Journal of the Taiwan Institute of Chemical Engineers 42 (2011) 298304

Contents lists available at ScienceDirect

Journal of the Taiwan Institute of Chemical Engineers

journal homepage: www.elsevier.com/locate/jtice

Fungal protease: Production, purication and compatibility with laundry

detergents and their wash performance
S. Savitha a,*, S. Sadhasivam a, K. Swaminathan b, Feng Huei Lin a
a
Institute of Biomedical Engineering, College of Engineering, National Taiwan University, Taipei, Taiwan
b
Microbial Biotechnology Division, Department of Biotechnology, Bharathiar University, Coimbatore-641 046, Tamil Nadu, India

A R T I C L E I N F O A B S T R A C T

Article history: An extracellular serine protease producing fungi were isolated from the efuent sample collected from a
Received 15 January 2010 sago industry in Salem and was identied as Graphium putredinis and Trichoderma harzianum.
Received in revised form 19 May 2010 Intergenerically developed fusant produced high amounts of protease in soya bean meal amended
Accepted 23 May 2010
minimal medium than the parents, G. putredinis and T. harzianum. The enzyme was puried by Sephadex
G 100 column chromatography. The proteases of G. putredinis and T. harzianum had optimum pH and
Keywords: temperature of 7.08.0 and 5060 8C respectively. At 37 and 60 8C, the parental proteases were
Casein
respectively stable for 1 day and 15 min and the fusant was stable for 2 days and 10 min. The Km and Vmax
Detergents
were 0.65, 1.25 and 0.40 mg/ml and 2.00, 1.60 and 2.60 IU/mg protein for G. putredinis, T. harzianum and
Fusant
G. putredinis fusant respectively with a molecular weight of 31, 20 and 33 kDa. The effect of metal ions showed that, at
Metal ions 5 mM concentration of Hg2+, the residual protease activity of parent and fusant was 5.47, 2.48 and 9.76%
Rin Advanced respectively; Cu2+, Ca2+ and Zn2+ did not greatly affect the enzyme activity. In 5 mM EDTA showed
Soya bean residual activity between 60.76 and 85.66%. PMSF (2 mM) completely inhibited the protease activity of
all the three fungi studied. All the three fungal enzymes retained maximum residual activity of 66.00,
63.80 and 76.74% with the commercial detergent Rin Advanced. With Kite, all the enzymes had more or
less same level of residual activity (5455%). With SDS and sodium perborate (0.2%) the residual
activities were 58.2573.82 and 61.5870.24% respectively. Wash performance analysis revealed that
fusant protease with Rin Advanced at 60 8C yielded good result.
2010 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

1. Introduction industrial demand of proteolytic enzymes, with appropriate

Proteases are amongst the most studied proteins. Proteases
catalyze the cleavage of peptide bonds in other proteins, are the class
of enzymes having tremendous applications in both physiological
and commercial elds. Microbial proteases represent one of the three
largest groups of industrial enzymes and account for approximately
60% of the total enzyme sale in the world and they are the leaders of
the industrial enzyme market worldwide (Rai and Mukherjee, 2010).
Among the world sale of industrial enzymes of about US $ 300600
million per annum, 75% of these are hydrolytic enzymes, of which
two-thirds are proteolytic enzymes. Protease constitute one of the
most important groups of industrial enzymes and have applications
in different industries viz. detergent, food, pharmaceutical, leather,
silk and for recovery of silver from used X-ray lms (Inhs et al., 1999;
Maheshwari et al., 2000; Outtrup et al., 1995).
Increasing demand of proteases with specic properties has lead
biotechnologists to explore newer sources of proteases. The
* Corresponding author. Tel.: +886 987653459; fax: +886 2312 3456.
E-mail address: savisbio@yahoo.com (S. Savitha).
specicity and stability to pH, temperature, metal ions, compatibili-
ty with detergent compounds like surfactants and organic solvents
continues to stimulate the search for new enzyme sources. Proteases
with high activity and stability in high alkaline range and high
temperatures are interesting for bioengineering and biotechnologi-
cal applications. In general, microbial proteases are extracellular in
nature and are directly secreted into the fermentation broth by the
producer, thus simplifying downstream processing of the enzyme as
compared to proteases obtained from plants and animals (Lageiro
et al., 2007). Alkaline proteases used industrially are produced by a
wide range of microorganisms including bacteria, moulds and
yeasts. Nowadays, the use of enzyme (protease)-based detergents is
preferred over the conventional synthetic ones in view of their
cleaning properties, better performance at lower washing tempera-
ture and the alleviation of pollution (Kirk et al., 2002).
In the present study, we investigated the efciency of proteases
recovered from Graphium putredinis, Trichoderma harzianum and
intergenerically fused fusant strain isolated from sago industry
efuent. The main objective of the present study was to make use
of fungal protease and to reduce the harsh chemicals employed in
laundry detergents, thus eliminating the toxicity in wash efuent.

