Annual Research & Review in Biology

8(3): 1-15, 2015, Article no.ARRB.19936

ISSN: 2347-565X, NLM ID: 101632869

SCIENCEDOMAIN international
www.sciencedomain.org

Degradation of Phenol Containing Wastewater by

Advance Catalysis System – A Review
Pratik M. Kolhe1, Sopan T. Ingle1 and Nilesh D. Wagh1*
1
School of Environmental and Earth Sciences, North Maharashtra University, Jalgaon – 425 001,
India.

Authors’ contributions

This work was carried out in collaboration between all authors. Author PMK designed the review
article, did the literature search and wrote the first draft of the manuscript. Authors STI and NDW
wrote the final drafting of the manuscript. All authors read and approved the final manuscript.

Article Information

DOI: 10.9734/ARRB/2015/19936
Editor(s):
(1) George Perry, Dean and Professor of Biology, University of Texas at San Antonio,
USA.
Reviewers:
(1) Ilham Zahir, University Sidi Mohamed Ben Abdellah, Morocco.
(2) Anonymous, Lagos State University, Nigeria.
(3) N. K. Kortei, University of Ghana, Ghana.
Complete Peer review History: http://sciencedomain.org/review-history/11469

Received 3rd July 2015

Review Article Accepted 31st August 2015
th
Published 19 September 2015

ABSTRACT

Phenols and their derivatives are broadly distributed as a characteristic pollutant due to its frequent
presence in effluents of many industrial processes. Most of the phenolic compounds are toxic to
living organisms as well as environment, even at low concentration. These phenol derivatives
introduced into the environment, they may accumulate in soil and water. This signifies enormous
environmental issues and if they enter into the food cycle through that polluted water, they can
cause numerous health problems to humans. They show adverse effects on human being which
are short term as well as long term effects. Enzymes are good biocatalysts. We discussed in this
study about an enzymatic treatment on effluent containing phenols. Phenol degrading enzymes
and their delivery systems in effluent shortly discussed. We focused only on the phenol degrading
peroxidase enzyme. Numerous researchers extracted the peroxidase from various plants and their
parts. Many researchers have reported that methods of biodegradation of phenols by peroxidase
with additives to retain the specificity of peroxidase through their whole reaction. In conclusion, the
plants having a great source of enzymes, such as horseradish roots, soybean seed hulls and turnip
roots are having rich sources of enzymes. The enzymes are time saving and inexpensive catalyst.

_____________________________________________________________________________________________________

*Corresponding author: E-mail: dr.nilesh31@gmail.com;

 Kolhe et al.; ARRB, 8(3): 1-15, 2015; Article no.ARRB.19936

There are no harmful products formed after completion of reaction. Hence, enzymatic treatment is
fully eco-friendly treatment.

Keywords: Phenol; enzyme; biocatalyst; biodegradation; peroxidase; eco-friendly.

1. INTRODUCTION (Central Pollution Control Board) guidelines the

permissible limit of phenol concentration in waste
1.1 Phenols water is 0.001 mg/L. As per MPCB (Maharashtra
Pollution Control Board) guidelines the
Phenol (C6H5OH) and their derivatives are mostly permissible limit of phenol concentration in waste
distributed as a pollutant because of its common water is 0.002 mg/L. The permissible limit of
presence in effluents of several industries such phenols relaxed in between the range of 0.1 to
as wood, resins, dye and plastic industries [1], 5 mg/L in industrial effluent and they are varied
iron, textiles, coal conversion, petroleum refining, by the agencies, viz. EPA, BIS (Bureau of Indian
steel as well as pulp and paper industries [2,3]. Standards), CPCB and MPCB. Hence, the
Even at low concentration, several numbers of treatment of wastewater containing phenol is a
phenolic compounds are harmful to living very crucial need.
organisms as well as the environment.
Therefore, they are classified as hazardous 1.2 Comparative Studies of Conventional
pollutants [4-6]. If these phenols introduced into and Biological Methods to
the environment, they may accumulate in soil Degradation of Phenols
and water. This signifying enormous
environmental issues [5] and if they enter into the Conventional methods are applied to remove
food cycle through that polluted water, they can phenolic compounds and their derivatives from
cause numerous health problems to human wastewater. These methods like as adsorption
being. The high dose can cause paralysis, on activated carbon, microbial degradation,
hemolytic anemia and liver damage [7]. There incineration, chemical oxidation, use of oxidizing
are some short term effects such as headache, agents such as UV and ozone, solvent extraction
burning eyes and respiratory irritation. Chronic [11]. But these methods have some
effects like anorexia, weakness, fatigue, muscle disadvantages like time-consuming procedures,
pain and weight loss [1]. If they persist in the low efficiency, high cost or generation of some
environment, they persevere to ending through products that are more harmful than the original
bioaccumulation, transportation in living things phenolic compounds. Enzymatic treatment has
and biomagnifications in the food chain. been proposed by many as an alternative
treatment technology to traditional methods [12].
OH Biological processes are achieving more
important over physicochemical process, as
biological systems are more effective and the
end products formed are non toxic [13]. Due to
these causes, more concentration has been
given to the improvement of alternative
methodologies for degradation of poisonous
organic pollutants containing water and soil, such
as phytoremediation [14].

