Sustainable Chemistry and Pharmacy 14 (2019) 100178

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Sustainable Chemistry and Pharmacy

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Extraction of amylase from the microorganism isolated from textile mill

effluent vis a vis desizing of cotton
Ruchika Aggarwal a, Tanmay Dutta b, Javed Sheikh a, *
a
Dept. of Textile Technology, Indian Institute of Technology, Delhi, India
b
Dept. of Chemistry, Indian Institute of Technology, Delhi, India

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

Keywords: Amylase is one of the most popular enzymes in industrial biotechnology and is used widely in different industries
Aspergillus such as food, detergent, textile, biofuel, and alcohol production. In textile wet processing, amylase is utilized for
Fungus a preparatory process called desizing where it helps in the removal of starch from the grey cotton fabric. Even
Desizing
though a wide variety of products based on amylase are available in the market, the extraction of amylase from
Textile effluent
Amylase
the novel source which can meet the requirements of desizing remains the quest of research. In the present work,
Solid state fermentation textile mill effluent was utilized as a novel source for the isolation of fungus (Aspergillus), which can produce
extracellular thermostable amylase. Temperature and pH optimization of amylase activity was studied, which
reveals the broad range temperature stability of the extracted amylase. Zymogram analysis of this enzyme has
shown the one isoform of amylase of approximately 55 kDa. The partially purified enzyme was applied on fabric
and the treated fabric was analyzed for the efficiency of desizing. The extracted amylase displayed a good level of
activity under the broad range of temperature and pH which can be considered as the ideal case for enzymatic
desizing. The grey fabric was efficiently desized using the extracted amylase which was confirmed through
TEGEWA ratings and absorbency of the desized fabric.

1. Introduction
Alpha-amylases (endo-1,4-α-D-glucan glucanohydrolase, E.C.
3.2.1.1) are extracellular endo acting enzymes that randomly cleave the
α-1,4 linkage between adjacent glucose units in the linear amylose chain
while bypass α-1,6 linkages and ultimately generate glucose, maltose,
and maltotriose units (Panneerselvam and Elavarasi, 2015).
Beta-amylase (1,4-α-D-glucan maltohydrolase, E.C. 3.2.1.2) on the other
hand, are exoenzymes that cleave the α-1,4 linkage from the nonre­
ducing end of the chains and generate maltose units (Saini et al., 2017).
Amylases are widely known enzyme of the enzyme family, as they
are present in animals, fungi, plants, unicellular eukaryotes, and pro­
karyotes. Generally, to meet the increasing industrial demand, micro­
organisms has stimulated the interest in the exploitation of extracellular
enzymatic activity in different microbes. This enzyme is continuously
consumed in various industries (Liu and Xu, 2008), such as food industry
where amylase is used for the liquefaction of starch to convert it into
fructose and glucose syrup. Addition of alpha-amylase increases the rate
of fermentation, therefore improves the texture and volume of the food
product (Raveendran et al., 2018). In the textile industry, amylase is
used for the wet processing known as desizing. This involves in the
removal of a protective layer of starch from the fabric. In pulp and paper
industry amylase is used for the modification of paper coated with starch
which makes the paper smooth, strong, and it also intensify the quality
of the paper. The utilization of amylase in the detergent industry en­
hances the detergent’s ability to remove tough stains and also makes it
environment-friendly (Sundarram and Murthy, 2014). Amylase is also
consumed in fuel and alcohol production. Sugar produced by starch
hydrolyzes is converted into ethanol using ethanol converting micro­
organisms (Souza, 2010). The production of amylase is outshining most
of the other enzyme present, as it accounts for the 30% of the enzyme
production market in the world (Zaferanloo et al., 2014).
Rising textile demands have a huge impact on the contribution of the
textile industry to enhance the economic progress of the country.
However, textile processing requires the utilization of a wide array of
hazardous chemicals e.g. acid, alkali, detergents or heavy metals and
involvement of intense energy consumption to successfully carry out
these processes which also produce a great load on our environment
(Ibrahim et al., 2016). Waste and effluent upon treatment can cause
toxicity to the aquatic and other forms of lives. To overcome these issues,