1876-1070/$ see front matter 2010 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.jtice.2010.05.012
 S. Savitha et al. / Journal of the Taiwan Institute of Chemical Engineers 42 (2011) 298304
Further, characterization of these enzymes and the effect of various
metal ions and inhibitors on the stability at higher temperatures
and in alkaline pH were carried out. The biochemical properties of
the produced proteases and its suitability for application in
laundry detergents were investigated. The compatibility of
detergents in wash performance was also determined and found
that it could be employed in commercial applications. To the best
of our knowledge, the fungus, G. putredinis was utilized for the rst
time in the literature for the production of alkaline protease
enzyme and its application in detergent industry.
2. Materials and methods
2.1. Growth and protease enzyme production
The fungi, G. putredinis and T. harzianum was isolated from the
efuent samples collected near the sago industries of Salem District,
Tamil Nadu, India and placed in microbial culture collection,
Microbial Biotechnology laboratory, Bharathiar University, India.
For growth and protease enzyme production, the fungi, G. putredinis,
T. harzianum and their intergenerically developed fusant (Savitha
et al., 2010) were grown in minimal medium amended with soya
bean meal (0.5%, w/v) (Sen and Satyanarayana, 1993) for 4 days on a
rotary shaker (125 rpm) at 27  2 8C. After 4 days, the fungal biomass
was ltered and the ltrate was centrifuged at 10,000 rpm for 20 min at
4 8C. The cleared culture ltrate was used as crude enzyme. Growth was
determined in terms of mycelial dry weight and chitin (Chen and
Johnson, 1983) and protein (Phillips and Gordon, 1989) contents of the
mycelial biomass.
2.2. Enzyme assay
The enzyme activity was estimated with casein as substrate
(Hagihara et al., 1958). The culture ltrate (1.0 ml) obtained from
soya bean meal amended medium was mixed with 5 ml of 1% (w/v)
casein in 0.05 M, pH 7.0 phosphate buffer and incubated for 10 min
at 45 8C in a water bath. After 10 min, the reaction was terminated
by the addition of 5.0 ml of 10% trichloroacetic acid (TCA) and
incubated at 30 8C for 30 min. Then the reaction mixture was
centrifuged at 10,000 rpm for 20 min at 30 8C. Absorbance of clear
supernatant was read at 275 nm using UVvis spectrophotometer
(Shimadzu-1601). The enzyme activity was expressed as mg of
tyrosine released per min under standard assay conditions.
2.3. Purication
The protease enzyme was puried from the culture ltrate by
ethanol precipitation and Sephadex G-100 column chromatography.
Three volumes of chilled ethanol was added to the culture
ltrate and left overnight at 4 8C for precipitation. The precipitate
was collected by centrifugation at 10,000 rpm for 30 min at 4 8C.
The supernatant was decanted and the pellet was extracted with
50 ml of sodium phosphate buffer (50 mM; pH 7.0). The buffer
extract was centrifuged at 10,000 rpm for 20 min at 4 8C to clear
the undissolved solid particles and lyophilized. The lyophilized
enzyme sample was applied to Sephadex G-100 column equili-
brated with 50 mM sodium phosphate buffer, pH 7.0 and eluted
with the same buffer containing 0.5 M NaCl at an elution rate of
10 ml/h. The protease activity and protein content of the culture
ltrate, buffer extract and the fractions obtained in the column
chromatography were estimated.
2.4. Characterization
Properties of the protease enzymes viz. optimum pH, tempera-
ture, thermostability at 37 and 60 8C and optimum substrate
299
concentration (Vmax and Km) were determined against casein (in
50 mM sodium phosphate buffer, pH 7.0) substrate. The molecular
weight was determined by SDS-PAGE (Laemmli, 1970). All assays
were carried out in replicates of ve.
2.5. Proteases as biodetergents
2.5.1. Effect of metal ions and inhibitors on protease activity
To determine the effect of metal ions and inhibitors on protease
activity, the enzyme reactions were performed by incubating the
reaction mixture containing 1.0 ml of enzyme, 4.0 ml of sodium
phosphate buffer (50 mM; pH 7.0), 1% casein substrate, 1.0 ml of
5 mM metal ion solutions (Hg2+, Cu2+, Ca2+, Zn2+)/inhibitors (EDTA
(ethylene diamine tetraacetic acid) 5 mM; PMSF (phenylmethyl-
sulfonyl uoride) 2 mM) at 50 8C for 30 min. After incubation, the
residual enzyme activity was assayed. The reaction mixture with
heat denatured enzyme served as control.
2.5.2. Compatibility of proteases with laundry detergents
The compatibility of proteases with commercial detergents, Rin
Advanced (Hindustan Lever Limited, Mumbai), Surf Excel (Hindu-
stan Lever Limited, Mumbai), Henko (Henkel Spic India Limited,
Germany), Kite (Dynavista Industries Pvt. Ltd, Pondicherry) and
Tide (Procter and Gamble Company, USA) (7 mg/ml, w/v), chemical
detergent SDS (0.2%, w/v) and the bleaching agent sodium
perborate (0.2%, w/v) were assessed. The reaction mixture
containing 1 ml of enzyme in 4 ml detergent/bleaching solution
was incubated at 50 8C for 1 h and the residual protease activity
was determined using casein as substrate. The enzyme activity
without any detergent was taken as 100% and heat inactivated
enzyme served as control.
2.5.3. Wash performance analysis
A white cotton cloth of the size 5  5 cm was stained with 25 mL
of human blood and subjected to the following washing processes.
1. Washing with distilled water at 37 8C for 30 min
2. Washing with distilled water at 55 8C for 30 min
3. Washing with 1% (w/v) commercial detergent solutions at 60 8C
4. Washing with crude enzyme (10%, v/v) at 60 8C
5. Washing with mixture of crude enzyme (10%, v/v) and 1% (w/v)
commercial detergent solution
After washing, the cloth was rinsed well with water to remove
excess of detergent/enzyme, dried and visualized.
3. Results and discussion
In the present work proteases from G. putredinis, T. harzianum
and fusant were isolated, characterized and their efciency in
enhancing the wash performance of commercial detergents was
analysed.
3.1. Growth and protease production
The present study revealed that, fusant grew faster than the
parental strains when grown in soybean amended medium.
Similar results were observed in protease production also (Table
1). The fusant produced 0.45 U/ml of protease; whereas the
parents, G. putredinis and T. harzianum could produce only 0.38 and
0.32 U/ml respectively. The fusant and T. harzianum produced
0.25 mg/ml of soluble proteins and G. putredinis produced 0.20 mg/
ml (Table 1). Hence, the results revealed that, fungus isolated from
starch industry efuent could produce only minimum amount of
proteases due to the presence of toxic compounds like cyanide and
cyanoglucosides in the efuent.
300 S. Savitha et al. / Journal of the Taiwan Institute of Chemical Engineers 42 (2011) 298304