1.3 Enzymes
Fig. 1. Structure of phenol Almost all enzymes are proteins, but all proteins
are not enzymes. Enzymes have several
The concentrations found in the effluents of beneficial characteristics. They are participating
above mentioned industries can range from as biocatalyst in various biological reactions [15].
hundreds to thousands of milligrams per liter [8] They are highly specific and produce only the
while the maximum allowable concentrations for expected products from the given reactants or
discharge into rivers defined by regulations can substrates. Enzymes may produce extensive
vary from 0.1 to 5 mg/l [9,10]. As per EPA transformations of structural and toxicological
(Environmental Protection Agency) and CPCB properties of contaminants and even their

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complete conversion into innocuous inorganic Enzymes are classified into six major classes,
end products. The reaction occurs in multiple i.e. oxidoreductases, transferases, hydrolases,
steps, as shown below: lyases, isomerases and ligases [19]. Peroxidase
is one of the subclass of oxidoreductases which
Enzyme + H2O2 Compound I + H2O (1) is segregated from other subclasses by the use
of H2O2 (hydrogen peroxide) as an electron
● acceptor. Peroxidases and laccases show a wide
Compound I + AH2 Compound II + A (2)
substrate range, especially with regards to
Compound II + AH2
●
Enzyme + AH + H2O (3) phenols and amines [20,21] and azo dyes also
[22,23]. The activity of an enzyme can be
In first step of the process, the active site with influenced by a change in the conditions such as
hydrogen peroxide occurs. The oxidation of temperature, pH and change in substrate
hydrogen peroxide takes place, generating concentration or binding of specific chemicals
compound I and water molecules. In second step that regulates its activity.
of the process, compound I oxidizes a substrate 1.3.1 Phenol degrading enzymes
molecule (AH2), producing a substrate radical
and compound II. Finally, a second substrate Many researchers work on oxidative enzymes to
molecule reduces compound II and returning the the degradation of phenol and their derivatives
enzyme to its initial form [16,17]. They may such as peroxidase, chloroperoxidase, lignin
perform processes for which no efficient peroxidase, Manganese peroxidase, Tyrosinase,
chemical transformations have been devised. Laccase and Catechol dioxygenase. Wide
Enzymes may present advantages over applications of peroxidase in different areas of
traditional technologies, and also over microbial clinical biochemistry, biotechnology,
remediation. Indeed, enzymes are not inhibited environmental sciences, food industry, etc.
by inhibitors of microbial metabolism. All these enhance the interest for further study on the
characteristics render enzymes eco-friendly enzyme [24]. Peroxidase is the group of
catalysts as well as enzymatic techniques, oxidoreductase which is the most commonly
environmentally friendly processes [18]. A variety used by various researchers to the removal of
of enzymes from plants, fungi, animals and phenol and their derivatives from industrial
microorganisms have been reported to play wastewater or effluent. These peroxidase group
important roles in an array of waste treatment consisting enzymes, their sources and their
applications. applications are shown in Table 1.

Table 1. Phenol degrading oxidative enzymes from plants (modified from duran and
esposito, 2000) [25]

Enzymes Source Applications

Peroxidase Horseradish Degradation of phenols, chlorophenols
Artromyces ramosus Degradation of phenols
Chloroperoxidase Caldariomyces funago Oxidation of Phenolic compounds.
Biosensor chlorophenol detection
Lignin peroxidase Phanerochaete Removal of aromatic compounds and
chrysosporium phenolic materials
Manganese peroxidase Phanerochaete Degradation of phenols and
chrysopsorium pentachlorophenol
Lentinula edodes Removal of chlorophenol
Tyrosinase Sigma Phenol biosensor. Degradation of
phenols, Oxidation of catechol and
polymerization of phenolic compounds
Agaricus bisporus Catechol oxidation
Laccase Trametes versicolor chlorophenols and urea derivatives
degradation
Cerrena unicolor Phenol detoxification, 2,4-dichlorophenol
degradation
Catechol dioxygenase Comamonas testosterone Chlorophenol oxidation

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2. EXTRACTION AND PURIFICATION OF (–15°C, –5°C, 0°C and 5°C). After incubation the
PEROXIDASE samples were equilibrated at room temperature
(23°C) for 1 hour, then centrifuged at 1500 rpm
An invention reported by Lakshminarayanan in for 8 minutes and the % transmittance at 700 nm
1976 titled as Method for isolating high purity was determined. Then they resulted that removal
peroxidase. This invention encompasses a of particles indicated by % transmittance was
method for isolating a peroxidase enzyme from more efficient when the sample was completely
plant tissue containing peroxidase. The invention frozen.
has as its critical step treating an aqueous
extract of said plant tissue having the pH Rehman et al. [28] concluded that the
-3
adjusted to 6 – 9 with at least 2.7 × 10 moles horseradish roots (HRR) were found as the best
per litre of zinc ions. Unexpectedly at this pH the source of peroxidase among the studied
zinc ion selectively precipitates contaminating vegetable sources. The optimum pH for enzyme
impurities from the extract. Thus, the critical step activity was measured at 6.0 for radish whereas
of this invention was used in conjunction with salt 6.5 for turnip, horseradish legumes (HRL) and
fractionation, solvent fractionation, dialysis, HRR. Enzyme was found stable even at a
reverse osmosis, electrophoresis, column temperature of 50°C showing relative activity
chromatography and other techniques for from 60 to 80%. The crude enzyme was purified
purifying the protein. They gave variety of eight by DEAE cellulose chromatography after
examples, in these eight examples most ammonium sulfate precipitation and the degree
common step is the deposition of various of purification were 14 folds. Enzyme extraction
concentrations of zinc solution at different pH. procedure contains following steps: 1) 100 gm
They used reverse osmosis in this process to get cutting pieces of fresh vegetables were added to
a concentrated enzyme solution. Lastly, they 400 ml of distilled water and then blended for 15
converted liquid purified enzyme into solid or minutes. 2) The content was centrifuged at 6000
powder from the purified enzyme by the addition rpm for 15 minutes and the supernatant was
of isopropanol solvent at –10°C and mix for 30 passed through filter paper. 3) The extract was
minutes, centrifuged and solvent was discarded. heated at 65°C for 3 min. to inactivate any
Then precipitate was treated with chilled acetone catalase present in the extract. 4) This extract
99% at –10°C, blended and filtered this mixture. was used as a crude peroxidase enzyme.
Then again washed with acetone and dried at
room temperature to gain the final product in the In 2002, Alyas and co-workers studied on the
form of powder of high purity peroxidase [26]. extraction and purification of peroxidase from
soybean seeds. The crude extract obtained by
A new method is freeze-thaw technique of the procedure used by Ambreen et al. [29] with
recovering peroxidase from seed hulls was some modifications. The purification of
discovered by Pokora et al. [27]. They mentioned peroxidase consists of partial purification by
some examples in their article. In the first ammonium sulfate precipitation technique and
example, they prepared two sets of six different purified by ion exchange and gel filtration
percentages of the concentration of soybean chromatography. The activity and specific activity
seed hull extract with water: 10%, 20%, 40%, of crude enzyme was recorded as 17.29 U/ml
60%, 80% and 100%. One set is placed in the and 1.586 U/ml respectively. And this crude
freezer (–20°C) and another set stored in the enzyme was subject to ammonium sulfate
refrigerator (5°C). After 2 hours, the samples precipitation for partial purification and the
were thawed at room temperature (23°C) and resulted activity and specific activity was 12.85
took the absorbance against water in the range U/ml and 5.68 U/mg respectively. After ion
of 1100 nm to 200 nm. They mentioned exchange chromatography through DEAE
transmittance from 1100 nm to 200 nm for both cellulose, fraction No. 43 exhibited maximum
freezer and refrigerator sample. They also activity of 18 U/ml and specific activity of 9.5
determine the specific activity and fold U/mg. This fraction was subsequently applied to
purification. Then they revealed that particulate sephadex G-75 column and after the election;
contamination can be removed quickly (2 hours) the activity and specific activity were enhanced to
by a freeze-thaw cycle and settling by the force 16.04 U/ml and 14.948 U/mg respectively [30].
of gravity. In second example, the example they
determined the effect of freezing on separation. The quantity and biological activity of peroxidase
For this determination, they prepared four isolated from different parts of the soybean plant
samples and cooled to various temperatures was compared by Sariri et al., 2003. Then they

