Biotechnological Approaches for Sustainable Development (2004)

M Sudhakara Reddy and Sunil Khanna (editors)

Allied Publishers Pvt. Ltd., New Delhi
ISBN: 81-7764-669-9
Pages: 186- 191

EXTRACELLULAR PROTEASE FROM BACILLUS

SP BSA-26: APPLICATION IN DEHAIRING
OF BUFFALO HIDE

S. S. NILEGAONKAR, V. P. ZAMBARE AND P. P. KANEKAR

Microbial Sciences Division,

Agharkar Research Institute,
G. G. Agarkar Road,
Pune 411 004.

ABSTRACT

A strain of Bacillus sp., BSA-26, isolated from buffalo hide was studied for
production of extracellular protease using wheat bran, soybean meal, starch (1%
each) and 0.3% CaCO3 medium, within 36h at pH 9.0 and 300C under shake
culture condition. Ammonium sulphate precipitated (60% saturation) enzyme
showed 100% dehairing of buffalo hide pieces soaked in tap water containing
0.3% calcium carbonate, within 21h. Visual and histopathalogical observations of
dehaired hide showed that the collagen fibers were well opened without damage.
The microscopic observations also revealed that hair was uprooted with leaving
empty follicle cavities in the hide. The experimental results indicated that protease
of Bacillus sp. BSA-26 bears potential application in leather industry.

INTRODUCTION

Leather manufacturing is a chemical processing of biological matrix i.e. animal skin/ hide.
The conventional process of dehairing uses lime and sodium sulphide (Money et al. 1996).
The 50% of chemical oxygen demand (COD) is due to the excess use of sulphide in
dehairing operation which threats environmental as well as health problems (Thanikaivelan
et al. 2004). A paradigm shift in leather sector would call for a bioprocessing of skin/ hides
at beam house stages. It is
 Extracellular Protease from Bacillus sp. BSA-26: Application in Dehairing of Buffalo Hide 187

possible to reduce quantity of sulphide using enzyme-assisted process (Alexander et al.

1988). Generally proteolytic enzymes are used in dehairing process. Their origin can be
animal (Green, 1952), bacterial (Paul et al. 2001; Raju et al. 1996), fungal (Tayler et al.
1987, Malathi et al. 1991, Gehring et al 2002) and plant (Adewoye et al. 1984). The
commercial bioproducts available for dehairing are Pelvit SPH, Truponat HL, Mystozyme
ECO-S, Basozyme L 10, Microdep C, Forezym LM, Biodart, Erhavit MC, Novolime etc.
(Thanikaivelan et al. 2004). Although studies on the use of enzymes for various stages of
leather processing are numerous, the commercial production and application of enzymes in
the leather industry is limited due to the high cost of production. This paper presents
microbial production of extracellular enzyme protease and its application in dehairing of
buffalo hide.

MATERIALS AND METHODS

Organism

The organism used for the protease production was a strain of Bacillus sp. BSA-26, isolated
from buffalo hide, collected from Municipal Corporation Slaughterhouse, Kondawa, Pune.
Bacillus sp. BSA-26 was screened for protease production on milk agar plate. The strain
was routinely maintained on Nutrient agar slants (pH 7) and in glycerol stocks.

Production of Enzyme

The enzyme was produced by submerged culture condition using different cheap nitrogen
sources like deolied soya cake, deoiled saffola cake, deolied rap seed cake, deoiled ground
nut cake, defatted soybean (MACS variety), soya flour (market), wheat bran, bengal gram
flour, bengal gram flour-wheat bran, bengal gram flour- ground nut cake, bengal gram flour-
soybean meal, wheat bran- ground nut cake, wheat bran- soybean meal and starch- soybean
meal (SS) medium. The media were inoculated with 24 h old inoculum (1%) and incubated
at pH 9.0 and 300C for 36 h under shake culture condition. The broth was centrifuged at
13,000 x g for 10 min to get cell free supernatant (CFS). The enzyme activity was
determined by protease assay. The CFS was then partially purified by 60% ammonium
sulphate saturation.

Protease Assay

Protease activity was measured by using caseinolytic assay method (Manachini et al. 1988).
The culture supernatant (1 ml) was incubated with 4 ml of 0.625% casein solution at 370C
for 30 min. The reaction was stopped by the addition of 5ml of trichloroacetic acid (5%) and
the casein hydrolysis was measured by modified Folin-Ciocalteu method, against inactive
enzyme. A standard graph was generated using standard tyrosine solution of 5-50 µg ml-1
(Lowery et al. 1951). One unit of protease activity was defined as the amount of enzyme,
which liberated one µg tyrosine per minute at 370C.
188 Biotechnological Approaches for Sustainable Development

Dehairing of Buffalo Hide

The salted buffalo hide pieces 2.5cm x 2.5cm or 3 g were soaked overnight in different
soaking buffer and solutions (400% v/w of salted hide) with and without calcium. The
soaking solutions were sodium carbonate buffer, sodium tetrahydroborate, hydrazine
hydrate, Sodium dodesyl sulphate (SDS) and tap water. The partially purified enzyme was
applied to the flesh side of the soaked hide pieces. For comparison, chemical dehairing was
also carried out with 10% lime and 2% sodium sulphide.