Table 1
Growth and protease production by G. putredinis, T. harzianum and fusant in soyabean amended medium.

Fungi Growth (mg) Enzyme production (IU/ml) Soluble proteins (mg/ml)

Mycelial dry weight Chitin content Mycelial protein

G. putredinis 91.30  5.14 10.09  1.28 12.60  0.88 0.38  0.19 0.20  0.02
T. harzianum 83.92  3.08 7.41  0.57 10.80  0.34 0.32  0.22 0.25  0.07
Fusant 102.50  1.99 13.73  0.94 14.36  0.61 0.45  0.05 0.25  0.03

Values are mean of ve replicates; standard deviation.

T. harzianum T39 and NCIM1185 produced 0.9 and 0.6 mU/ml of
protease respectively when cultivated on the surface of bean leaves
(Elad and Kapat, 1999) similar to our present studied protease from
casein. Although Bacillus was a known producer of protease, Do
Nascimento and Martins (2004) reported a low level of protease
production (1.93 U/mg protein) by thermophilic Bacillus sp. which
was nearer to our present study. Naidu and Lakshmi Devi (2005)
observed that Bacillus sp. K 30 produced 81.1 U/ml of protease on
wheat bran, 62.0 U/ml on starch, 42.1 U/ml on rice bran and 40.0 U/
ml on casein when they were used as carbon sources; the best
nitrogen sources were beef extract (75.0 U/ml), yeast extract
(65.0 U/ml) and tryptone (62.0 U/ml) and reported a lesser enzyme
production in casein when compared to other sources. Verma et al.
(2007) reported that addition of carbon and nitrogen sources to
starch industry waste water increased the production of protease
from 0.4985 to 2.43 IU/ml by Trichoderma viride. This result
correlates with the present studied protease since the employed
organisms were also isolated from starch industry efuent.
3.2. Enzyme purication
In the present study, protease recovery by ethanol precipitation
was 36.49 and 29.39% with 8.63 and 11.50-fold purication for the
parents G. putredinis and T. harzianum respectively and 33.48% with
18.88-fold for the fusant strain (Table 2). Then the nal purity of the
enzymes from both parent and fusant strains was observed by
Sephadex G-100 column chromatography. In Sephadex G-100
column chromatography, the fraction 8 showed maximum protease
activity. In this fraction, the specic activity was 14.85 IU/mg protein
and the protein content was 0.14 mg/ml. Similarly, in T. harzianum
and fusant, Sephadex G-100 column chromatography showed a
specic activity of 14.50 IU/mg protein and 27.00 IU/mg protein
with a protein content of 0.10 and 0.08 mg/ml respectively (Table 2).
De Marco and Felix (2002) puried protease from T. harzianum
by ammonium sulfate precipitation and hydrophobic (Phenyl-
Sepharose column) chromatography with a specic activity of 29.3
and 119 U/mg, purication fold of 1 and 4.06 and yield of 80 and
27% respectively; the puried enzyme had a molecular weight of
18.8 kDa which was similar to our present studied proteases with
yield of 36.49, 29.39 and 33.48% and purication fold of 8.63, 11.50
and 18.88 in G. putredinis, T. harzianum and fusant respectively.
An extracellular bleach stable protease from Aspergillus clavatus
ES1 was isolated from wastewater. The protease of ES1 strain was
puried to homogeneity by acetone precipitation, Sephadex G-100
gel ltration and CM-Sepharose ion exchange chromatography
with a 7.5-fold increase in specic activity and 29% recovery (Hajji
et al., 2007) which were similar to our present studied yield and
fold. Wang et al. (2007) puried Vibrio uvialis TKU005 protease by
DEAE-Sepharose, Sephacryl S-200 and Sephacryl S-100 column
chromatography; this enzyme initially had a specic activity of
0.016 in DEAE with a purication fold and recovery of 2.5 and 31%.
In Sephacryl S-200 chromatography, two protein peaks (FI and FII)
were observed with a specic activity of 0.008 and 0.030 U/mg;
purication folds of 2.7 and 10 and yield of 1.2 and 9.1%. In
Sephacryl S-100 chromatography, the specic activity was
increased to 0.025 and 0.070 U/mg protein with purication of
8.3 and 23 folds and yield of 0.8 and 5.2% respectively in the I and II
fractions. When compared to our present studied protease, the
specic activity, purication fold and yield percentage of V. uvialis
TKU005 protease was found to be very less.
3.3. Properties
The pH affects the ionization of amino acids, which dictate the
primary and secondary structure of enzyme and hence control its
activity. A sharp peak was obtained for both the parent and fusant
strains with the optimum values at pH 7.0 and pH 8.0, respectively
(Fig. 1). The temperature also inuences enzyme reaction rate. The
ideal assay temperature found in this study was 50 8C for the
parent and 60 8C for fusant strain (Fig. 1). The results of
thermostability studies revealed that, at 37 and 60 8C, the parental
proteases were respectively stable for 1 day and 15 min and the
fusant was stable for 2 days and 10 min. Vmax and Km values of G.
putredinis, T. harzianum and fusant enzymes for casein substrate

Table 2
Purication of proteases from the culture ltrates.