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resulted that the biological activities of as acetone, methanol and ethanol, which widen
peroxidase extracted from the leaves and seed the applicability of SBP for the treatment against
coat were similar seed coat contained higher a variety of organic pollutants present in
quantities of peroxidase than the leaves. In industrial and petroleum waste waters and its
extraction procedure, seeds were soaked in application may be advantageous as a biosensor
distilled water at room temperature for 24 hours, or for lower cost industrial wastewater treatment
after soaking grind the soaked seeds and the compared to other peroxides such as HRP. SBP
husk was separated by filtration. The extracts is fairly active in organic solvents and exhibited
were filtered using four layers of cheesecloth to optimal activity in the presence of 20% (v/v)
remove suspended fibrous solid particles. Crude acetone. Increasing the organic solvent content
extracts were also prepared using 10 mM resulted in a reduction in SBP activity. Two
phosphate buffer by exactly the same procedure. methods were used for extraction of peroxidase
But the activity and stability of the enzyme were from soybean seed hulls in this study: In first
similar in both the extracts. Peroxidase activity method 25 g of soybean hulls were soaked in
was determined at room temperature with 200 ml phosphate buffer (0.1M NaH2PO4/
spectrophotometer following the formation of Na2HPO4, pH 6.0) in 4°C for 24 h. The extract
-1
tetraguaiacol (Amax = 470 nm, ε = 26.6 mr was then filtered through four layers of cheese
cm-1) in a 3 ml reaction mixture containing 1 ml of cloth; the filtrate was clarified by centrifugation at
2-methoxyphenol (guaiacol); 1 ml of 3 mM H2O2 6000 rpm for 15 min in 4°C to remove cell debris.
and 50 ml of enzyme extract. The reaction was The final supernatant was collected and stored at
carried out for 3 min. One unit of peroxidase 4°C and used as a source of crude SBP enzyme.
activity (U) represents the amount of enzyme An enzyme solution was warmed to room
catalyzing the oxidation of 1 mmol of guaiacol in temperature immediately prior to use. In the
1 min. They were protein determined by second method 1g of defatted soybean flakes
Comassie brilliant blue G-250 method using was mixed with 0.1M phosphate buffer, pH 8.0,
bovine serum albumin as standard [31]. for 2h in 4°C (1:25, w/w) and then centrifuged at
5000 rpm for 30 min. The supernatant was
Liu and co-workers reported ‘A novel process of maintained in 4°C. But the enzyme extracted by
purifying soybean hull peroxidase’ in 2005. They first method having more Activity (U/ml), Protein
mostly focused only on purification of peroxidase. concentration (mg/ml) and Specific activity
They concluded that the RZ value (U/mg) [33].
(Reinheitszahl) of soybean hull peroxidase (SBP)
reached 1.32 and the recovery of enzyme activity A comparative study of peroxidase purification
was 65% after purification. The satisfactory from apple and orange seeds was studied by Zia
recovery of activity and RZ value as well as the et al. [34]. They revealed that orange seed
simplicity of the procedure make this strategy a peroxidase had more activity than apple seed
useful alternative for the purification of SBP. peroxidase in crude extract and each step of
They used a new process is the enzyme solution purification. Apple and orange seeds were kept
was purified by ammonium sulfate–acetone separate from the fruits, dried and soaked in 200
cooperation precipitation, the acetone ml of 0.1 M phosphate buffer of pH 6.0 over night
precipitation and zinc sulfate precipitation. The and thoroughly homogenized by blending for 15
ammonium sulfate–acetone cooperation to 20 min. The contents were centrifuged at
precipitation, namely, the two-phase system of 10,000g for 15 min to eliminate cell debris. The
organic solvent- inorganic salt, was adopted in supernatant was removed carefully from the
their process [32]. sediments and filtered through Whatman No. 1
filter paper to obtain more clarity of the crude
Ghaemmaghami and co-workers in 2010 enzyme extracted. Enzyme activity was
concluded that SBP extracted from soybean determined using a UV–Vis spectrophotometer at
seed hulls is a highly robust enzyme and the the wavelength of 470 nm according to the
enzyme exhibited the highest activity and stability method of Rad et al., 2007 with minor
at pH 6.0 and retained over 75% of the maximum modifications. A mixture of pyrocatechol (170
activity for 12 hours. The activity of SBP was mM) and aniline (2.5 mM) was prepared in 0.2 M
found to be 2.5 times higher at an elevated phosphate buffer solution of pH 6.5. For each
temperature of 65°C compared to the activity at blank and sample cuvette, 500 µl of the earlier
room temperature. The activity retains over 95% mentioned mixture solution and 500 µl of
for 30 min at 75°C. Also, this enzyme is fairly hydrogen peroxide (35%) was pipetted and
active in the presence of organic solvents such incubated at 25°C for 3 to 4 min. Then, 50 µl of

