i need a rough draft.

Scientific African 10 (2020) e00608

Contents lists available at ScienceDirect

Scientific African

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

Peroxidase from waste cabbage ( Brassica oleracea capitata L .)

exhibits the potential to biodegrade phenol and synthetic

dyes from wastewater

Enoch B. Joel a , ∗, Simon G. Mafulul a , Hadiza E. Adamu

a , Lazarus J. Goje

b , Habibu Tijjani c , Adedoyin Igunnu

d , Sylvia O. Malomo

d

a Department of Biochemistry, Faculty of Basic Medical Sciences, College of Health Sciences, University of Jos, Jos, Nigeria b Department of Biochemistry, Faculty Science, Gombe State University, Gombe, Nigeria c Department of Biochemistry, Bauchi State University, Gadau, Nigeria d Department of Biochemistry, Faculty of Life Sciences, University of Ilorin, Ilorin, Nigeria

a r t i c l e i n f o

Article history:

Received 28 July 2020

Revised 1 October 2020

Accepted 23 October 2020

Keywords:

Waste cabbage

Brassica oleracea

Peroxidase

Biodegradation

Phenol

Azo dyes

a b s t r a c t

Peroxidases are well known for their ability to biodegrade some recalcitrant organic pollu-

tants like phenol and their derivatives resulting in a reduction in their toxicity. The present

study was designed to extract, characterize, and evaluate the potential of partially purified

peroxidase from discarded and decaying waste cabbage leaves in the biodegradation of

phenol and some common synthetic azo dyes. This was done by first partially purifying the

crude extract of waste cabbage peroxidase (WCP) using ammonium sulfate precipitation,

dialysis, and gel filtration chromatography. Thereafter, the experimental determination of

protein concentration, peroxidase activity, and biodegradation of phenol and azo dyes was

done spectrophotometrically. The results showed a purification fold of 87.65 with a 34.92%

yield. The partially purified peroxidase had its optimum activity at temperature 30 °C, pH

5.5 while showing broad substrate preference with ABTS been the substrate. The stability

studies also showed that WCP was stable over a wide range of pH (4.0–7.0) and 41% of

its original activity was retained at 80 °C indicating that it is a thermostable enzyme. The

kinetic data of WCP showed K m

values of 1.24, 17.89, and 19.24 mM and V max values of

1111.11, 909.09, and 588.24 mM /minutes for ABTS, guaiacol, and o-dianisidine respectively.

Three metal ions, Hg 2 + , Cu 2 + , Ni 2 + , organic solvent (acetone), EDTA, and urea inhibited

peroxidase activity; whereas Mn 2 + and Zn 2 + showed slight activation. The partially puri-

fied WCP exhibited high efficiency for the biodegradation of synthetic azo dyes and phenol

at the lab-scale. After 48 h incubation, the waste cabbage peroxidase efficiently catalyzed

the decolorization of tested azo dyes at varying degrees; azo blue 5, azo purple, azo yellow

6, and citrus red 2, with a percentage decolorization of 85.1, 69.1, 46.2 and 42.9%, respec-

tively. The waste cabbage peroxidase also shows up to 91.1% efficiency for degradation of

phenol in aqueous solution after 60 min. Findings from this study provide promising evi-

dence on the possibility of utilizing/recycling a readily abundant waste cabbage to useful

bioproducts like peroxidase enzyme with the ability to biodegrade azo dyes and phenol at

a small scale in the laboratory. Moreover, the findings from this study increase the prospect

of waste cabbage peroxidase for the treatment of industrial effluents containing dyes and

∗ Corresponding author.

E-mail address: [email protected] (E.B. Joel).

https://doi.org/10.1016/j.sciaf.2020.e00608

2468-2276/© 2020 The Authors. Published by Elsevier B.V. on behalf of African Institute of Mathematical Sciences / Next Einstein Initiative. This is an

open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ )

E.B. Joel, S.G. Mafulul, H.E. Adamu et al. Scientific African 10 (2020) e00608

phenolic pollutants. The approach of transforming waste from one source into a useful bio-

catalyst that can potentially be exploited to treat waste pollutants from a different source

offers a chain of green technology.

© 2020 The Authors. Published by Elsevier B.V. on behalf of African Institute of

Mathematical Sciences / Next Einstein Initiative.

This is an open access article under the CC BY license

( http://creativecommons.org/licenses/by/4.0/ )

Introduction

One of the major environmental challenges, facing the world today is pollution, which is the contamination of soil, wa-

ter, and air by toxic chemicals [1–4] Phenol and azo dyes are hazardous pollutants released from industrial effluents such as

textile, leather, food, and cosmetic, petroleum/petrochemical pose a threat to the environmental safety [5] . In Nigeria, azo

dye residues, phenol, and other phenolic derivatives arising anthropogenic practices such as industrial activities, petroleum

and petroleum derivatives (such as gasoline, diesel, and kerosene spills), extensive use of pesticides/herbicides in modern

agriculture, and extensive use of synthetic azo dyes as a colorant in the food and textile industries constitute an impor-

tant environmental concern to human health [3 , 6] . Due to the poor wastewater treatment system in Nigeria, this effluent

containing harmful organic pollutants are usually discharged untreated or partially treated in the mainstream of water re-

sources or land sites [7–10] . And even at low concentrations, the azo dye residues and phenolic pollutants can persist in the

environment for long which becomes noxious to terrestrial and aquatic life and in turn, affects human health [2 , 11] . There-

fore, the treatment of industrial effluents containing reactive azo dyes and other phenolic pollutants has become necessary

before they can be discharged into the ecosystem [11] . Numerous other physicochemical methodologies have been utilized

in the post-treatment of azo dyes and other phenolic derivatives from industrial effluents, which include coagulation, ad-

sorption, degradation by ozonation reaction, precipitation, chemical degradation, and irradiation [2 , 12] . However, existing

physicochemical methods are usually expensive and commercially unattractive, time-consuming procedures, not capable of

treating a variety of pollutants, and sometimes generate some byproducts that are more harmful than the parent pollutant

thus creating disposal problems [2 , 13–15] . Biological treatment methods of waste pollutants such as microbial and enzyme-

mediated biodegradation provide a cost-effective, eco-friendly alternative to existing physicochemical technologies applied

to treat different kinds of azo dye residues and phenolic pollutants [14 , 15] .