Sample Volume Activity Protein Total Total Specic Yield (%) Purication
(ml) (IU/ml) (mg/ml) activity (IU) protein (mg) activity (IU/mg) factor (fold)

G. putredinis
Cf 300 0.38 0.22 114 66.0 1.72 100 1.00
Ep 30 1.53 0.40 45.9 12.0 3.82 40.26 2.22
Sephadex G 100 20 2.08 0.14 41.6 2.80 14.85 36.49 8.63

T. harzianum
Cf 300 0.33 0.26 99.0 78.0 1.26 100 1.00
Ep 30 1.29 0.31 38.7 9.30 4.16 39.09 3.30
Sephadex G 100 20 1.45 0.10 29.0 2.00 14.50 29.39 11.50

Fusant
Cf 300 0.43 0.30 129 90.0 1.43 100 1.00
Ep 30 1.91 0.43 57.3 12.9 4.44 44.41 3.10
Sephadex G 100 20 2.16 0.08 43.2 1.60 27.0 33.48 18.88

Cf: culture ltrate; Ep: ethanol precipitation.

[()TD$FIG] [()TD$FIG]
S. Savitha et al. / Journal of the Taiwan Institute of Chemical Engineers 42 (2011) 298304 301

Fig. 2. SDS-PAGE of G. putredinis, T. harzianum and fusant proteases.

conformation of the enzymes at higher temperatures (Do

Fig. 1. Effect of pH and temperature on protease activity.
were determined. The molecular weight for most of the protease
enzymes were reported to be in the range of 2741 kDa. It was
observed that the puried proteases from both parent and fusant
strains migrated as a single band of 31, 20 and 33 kDa respectively
in SDS-PAGE suggesting that the puried proteins were homoge-
neous (Fig. 2; Table 3). The molecular weight of protease enzymes
were reported to be in the range of 2741 kDa (Kumar et al., 2005;
Wang et al., 2007).
The pH stability of the alkaline protease from B. subtilis CN2 was
reported to be in the range of 711 (Uchida et al., 2004). The
optimum temperature for Bacillus pumilus (Aoyama et al., 2000)
and B. subtilis CN2 protease (Uchida et al., 2004) was 50 8C which
was similar to our parental protease. However, a higher optimum
temperature of 60 8C was observed in Bacillus sp. SMIA-2 (Do
Nascimento and Martins, 2004) and in another Bacillus strain
(Horikoshi, 1990; Banerjee et al., 1999). It was observed that the
optimum pH and temperature for most of the fungal proteases
were in the range of 7.09.0 and 4060 8C. These observations
revealed that the optimum pH for most of the acid proteases was in
the range of 3.06.0 and for alkali proteases it was 7.010.0. The
reported optimum temperature varies from 50 to 60 8C (Table 4).
3.4. Proteases as biodetergents
Nascimento and Martins, 2004). Palmieri et al. (2001) stated that
activation or inhibition of proteolytic enzymes by metals could
change the turnover rate of extracellular enzymes. Many alkaline
proteases were reported to be inhibited by mercury and silver
(Banerjee et al., 1999; Beg and Gupta, 2003). However, metal ions
such as Mn2+, Mg2+, Ca2+ and Co2+ increased or stabilized the
activity of these enzymes conrming that these cations take part in
the stabilization of protease structure and are required for
protection against thermal denaturation (Paliwal et al., 1994).
In the present study, the stability of protease activity against
several metal ions and inhibitors was tested. At 5 mM concentra-
tion of Hg2+, the residual protease activity of G. putredinis, T.
harzianum and fusant were 5.47, 2.48 and 9.76% respectively; Cu2+,
Ca2+ and Zn2+ did not greatly affect the enzyme activity. In 5 mM
EDTA, 60.7685.66% of the residual activity was observed after
30 min incubation. PMSF (2 mM) completely inhibited the prote-
ase activity of all the three fungi studied. These observations
revealed that the proteases of G. putredinis, T. harzianum and fusant
were serine proteases because the enzyme activity was totally
inhibited by PMSF which is a known inhibitor of serine protease
and was not inhibited by EDTA and Zn suggesting that it is not a
metallo and cysteine protease (Table 5).
Damare et al. (2006) reported that the metal ions, Ni2+, Zn2+,
Hg2+, Cu2+ and Fe2+ at 1 mM concentration inhibited the activity of
protease obtained from deep-sea fungi by 72, 81, 55, 0 and 74%
respectively and EDTA (ethylene diamine tetraacetic acid) at 5 and
Table 3
Properties of protease enzymes.
Properties G. putredinis T. harzianum Fusant
Optimum pH 7.0 7.0 8.0
Optimum temperature (8C) 50 50 60
Vmax, IU/mg protein (Casein) 2.00 1.60 2.60
Km, mg/ml (casein) 0.65 1.25 0.40

Thermostability
3.4.1. Effect of metal ions and inhibitors on protease activity 37 8C (days) 1 1 2
60 8C (min) 15 15 10
Generally metal ions protect the enzymes against thermal
Molecular weight, kDa (SDS-PAGE) 31 20 33
denaturation and play a vital role in maintaining the active
302 S. Savitha et al. / Journal of the Taiwan Institute of Chemical Engineers 42 (2011) 298304

Table 4
Comparison of biochemical properties of other reported microbial proteases.