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the crude enzyme extract and 50 µl phosphate extraction procedure was same as seen earlier in
buffer solutions were added to the sample and this article with some modifications. The enzyme
blank cuvettes, respectively. Increase in was purified from the stem juice by ultrafiltration
absorbance was recorded from 4 to 5 min and anion-exchange column chromatography on
intervals. Protein was determined by the method DEAE with 8-fold purification and purification
of Lowry et al., 1951 using bovine serum albumin yield of 65%. They also reported that the pH and
as standard. Partial purification of the crude temperature optimum of the enzyme was 4.5 and
enzyme extract was done by ammonium sulfate 25°C respectively. The enzyme in combination
precipitation and ion exchange chromatography. with H2O2 liberated bromine and iodine in the
It was observed that after partial purification, the presence of KBr/KI respectively. All these
enzyme activity was increased as compared to enzymatic characteristics were similar to those of
crude enzyme extract. Peroxidase from orange fungal MnP.
seed was purified up to 17.17 fold with specific
activity of 10.17 U/mg and that from apple seed In 2012, Khatun et al [37] revealed that Moringa
was 6.82 fold with specific activity of 7.53 U/mg oleifera L. leaves were available in large
after diethyl amino ethyl (DEAE) cellulose quantities in almost all seasons, purified
chromatography. Further purification was peroxidase from these leaves was more stable
obtained through gel filtration chromatography by and active in acidic pH and the activity remains
using sephadex G-75 column. Peroxidase from 90% at 60°C for 30 min incubation. Silva et al.
orange and apple seeds got purified up to 30.64 extracts peroxidase from five vegetables and
and 8.34 fold with their specific activity of 18.16 determine the enzyme activity, then they
and 9.20 U/mg, respectively. It is more evident concluded that the enzyme activity more when
that peroxidase is the most heat stable enzyme; NaCl added into the enzyme extract [16].
therefore, it is concluded that it may be Peroxidase was purified 164-fold from the leaves
potentially useful for industrial purposes [34]. of Moringa oleifera L. with a recovery of 28% by
ammonium sulfate precipitation, DEAE-cellulose
Shazia et al., 2012 studied on Production and column chromatography, Sephadex G-200
purification of horseradish peroxidase (HRP) in column chromatography and Con-A column
Pakistan. From their results, the activity of the chromatography by them. The extraction
crude HRP extracts was 6.3027 U/ml and procedure was identical as seen earlier in this
specific activity of 0.8586 U/mg. It was shown article with some modifications. After Con-A
that the activity was increased to 6.6928 U/ml column chromatography specific activity and
and 12.77 U/mg specific activities by (NH4)2SO4 purification fold were 346.43 and 164.18
precipitation. Protein contents were decreased respectively. They also studied on some metal
from 7.339 mg/ml of crude extract to 0.524 mg/ml ions out of them Ni2+, Pb2+, Zn2+, Al3+, Mg2+, Cu2+,
which indicate that unwanted proteins have been 2+ 2+
Co and Cd exhibited low inhibitory effect
removed. An enzyme fraction having the highest while Fe , Fe3+ and Hg2+ exhibited strong
2+
activity after dialysis was passed through a inhibitory effects.
Sephadex G-75 column for gel filtration
chromatography. Maximum activity of 9.9452
U/ml was obtained in the fourth fraction during Anbuselvi and co-worker purify peroxidase from
the experiment, with 0.253 mg/ml of protein two varieties of Tulsi and Neem and also studied
contents and 39.30 U/mg of specific activity. So, their characteristics in 2013. They were studied
the HRP enzyme was purified up to 45.77 fold on Ocimum tenuiflorum, Ocimum gratissimum
[35]. varieties of tulsi and Azadirachta indica, Melia
azadirachta varieties of neem for peroxidase
Yadav et al. [36] worked on the purification and analysis. Peroxidase extracted by following
characterization of Mn-Peroxidase (MnP) from procedure: 500 mg of all the four leaf samples
Musa paradisiacal (banana) stem juice which is was weighed and ground with the addition of 1
an agro-waste easily available after harvest of ml of Phosphate buffer (pH7). This was then
banana fruits. Earlier MnP was isolated and centrifuged at 10000 rpm for 15 minutes at 4°C
purified only from fungal sources, but for the first and the supernatant was passed through filter
time it was purified from available plant source. paper. It was heated in a water bath at 65°C for 3
The purification procedure was simpler than that minutes to inactivate catalase in the extract and
reported for the fungal MnPs. They reported that cooled promptly by placing in an ice bucket for
the purification procedure was simpler than that 10 min. The optimum pH and temperature of
reported for the MnPs from fungal sources. The peroxidase were found to be 6.5 and 40°C. The