The ability of the microorganism to degrade different azo dyes, phenol, and other aromatic pollutants has been widely at-

tributed to their unique ability to secrete and utilize intracellular oxidoreductive enzymes such as peroxidases [6 , 10] . Hence,

the direct use of extracted peroxidase for biodegradation of poisonous organic pollutants may be a better option because

enzymes are easy to work with and can degrade a wide range of pollutants generating non-toxic products [9 , 16] . Oxidore-

ductive enzymes especially peroxidases Peroxidases are unique biocatalyst with the potential ability to react with a broad

range of organic environmental pollutants (such as azo dyes and phenolic compounds) in the presence of H 2 O 2 , thereby

remove them by precipitation or the cleavage of the aromatic ring structure, transforming them into other nontoxic byprod-

ucts [17–20] . Peroxidases (E.C. 1.11.1.7) are ubiquitous enzymes widely distributed in plants, animals, and micro-organisms

[21 , 22] . They are heme-containing enzymes that utilize hydrogen peroxide as an oxidant to catalyze the oxidation reaction of

broad electron donor substrates (e.g. phenols, aromatic amines, indoles, and sulfonates) [23–25] . Peroxidases have attracted

industrial attention due to its multiple applications which include bioremediation of wastewater such as decolorization of

dyes as effluents of textile industries [26] , and removal of carcinogenic phenolic pollutants from industrial effluents [27 , 28] .

Peroxidases are also applicable in clinical diagnosis and laboratory experiments such as enzyme-linked immunoassay (ELISA)

kits [29 , 30] , preparation of biosensor [31 , 32] treatment of cancer [33] , synthesis of aromatic chemicals and polymeric mate-

rials, and removal of peroxides from foodstuffs in food industries [30 , 34 , 35] .

Peroxidases have been identified as one of the suitable enzymes for the treatment of phenolic contaminants and related

compounds [36] . However, most of the studies carried out using purified HRP usually imposed a high cost [36] . This has

necessitated the search for alternative peroxidases from other cheap and local sources with the capability for biodegradation

of phenolic pollutants from traditional and industrial effluents in developing countries like Nigeria. Although several other

works on the use of peroxidases in this regard have been reported and several attempts have been made to search for local

sources of peroxidases as an alternative to the commercially available peroxidases like horseradish peroxidase [25] , artichoke

peroxidase [37] , and Schizophyllum fungal peroxidase [38] . Given this peroxidase activity has been investigated in a range of

vegetables and fruits such as water Spinach [39] , Broccoli [40] , moringa leaves [41] , oranges [42] , papaya [43] , and cabbage

[44 , 45] . However, the current global system aimed at minimizing competition with fresh foodstuffs like vegetables and fruits

due to growing malnutrition and food insecurity. Hence, rather than resorting to fresh vegetables and fruits for isolation of

peroxidase, exploring waste vegetables and fruits as a source of peroxidase would be a better option.

Cabbage ( Brassica oleracea var. capitata ) is a well-known vegetable that has been widely studied for nutritional value and

bioactive substances [46] . It is one of the major vegetable food crops cultivated on the Jos plateau, Nigeria because of the

near temperate climate. It is extensively consumed in this area and across the globe as a food [47 , 48] . It is usually grown

2

E.B. Joel, S.G. Mafulul, H.E. Adamu et al. Scientific African 10 (2020) e00608

and harvested in large quantities to supply other parts of the country. Most often the supply for perishable vegetables like

cabbage usually exceeds its demand, which leads to large amounts being rotten and wasted due to poor storage systems.

Furthermore, over 60 percent of the global total food losses and wastages are from fruits and vegetables and this is common

in developing countries like Nigeria due to poor market chain and storage facilities [49] . Due to poor waste management

strategies, the vegetable and fruit wastes are incriminated for a high quantity of pollution and constitute a source of envi-

ronmental nuisance in the municipal of developing countries like Nigeria [50] . Though Government policies are being put in

place for adequate storage facilities to prevent these spoilages and wastages but yet to be fully implemented. Hence, consid-

ering other possible ways for the utilization of these wastes for the production of valuable products like peroxidase enzyme

has become necessary. Peroxidase from fresh cabbage leaves has been characterized and tested for decolorization some syn-

thetic dyes [44 , 45 , 51] in an attempt to search for a cost-effective alternative to the commercially available peroxidases such

as horseradish peroxidase [25] , artichoke peroxidase [37] , and Schizophyllum fungal peroxidase [38] . Even though, isolation

of peroxidase from cabbage and their application for decolorization dyes are known. However, fresh cabbage may not be a

viable source because all effort s have to be made to minimize competition with food consumption.