Microorganism pH Temperature (8C) Vmax (IU/mg protein) Km (mg/ml) Molecular References

weight (kDa)

Aspergillus fumigatus CBS113.26 9.0 3742 0.62 mM 33 Larcher et al. (1992)

Ophiostoma piceae 387N 8.0 40 33 Abraham and Breuil (1996)
A. fumigatus TKU003 8.0 40 124 Wang et al. (2005)
Rhizopus oryzae 5.5 60 5 34 Kumar et al. (2005)
Aspergillus clavatus ES1 8.5 50 32 Hajji et al. (2007)
A. clavatus CCT2759 9.5 40 35 Tremacoldi et al. (2007)
Thermoplasma volcanium 3.0 55 Kocabiyik and O zel (2007)
Vibrio uvialis FI 9.0 60 0.26 2.21 41 Wang et al. (2007)
TKU005 FII 0.27 3.92 39
Bacillus mojavensis A21 8.5 60 20 Haddar et al. (2009)
Graphium putredinis 7.0 50 2.00 0.65 31 Present study
Trichoderma harzianum 7.0 50 1.60 1.25 20 Present study
Fusant 8.0 60 2.60 0.40 33 Present study

100 mM concentrations and PMSF (phenyl methyl sulfonyl
uoride) at 2 mM concentration showed a residual activity of 0,
99 and 93%. Hajji et al. (2007) studied the effect of metal ions and
EDTA (5 mM concentration) on A. clavatus ES1 protease. The metal
ions Ca2+, Zn2+, Cu2+, Mg2+, Mn2+, Ba2+ and Co2+ at 5 mM
concentration retained 124, 16, 62, 108, 56, 77 and 0% of residual
activity; EDTA at 5 mM concentration retained about 92% of
residual activity. The high activity of ES1 protease in the presence
of EDTA was reported to be useful for application in detergent
additives.
The activity of protease from V. uvialis was found to be
inhibited by the serine protease inhibitor PMSF (6365% inhibition
at 1 mM) and the metal chelator EDTA (84% inhibition at 2.5 mM)
and not by the classic metalloprotease inhibitor 1, 10 phenanthro-
line (Venugopal and Saramma, 2006). Kamoun et al. (2008) have
also obtained similar results with 5 mM PMSF which caused 100%
inactivation of enzyme preparation suggesting that the alkaline
protease from Bacillus licheniformis RP1 strain belongs to the family
of serine proteases and the metalloenzyme inhibitor EDTA (5 mM)
also inhibited the enzyme activity by 70% similar to the T.
harzianum protease. Indeed, serine proteases are known to contain
two Ca binding sites and the removal of Ca2+ from the strong
binding site is associated with signicant decrease in thermal
stability (Lee and Jang, 2001).
3.4.2. Compatibility of proteases with laundry detergents and their
wash performance
Microbial enzyme-based detergents today occupy a place of
prominence among different categories of detergents. Use of
alkaline proteases has increased remarkably in detergent industry
because they are both stable and active under harsh conditions,
such as high temperatures, pH and in the presence of surfactants or
oxidizing agents (Joo et al., 2001). The addition of proteases
together with amylases, cellulases and lipases helped to improve
the cleaning efciency of the detergents with savings in energy as
an additional advantage. But the use of enzymes in detergents is
not a straightforward approach; besides high temperature and
alkalinity, the enzyme must withstand the presence of ionic and
non-ionic detergents, surfactants, bleach chemicals, sequestering
agents, etc. (Anwar and Saleemuddin, 1998; Kalisz, 1988). The
most recent application of enzyme in detergent industry is the use
of mannanase, which helps in the removal of various food stains
containing guar gum, a commonly used stabilizer and thickening
agent in food products (Kirk et al., 2002).
In the present study, all the three fungal enzymes retained
maximum residual activity with the commercial detergent Rin
Advanced. In G. putredinis, T. harzianum and fusant the residual
activities were 66.00, 63.80 and 76.74% respectively for Rin
Advanced. With other commercial detergents, Surf Excel and
Henko, the residual activity was in the range of 53.0071.00%, the
highest being in fusant protease. G. putredinis and T. harzianum
proteases with Tide showed the lowest activity of 4849% but the
fusant showed higher residual activity of 62.67%. With Kite, all the
enzymes had more or less same level of residual activity (5455%).
With SDS and sodium perborate (0.2%) the residual activities were
58.2573.82 and 61.5870.24% respectively (Table 6). The stability
of enzymes in the presence of EDTA suggested that metal cofactors
are not required for enzyme activity. This property of the enzyme
was very useful for application as detergent additive since
chelating agents, which function as water softeners and are
involved in the removal of stains are components of the detergent
and then specically bind cations (Beg and Gupta, 2003). Among
the three proteases studied, fusant strain exhibited maximum
residual activity with all the detergents and additives.
Alkaline proteases in laundry detergents play a specic catalytic
role in the hydrolysis of protein stains such as blood, milk, human
sweat, etc. The increased usage of the protease as a detergent
additive is mainly due to its cleaning capabilities in environmen-
tally acceptable, non-phosphate detergents (Gouda, 2006; Mei and
Jiang, 2005). Alkaline proteases from various Bacillus sp. such as
Bacillus brevis (Banerjee et al., 1999), Bacillus stearothermophilus
(Dhandapani and Vijayaragavan, 1994) and Bacillus sp. SSR1 (Singh