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ammonium sulfate precipitation and gel filtration inactivation of tomato peroxidase required 6–10
were used by authors for purification. Protein min at 90°C at pH 6.0.
profiling was done in both Native PAGE and
SDS-PAGE. They also concluded that from this 3. DELIVERY SYSTEMS FOR ENZYMES
study it was found that the protein content and its IN EFFLUENT TREATMENT
enzyme activity were different for varieties within
the same species. This study was helpful in Some researchers worked on extraction of
understanding the varietal difference within the enzymes from plants, microorganisms and
same species. Local availability of these plants animals, their purification and characterization
and reasonably high specific activity of the before 1980. These extracted enzymes were
enzymes isolated from these medicinal plants applied in various fields after the decade of 1980.
makes it a better choice for the production of Since last two decades, due to the increasing of
peroxidase for its use as an antioxidant [38]. awareness about the environment in the whole
world, many scientists use enzymes in
Four vegetables viz. potato, carrot, eggplant and
environmental sciences to clean the
tomato were studied by Suha et al. [39] for
environment. Therefore, researchers studied the
thermostability at different pH levels of
different techniques of the delivery of enzymes in
peroxidase extracted from these four vegetables.
waste water for degradation of numerous
All vegetables investigated contained peroxidase
pollutants. The delivery system is selected, must
enzyme. Extraction of peroxidase procedure
be proper to the intention, easy, cost effective
follows by following steps: Fresh fruits of potato,
and efficient. However, keep in mind to ensure
carrot, eggplant and tomato were washed
that the activity of the enzyme is not adversely
thoroughly with distilled water and cut into
affected due to the mode of delivery [40].
pieces. Then fruits were homogenized with ice
cold 10 mM sodium phosphate buffer of pH 5, 6,
7 or 8. The ratio of quantity of fruits taken to that 3.1 Direct Use of Biological Source
of buffer was maintained constant at 1:1 (w/v).
The crude extract was filtered through cheese Directly introducing an enzyme into the effluent is
cloth and centrifuged to remove traces of fibrous to provide tissues or cells that produce the
particles and cell debris. Supernatant was stored enzyme. This mode of enzyme delivery system is
at 4°C and used as a stock solution for further accepted when suitably adapted microbial strains
experiments. Then give heat treatment to an are used to co-metabolize target pollutants or
enzyme. They did not purify that crude extracted when the cell producing the enzyme is
enzyme. From this study, they concluded that introduced directly into the wastewater. There
peroxidase of high activity was extracted at pH are two types of direct use of biological source
5.0 from potato and tomato while those of carrot which are use of plant tissues or entire plant and
and eggplant was extracted at pH 6.0. But potato use of microbial cells [41].
tuber contained a higher level of peroxidase
whereas carrot had lower levels at all pH values. 3.2 As Cell-free Enzyme Extracts
The activity of the enzyme is dependent on the
pH value of the medium. The results showed that Extracted enzymes or cell-free enzymes are
the rate of loss of peroxidase activity from the preferred for use over the integral organism,
vegetables increased with both increases in particularly when the effluent to be treated which
temperature and heating time. Biphasic cannot support growth. The extracted enzymes
inactivation curves were observed for the could be used in either pure form or crude
enzymes extracted from all samples, where the extract. Well, it is comparatively easier to
initial heat inactivation is rapidly followed by standardize optimum treatment conditions with
much slower inactivation periods. The rate of extracted enzymes [42]. The use of extracted
loss of peroxidase activity was shown to be pH enzymes has simple for handling and storage
dependent. Potato peroxidase was noted to be over microbial cultures. The process of crude
more stable to heat. A less severe heat treatment enzyme extract preparation includes grinding and
is required to inactivate carrot, eggplant and homogenizing the source tissue or cells with a
tomato peroxidases. Complete inactivation of suitable buffer solution followed by filtration
carrot peroxidase was accomplished within 4–10 [43,44]. Crude enzyme extracts are
min at 80°C and within 2–10 min at 90°C at pH comparatively inexpensive over pure enzymes.
8.0, while peroxidase inactivation in eggplant Crude enzyme extracts can also effectively
required 8–10 min at 90°C at pH 8.0. Complete degrade pollutants from wastewater.

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3.3 In Immobilized Form are capable of transporting them through an

aqueous medium. In contemporary times,
An enzyme is deemed to be immobilized when it research on carbon based nanotechnology, such
is physically confined to a certain region of as the carbon nanotube is gaining force. The
space, retaining its catalytic activity and the nanotubes carrying oxidative enzymes such as
capacity to be used repeatedly or continuously. laccases or peroxidases could be synthesized for
The use of immobilized enzymes in effluent utilization in the treatment of recalcitrant
treatment has many significant advantages over pollutants in wastewater. In the future, carbon
the use of free enzymes, including increased nanotubes will widely use in water treatment.
stability, localization, the ease of handling, Fig. 2 (a) shows structure of nanosponge before
reusability and a consequent decrease in running introducing target and after treatment. The upper
cost [42,45]. The HRP enzyme has proved to be layer in Fig. 2 (b) indicates attached toxic
an adjustable molecule that can be used in the material [41,48].
form of cell-free crude extract [46] or in an
immobilized form entrapped in calcium alginate
capsules at a laboratory scale [47].

Some methods of immobilization such as

adsorption, covalent binding, entrapment,
encapsulation, membrane confinement and
chemical coupling can adversely affect the
catalytic activity of certain enzymes.
Immobilization procedures need to be optimized
to minimize the loss of enzyme activity and
achieve maximum reusability. This method of Fig. 2. Structure of nanosponge:
enzyme delivery holds great potential for the (a) Before use (b) After use
continuous treatment of large volumes of
effluent. 4. BIODEGRADATION OF PHENOL
USING FREE ENZYME AND
3.4 In the Form of Different Nanoparticles IMMOBILIZED ENZYME

Nanotechnology is one of the highly acceptable In 1996, Tatsumi et al. studied on the removal of
in wastewater treatment methods, which can chlorophenols from wastewater by immobilized
effectively decontaminate xenobiotics in the HRP. Method of Immobilization of enzyme by
environment. Use of nanoparticle in Reactive physical adsorption on magnetite was more
Remediation Technology having a great interest effective than crosslinking method. Kalssom et
in wastewater treatment. Since it involves the al. also reported that efficient dye degradation by
complete degradation of pollutants into carbon the SBP immobilized in polyacrylamide matrix
dioxide and water, which are inoffensive products [49]. Besides, it was discovered that HRP was
[41]. adsorbed on magnetite. The enzyme had a
specific activity of 100 units/mg. Crude
Remediation of polluted wastewater can be peroxidase was prepared from horseradish and
achieved by using a combination of concentrated by ultrafiltration. Freeze drying
nanotechnology and enzyme technology called method was used to obtain a crude enzyme
as Single Enzyme Nanoparticle technology powder. The enzyme activity was observed
(SEN). A SEN may be elaborated as an enzyme about 2.5 U/mg. Peroxidase was immobilized on
enclosed by a protective cover which is a few magnetite by both chemically and physically. The
nanometers thick. SENs are able to withstand immobilized protein was calculated before and
drastic conditions of temperature, pH, after immobilization. The degradation of each
contaminant concentration and salinity as chlorophenol from the wastewater by peroxidase
compared to free enzymes. can be seen in Table 2. They utilized the same
enzyme activity (0.2 U/ml) for both soluble and
Another type of novel nanoparticle is immobilized enzymes. The results specify that
nanosponges. These are materials containing the immobilized enzyme was more effective than
microscopic particles with nano-sized cavities. the soluble and each chlorophenol was degraded
These particles can encapsulate or can be to almost 100%. For soluble peroxidase, 2,4,6-
embedded with various types of substances and trichlorophenol was more reactive than 2,4,5-