It has been reported many fruit and vegetable wastes contain several exogenous enzymes many other re-usable products

of high value with different industrial applications, with adequate technology, such agro-waste residual matter can be con-

verted into cost-effective commercial products [52 , 53] . And in Nigeria cabbage and other vegetables usually rot/decay and

become waste in the market due to lack of storage facilities. Therefore, exploring the utilizing of such agro-waste residual

matter as a potential source for extraction re-usable substances of high value (particularly enzymes like peroxidases) could

be a more cost-effective source for value-added peroxidase enzymes and would have the potential for industrial application

such as wastewater treatment. Considering the waste cabbage as a source of peroxidase could be more viable because it

is not in competition with food consumption and is a way of recycling agro-waste pollution that constitutes a municipal

environmental nuisance. This study attempts to explore the discarded decaying waste cabbage as a better alternative to

fresh cabbage as a potential cost-effective source of peroxidase. Therefore; this work was design to isolate, characterized

the biochemical properties of waste cabbage peroxidase, and testes its potential ability to biodegrade azo dyes and phenol

from aqueous solution. This kind of study will provide evidence for exploration of agro-based waste as a source of useful

products like peroxidase enzyme that can be applied for actual and large-scale treatment of industrial effluents containing

azo dyes and phenolic pollutants. This approach offers a chain of green technology since waste from one source is being

transformed into a useful biocatalyst for waste treatment from another source [36] .

Materials and methods

Materials and reagents

The decaying waste cabbage ( Brassica oleracea var. capitata ) was collected from Farin Gada vegetable Market, Jos, Plateau

State, Nigeria. Ammonium sulfate, Ciocalteu reagent, bovine serum albumin, ethylene diamine tetraacetic acid (EDTA), ace-

tone, urea, substrates O-dianisidine, guaiacol and 2, 2 ′ -Azino-bis (3-Ethylbenzthiazoline-6-Sulfonic Acid) [ABTS], Sephadex

G-75, azo citrus red 2, azo purple, azo yellow 6, and azo blue 5, and phenol were procured from Sigma Aldrich. All these

and other chemicals used in this study were of analytical grade and obtained from commercial sources.

Extraction of waste cabbage peroxidase

Extraction of crude peroxidase from waste cabbage

Peroxidase was extracted from waste cabbage leaves using the method of [54] with slight modifications. Waste cabbage

leaves were weight (50 g) and homogenized with 200 ml of 0.1 M Tris-HCl buffer, pH 7.5 for 10 min. The homogenate was

filtered with a clean cheesecloth arranged in two layers and the filtrate was subjected to centrifugation using a refrigerated

centrifuge (4 °C) at 10,0 0 0 rpm for 15 min. The supernatant was carefully collected and filtered into a clean tube through

Whatman No. 1 filter paper and the clearer filtrate was used as crude homogeneous waste cabbage peroxidase (WCP).

Thermal treatment of crude extract of waste cabbage peroxidase

The extracted crude waste cabbage peroxidase was incubated at 65 °C for 5 min using a water bath and cooled on ice

for 25 min to selectively inactivate any contaminating traces of catalase moieties in the crude homogeneous sample.

Peroxidase assay and protein determination

The total protein concentration was determined by the Lowry method [55] using Folin’s Ciocalteu phenol reagent with

graded concentrations of bovine serum albumin (BSA) as the standard. The straight-line equation of the plot of the net

absorbance values at λ= 595 nm versus the concentrations of BSA was used to determine the protein concentration of the

unknown sample(s).

Peroxidase activity was assayed via time course spectrophotometric, rate determination using ABTS as substrate according

to the method of [56] with minor modifications. An aliquot of 2.7 ml of 0.1 M Tris-HCl buffer solution (pH 7.5) 100 μl

of crude enzyme extract and 100 μl of a substrate (3.0 mM ABTS) were pipetted into clean cuvettes. The reaction was

3

E.B. Joel, S.G. Mafulul, H.E. Adamu et al. Scientific African 10 (2020) e00608

initiated by the addition of 100 μl 3% of hydrogen peroxide and the increase in the absorbance was monitored using UV–VIS

spectrophotometer (model-CHEBIOS s.r.l., Rome, Italy), as the amount of ABTS •+ radical produced at the 20-second interval

for 3 min (as a function of peroxidase activity) at 316 nm ( Ɛ416 nm = 36 mM

−1 cm

−1 ). The absorbance values shown were

zeroed with those obtained in reactions that did not include the partially purified WCP.

The corresponding change in absorbance values was used to calculate peroxidase activities ( Table 1 legend for conver-

sions formula). Peroxidase activity is, therefore, defined as the amount of ABTS substrate converted to ABTS •+ radical (prod-

uct) per minute.

Partial purification of crude peroxidase from waste cabbage

Ammonium sulfate precipitation of crude waste cabbage peroxidase and dialysis

The principle of ammonium sulfate precipitation is that at higher salt concentrations, protein solubility usually decreases,

leading to precipitation which is termed salting-out. Graded concentrations of ammonium sulfate salt that correspond to 40-

90% was added to the crude WCP and subjected to stirring for complete precipitation and allowed to stay for about 4 h in

the fridge. The resulting precipitate was collected by centrifugation at 40 0 0 rpm for 15 min at 4 °C and pellets were re-

dissolved in a small amount of extraction buffer- 0.1 M Tris-HCl buffer solution (pH 7.5). Each of the individual percentage

saturation was then analyzed successively and the concentration with the highest activity was subjected to further purifica-

tion.

Dialysis through a semi-permeable membrane dialysis tubule is usually carried out after salting out to separate the

protein enzyme from salt and other small molecules. The re-suspended pellets obtained from 75% saturation was poured in

a dialysis tubule sealed securely and dialyzed against 0.1 M Tris-HCl buffer solution (pH 7.5) by constant magnetic stirring

for 12 h with 4 h interval for change of the extraction buffer. The dialyzed WCP was used for further purification.

Gel filtration chromatography

The dialyzed WCP was subjected to further purification by gel filtration chromatography using Sephadex-G-75 as a col-

umn. The glass column having an inner diameter of 1.5 cm was packed with a column of 15 cm height. The 2 ml of dialyzed

peroxidase was loaded on the column and eluted with phosphate buffer at pH 7. Fractions of purified enzyme were collected

at a flow rate of 1 ml per tube and the peroxidase activity with protein concentrations were determined as described ear-

lier in section 2.3 . The fractions with significant activities were pooled together and used as the purified WCP for a further

experiment involving biochemical characterization of WCP properties and potential application in biodegradation of phenol

and azo dyes.