* Corresponding author.
E-mail address: jnsheikh@iitd.ac.in (J. Sheikh).

https://doi.org/10.1016/j.scp.2019.100178
Received 14 June 2019; Received in revised form 30 August 2019; Accepted 1 September 2019
Available online 7 September 2019
2352-5541/© 2019 Elsevier B.V. All rights reserved.
R. Aggarwal et al.
the use of enzymes in the textile industry is rapidly growing and in­
dustrial enzymology is drawing global recognition (Yusuf, 2018). It is
becoming an integral part of the textile industry as it can be used in
textile waste treatment as well as in the textile manufacturing (Mojsov
et al., 2016; Forgiarini and de Souza, 2007; Doshi and Shelke, 2001).
Textile chemical processing with the use of enzymes is drawing world­
wide attention due to their distinctive substrate specificity, through
which catalysis happens in an environment-friendly manner to produce
the desired product (Ibrahim et al., 2008). Aside from that
enzyme-driven catalytic reactions are non-toxic, stereospecific, and
involve mild condition for handling (Ibrahim et al., 2019). A principal
advantage of the usage of enzymes is the drastic reduction of energy
consumption in textile processing (Khushboo et al., 2016).
A gelatinous substance, generally starch is applied on the warp
thread of the cotton fiber due to which it sustains the mechanical stress
and it also prevents it from breaking during weaving. This process is
called sizing and the substance is the size. For the improved and uniform
wet processing of the fiber, starch-based size is needed to be removed
prior to dyeing and printing. This is known as desizing, which is the key
action of the pretreatment. The widely used enzyme for this process is
hydrolytic amylase. These enzymes catalyze the breakdown of the starch
chain without damaging the support material as their action is specific
to starch.
Till date, the soil is the most frequent source for the isolation of
amylase producing microorganism. Soil collection has been reported
from a variety of locations, either from the sea coast, garbage area or
dump yards (Chakraborty et al., 2009; Mukherjee et al., 2017; Chimata
et al., 2011). Other starchy material like rotten fruits, plants and vege­
tables has also been reported for isolation of amylase producing mi­
crobes (Xian et al., 2015; Obafemi et al., 2018).
This paper reports the textile effluent as the source for microbe
cultivation and enzyme isolation. Textile effluent as a novel source has
seldom a choice for the isolation of microbes. The motive of the selection
of effluent is to produce an enzyme with a wide range of temperature,
pH activity and stability in the presence of various salts or chemicals. For
the isolation of enzyme, low-cost agriculture waste has been used for
microbial growth in solid state fermentation. In the present report
optimization of temperature, pH and thermal stability have shown for
the crude enzyme extract. There are limited reports highlighting the use
of extracted enzymes for desizing of cotton. Thus, an attempt has been
directed towards the extraction of amylase from the fungus isolated from
the effluent arising from textile mill and the extracted amylase was
utilized for desizing of cotton. The efficacy of the isolated amylase in
desizing of cotton was studied in comparison with the commercially
available amylase.
2. Materials and methods
2.1. Materials
Chemicals like MnSO4, MgSO4, CaCl2.2H2O, K2HPO4, NH4NO3,
(NH4)2NO3, starch were purchased from Merck (Germany), while others
like agar-agar, NaCl, KCL, ZnSO4, NaH2PO4, Na2PO4, gram’s iodine were
from Fisher scientific. Organic Wheat bran was purchased locally.
2.2. Microorganism and culture conditions
The textile effluent was collected from the local textile industry (Pale
yellow in color, foul smell, pH 8.8, TDS 8010 ppm, BOD 187 ppm).
Microorganisms were isolated by salt and substrate enrichment method.
The isolation salt medium contained (g/L): NaCl 1.0; KCL 0.5; MnSO4
0.5; MgSO4 0.4; CaCl2.2H2O 0.5; K2HPO4 0.3; NH4NO3 0.5; (NH4)2NO3