Histopathology of Hide

Histopathology of control, chemically and enzymatically dehaired hide was studied. The
hide was dehydrated with 80, 95 and 100% (v/v) of alcohol gradients followed by xylene
and then embedded in paraffin. The longitudinal sections of hide embedded in paraffin wax
were obtained using microtome. The sections were fixed on slides using starch paste
containing thymol, which acts as preservative. The sections were stained using Harris's
haematoxylin stain followed by 0.5% (v/v) HCl and diluted ammonia (John et al. 2002).
The slides were observed microscopically for hair follicle, collagen layer, epidermis etc.

RESULTS AND DISCUSSION

Protease Production

Bacillus sp. BSA-26 exhibited zone of clearance of 30 mm on milk agar plate. The culture
produced the enzyme protease in production media containing 1% starch, 1% nitrogen
source and 0.3% CaCO3. The effect of different cheap nitrogen sources on production of
protease are detailed in Table.1. The wheat bran- soybean meal- starch (1% each) and 0.3%
CaCO3 showed maximum protease production at 300C under shake culture condition at 36 h
followed by wheat bran- groundnut cake- starch.

Dehairing of Buffalo Hide

The enzyme obtained from Bacillus sp. BSA-26, was used for dehairing of buffalo hide.
From Table 2 it is seen that, dehairing was observed at the end of 21h. The dehairing was
irrespective of the soaking agent such as tap water or hydrazine hydrate or SDS. During
soaking 100% dehairing was noted in presence of calcium while 80% dehairing in absence
of calcium. On the other hand Gehring et al. (2002) reported protease from Streptomyces
griseus that dehaired the cattle hide after soaking the hide in carbonate buffers.

The enzymatically dehaired hide or pelt surface appearance was smooth, silky and white.
Histopathalogical sections showed that the epidermis was totally removed. It was also
observed that intact hair was removed with hair root leaving empty cavities in the hide. The
collagen layer of hide was not damaged but it was well opened which is necessary for
penetration of
 Extracellular Protease from Bacillus sp. BSA-26: Application in Dehairing of Buffalo Hide 189

tanning material in leather manufacturing. The enzyme may have ability to hydrolyze the
cementing substance present around root hair so as to remove the intact hair with hair
follicle, similarly noted by Thanikaivelan et al. (2004).

Table 1.
Effect of different nitrogen sources on protease production from BSA-26.
Sr. No. Nitrogen sources Enzyme activity (U/ml/min)
1 Deoiled soya cake-starch 95.85
2 Deoiled saffola cake-starch 72.09
3 Deoiled rap seed cake-starch 71.28
4 Deoiled ground nut cake-starch 119.54
5 Defatted MACS- soybean meal-starch 59.96
6 Wheat bran-starch 75.54
7 Soya flour (market) –starch 81.32
8 Bengal gram flour-starch 38.21
9 Bengal gram flour+ wheat bran-starch 160.80
10 Bengal gram flour+ ground nut cake-starch 118.41
11 Bengal gram flour+ soybean meal-starch 90.23
12 Wheat bran + ground nut cake-starch 142.89
13 Wheat bran+ soybean meal-starch 176.05
14 Soybean meal-starch 140.73

Table.2.
Effect of soaking condition on dehairing of buffalo hide

Sr. soaking buffer/ Concent Calcium carbonate Percent dehairing of

No. chemical solution
1 Sodium carbonate
buffer
2 Sodium
tetrahydroborate
3 Hydrazine hydrate
4 SDS
5 Tap water
pH
- -ration ( 0.3% w/w of hide) buffalo hide at 21 h
0.1 M 9.5 + 0
- 0
0.1 % 9.3 + 0
- 0
0.25 % 9.4 + 100
- 80
0.1 % 7.2 + 100
- 80
- 7.0 + 100
- 80
190 Biotechnological Approaches for Sustainable Development

The Bacillus sp. BSA-26 has the maximum protease production in medium containing wheat
bran- soybean meal- starch (1% each), and 0.3% CaCO3, pH 9.0 within 36 h under shake
culture condition. The protease has dehairing ability of buffalo hide in presence of calcium
ions. The use of this enzyme in dehairing process will reduce the cost of process and pollution
load of the tannery effluent indicating its importance in the pretanning operations of leather
industry.

ACKNOWLEDGMENT

We greatly thank CSIR- NMITLI (Govt. of India) for providing financial support.

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