Biochemical characterization of partially purified waste cabbage peroxidase properties

Determination of the effect of pH on waste cabbage peroxidase activity and stability

To determine the optimum pH of WCP, peroxidase activity was assayed for at different pH values. The Reaction mixture

contained 3% of H 2 O 2 , 0.1 M buffers of varying pH (2–9), enzyme, and 3 mM ABTS carried out for 3 min (change in ab-

sorbance measured at 20-second intervals). To achieve this, different buffers of uniform concentration (0.1 M) were prepared

and used as assay buffers and these include a glycine-HCl buffer (pH 2.0 to 5.0), phosphate buffer (pH 6 to 7), and Tris-HCl

(pH 8.0 to 10). To determine the pH stability the residual peroxidase activity was assayed after 24 hours incubation at room

temperature in a series of assay buffers with varying pH varying from 2.0 to 9.0. Thereafter peroxidase activity was assayed

as usual ( see Section 2.3 ).

Determination of the effect of temperature on waste cabbage peroxidase activity and stability

To determine the optimum temperature of WCP, peroxidase activity was assayed at varying temperatures (10 to 90 °C) in

a reaction mixture containing 3% of H 2 O 2 , 0.1 M Phosphate buffer solution (pH 6.0), enzyme, and 3 mM ABTS carried out

for 3 min (change in absorbance measured at 20-second intervals). The temperature was regulated by using a water bath.

The thermal stability of the waste cabbage peroxidase was determined by incubating the enzyme without the substrate at

50 °C, 60 °C, 70 °C, and 80 °C for 1 hour and then cooled on ice for 5 min. After cooling peroxidase activity was assayed as

usual ( see Section 2.3 ).

Kinetic constants/substrate specificity of waste cabbage leaves peroxidase

To determine the kinetic parameters (K m

and V max ) of the WCP, peroxidase activity was assayed at varying concentrations

(1.0–10 mM) of three well-known peroxidase substrates (ABTS/guaiacol/O-dianisidine) with a suitable amount of purified

enzyme, and 0.1 M Phosphate buffer (pH 6.0). Reactions were initiated by the addition of 3% of H 2 O 2 and absorbance

at 416 nm was monitored for 3 min (at 20-second intervals) and converted to peroxidase activities ( Table 1 legend for

conversions formula). The reciprocal of peroxidase activity and substrate concentrations were plotted (Lineweaver-Burk plot)

and the kinetic parameters of the partially purified peroxidase for the three substrates were calculated from the equation of

the straight line of the Lineweaver-Burk plots as follows:

1 / V= ↑

Y=

( KM / Vmax ) × ↑

M ×

(1 / [S])+ ↑

X+

(1 / Vmax )

↑

C

( Lineweaver − Burk equation ∗) ( Equation of straight line )

4

E.B. Joel, S.G. Mafulul, H.E. Adamu et al. Scientific African 10 (2020) e00608

Where: K m

= Michaelis Menten constant for a particular substrate, V = Enzyme activity (Initial rate of reaction),

V max = Maximum velocity (maximum rate of reaction) obtained for a particular substrate concentration, pH and tempera-

ture, and [S] = concentration of substrate.

Determination of the effect of chemicals and metal ions on waste cabbage peroxidase activity

The effect of divalent metal ion, EDTA, acetone, and urea on peroxidase activity was determined by pre-incubating the

enzyme with either divalent metal ion (Mg 2 + , Fe 2 + , Zn

2 + , Co 2 + , Ni 2 + ), EDTA, acetone, or urea to a final concentration of

5 mM for 30 min at room temperature. Thereafter peroxidase activity was assayed for as usual (see Section 2.3 ). The perox-

idase activity in the absence of divalent metal ion, EDTA, acetone, and urea were taken as the control experiments.

Application waste cabbage peroxidase in biodegradation of phenol and synthetic dyes

Waste cabbage peroxidase mediated decolorization of synthetic dyes

The four tested synthetic azo dyes were selected for this study based on the availability at the time of purchase. The

aqueous solution of each azo dye was prepared to a uniform concentration of 5 mM. To determine the maximum wavelength

for each dye, the prepared solution of each dye was scanned using a UV/Visible Spectrophotometer (200–850 nm range).

Thereafter, the initial absorbance was obtained for different dyes (azo citrus red 2, azo purple, azo yellow 6, and azo blue 5)

after the addition of buffer to each of the dye solutions.

The influence of WCP on the decolorization of azo dyes was investigated at the optimum reaction condition of the charac-

terized WCP and maximum wavelength for each dye. The reaction mixture consisted of a fixed concentration of dye, partially

purified enzyme, 100 mM phosphate buffer, pH 5.5, and 3% H 2 O 2 . The reaction mixtures were incubated at 30 °C, and the

final absorbance readings were taken at varying time intervals (30 min, 1, 24, and 48 h). The percentage of decolorization

was thus calculated as follows:

Percentage Decolorization =

A i −A f

A i

∗ 100

Where A i = initial absorbance before decolorization.

A f = final absorbance after incubation.

Note that all reactions with partially purified WCP were carried out at optimum conditions of the enzyme obtained from

the biochemical characterization to guarantee the high efficiency of dye decolorization.