0.5; ZnSO4 0.7; substrate (starch) 2.5 g (Kumar et al., 2012). The sample
was added (2 ml/L) to the above salt medium and incubated at room
temperature for 120 h. 0.2 ml of inoculum from the enriched salt me­
dium was further spread on the substrate (starch) agar plate (salt
2
Sustainable Chemistry and Pharmacy 14 (2019) 100178
medium, 2% Agar) and incubated at 37 � C for 92 h. Resultant fungal
colonies were purified by inoculation on slant agar tube of the salt
medium. The microorganism was maintained over potato dextrose agar
slants (PDA), stored at 4 � C and subculture after every 15 days. Culti­
vation for production of amylase was done in solid-state fermentation.
2.3. Amylase production and partial purification
The moisture content of the solid matrix was estimated by recording
the dry weight of the 10 g wheat bran. Then to fix the moisture content,
it was autoclaved and soaked with the desired amount of water. Wheat
bran was weighed again and the moisture content was calculated as
follows:
The percent moisture content of the solid medium
¼ ðwt: of the wheat bran dry wt:Þ * 100 = dry weight
Spores were collected from PDA slants in 10 ml of deionized water
and dropwise spread over the solid matrix of wheat bran (per 10g)
uniformly, maintaining the moisture content of the solid bed. Microor­
ganisms were then allowed to grow at 37 � C for 3–4 days. The enzyme
was extracted from wheat bran, by agitating it with 50 ml of phosphate
buffer saline (PBS) of pH 7.2 on a shaker for 30 min with 120 rotations
per minute (RPM). The fungal biomass and suspended material were
separated by centrifugation at 10,000 RPM for 30 min and filtered su­
pernatant was used as the crude enzyme extract. For the partial purifi­
cation ammonium sulfate was added slowly to crude enzyme extract
after centrifugation to obtain 80% saturation. The solution was stirred
for 40 min and left for 1 h at 4 � C. Then the solution was subjected to
centrifugation for 40 min at 6000 rpm, the precipitate was collected and
dissolved in 10 mM PBS, pH 7.2 and dialyzed overnight with the same
buffer at 4 � C. The dialyzed sample was further assayed for its amylase
activity.
2.4. Amylase assay
Amylase activity was measured using the method described by Miller
(Miller, 1959) with few modifications. The starch solution was boiled for
5 min before use to make a clear solution. 0.1 ml of crude enzyme extract
was added to 5 ml of 1% starch solution dissolved in deionized water.
This reaction mixture is then incubated for 15 min at 37 � C. 1 ml of the
reaction mixture was picked up after 15 min and to terminate the re­
action, 1 ml of dinitrosalicylic acid (DNS) reagent was added. Subse­
quently, the tubes were placed in a water bath at 100 � C for 8–10 min.
Finally, optical density (OD) of samples was taken at 540 nm against a
blank containing reaction mixture except for crude enzyme. One unit of
amylase activity was defined as the amount of enzyme that liberated
1 μmol of reducing sugar (glucose) equivalent per min from starch under
assay condition.
Optimization of temperature for enzyme activity was done by incu­
bating 1% starch solution with 0.1 ml enzyme for 15 min at different
temperature of 30 � C, 40 � C, 50 � C, 60 � C, 70 � C, 80 � C, and 90 � C. For
studying the effect of pH on enzyme activity and optimizations of pH,
1% of a starch solution of pH 3.5–10.5 was used. They were: 50 mM
sodium acetate buffer (pH 3.6–5.5), 50 mM sodium phosphate buffer
(pH 6.5–7.5) and 50 mM glycine–NaOH buffer (pH 8.5–10.5) (Dutta
et al., 2008). 1% of starch solution was incubated with 0.1 ml of crude
enzyme extract for 15 min.
To determine the thermostability of amylase, enzyme extract was
incubated at 60 � C, 70 � C, and 80 � C for 1 h. At different time interval of
15 min, 30 min, and 1 h, aliquots were taken and activity was deter­
mined using DNS assay.
2.5. Zymogram analysis
Zymogram of amylase was performed according to Upadhyay,
R. Aggarwal et al. Sustainable Chemistry and Pharmacy 14 (2019) 100178

