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Application of an alkaline protease in biological waste processing: an eco-

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Shanmugavel. M*1, S. Vasantharaj1, S. Saathiyavimal2 and A. Gnanamani1

1
Central Leather Research Institute, Adyar, Chennai, Tamilnadu, India.
2
Department of Biotechnology, Kongunadu Arts and Science College, Coimbatore, Tamilnadu, India
*Author to whom corresponding should be addressed
Received: Dec 2015; Accepted Jan 2016
Email: shanmugavel_m_2001@yahoo.com

Abstract: Aspergillus tamarii MTCC5152 produces an alkaline protease produced by SSF. Proteases
have a wide variety of industrial applications. The complete blood stain removal was observed in cotton
cloths in 10 min incubation at room temperature. This protease has the potential to break down gelatin
layer of used X-ray films, chitin from shrimp shell and blood stain from cotton cloth. Complete
breakdown of gelatin was observed after 2h at pH 8.5 in 55°C temperature. Shrimp shell waste are a rich
source of protein such as chitin. These waste were treated with protease enzyme to extract chitin. The
obtained chitin was characterized by Fourier transform infrared spectrometer analysis.
Keywords: Aspergillus tamarii MTCC5152, SSF, Proteases, FTIR
1. INTRODUCTION increases the consumption of surface-active
substances, thereby improving the ecological
Protease constitutes one of the most
situation. Recovery of silver by burning the
important groups of industrial enzymes,
films creates environmental pollution and
accounting for more than 65% of total
health hazards. On the other hand, enzyme
industrial enzyme market and it has diverse
from microbial source breaks the gelatin
applications in a variety of industries, such
layer embedded with silver in films creating
as detergent, food, pharmaceutical, leather,
pollution free stripping. Enzymatic method
silk, silver recovery and production of
although slow is free from pollution and
protein hydrolysates (Banik and Prakash,
cost-effective too. Decomposition of x-ray
2004). Proteases are also envisaged as
films by partially purified extracellular
having extensive applications in
alkaline protease produced by Streptomyces
development of ecofriendly technologies as
avermectinus NRRLB-8165 was also
well as in several bioremediation processes
reported (Chwojnowski and Lada, 1985).
(Bhaskar et al., 2007; Wang et al., 2008;
The stripping methods using proteolytic
Ahmed et al., 2008). Proteases are capable of
enzymes are obtained from various
cleaving proteins into peptides and amino
microorganisms (Horikoshi, 1999; Liu,
acids, they are characterized by their optimal
1989; Masui et al., 1999; Messerschmidt,
pH (acid, neutral or alkaline), their
1988; Mueller et al., 1990), inorganic and
temperature, their ability to hydrolyze
organic chemicals (Kibata and Nakamura,
specific proteins has received great attention
1999; Rettig and Buser, 1988; Wang et al.,
as a viable alternate to chemical approach
1997; Muzzarelli et al., 2012) and
(Chwojnowski and Lada, 1985; Fujiwara et
mechanical methods have also been used
al., 1991).
more often for recovery of the silver than
Addition of alkaline protease to burning and oxidation methods (Franco ad
detergents considerably increases (35-40%) Peter, 2011). Like that, Chitin, one of the
the cleaning effect (particularly in removing most abundant renewable biopolymers have
stains containing proteins, e.g., blood) and been applied to prepare from shrimp shell