Waste cabbage peroxidase mediated biodegradation of synthetic phenol

The effectiveness of the purified WCP biodegradation/removal phenol was tested. Phenol concentrations were quantified

at the initial and final stages using the 4-aminoantipyrene (4-AAP) method. The standard curve for pure phenol samples

without any enzyme was prepared. Experiments were carried out in 75 ml beakers. Varying volumes of partially purified

waste cabbage peroxidase and a particularly fixed concentration of H 2 O 2 and phenol (10 mg/L) were added into the phos-

phate buffer (pH 6.0). The mixture was shaken vigorously and allow to stand for 60 min at room temperature. Thereafter,

4.0 ml of 0.25 M sodium bicarbonate and 0.9 ml of 20.8 mM 4-aminoantipyrene were added and shaken vigorously, then

0.9 ml of 83.4 mM potassium ferricyanide was added, mixed by shaking again and allowed to stand for 9 min. Absorbance

was measured at 510 nm using an ultraviolet-visible (UV–VIS) spectrophotometer and converted to concentration using the

calibration curve. The efficiency of phenol removal (% removal) was thus calculated as follows:

% Phenol removal = ( C initial − C final ) / C initial ∗ 100

Where C initial = initial concentration (mg/L) and C final = final phenol concentration (mg/L).

Data/Statistical analysis

All data were analyzed using Microsoft Office (Excel) and values represent the means of results from three replicate

experiments.

Results

Purification of waste cabbage peroxidase

Peroxidase from waste cabbage leaves was purified to homogeneity by ammonium sulfate salting out, dialysis, and gel

filtration chromatography. The result of ammonium sulfate precipitation showed maximum peroxidase activity at 75% pre-

cipitation. The elution profile of the waste cabbage peroxidase purification scheme is as shown in Fig. 1 . The results obtained

for the degree of purity of WCP at each purification step are summarized in Table 1 . The proteins were eluted and five major

peaks (F4, F5, F6, F7and F-8) and three minor peaks (F1, F2, and F3) which indicate the presence of more than one protein.

It was found that only three major peak fractions in the same region contain peroxidase activity and the three active peaks

were pooled and used as purified WCP for biochemical characterization WCP and its effectiveness in the biodegradation of

5

E.B. Joel, S.G. Mafulul, H.E. Adamu et al. Scientific African 10 (2020) e00608

Fig. 1. Gel filtration chromatographic elution profile of waste cabbage peroxidase purification on the Sephadex G-75 column. The dialyzed fraction was

loaded on the Sephadex G-75 column pre-equilibrated with elution buffer 100 mM Tris-HCl buffer, pH 7.5. The protein elution profile was monitored at

280 nm.

Table 1

Summary of purification steps the degree of purity recorded for waste cabbage peroxidase.

Purification step Total enzyme Activity (U) Total Protein (mg) Specific activity (Umg −1 ) Recovery (%) Purification fold

Crude extraction 764.66 438.34 1.74 100.00 1.00

(NH 4 ) 2 SO 4 Precipitation 432.00 63.81 6.77 56.50 3.88

Dialysis 329.00 9.76 33.71 43.03 19.32

Gel filtration (Sephadex G-75) 267.00 1.75 152.89 34.92 87.65

phenol and azo dyes. This study recorded 87.62 with a high 34.92% as purification fold and purification yield respectively

for the purified waste cabbage peroxidase.

The above parameters were calculated as follows;

Peroxidase Activity ( U / ml ) =

�A / min × V × Df

36 × v × d

Where; �A/min. = Change in absorbance per minute, V = Total reaction volume (3 ml), Df = dilution factor, v = Volume

of enzyme source (0.1 ml), d = Lightpath (1 cm), 36 mM

−1 .cm

−1 = is micromolar extinction coefficient of ABTS at 416 nm.

Specific activity (U/mg): measure of enzyme’s purity = Enzyme activity (U/ml) /Total protein (mg/ml)

The percentage yield of a step = Total units of purified enzymes/Total units of crude enzymes

Purification fold (Measure of how effective the step is.) = specific activity purified enzyme/ specific activity crude en-

zymes.

Biochemical characterization of partially purified waste cabbage peroxidase

Effect of pH on activity and stability of waste cabbage peroxidase

The results of pH on peroxidase activity showed that the partially purified waste cabbage peroxidase exhibited high

activity between pH 3.5–6.5 reaching optimal at around pH 5.5 ( Fig. 2 ). To determine the pH stability of WCP, the residual

activity was carried out with ABTS as substrate, after 24 h incubation at room temperature in a series of buffers of varying

pH values ranging from pH 2.0 to 9.0. The result of the pH stability experiment suggests that the partially purified peroxide

was stable over a broad range of pH (4.0 −7.0) ( Fig. 2 ).

Effect of varying temperature on the activity of waste cabbage peroxidase

The partially purified peroxidase from waste cabbage showed an optimum temperature of 30 °C ( Fig. 3 ). A rapid and

progressive increase in peroxidase activity with an increase in temperature, and reaches a peak at a temperature of 30 °C.

The sharp decline in peroxidase activity with as the temperature progresses beyond 30 °C with a near or total loss of activity

at a temperature of 60–90 °C.

6

E.B. Joel, S.G. Mafulul, H.E. Adamu et al. Scientific African 10 (2020) e00608

Fig. 2. The effect of varying pH on the activities of waste cabbage peroxidase. The change in A416 was converted to peroxidase activity (see Table 1 legend)

and expressed as relative activity (percentage) taking optimum activity as 100%.

Fig. 3. The effect of varying temperature on the activity of waste cabbage peroxidase.

Thermal stability of waste cabbage peroxidase

The thermal stability of waste cabbage peroxidase is as shown in Fig. 4 . After incubation at 50, 60, 70, and 80 °C for 3 h,

the results showed that waste cabbage leave peroxidase was highly stable at the tested temperatures ( Fig. 4 ) with up to 41%

original activity retained at 80 °C after 3 h incubation.