Mukesh K., et al. with some modification (Upadhyay et al., 2005). Crude Table 2
enzyme extract was subjected to electrophoresis on 10% SDS-PAGE Various experimental conditions used for desizing using Pad-steam Process.
containing 1% starch (resolving gel). After electrophoresis, the gel was Sr. Enzyme Enzyme conc. in gpl Salt conc. in gpl Sample
washed with PBS buffer (pH 7.2) and incubated at 70 � C for 15 min. The No. (grams per litre) (grams per litre) code
gel was further stained with Gram’s iodine solution for 10 min and 0 Control – – PC
washed 3 times with deionized water. 1. Comm. 3 gpl – PC 1
enzyme
2. Comm. 5 gpl PC 2
2.6. Desizing of grey cotton fabric using amylase –
enzyme
3. Isolated 3 gpl – PI 1
2.6.1. Exhaust process enzyme
Grey fabric was desized using a standard method followed in the 4. Isolated 5 gpl – PI 2
textile mills. Fabrics were treated with amylase enzyme in rota dyer enzyme
5. Comm. 3 gpl 5 gpl PC 3
machine (Texlab, India) using the material to liquor ratio of 1:10. In
enzyme
order to optimize the parameters of the process, desizing was carried out 6. Comm. 5 gpl 5 gpl PC 4
at various pH and temperature conditions (Table 1). Desizing process enzyme
was carried out for 45mins and the fabrics were given a hot wash (95 � C 7. Isolated 3 gpl 5 gpl PI 3
enzyme
for 10 min) followed by a cold wash (30 � C for 10 min). The fabrics were
8. Isolated 5 gpl 5 gpl PI 4
dried in hot air oven (Khera, India) at 102 � C for 5mins. Effect of tem­ enzyme
perature was studied by using various temperatures from 40� C-90 � C by
taking enzyme 1% and 1 h treatment time (Jothi, 2015). Effect of pH on
the fabric by enzyme was determined by varying the pH from 4.5, 5.5, 7, 120 h, it was spread over the starch agar plates. Few fungal colonies
8.5 and 9 by keeping enzyme concentration fixed as 1% and treatment were observed within 4–5 days. One fungal colony was chosen for its
time 1 h (Jothi, 2015). highest activity after it underwent secondary screening. For secondary
screening, the fungal colony was stained with Gram’s iodine solution. As
2.6.2. Pad-steam process shown in fig 1, the interaction of starch with iodine solution causes the
The grey cotton fabric was padded (75% expression) with a solution intense blue-black color. While the clear hollow around fungal colony
containing enzyme, wetting agent and salt (Table 2). The padded fabric represents hydrolysis of starch due to its capability of producing extra­
was steamed at 102 � C for 5 min followed by hot wash (95 � C for 10 min) cellular amylase.
and cold wash (30 � C for 10 min). The fabrics were dried in hot air oven The growth kinetic profile of enzymatic production of Aspergillus in
(Khera, India) at 102 � C for 5mins. solid state fermentation using wheat bran as a substrate was obtained
after its cultivation for 7–8 days at 37 � C. The highest enzyme activity
2.6.3. Efficiency of desizing was observed after 5 days of culture. The strain belongs to genus
The initial and final weights of the fabric were utilized to calculate Aspergillus, identified by IMTech, Chandigarh. According to ITS4R þ
the removal of starch. ITS5F sequencing, the strain has shown 100% similarity to Aspergillus
Percentage of starch removed ¼ initial weight final weight
�100 (Chimata tubingensis, A. niger and A. costaricaensis. The enzyme was further
initial weight
partially purified using ammonium sulfate precipitation, followed by
et al., 2011)
dialysis. Amylase extract showing the optimum temperature at 70 � C
The degree of starch residue on the fabric was assessed by TEGEWA
with minimal change in the activity at temperature 60� C–90 � C (Fig. 2).
rating, where 1 indicates no desizing and 9 shows the complete desizing
This enzyme has assessed to be acidophilic, an enzyme has an optimum
or removal of the starch (Wurster et al., 1987).
pH of 3.5; from pH 3.5–5.5, enzyme activity had changed negligibly
The degree of whiteness was evaluated by measuring the CIE yel­
(Fig. 3). While from 7-10.5 pH, 80% of the activity was lost. The lowest
lowness index using Gretag Macbeth Colour-Eye 7000A
enzyme activity at pH 10.5 was 2.29 � 0.5 U/ ml. The Maximum activity
spectrophotometer.
observed was 8.71� 0.5 U/ ml. Zymogram analysis of the crude enzyme
Absorbency test was performed using the AATCC method 79-1995
preparation from Aspergillus revealed the presence of only one isoform of
(AATCC, 1998). Under standard conditions, a drop of water was put
approximately 55 kDa (Fig. 4). Thermal stability studies of this enzyme
on the surface of the fabric and the time required for absorption was
have shown that the enzyme is stable enough at a higher temperature
measured using a stopwatch. Lesser absorbency time indicates better
(from 60� C-80 � C) for 1 h of incubation. It is observed (Fig. 5), that the
absorbency.
enzyme is losing its activity with the course of time. As the time of in­
cubation increases protein starts to degrade because high temperature
3. Results and discussion