Table 6
Table 5 Effect of commercial detergents and bleaching agents on protease activity.
Effect of metal ions and inhibitors on protease activity.
Detergents Residual activity (%)
Metal ions (5 mM) Residual activity (%)
G. putredinis T. harzianum Fusant
G. putredinis T. harzianum Fusant
Rin Advanced 66.00  1.09 63.80  1.80 76.74  0.36
HgCl2 5.47  0.69 2.48  1.05 9.76  1.16 Surf Excel 60.50  2.07 54.30  2.33 70.34  3.30
CuSO4 30.51  2.77 24.19  4.80 39.47  1.99 Henko 60.30  2.14 53.90  0.68 63.31  1.71
CaCl2 61.09  1.69 47.80  1.78 88.14  3.13 Kite 55.00  3.58 54.12  0.55 54.36  0.91
ZnCl2 32.42  2.01 36.66  3.12 52.95  1.40 Tide 49.50  2.75 48.53  1.50 62.67  3.94
EDTA 78.33  1.90 60.76  2.22 85.66  1.90 SDS (0.2%) 58.25  0.87 61.33  1.11 73.82  3.17
PMSF (2 mM) 0 0 0 Sodium perborate (0.2%) 61.58  1.06 60.09  0.69 70.24  1.69

Values are mean of ve replicates. Values are mean of ve replicates.

 S. Savitha et al. / Journal of the Taiwan Institute of Chemical Engineers 42 (2011) 298304 303

et al., 2001) have reported to be used in laundry detergent
formulations. Proteases used in detergent formulations should
have considerably high level of activity in the presence of soaps,
alkali and high temperature (Banik and Prakash, 2004).
The efciency of protease from Spilosoma obliqua for removal of
blood stains from cotton cloth was studied in the presence and
absence of detergents (Anwar and Saleemuddin, 1997). Matta and
Punj (1998) reported that protease from Bacillus polymyxa B-17 lost
only 10% of its activity on treatment with 1 mM SDS; the enzyme
also exhibited strong stability against bleaching agent, hydrogen
peroxide and relatively stable in presence of commercial detergents.
[()TD$FIG]
Naja et al. (2005) reported that protease from Pseudomonas
aeruginosa PD-100 efciently removed blood stains from cotton
cloth without the addition of detergent. The effect of detergents on V.
uvialis protease was studied with commercial detergents, Ariel,
Henko, Rin, Sunlight, Surf and Tide. A 42% residual activity was
observed in Ariel and Henko, 51% with Rin, 47% with Sunlight, 61%
with Surf and 47% with Tide respectively. The maximum stability of
the enzyme was reported with the detergent Surf (Venugopal and
Saramma, 2006). Gouda (2006) reported that protease from marine
Bacillus sp. was very stable against non-ionic surfactants (Triton X-
100 and Tween 80) and stable in anionic detergent, SDS. The strong
anionic surfactant (SDS) at 0.1 and 0.5% caused a moderate inhibition
9 and 27%, respectively in B. licheniformis RP1 (Kamoun et al., 2008).

Fig. 3. Wash performance analysis of G. putredinis, T. harzianum and fusant proteases.