www.ijbsans.com 19 J. Biosci. Nanosci., 3(2):2016

powder by chemical (Nakagawa et al., 2011; 2.2 SSF and growth conditions:
Ghorbel et al., 2011) enzymatic (Jung et al., Erlenmeyer flasks (250 mL) containing 10 g
2007; Gildberg and Stenberg, 2011) and of solid substrates were moistened with
microbiological methods (Banerjee et al., water (70% v/w) and sterilized at 121°C for
1999; Frost and Moss, 1987) sectors of food, 15 min. After cooling, the flasks were
agriculture, medicine and materials (Abdou inoculated with 2% v/w inoculum containing
et al., 2008; Chandumpai et al., 2004; 106 spores/mL. The contents of each flask
Valdez et al., 2010). The enzymatic method were mixed thoroughly with a sterile glass
includes the use of trypsase, papain, and rod for uniform distribution of fungal spores
pepsase (Manjusha, 2011). After enzymatic in the medium and incubated at 26-280C for a
treatment of shrimp shell the experimental period of 72-96 h at 90-95% humidity. After
liquor is found to contain high amounts of the incubation period, the contents from each
protein and amino acids, indicating a high flask were extracted and the enzyme prepared
nutritional value which can be used for food, from the fungus showed 140 µ/g protease
feed or as a nitrogen source in growth media activity. The crude enzyme was loaded in to the
for microorganisms (Nakiboglu et al., 2001). ultra-concentrator and the nitrogen gas was
In fact, the potentiality of chitin as a applied to give the pressure. The tenfold
storehouse of proteins and amino acids has concentrated enzyme was collected.
prompted the present researchers to 2.3 Blood stain removal: Application of
undertake the study. protease as a detergent additive in blood
stain removal was studied on white cotton
Use of these chemicals can seriously cloth pieces (05 Nos) (5cm × 5 cm) stained
pollute the ecological environment, produce with human blood and oven dried at 95-
abundant waste, and damage human health. 100°C for 5 min. The stained cloth pieces
Moreover, the addition of acid and alkali can were taken in separate trays. The following
hydrolyze the polymer, resulting in sets were prepared and studied:
inconsistent physiological properties of the
final product. Along with increased demands 1. Tray with distilled water (100 ml) + blood
on environment friendly society and rapid stained cloth (control).
development of fermentation technology, 2. Tray with distilled water (100 ml) + blood
more eco-friendly processes using enzymatic stained cloth + 2 ml of crude enzyme extract.
and microbiological methods for producing
chitin have attracted great interests. In this 3. Tray with distilled water (100 ml) + blood
study an attempt was made to (i) apply stained cloth + 1 ml of commercial detergent
purified protease as a cleansing additive in (Surf excel- 5 mg/ml) + 1ml of crude
blood stain removal, (ii) the hydrolysis of enzyme extract.
gelatin layers from used X-ray films (iii) 4. Tray with distilled water (100 ml) + blood
chitin extraction from shrimp shell waste stained cloth + 2 ml of commercial detergent
were investigated using proteases produced (Surf excel-5 mg/ml).
by Aspergillus tamarii.
5. Tray with distilled water (100 ml) + blood
2. MATERIALS AND METHODS stained cloth + 1 ml of commercial detergent
2.1 Isolation and identification of fungi: (Surf excel- 5 mg/ml) + 2 ml of crude
Fungal cultures isolated from tannery soil enzyme extract.
were screened for protease production on 1% The trays were incubated at 55◦C for 10 min.
(w/v) skimmed milk agar plate. Colonies The cloth pieces were taken out from each
causing a clear zone of proteolytic activity set at regular intervals of 5 min, rinsed with
were selected and identified as Aspergillus water, dried and visually examined. The
tamarii and deposited at the Institute of distilled water treated blood stained cloth
Microbial Technology, Chandigarh, India. pieces were considered as control.
The fungus was cultivated in Czapek-Dox
2.4 Gelatin layer removal from the waste
agar slants at 30°C for 7 days.
x-ray film: The used X-ray films were

www.ijbsans.com 20 J. Biosci. Nanosci., 3(2):2016

washed with the distilled water and wiped (1 %). Formation of clear zone on plates by
with cotton impregnated with ethanol. The the growth of Aspergillus tamarii.
washed film were oven dried at 40˚C for 30 3.2 Application of purified protease on
min and adjusted at different concentration blood stain removal: The protease purified
of crude enzyme extract (10ml and 15ml) from the present study was investigated for
and different pH (6.5 to 10.5). Optimized its application as cleansing additive in blood
enzyme concentration and pH for the stain removal and the results revealed that
gelatin layer removal was standardized. The blood stains were completely removed with
pH adjusted with 2N NaOH or 2N HCl. combination of detergent (7 mg/ml) and
2.5 Chitin extraction from shrimp shell crude enzyme extract within 10 min (c) and
waste: Shrimp Shell wastes were mixed with no stains removal when rinsed with distilled
water at a ratio of 1:2 (w/v), minced then air water (b), enzyme (e) and detergent (f) (Fig.1
dried, grounded and stored in the refrigerator. a, b, c, d). Banerjee et al. (1999) reported that
These materials were grinded and the thermostable alkaline protease from Bacillus
experiments were carried out on 5g of powder brevis removed blood stains with
with 4ml and 6ml crude enzyme extract. The combination of 7 mg/ml detergent + enzyme
pH of the mixture was adjusted to 8.5. After (2 ml) for 25 min. Thus, the results clearly
incubation for 2 h at 55 °C, the reaction was indicated that addition of enzyme with
stopped by heating the solution at 90°C for 20 commercial detergent significantly enhanced
min to inactivate the enzyme. The shrimp blood stains removal from cotton cloths and
shell waste protein hydrolysates were then these properties indicated its possible use in
centrifuged at 5,000×g for 20 min to separate the manufacture of cleaning detergent
insoluble and soluble fractions. The solid industry.
phase was washed and then dried for 1 h at 60
°C.
2.6 Demineralization: Demineralization was
carried out in a dilute HCl solution. Solid
fractions obtained after hydrolysis by crude
protease were treated with 2N HCl in 1:10
(w/v) ratio for 6 h at room temperature
(37 °C) under constant stirring. The chitin
product was filtered through four layers of
gauze with the aid of a vacuum pump and
washed to neutrality with deionized water and
then freeze-dried. The residual minerals were
estimated by FT-IR analysis.
Fig .1. Blood stain removal activity of protease
2.7 Fourier Transform Infrared attenuated from Aspergillus tamarii a) control, b) blood-
total reflectance (FT-IR ATR): A Bruker stained cloth washed with detergent only c) blood-
Tensor 27 FT-IR spectrophotometer (CLRI, stained cloth washed with crude enzyme extract
and d) blood-stained cloth washed with crude
Chennai) equipped with an the FT-IR ATR enzyme extract and detergent.
spectra (64 scans, 4 cm-1 resolution) were
recorded using a single reflection horizontal 3.3 Gelatin- Silver layer removal from
ATR accessory with a spherical Ge crystal X-ray sheets: Four major types of proteases
fixed at an incident angle of 45o. A sample are distinguished based on their pH
with a 2-mm diameter was measured. requirement for optimum activity i.e.,
alkaline (serine) proteases, thiol proteases,
3. RESULTS AND DISCUSSION acid (carboxyl) proteases and neutral
3.1 Plate assay: The ability of fungi (metallo) proteases. Alkaline proteases have
producing protease enzymes was confirmed a serine residue at the active site and they
by plate assay. The fungi was grown on exhibit activity in the neutral-alkali region
medium containing skimmed milk agar plate range of pH with optima between at values