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 Kolhe et al.; ARRB, 8(3): 1-15, 2015; Article no.ARRB.19936

trichlorophenol. Such an effect was not observed Wilberg et al. [52] reported that the SBP activity
in immobilized peroxidase. The removal of 2,4,5- in fresh hulls was greater than in aged hulls and
trichlorophenol was observed with a lowest was preserved at –10°C. They purchased all
removal rate of only 36%. chemicals from various places and soybean seed
hulls extract obtained in two steps: - 1) pH 6
Table 2. Removal of chlorophenols from the phosphate buffer extractions and 2) Freeze-thaw
wastewater by soluble and immobilized technique. A linear relationship with a slope of
peroxidase 0.8 U cm-3 mmol dm-3 between minimum low
purity SBP (LP-SBP) dosage and initial phenol
Chlorophenol Soluble Immobilized concentration was found for 95% phenol removal
peroxidase peroxidase efficiency. This relationship remained unaltered
-3
(%) (%) when 1000 mg dm of PEG-6000 was added to
the solution. Minimum LP-SBP dosage was 1.7
p-Chlorophenol 58 100
times lower than those published by Kinsley and
2,4-Dichlorophenol 82 100
Nicell using a medium purity SBP (MP-SBP).
2,4,5- 36 99 A retention time of about 100 min was sufficient
Trichlorophenol to achieve yields of 95%. This retention
2,4,6- 97 98 time decreased with increasing phenol
Trichlorophenol concentration.
2,3,4,6- 81 99
Trichlorophenol Cheng and co-workers deliberate on the HRP
Pentachlorophenol 55 97 immobilized on aluminium-pillared interlayered
clay for the catalytic oxidation of phenolic
In conclusion, peroxidase was very simply wastewater in 2006. HRP was immobilized on
immobilized on magnetite by physical adsorption. aluminium-pillared interlayered clay (Al-PILC) to
HRP was immobilized from crude HRP and the obtain enzyme-clay complex for the treatment of
enzyme was purified. The immobilized phenolic wastewater. That immobilized HRP
peroxidase can effectively degrade phenols used for phenol removal by precipitation or
because of the binding of colored reaction transforming to other products. The addition of
products to the immobilized enzyme. In the PEG in reaction mixture could expressively
treatment of chlorophenolic wastewater, about improve the phenol degrading efficiency and
90% of TOC and AOX were found to be removed reduce the amount of immobilized enzyme
by immobilized peroxidase [50]. required to attain high removal efficiency of over
90%. The complete oxidation of phenol could
within the short retention time when the molar
Kinsley and Nicell worked on the treatment of ratio of H2O2/phenol and the mass ratio of
aqueous phenol with SBP in the presence of PEG/phenol were 1.5 and 0.4 respectively. HRP
polyethylene glycol (PEG) in 2000. They immobilized on Al-PILC had better storage
purchased all chemicals as well as medium stability than the free enzyme. However, the
purity SBP as a dry powder from various places. reusability of the immobilized enzyme was not
SBP catalyzes the oxidation and polymerization satisfactory. Besides, they reported that the
of aromatic compounds in the presence of immobilized enzyme lost its catalytic
hydrogen peroxide. Studies were undertaken to performance in the fourth repeated test [3].
characterize the use of PEG as an additive to
increase the functional life of the enzyme [51]. Nair and co-workers in 2008 studied on
The effectiveness of PEG increased with its biodegradation of phenol. During the past three
molecular weight, with maximum protection decades, the use of microbial strain as a catalyst
accomplished with PEG of molecular weight of in the biodegradation of organic compounds has
35,000. Linear relationships were found between highly developed significantly. It has been found
the quantity of phenol to be treated (1.0 - 10 mM) that large numbers of microbes exist in almost all
and the optimum doses of SBP and PEG natural environments, particularly in the
required for greater than 95% removals. lithosphere. Not only natural, but also synthetic
Observations indicate that it is the interaction organic chemicals are casually biodegradable in
between the PEG and the polymeric products a natural environment. Therefore, they produce
that results in the protection of SBP. Following this review article, especially on soil
treatment, approximately 25% of the optimum microorganisms and they focused only on phenol
PEG dose remained in the supernatant [10]. degrading enzymes secreted by microorganisms.