Substrate specificity and kinetics studies of waste cabbage peroxidase

To determine the substrate preference and kinetics of WC, peroxidase activity was assayed for at varying concentrations

(1.0–10 mM) of three tested substrates (O-dianisidine, guaiacol). Figs. 5 , 6 , and 7 showed the Lineweaver-Burk plot using

ABTS, guaiacol, and O-dianisidine respectively. Findings from this study showed that the maximum velocity (V max ) of ABTS

by waste cabbage peroxidase was highest followed by guaiacol and O-dianisidine with the least ( Table 2 ). On the other hand,

Km values follow the reverse order O-dianisidine > guaiacol > ABTS. This trend of Km values suggests that the affinity of the

partially purified enzyme towards the tested substrates follows this trend; ABTS > guaiacol > O -dianisidine.

7

E.B. Joel, S.G. Mafulul, H.E. Adamu et al. Scientific African 10 (2020) e00608

Fig. 4. Thermal stability of waste cabbage peroxidase. Peroxidase activity was expressed as residual activity (percentage) taking WCP activity without the

pre-incubation for 1 hour as 100% (control).

Fig. 5. Analysis of the effects of ABTS on the activities of waste cabbage peroxidase. (A) . A plot of enzyme activity versus ABTS concentrations. (B) .

Lineweaver-Burk plot of ABTS hydrolyzes catalyzed by waste cabbage peroxidase. The reciprocal of peroxidase activity and substrate concentrations were

calculated (Lineweaver-Burk plot). 8

E.B. Joel, S.G. Mafulul, H.E. Adamu et al. Scientific African 10 (2020) e00608

Fig. 6. Analysis of the effects of Guaiacol on the activities of waste cabbage peroxidase. (A) . A plot of enzyme activity versus Guaiacol concentrations.

(B) . Lineweaver-Burk plot of Guaiacol hydrolyzes catalyzed by waste cabbage peroxidase. The reciprocal of peroxidase activity and substrate concentrations

were calculated (Lineweaver-Burk plot).

Table 2

Kinetic parameters of waste cabbage peroxidase.

The K m and V max values were calculated from the

equation of the straight line of the Lineweaver-Burk

plots ( Figs. 5 , 6 , and 7 ) (see section 2.9 for the

translated formulae and equation).

Substrate Kinetic Parameters

K m (mM) V max (mM/min.)

O-dianisidine 19.24 588.24

Guaiacol 17.82 909.09

ABTS 1.24 1111.11

Effects of metal ions, organic solvent (acetone), and chemicals (EDTA and urea) on waste cabbage peroxidase

To determine the effects of metal ions, acetone, EDTA, and urea on peroxidase activity, the WCP was pre-incubating with

an individual divalent metal ion, EDTA, acetone, and urea a final concentration of 5 mM for 30 min at 30 °C; the perox-

idase activity in the absence of metal ion, EDTA and acetone was taken as the control. Table 3 showed the effect of the

divalent metal ions (Hg 2 + , Zn

2 + , Cu

2 + , Mn

2 + , Ni 2 + ), acetone, and chemicals (EDTA and urea) in cabbage waste peroxidase.

The result suggests that the tested three metal ions, Hg 2 + , Cu

2 + , Ni 2 + and, organic solvent and chemicals exerted an in-

9

E.B. Joel, S.G. Mafulul, H.E. Adamu et al. Scientific African 10 (2020) e00608

Fig. 7. Analysis of the effects of O-dianisidine on the activities of waste cabbage peroxidase. (A) . A plot of enzyme activity versus O-dianisidine concen-

trations. (B) . Lineweaver-Burk plot of O-dianisidine hydrolyzes catalyzed by waste cabbage peroxidase. The reciprocal of peroxidase activity and substrate

concentrations were calculated (Lineweaver-Burk plot).

Table 3

Effects of metal ions, organic solvent (acetone), and chemicals (EDTA and urea)

on waste cabbage peroxidase. Residual peroxidase activities (%) were calculated

as = [(peroxidase activity in the presence of metal ion, EDTA, acetone, urea /the

peroxidase activity in the absence of metal ion, EDTA, acetone, urea)] ∗100.

Reagents (5 mM) Peroxidase activity (mmole/min.) Residual activity (%)

Control (none) 2.755 100

Hg 2 + 2.02 73.3

Mn 2 + 3.175 115.2

Zn 2 + 2.835 102.9

Cu 2 + 1.515 55.0

Ni 2 + 2.23 80.9

EDTA 2.65 96.2

acetone 1.76 63.9

Urea 1.51 54.8

10

E.B. Joel, S.G. Mafulul, H.E. Adamu et al. Scientific African 10 (2020) e00608

Fig. 8. Percentage decolorization of various synthetic dyes by waste cabbage leaves peroxidase.

Table 4

Effect of waste cabbage peroxidase on the degradation of phenol. The phenol concentrations were estimated using the equation of

the straight line of the calibration curve for phenol ( Fig. 9 ). See section 2.11.2 for the calculation for percentage phenol degradation.

Initial phenol conc.

(mg/L)

Partially purified cabbage

peroxidase (mL)

Final Phenol

conc. (mg/L)

Residual Phenol conc.

(%)

% Phenol degradation

or removal

10 0 9.9 100.0 1.2

10 1 6.2 62.6 38.2

10 2 4.0 40.4 60.1

10 4 1.5 15.4 84.8

10 8 0.9 14.4 91.1

hibitory effect. The peroxidase activity was slightly enhanced by Mn

2 + and Zn

2 + with residual activity of 115.2% and 102.9%

respectively.