Pale yellow enzyme effluent was collected from the textile industry
at room temperature. Synthetic media was used for the enrichment
process of the effluent. After incubation of the effluent in media for

Table 1
Various experimental conditions used for desizing using exhaust process.
Sr. No. Enzyme pH Temp Sample code

0 Control – – C
1. Comm. enzyme 5.5 55 � C CE 1
2. Isolated enzyme 5.5 55 � C IE 1
3. Isolated enzyme 4.5 55 � C IE 2
4. Isolated enzyme 9.0 55 � C IE 3
5. Comm. enzyme 5.5 70 � C CE 2 Fig. 1. Black-spored fungal colony on Agar plate, containing starch as a sub­
6. Isolated enzyme 5.5 70 � C IE 4 strate. For secondary screening, potent Amylase producing fungus was treated
7. Isolated enzyme 4.5 70 � C IE 5
with iodine solution. The clear zone is showing the utilization of starch by
8. Isolated enzyme 9.0 70 � C IE 6
the fungus.

3
R. Aggarwal et al.
Fig. 2. Temperature optimum curve of an Amylase was shown in the figure. To
determine the optimum temperature, 0.1 μl crude enzyme extract was incu­
bated with 1% of the substrate at differed temperature for 15 min. Relative
enzyme activities (% of maximum) were plotted against different temperature.
All the results were expressed in mean � SD.
Fig. 3. pH optimum curve of an Amylase was shown in the figure. Relative
enzyme activities (% of maximum) were plotted against pH. All the results were
expressed in mean � SD.
tends to disrupt the active site of the enzyme.
Textile effluent as a novel source for microorganism isolation is one
of the rarest choices. None of the studies in the literature reported the
Fig. 4. Zymogram analysis of amylase. 10 μl of crude extract was subjected to
SDS-PAGE electrophoresis containing 1% starch in running gel. Subsequently,
the gel. was stained with Gram’s iodine, followed by destaining.
4
Sustainable Chemistry and Pharmacy 14 (2019) 100178
Fig. 5. Thermostability of amylase was determined by incubating enzyme at
temperature 60 � C, 70 � C and 80 � C for 1 h. At different time interval aliquots
were taken and enzyme activity was measured using DNS reagent. Relative
enzyme activities (% of maximum) were plotted. All the results were expressed
in mean � SD from three separate experiments.
enzyme isolation from textile effluent and its application in textile wet
processing. Solid state fermentation has found to be an appreciable way
of enzyme isolation by using agro-industrial waste as a bed material and
production of value-added products in an eco-friendly way (Pandey
et al., 1999). Solid state fermentation (SSF) has a great deal in the fungal
growth process, as its hyphal development makes it penetrate the solid
substrate and let them colonize effectively (te Biesebeke et al., 2002).
Other advantages of SSF includes resistant to bacterial contamination as
well as requirement of low solvent volume, therefore the final product
will be relatively less diluted (Singhania et al., 2010). Wheat bran is
substantial as well as eco-friendly supportive material for fungal growth