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 Kolhe et al.; ARRB, 8(3): 1-15, 2015; Article no.ARRB.19936

Biodegradation of materials involves allowing solution. Fig. 5 containing compounds were used
adsorption of the substrate, initial proximity, as substrate for study.
secretion of extracellular enzymes to polymerize
the pollutants. The effectiveness of Table 3. Peroxidase involved in the
biodegradation of contaminants is influenced on biodegradation of phenolic compounds
the basis of the organic pollutant, the nature of
the influencing factors, the enzyme involved, the Sr. Type of Enzyme References
nature of the organism and the mechanism of no. phenol
degradation. 1 Phenol Horseradish [54]
peroxidase
Table 3 shows peroxidase enzyme involved in 2 Phenol Horseradish [55]
the biodegradation of phenol and phenolic peroxidase
derivatives. They also investigated the 3 Phenol Peroxidase [56]
mechanism of phenol biodegradation and 4 Bis-phenol Peroxidase [57]
reported two pathways of phenol degradation viz. 5 Lignophenols Peroxidase [58]
Meta and Ortho pathway of phenol degradation
in Fig. 3 and 4 [53]. The effect was investigated in the removal
process by some parameters. The positive
In 2009, Hejri and Saboora concluded that an regression showed between enzyme
increase in hydrogen peroxide up to the optimal concentration and degradation of phenols by the
amount leads to an eminent degradation of application of various concentrations of the
phenolic compounds. More concentrations of enzyme in the reaction. To examine the optimum
hydrogen peroxide inhibited the reaction. The pH for enzyme activity resulted that removal of
effect of enzymatic removal increased in the phenols was enhanced in neutral pH.
presence of PEG as an additive. The Additionally, this study resulted that the integral
polymerized products were in innocuous form soybean seed were effective in the removal of
and can be easily filtered from treated the phenolic compounds in synthetic wastewater [4].

OH OH OH O COOH CH3 CHO

OH Aldehyde
COOH COOH C O
CHO CH2
2-hydroxy muconic Oxopent 4-enoate CH3 C COO-
Phenol
semialdehyde Pyruvate
COOH
Oxaloacetate

Fig. 3. Meta pathway of phenol degradation

OH OH
OH COOH COO
COOH TCA Cycle
O
COOH
Phenol Catechol Cis-Cis muconic acid Muconoactone oxo-adipate

Fig. 4. Ortho pathway of phenol degradation

OH OH OH

CH3

CH3
Phenol o - cresol m- cresol

Fig. 5. Chemical structures of phenol, o-cresol and m-cresol

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 Kolhe et al.; ARRB, 8(3): 1-15, 2015; Article no.ARRB.19936

A review article prepared by Rao et al. [18] respectively. This shows that phenol degradation
named as Role of enzymes in the remediation of occurred due to enzyme action [1].
polluted environments. They reported that the
environmental pollution is growing more due to Wash and weigh 300 gm of Turnip

the indiscriminate and frequently deliberate

Cut and mince
absolve of hazardous substances. Research
efforts have been dedicated to develop new, cost
Add 300 ml water and stir thoroughly with glass rod for 20 mins
effective, low-technology, eco-friendly treatments
capable of reducing pollution in the atmosphere, Transfer supernatant to Buchner funnel
hydrosphere and lithosphere. The biological
agent, enzymes has a vast potentiality to Take Pulp in cotton cloth
effectively polymerize, transform and detoxify
pollutants because they have been endorsed to Squeeze and mix with filtrate
be able to transform pollutants and are
potentially suitable to restore polluted Measure total volume
environments. This review examined some
pollutants and enzymes capable of bio Add 70 g of powdered (NH4)2SO4 / 100 ml of filtrate

transforming them into innocuous products. The

Stir well and allow standing for 5 min
enzymatic processes renovated and
implemented in some detoxification treatments
Peroxidase precipitate out
examined in details. Not only advantages, but
also drawbacks that are present in the spacious Sink to bottom Rise to surface
application of enzymes in the in situ restitution of
polluted environments will be discussed. Decant the supernatant, Transfer to separating
filter the rest through funnel allow to stand
Buchner funnel and run off the liquid
Pradeep et al. [2] revealed that the phenol
degraded by free enzymatic treatment. They Place the filter paper in vacuum desiccator and allow to dry overnight
used HRP, Radish Peroxidase and SBP for their
study, their phenol removal efficiency recorded Detach the dried precipitate
for 100 mg/L as 84%, 76% and 72% respectively.
From a comparative study, peroxidase extracted Store it in refrigerator
from HRR was able to polymerize phenol more
efficiently than the enzymes extracted from Fig. 6. Schematic diagram of extraction of
soybean hulls and radish roots. The ambient peroxidise
room temperature during the study period ranged
from 27-32°C. The HRP also proved to be Removal of Phenol has been studied using free
valuable in the removal of phenol at and immobilized HRP by Pradeep et al. [13]. The
concentrations between 100 mg/L to 300 mg/L whole study performed in between 27-32°C
when compared with Radish Peroxidase and which is the ambient room temperature. A phenol
SBP [2]. concentration of 100-500 mg/L was kept to both
free and immobilized HRP. Free enzyme studies
Shruthi et al., 2012 resulted that the were carried out in conical flasks while
concentration of phenol decreased with an immobilized HRP enzyme bead reactor used for
increase in the concentration of enzyme extract removal of phenol. Free HRP removed 84% of
& H2O2. The turnip root extract (Peroxidase) 100 mg/L phenol whereas Immobilized HRP
degrade phenol more efficiently. Chemical removed 62% with the same phenol
methods, for instance, ozone treatment is costly concentration. Free enzyme showed a better
and chlorine oxidation may give more toxic degradation than immobilized enzyme due to the
compounds than the phenol itself. Fig. 6 shows availability of the most active sites in the free
the extraction of peroxidase from turnip roots. enzyme than immobilized enzymes. Reduction in
Table 4 shows determination of phenol, after the phenol degradation could be observed with the
treatment of 100 mg/ ml and 80 mg/ ml phenol increase in phenol concentration. The
with three different concentrations of the crude experimental setup of immobilized enzyme bed
enzyme. Maximum phenol degradation was reactor (IEBR) as shown in Fig. 7. The plastic
observed with high concentration of the enzyme column of 20.3 cm having a sampling port at the
extract i.e. 0.8 ml. The resulting degradation of bottom of the column was fitted to the iron stand.
100 mg/ml and 80 mg/ml at 91% and 94% Immobilized enzyme beads were filled upto