304 S. Savitha et al. / Journal of the Taiwan Institute of Chemical Engineers 42 (2011) 298304
3.4.3. Wash performance analysis
The wash performances of the proteases isolated in the present
study were assessed by their ability to remove blood stain from
white cotton cloth. Enzyme with various combinations of
commercial detergents and additives were tried. The visual
comparison of the washed cloth revealed that washing with
distilled water at temperatures of 37 and 55 8C removed some
amount of blood stain from the cotton cloth. The combinations of
commercial detergents (7 mg/ml) and crude enzyme preparations
(10%, v/v) yielded fairly good results. But, the best results were
obtained in the combination of fusant protease with Rin Advanced
at 60 8C. As shown in Fig. 3, the supplementation of the enzyme
preparation (10%, v/v) in detergent (Rin Advanced 1%, w/v)
signicantly improved the removal of the blood stains from the
white cotton cloth effectively (Fig. 3) and therefore it could be used
as an alkaline protease in detergent powder or solution. The results
showed that both the detergent and the protease enzyme can act
synergistically in efcient removal of blood stain.
4. Conclusion
It was envisaged that proteases from G. putredinis, T. harzianum
and fusant can be used as potential additives in the commercial
detergents to enhance the wash performance. The proteases were
stable and highly reactive even in the combination of conventional
and essential detergent additives like EDTA and sodium perborate.
This property of the enzyme is very essential for its application as
detergent additive. Use of proteases in detergent will help in
formulating efcient detergents for removal of various stains, so
that volume of detergents used will be very much reduced which in
turn could provide a safer environment in the pollution loaded
world. Our study shows that the extracellular proteolytic enzyme
produced by the fusant could be of an industrial value. Moreover,
the proteases were compatible with most of the tested laundry
detergents and showed a good washing performance.
References
Abraham, L. D. and C. Breuil, Isolation and Characterization of a Subtilisin-like Serine
Protease Secreted by the Sap-staining Fungus Ophiostoma piceae, Enzyme Microb.
Technol., 18, 133 (1996).
Anwar, A. and M. Saleemuddin, Alkaline-pH Acting Digestive Enzymes of the
Polyphagous Insect Pest Spilosoma obliqua: Stability and Potential as Detergent
Additives, Biotechnol. Appl. Biochem., 25, 43 (1997).
Anwar, A. and M. Saleemuddin, Alkaline Protease: A Review, Bioresour. Technol., 64,
175 (1998).
Aoyama, M., M. Yasuda, K. Nakachi, N. Kobamoto, H. Oku, and F. Kato, Soybean-Milk-
Coagulating Activity of Bacillus Pumilus Derives from a Serine Proteinase, Appl.
Microbiol. Biotechnol., 53, 390 (2000).
Banerjee, U. C., R. K. Sani, W. Azmi, and R. Sani, Thermostable Alkaline Protease from
Bacillus brevis and Its Characterization as a Laundry Detergent Additive, Process
Biochem., 35, 213 (1999).
Banik, R. M. and M. Prakash, Laundry Detergent Compatibility of the Alkaline Protease
from Bacillus cereus, Microbiol. Res., 159, 135 (2004).
Beg, Q. K. and R. Gupta, Purication and Characterization of an Oxidation-stable,
Thiol-dependent Serine Alkaline Protease from Bacillus mojavensis, Enzyme
Microb. Technol., 32, 294 (2003).
Chen, G. C. and B. R. Johnson, Improved Colorimetric Determination of Cell Wall Chitin
in Wood Decay Fungi, Appl. Environ. Microbiol., 46, 13 (1983).
Damare, S., C. Raghukumar, U. D. Muraleedharan, and S. Raghukumar, Deep-sea Fungi
as a Source of Alkaline and Cold-tolerant Proteases, Enzyme Microb. Technol., 39,
172 (2006).
De Marco, J. L. and C. R. Felix, Characterization of a Protease Produced by a
Trichoderma Harzianum Isolate which Controls Cocoa Plant Witches Broom
Disease, BMC Biochem., 3, 3 (2002).
Dhandapani, R. and R. Vijayaragavan, Production of a Thermophilic, Extracellular
Alkaline Protease by Bacillus stearothermophilus AP-4, World J. Microbiol. Biotech-
nol., 10, 33 (1994).
Do Nascimento, W. C. A. and M. L. L. Martins, Production and Properties of an
Extracellular Protease from Thermophilic Bacillus sp., Braz. J. Microbiol., 35, 91 (2004).
Elad, Y. and A. Kapat, The Role of Trichoderma harzianum Protease in the Biocontrol of
Botrytis cinerea, Eur. J. Plant Pathol., 105, 177 (1999).
Haddar, A., A. Bougatef, R. Agrebi, A. S. Kamoun, and M. Nasri, A Novel Surfactant-
stable Alkaline Serine-protease from a Newly Isolated Bacillus mojavensis A21,
Purication and Characterization, Process Biochem., 44, 29 (2009).
Gouda, M. K., Optimization and Purication of Alkaline Proteases Produced by Marine
Bacillus sp. MIG Newly Isolated from Eastern Harbour of Alexandria, Polish J.
Microbiol., 55, 119 (2006).
Hagihara, B., H. Matsubara, M. Nakai, and K. Okunuki, Crystalline Bacterial Proteinase.
I. Preparation of Crystalline Proteinase of Bacillus subtilis, J. Biochem., 45, 185 (1958).
Hajji, M., S. Kanoun, M. Nasri, and N. Gharsallah, Purication and Characterization of
an Alkaline Serine-protease Produced by a New Isolated Aspergillus clavatus ES1,
Process Biochem., 42, 791 (2007).
Horikoshi, K., Enzymes of Alkalophiles,Microbial Enzyme and Biotechnology, p. 275, St.
Louis, MO, 2nd ed., Elsevier, Amsterdam (1990).
Inhs, D. A., W. Schmidt, and F. R. Richter, Proteolytic Enzyme Cleaner, U.S. Patent