Application waste cabbage leaves peroxidase in biodegradation of phenol and synthetic dyes

Waste cabbage peroxidase mediated decolorization of synthetic dyes

The results showed that the absorbance peaks of the tested dyes were recorded at the following wavelengths alone azo

Yellow 6 (534 nm), azo Citrus Red 2 (525 nm), azo Purple (268 nm), and azo Blue 5 (648 nm). To investigate the abil-

ity of partially purified waste cabbage leaves peroxidase to decolorize different types of hazardous dyes, citrus red, azo

purple, azo yellow, and azo blue after 30 min, 1 hour, 24 h, and 48 h. The results showed that the waste cabbage perox-

idase was able to decolorize all the tested dyes at varying degrees ( Fig. 8 ). The result suggests that the partially purified

waste cabbage peroxidase was very efficient in the decolorization of azo dyes. It was observed that the% decolorization

of all the tested azo dyes increased with an increase in the incubation period. The extent of decolorization achieved with

different classes of dyes followed this trend, azo blue 5 (85.1) > azo purple (69.1) > azo yellow 6 (46.2) > azo citrus red

2 (42.9).

Waste cabbage peroxidase mediated phenol degradation

The calibration curve of the phenol standard ( Fig. 9 ) was used to quantify the residual phenol concentration after treat-

ment with WCP. The sharp increase in phenol conversion as the volume of the partially purified waste cabbage peroxidase

was increased ( Table 4 ). The optimum reaction conditions for waste cabbage leaves peroxidase were used to guarantee the

high efficiency of phenol degradation.

The result suggests that the partially purified waste cabbage leaves peroxidase was very efficient in the degradation of

phenol with over 60% phenol degradation observed after treatment with ≥ 2 ml of waste cabbage peroxidase.

11

E.B. Joel, S.G. Mafulul, H.E. Adamu et al. Scientific African 10 (2020) e00608

Fig. 9. Calibration curve of phenol standard. The plot of the corresponding absorbance values against the concentrations of phenol (calibration curve) was

used to extrapolate the residual phenol concentration after treatment with WCP.

Discussion

Peroxidase from waste cabbage was extracted, partially purified, characterized biochemical properties, and its potential

to biodegrade phenol and synthetic azo dyes from aqueous solution evaluated. Previously studies revealed that peroxidases

from different sources have variable optimum temperature and pH. The 30 °C optimum temperature for waste cabbage leaves

peroxidase reported in this work agrees with the previous report of Abbas [57] for fresh cabbage leaves peroxidase, Broccoli

( Brassica oleracea l. Var. Italica ) Stems peroxidase [40] , Jatropha curcas leaves peroxidase [58] which all displayed optimum

activity at 30 °C. [59] , also reported an optimum temperature range around 25- 40 °C for garlic ( Allium sativum ). Peroxidases

purified from other sources, however, have relatively higher optimum temperatures such as Calotropis Procera leaves peroxi-

dase [60] and M. oleifera leaves peroxidase [41] . Results for thermal stability profiles suggest that waste cabbage peroxidase

is a thermostable enzyme up to 80 °C, with 41% original activity retained after 3 h incubation. Thermal stability decreases as

the temperature increases. The residual peroxidase activity reported for date palm leaves ( Phoenix dactylifera L.) peroxidase

was higher than the observed 15% for this study at 80 °C after 60 min incubation period [61] . Previous findings have shown

that the inactivation of peroxidases at higher temperatures is likely to be a result of the unfolding of the tertiary structure

enzyme [61] .

The optimum pH and stability of WCP were comparable to that of peroxidase of date palm leaves ( Phoenix dactylifera L.)

[61] . Previous other studies also suggest that most peroxidases isolated from different sources exhibit optimum activity

in the pH range of 4.5- 6.5 [62–64] . Findings from this study showed a sharp decrease in extreme acidic and alkaline

pH peroxidase activity and stability. The pH usually affects the ionic state of the side chain of the enzyme’s amino acids.

Therefore, the effect at pH on the peroxidase activity and stability could be due to changes in the ionic state of amino acids

side chain at the active site which invariably affects heme-binding at low pH. Also, a decrease in activity and stability at

high and low pH values could be as a result of ionic changes in the heme group [61 , 65] .

The inhibitory effects of the tested metal ions (Hg 2 + , Cu

2 + , Ni 2 + ) and chemicals follow a similar trend with earlier reports

of [66] and [67] for Calotropis procera leaves peroxidase and Moringa oleifera leaves peroxidase respectively. EDTA is a well-

known chelating agent. This inhibitory effect exerted by EDTA could be by chelating iron (ii) atom (Fe 2 + ) at the active center

of the enzyme [68] . The inhibitory effect of Hg 2+ and other metal ions such as Cu

2 + , Ni 2 + may be as a result of binding to

SH groups present in the actives side of the enzyme thereby causing irreversible inactivation [69] . The activation of partially

purified peroxidase activity by Mn

2 + and Zn

2 + is in agreement with the previous reports of Al-Senaidy and Ismael [70] for

date palm leaves ( Phoenix dactylifera L .) peroxidase. Also, this enzyme is fairly stable in the presence of an organic solvent,

acetone, which further widens the applicability of waste cabbage leaves peroxidase for the treatment against a variety of

organic pollutants present in industrial and crude oil spilled wastewaters.

The kinetic data revealed that waste cabbage leaves peroxidase obeyed first-order reaction kinetics. The high turnover

rate and low K m

value of the partially purified waste cabbage leaves peroxidase towards ABTS follows a similar trend with

the substrate specificity result of this study that ABTS is the best substrate followed by guaiacol then O-dianisidine. Lower

Km values suggest that the enzyme has a high apparent affinity toward a substrate [61] . Although the K m

values were

higher than the ones reported for spring cabbage peroxidase [71] , for date palm leaves ( Phoenix dactylifera L .) peroxidase

12

E.B. Joel, S.G. Mafulul, H.E. Adamu et al. Scientific African 10 (2020) e00608

[61] and Moringa oleifera leaves peroxidase [41] . However, high K m

values were reported for peroxidases from Calotropis

procera leaves [66] , Zea mays L waste [72] , garlic Allium sativum [73] wheat ( Triticum aestivum ssp. vulgare) [74] .