(Ghosh and Ghosh, 2012). Out of different industrial waste available,
wheat bran has found to have a great yield of amylase using Aspergillus
(Khan and Yadav, 2011).
Rhizopus spp., and Aspergillus spp. is a known predominating form of
fungus found in alkaline effluents (Faryal and Hameed, 2005). Asper­
gillus has already been reported to be a promising fungus for Amylase
production (Sivaramakrishnan et al., 2006). Most amylase produced
from fungus showed the optimum temperature ranging from 40� C-55 � C
(Asrat and Girma, 2018; Shanmugasundaram et al., 2015) but this iso­
lated amylase can survive at a higher temperature, which has already
been observed in case of some thermophilic Amylase, isolated from
bacteria (Burhan et al., 2003; Saito, 1973). These thermostable prop­
erties are generally arising due to the presence of a large number of weak
interactions. Conformation stability also plays an important role in
defining the upper limit of the enzyme thermal stability (Daniel et al.,
1996). While a change in pH changes the interactions of amino acids of
the proteins due to their protonation and deprotonation. Protonation
and deprotonation of amino acids in the active sides is the basic reason
for enzyme stability at various pH. Different studies have been con­
ducted where isolated amylase has shown higher activity toward acidity
pH (Mukherjee et al., 2017).
3.1. Desizing of cotton using extracted amylase
The desizing of grey cotton fabric was carried out using commercial
and isolated amylase and the results are summarized in Table 3. In
general, grey cotton is a heterogeneous system where the starch layer is
attached to the cotton fabric unlike in the case of biochemical testing. In
such cases, the efficiency of removal of starch is dependent on the pa­
rameters of the enzymatic process like enzyme concentrations, pH, and
the temperature which ensures the best activity of amylase to act on
starch. Apart from this temperature is also important to solubilize the
starch film in water irrespective of the degradation of starch ensured by
enzyme activity. Hence the removal of starch is a resultant of the
R. Aggarwal et al. Sustainable Chemistry and Pharmacy 14 (2019) 100178

Table 3 Table 4
Table showing results of effect of various experimental conditions (exhaust Table showing results of effect of various experimental conditions (Continuous
process) on desizing efficiency. process) on desizing efficiency.
Sample Weight Loss TEGEWA Yellowness Absorbency (in Sample Weight Loss TEGEWA Yellowness Absorbency (in
code (%) rating index sec) code (%) rating index sec)

C – 1 32.20 >5mins PC – 1 30.40 >5mins

CE 1 18.62% 7 25.26 16 s PC 1 18.21% 7-8 25.91 14 s
IE 1 17.76% 6-7 24.14 19 s PC 2 20.13% 7-8 27.29 16 s
IE 2 16.43% 5-6 26.52 20 s PI 1 10.61% 5-6 26.17 21 s
IE 3 15.87% 5-6 25.98 21 s PI 2 11.24% 4-5 28.97 23 s
CE 2 16.36% 7 24.62 17 s PC 3 16.31% 8 27.32 19 s
IE 4 16.42% 4-5 26.15 20 s PC 4 15.26% 7-8 28.13 21 s
IE 5 16.51% 5-6 26.67 21 s PI 3 12.37% 6-7 32.71 22 s
IE 6 16.33% 5-6 23.91 20 s PI 4 11.42% 4-5 27.03 23 s