11
 Kolhe et al.; ARRB, 8(3): 1-15, 2015; Article no.ARRB.19936

15.2 cm of the plastic column. Phenol and H2O2 Xanthium strumarium, Cyperus rotandus and
were poured from the top of the column. Trianthema portulacastrum for biodegradation of
phenolic compounds in wastewater. Four models
of synthetic wastewater at concentration of 10
mM were prepared in the laboratory as follows:
model (A) composed of (α-naphthol + quinol +
catechol + resorcinol), model (B) composed of
(resorcinol + quinol + phenol + β-naphthol),
model (C) composed of (tannic acid + pyrogallol
+ gallic acid + α-naphthol) and model (D)
composed of (catechol + gallic acid + β-naphthol
+ phenol). The tested enzymes showed a wide
range of substrate specificity and different rates
of enzymatic activities. It is evident that the
peroxidases enzymes were very active towards
most of the phenolic compounds. When using
pyrogallol as substrate, the peroxidase from
C. rotandus showed high specific activity (1.75
U/mg-1) and high Km value (4.19 mM pyrogallol).
All extracts showed marked ability to degrade
Fig. 7. Immobilized enzyme bed reactor (all phenolic pollutants in the tested wastewater. The
dimensions are in cm) highest rate of degradation was noticed when
crude peroxidase from C. rotandus was added to
Enzyme beds prepared by the following both industrial and synthetic wastewater. This
procedure: Took 4 gm of sodium alginate and study revealed that C. rotandus is the most
100 ml distilled water in a beaker; that beaker interesting source of peroxidase enzymes for the
was kept on a hot water bath to dissolve sodium eliminating or reducing phenolic pollutants in
alginate. Sodium alginate solution was cooled wastewater [7].
and 4% crude enzyme was mixed. 0.2 M of
CaCl2 solution was placed on a magnetic stirrer Crude peroxidase extracted from fresh soybean
and a mixture of sodium alginate and enzyme seed hulls having more potential to degrade the
was added drop by drop with the help of a phenol from synthetic wastewater this was
burette. The beads have a uniform size about 8 observed by Kolhe et al. [59]. They extracted
mm in diameter and stored at 4°C prior to use peroxidase having 6.091 U/ml activity, 2.325
[13]. mg/ml protein content and 2.62 U/mg specific
activity. They examined some parameters like
pH, concentration of enzyme and concentration
Crude peroxidases from five weed plants to
of phenol. Then they resulted that 0.2 ml H2O2
utilize to biodegradation of phenols in wastewater
and 0.4 ml crude peroxidase at neutral pH could
studied by Hamad and Ahmed in 2013. They
be the most favourable condition for the phenol
were extracting crude peroxidases from
degradation in aqueous medium [59].
Portulaca oleracea, Sonchus oleraceus,

Table 4. Determination of phenol after the treatment of 1 ml and 0.8 ml phenol and crude
enzyme

Sr. Phenol Conc. H2O2 Enzyme Incubation FeCl3 D/W OD at

no. (ml) (mg) (ml) extract at room (ml) (ml) 540 nm
(ml) temp for
1 1.0 100 0.50 0.2 30-40 mins 2.0 5.0 0.64
2 0.10 0.4 0.37
3 0.20 0.8 0.09
4 0.8 80 0.50 0.2 0.40
5 0.10 0.4 0.26
6 0.20 0.8 0.04

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 Kolhe et al.; ARRB, 8(3): 1-15, 2015; Article no.ARRB.19936

5. CONCLUSION enzymatic treatments. J Sci. (University of

Tehran), JUST. 2009;35(1):13-9.
We have concluded that the plants having a 5. Gianfreda L, Sannino F, Rao MA, Bollag
great source of enzymes, such as horseradish JM. Oxidative transformation of phenols in
roots, soybean seed hulls and turnip roots are aqueous mixtures. Water Res. 2003;37:
having rich sources of enzymes such as 3205–15.
Peroxidase, Chloroperoxidase, Mangnese 6. Aksu Z. Application of biosorption for the
peroxidase, Laccase and Catalase. The removal of organic pollutants: A review.
enzymes are having a wide range of degradation Proc Biochem. 2005;40:997–1026.
of pollutants. Some authors studied on the 7. Hamad IS, Ahmed AAA. Biodegradation of
delivery systems for enzymes in effluent phenols in wastewater using crude
treatment. The efficiency of enzymes depends on peroxidases from five weed plants. J Chem
the factors affecting parameters such as pH, Pharmaceut Res. 2013;5(4):60-5.
temperature, retention time, purification of an 8. Cooper VA, Nicell JA. Removal of phenols
enzyme, concentration of enzyme and from a foundry wastewater using
concentration of pollutants in wastewater to horseradish peroxidase. Water Res. 1996;
degradation of phenols. But enzymes are having 30:954-64.
great efficiency to degradation of phenol and 9. Fuller RK, Tomlin JL. Phenol destruction in
their derivatives. The enzymes are time saving foundry wastewater. Modern Casting June.
and inexpensive catalyst. There are no harmful 1988;25-7.
products formed after completion of reaction. 10. Kinsley C, Nicell JA. Treatment of aqueous
Hence, enzymatic treatment is fully eco-friendly phenol with soybean peroxidase in the
treatment. presence of polyethylene glycol.
Bioresource Tech. 2000;73:139-46.
ACKNOWLEDGEMENTS 11. Miland E, Smyth MR, Fagain OC. Phenol
removal by modified peroxidases. J Chem
This work was supported by UGC, New Delhi, Tech Biotechnol. 1996;67:227–36.
Government of India through BSR (RFSMS) 12. Mossallam KF, Sultanova FM, Salimova
fellowship [F.7-289/2009 (BSR)] to the PMK is NA. Enzymatic removal of phenol from
gratefully acknowledged. PMK is also thankful to produced water and the effect of petroleum
North Maharashtra University, Jalgaon for whole oil content. Thirteenth International Water
necessary help. Technology Conference, Hurghada, Egypt.
2009;1009-1020.
13. Pradeep NV, Anupama, Hampannavar US.
COMPETING INTERESTS Polymerization of Phenol using Free and
Immobilized Horseradish Peroxidase. J
Authors have declared that no competing Env Earth Sci. 2012;2(1):31-6.
interests exist. 14. Pletsch M, Santos DAB, Charlwood BV.
Novel biotechnological approaches in
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