Phenol, substituted-phenol derivatives in azo dyes constitute hazardous compounds found in wastewaters of a wide va-

riety of industries [75] . Peroxidases have been reported to decrease environmental pollution via oxidation degradation phe-

nols, cresols and chlorinated phenols, and synthetic textile azo-dyes present in industrial effluent [18 , 76] . Enzymatic treat-

ment of phenolic pollutants is usually by the transformation of total phenol concentration into less biodegradable polymeric

compounds that could be removed by coagulation [77] . Findings from this study have shown significant potential and ca-

pacity of waste cabbage peroxidase for biodegradation of phenol from aqueous solution at the laboratory scale. The high

efficiency of this enzyme in phenol removal observed in this study is under optimum reaction conditions for the enzyme

obtained from the characterization studies. The reduction in phenol concentration with an increase in the volume of the

partially purified WCP indicates that it is caused by peroxidase oxidation of phenol. [78] , also reported on the role of perox-

idase as an enzymatic method for the removal of phenol from industrial effluent. However, the% removal of phenol by waste

cabbage leaves peroxidase was slightly lower than the one’s reports by [79] for horseradish ( cochlearia armoracia l) peroxi-

dase and [80] for immobilized turnip peroxidase. The slightly higher% removal of phenol in the previous reports compared

to this could be due to immobilization, presence of polyethylene glycol, or longer reaction period.

The rate of dye decolorization by waste cabbage leaves peroxidase varies due to the nature of the tested dyes. The high

efficiency of decolorization azo dyes as seen in this study was also reported by [81] for peroxidase partially purified from

garlic . After 4 h of incubation with Momordica charantia peroxidase, 23% decolorization of tannery effluent dyes was also

reported by [82 , 83] , reported 90% decolorization of naphthol blue after 5minutes by horseradish peroxidase. Husain et al.

[19] , also reported 85% decolorization of textile effluent dyes fenugreek peroxidase after 5 h of incubation. Therefore, the

variations in the time course of removal of these dyes as reported by various researchers might be due to the structural

barrier and electron localization among the dyes and the level of purification and concentration of peroxidase used for

decolorization.

The future research perspectives of this work

This study successfully demonstrated the possibility of recycling decaying waste cabbage contributing to an environ-

mental nuisance as a cost-effective source of a valuable enzyme, peroxidase that could potentially be explored for the

biodegradative treatment of toxic dye and phenolic pollutants present in industrial effluents such as oil spilled contaminated

water and soil in the Niger Delta of Nigeria. Nevertheless, to exploit the WCP for large-scale practical industrial application,

further research may focus on the following:

i Advanced purification of WCP, immobilization, and optimization of degradation reaction conditions to better understand

the factors affecting the performance of this enzyme in biodegradation of dyes and phenolic pollutants.

ii Analysis of degraded products of azo dyes and phenol by TLC, HPLC, FTIR, and GC–MS to ascertain the possible mecha-

nism of WCP mediated biodegradation.

iii Employ WCP for biodegradation of dyes and phenols from actual industrial effluent as well as the assessment of toxicity

of the degraded products and chemical oxygen demand (COD) and biological oxygen demand (BOD), and total oxygen

capacity (TOC) during treatment to understand the applicability of the developed process.

Conclusion

The present study demonstrated the prospect of transforming a heap agro-based waste in the agro market that consti-

tutes a source of environmental nuisance into a valuable product like peroxidase enzyme that could be deployed for biore-

mediation of industrial effluent. This study successfully extracted and partially purified peroxidase from a readily abundant

waste cabbage. Characterization of biochemical properties that the partially purified enzyme had its optimum activity at

temperature 30 °C, pH 5.5 while showing broad substrate preference. The WCP was stable over a wide range of pH (4.0 −7.0)

and its ability to retained 41% of its original activity at 80 °C indicates that it is a thermostable enzyme. The kinetic data

of WCP showed K m

values of 1.24, 17.89, and 19.24 mM and Vax values of 1111.11, 909.09, and 588.24 mM /minutes for

ABTS, guaiacol, and o-dianisidine respectively. Three metal ions, Hg 2 + , Cu

2 + , Ni 2 + , organic solvent (acetone), EDTA, and urea

inhibited peroxidase activity; whereas Mn

2 + and Zn

2 + showed slight activation. The partially purified WCP exhibited high

efficiency for the biodegradation of the tested synthetic azo dyes and phenol at the lab-scale. After 48 h of incubation, the

waste cabbage peroxidase efficiently catalyzed the decolorization of the different dyes such as azo blue 5, azo purple, azo

Yellow 6, and citrus red 2, with a percentage decolorization of 85.1, 69.1, 46.2 and 42.9%, respectively. The waste cabbage

peroxidase also shows up to 91.1% efficiency for degradation of phenol in aqueous solution after 60 min of reactions. The

significant increase in the% degradation of tested azo dyes and phenol from aqueous solution with an increasing volume of

the WCP suggests that this enzyme was responsible for the observed changes. Findings from this study provide promising

evidence on the possibility of utilizing waste cabbage as a good source peroxidase with the ability to biodegrade azo dyes

and phenol at a small scale in the laboratory. This study, therefore, increases the possibility of recycling waste cabbage and

other agro-waste for isolation of peroxidase as well as other bioproducts that can be useful for the treatment of industrial

effluents containing dye and phenolic pollutants.

13

E.B. Joel, S.G. Mafulul, H.E. Adamu et al. Scientific African 10 (2020) e00608

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have

appeared to influence the work reported in this paper.

Funding

This research was not supported by any funding source.

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