5961366 (1999).
Joo, H. S., G. C. Park, K. T. Kim, S. R. Paik, and C. S. Chang, Simple Methods for Alkaline
Protease Purication from the Polychaeta, Periserrula leucophryna, Process Bio-
chem., 37, 299 (2001).
Kalisz, H. M., Microbial Proteinases, Advances in Biochemical Engineering/Biotech-
nology, A. Fiechter, Ed., Springer, Berlin (1988).
Kamoun, A. S., A. Haddar, N. E. H. Ali, B. G. Frikha, S. Kanoun, and M. Nasri, Stability of
Thermostable Alkaline Protease from Bacillus licheniformis RP1 in Commercial Solid
Laundry Detergent Formulations, Microbiol. Res., 163, 299 (2008).
Kirk, O., T. V. Borchert, and C. C. Fuglsang, Industrial Enzyme Applications, Curr. Opin.
Biotechnol., 13, 345 (2002).
Kocabiyik, S. and H. O zel, An Extracellular-pepstatin Insensitive Acid Protease
Produced by Thermoplasma volcanium, Bioresour. Technol., 98, 112 (2007).
Kumar, S., N. S. Sharma, M. R. Saharan, and R. Singh, Extracellular Acid Protease from
Rhizopus oryzae: Purication and Characterization, Process Biochem., 40, 1701 (2005).
Laemmli, V. K., Cleavage of Structural Protein during the Assembly of the Head of the
Bacteriophage T4, Nature, 227, 680 (1970).
Lageiro, M. M., M. J. Moura, A. Reis, and M. J. Costa-Ferreira, Microbial Proteases
Application in Leather Industry, J. Biotechnol., 131, 239 (2007).
Larcher, G., J. P. Bouchara, V. Annaix, F. Symoens, F. Chabasse, and G. Tronchin,
Purication and Characterization of a Fibrinogenolytic Serine Proteinase from
Aspergillus fumigatus Culture Filtrate, FEBS Lett., 308, 65 (1992).
Lee, S. and D. J. Jang, Progressive Rearrangement of Subtilisin Carlsberg into Orderly
and Inexible Conformation with Ca2+ Binding, Biophys. J., 81, 2972 (2001).
Maheshwari, R., G. Bharadwaj, and M. K. Bhat, Thermophilic Fungi: Their Physiology
and Enzymes, Microbiol. Mol. Biol. Rev., 64, 461 (2000).
Matta, H. and V. Punj, Isolation and Partial Characterization of a Thermostable
Extracellular Protease from Bacillus polymyxa B-17, Int. J. Food Microbiol., 42,
139 (1998).
Mei, C. and X. Jiang, A Novel Surfactants and Oxidation-stable Alkaline Protease from
Vibrio metschnikovii DL 33-51, Process Biochem., 40, 2167 (2005).
Naidu, K. S. B. and K. Lakshmi Devi, Optimization of Thermostable Alkaline Protease
Production from Species of Bacillus Using Rice Bran, Afr. J. Biotechnol., 4, 724 (2005).
Naja, M. F., D. Deobagkar, and D. Deobagkar, Potential Application of Protease Isolated
from Pseudomonas aeruginosa PD100, Electron. J. Biotechnol., 8, 197 (2005).
Outtrup, H., C. Dambmann, M. Christiansen, and D. A. Aaslyng, Bacillus sp. JP 395,
Method of Making and Detergent Composition, U.S. Patent 5466594 (1995).
Paliwal, N., S. P. Singh, and S. K. Garg, Cation-induced Thermal Stability of an Alkaline
Protease from a Bacillus sp., Bioresour. Technol., 50, 209 (1994).
Palmieri, G., C. Bianco, G. Cennamo, P. Giardina, G. Marino, M. Monti, and G. Sannia,
Purication, Characterization and Functional Role of a Novel Extracellular Prote-
ase from Pleurotus ostreatus, Appl. Environ. Microbiol., 67, 2754 (2001).
Phillips, M. W. and G. L. R. Gordon, Growth Characteristics on Cellobiose of Three
Different Anaerobic Fungi Isolated from the Ovine Rumen, Appl. Environ. Micro-
biol., 55, 1695 (1989).
Rai, S. K. and A. K. Mukherjee, Statistical Optimization of Production, Purication and
Industrial Application of a Laundry Detergent and Organic Solvent-stable Subtili-
sin-like Serine Protease (Alzwiprase) from Bacillus subtilis DM-04, Biochem. Eng. J.,
48, 173 (2010).
Savitha, S., S. Sadhasivam, and K. Swaminathan, Regeneration and Molecular Char-
acterization of an Intergeneric Hybrid Between Graphium putredinis and Tricho-
derma harzianum by Protoplasmic Fusion, Biotechnol. Adv., 28, 285 (2010).
Sen, S. and T. Satyanarayana, Optimization of Alkaline Protease Production by
Thermophilic Bacillus licheniformis, Ind. J. Microbiol., 33, 43 (1993).
Singh, J., N. Batra, and R. C. Sobti, Serine Alkaline Protease from Newly Isolated Bacillus
sp. SSR1, Process Biochem., 36, 781 (2001).
Tremacoldi, C. R., R. Monti, H. S. Selistre-De-Araujo, and E. C. Carmona, Purication
and Properties of an Alkaline Protease of Aspergillus clavatus, World J. Microbiol.
Biotechnol., 23, 295 (2007).
Uchida, H., D. Kondo, S. Yamashita, T. Tanaka, H. L. Tran, H. Nagano, and T. Uwajima,
Purication and Properties of a Protease Produced by Bacillus subtilis CN2 Isolated
from a Vietnamese Fish Sauce, World J. Microbiol. Biotechnol., 20, 579 (2004).
Venugopal, M. and A. V. Saramma, Characterization of Alkaline Protease from Vibrio
uvialis Strain VM10 Isolated from a Mangrove Sediment Sample and Its Applica-
tion as a Laundry Detergent Additive, Process Biochem., 41, 1239 (2006).
Verma, M., S. K. Brar, R. D. Tyagi, R. Y. Surampalli, and J. R. Valero, Starch Industry
Wastewater as a Substrate for Antagonist, Trichoderma viride Production, Bior-
esour. Technol., 98, 2154 (2007).
Wang, S. L., Y. H. Chen, C. L. Wang, Y. H. Yen, and M. K. Chern, Purication and
Characterization of a Serine Protease Extracellularly Produced by Aspergillus
fumigatus in a Shrimp and Crab Shell Powder Medium, Enzyme Microb. Technol.,
36, 660 (2005).
Wang, S. L., Y. H. Chio, Y. H. Yen, and C. L. Wang, Two Novel Surfactant-stable Alkaline
Proteases from Vibrio uvialis TKU005 and Their Applications, Enzyme Microb.
Technol., 40, 1213 (2007).