enzymatic activity, temperature, agitation, and the subsequent washing.
In order to study the optimization of parameters for best enzymatic
activity, the pH of the process was varied at general temperatures of
desizing i.e. 60 � C and 70 � C. The TEGEWA rating of the desized fabric,
indicating the intensity of blue color developed with iodine solution, are
presented in Table. At 60 � C, the isolated enzyme showed a slightly
inferior TEGEWA rating (1 scale lower) as compared to that of Com­
mercial enzyme-treated sample. The variation of pH also resulted in
varied TEGEWA ratings; however, all the ratings were in the acceptable
range.
In general, TEGEWA rating of 4 is acceptable for desized fabric which
would further improve with subsequent bleaching operations.
The cotton fabric desized at pH of 5.5 showed a better rating as
compared to that of other samples. This is in well accordance with the
literature where the enzymatic desizing is recommended at pH 5.0–6.0.
The enzyme was found acidophilic, and the better activity in such pH
ranges were also shown by assay. Even though the better activity was
shown at highly acidic pH, the textile substrate especially cotton is
degraded by strongly acidic conditions. Apart from this, the strong acids
can also depolymerize starch and could result in desizing. Thus the
enzymatic processes of desizing are restricted to the mildly acidic side.
The better desizing was achieved at pH 4.5 in case of desizing at 70 � C.
Thus the pH range of 4.5–5.5 can be recommended for desizing using
isolated amylase. Even though the assay test showed the best activity of
amylase at 70 � C; no advantage was achieved by raising the temperature
of desizing from 60 to 70 � C. In case of alkaline pH, the TEGEWA ratings
were equivalent to that of the acidic process. This is highly encouraging
as the further purification processes like scouring and bleaching are
carried out in alkaline pH. The enzymatic scouring using pectinase is
also preferred in mildly alkaline pH and the processes thus can be
combined.
The result of enzymatic desizing was reflected clearly in the absor­
bency of desized cotton. Even though starch is a hydrophilic polymer,
the sizing formulation includes various hydrophobic substances which
ultimately results in hydrophobic film on the cotton fabric. Apart from
this, various natural impurities are present in cotton which are hydro­
phobic in nature. This makes the penetration of water into the grey
fabric more difficult. Desizing removes the size layer by degradation of
starch and thus imparts better absorbency. As indicated by the results in
Table 4, the trends of desizing efficiency (indicated by TEGEWA scale)
and absorbency were identical. There was no clear trend in the yel­
lowness index of desized cotton. The layer of starch on cotton imparts
yellowness the cotton fabric, the extent of which was reduced after
desizing indicating the removal of starch.
Results in Table 4 indicate the suitability of extracted enzyme for
desizing of cotton using pad-steam method. As the steaming is carried
out at a high temperature, most of the enzymes do not work efficiently.
This needs a specialized thermostable amylase. As indicated by
TEGEWA ratings and weight loss values, the extracted amylase showed
efficient activity. The weight loss values were comparable with those of
exhaust processes. The addition of common salt (NaCl) as mentioned in
the Table 2, which is generally recommended in enzymatic processes,
resulted in the better activity of amylase. Salt, in general, enhances the
thermal stability of enzymes and thus works in a synergistic way with
amylase.
Absorbency values were in well accordance with the removal of
starch and weight loss. Higher the weight loss, higher and TEGEWA
rating and better is the absorbency (lower time in seconds).
The objective of isolation of the fungus from effluent and subsequent
extraction of amylase was to get better stability of the enzyme in the
presence of various chemicals and at the higher temperature. The results
obtained were promising and the particular objective was more or less
achieved.
4. Conclusion
The successful isolation of stable Aspergillus fungus was carried out
from textile mill effluent as a novel source. The amylase was extracted
from the isolated fungus using fermentation technique. The study
confirmed the ideal enzyme activity profile for desizing of cotton. The
treatment of the grey cotton using extracted amylase also confirmed the
suitability for its use. The effective removal of starch was achieved using
the novel amylase.
Acknowledgement
Authors gratefully acknowledge IIT Delhi for Faculty Inter-
disciplinary Research grant (FIRP) to Dr. Javed Sheikh and Dr. Tan­
may Dutta.
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