www.ijbsans.com 21 J. Biosci. Nanosci., 3(2):2016

pH 8-11 (Chung et al., 2004). Nakiboglu et to that of the commercial chitin obtained
al. (2001) too selected 50°C as the stripping (Chung et al., 2004).
100.0
temperature for the enzyme from Bacillus 90
3836.59
3740.27

subtilis ATCC 6633. Maximum gelatin 80

2137.26

2358.15
hydrolysis was observed in the initial 15 min 70

in above study. The protease from Vibrio sp. 60

1259.70 898.70

(V26) took 25 min for the complete removal

%T
50

698.22
of gelatin whereas; the protease from a 40

2926.89
603.82

30
fungal isolate Conidiobolus coronatus 3109.00
2881.51
1316.81
1380.17 1155.61
20

ATCC PTA-4132 took only 6 min. Fungal 10

3439.52 1558.39
1639.84
1072.12
1020.11

strains are the major source for alkaline, acid 1.0

4000.0 3000 2000 1500 1000 400.0
and neutral proteases. Alkaline protease cm-1

produced by Aspergillus tamarii was Fig.5: FT-IR spectrum of Chitin standard

100.0

hydrolysed the gelatin layer completely in 90

15min at 55°C at the given experimental 80

2138.68

conditions i.e., 10 ml of crude enzyme 70

2354.44
897.04
1381.09
extract at the pH8.5. (Fig.2- 4). 60
1256.80

%T
50
696.19
40 1152.66
583.33
30

1315.78
20 2927.07 1427.21
3099.52 1553.32 1072.09
10 1015.38
3441.15 1643.22
0.0 3260.66
4000.0 3000 2000 1500 1000 400.0
cm-1

Fig. 6: FT-IR spectrum of Chitin (6ml protease

Fig.2: X-rays only with Buffer (a) Borate buffer, used )
pH 9.5 and (b) Borate Buffer, pH 8.5 100.0

90

80

70
2341.23
2146.17
60
1257.38
1422.48
%T

50
1200.00
3106.26
40 1555.63
899.05
30
699.23
597.19
20
1315.76
10 2927.21 1379.11 1155.44
3442.34 1073.04
1649.92
0.0 3260.66
4000.0 3000 2000 1500 1000 400.0
cm-1

Fig. 7: FT-IR spectrum of Chitin (4ml protease

Fig. 3: Used X-ray films treated with enzyme at
used )
various pH. 100.0

90

80

70
898.32
60 2142.18 1257.37
2359.88
%T

50

40 697.03
Fig. 4: Effect of different enzyme concentration on 30 1552.78 600.62

gelatin layer removal 20 2930.42 1316.62

1380.26 1073.13
3.4 FT-IR Spectrum for chitin extraction: 10 3273.943109.00
1156.07 1025.38

1644.90
The FT-IR spectrum (Fig. 5 - 8) of chitin 0.0
4000.0 3000 2000 1500 1000 400.0
extracted was compared with that of chitin cm-1

standard and it is found that peaks obtained Fig. 8: FT-IR spectrum of Chitin (only
in the experimental chitin is in similar range demineralised)

www.ijbsans.com 22 J. Biosci. Nanosci., 3(2):2016

4. CONCLUSIONS Liu X (1989) Method of recovering silver from
waste emulsion and film. Chinese Patent, CN
Considering the production cost and
1037547 (IPC C22B-011/04).
reutilization of this fungal protease will be Masui A, Fujiwara N, Takagi M and Imanaka T
inexpensive bio resources not only solves (1999) Feasibility study for decomposition of
environmental problems but also promotes gelatin layer on X-ray film by thermostable
the economic values of the marine wastes. alkaline protease from alkaliphilic Bacillus
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stain removal, gelatin-silver layer removal silver from waste photographic film and
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W, Kochanowskii H, Herbert SD, Mueller G,
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mechanisms of enzyme action, amino acid G03C-011/24).
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