indus-
try for dehairing of animal hide without damaging the
collagen layer, resulting in a better quality product and
avoiding the pollution problem associated with the use of
chemicals.
Acknowledgements
The work was carried out under NMITLI project on,
“Biotechnology for leather towards cleaner processing.”
sponsored by CSIR, Govt. of India. The authors also thank
Mrs. Smita Kale, consultant, State Family Planning
Bureau, Pune, for her help in the statistical analysis.
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