Peroxidase (EC. 1.11.1.7), an oxidoreductase, has iron porphyrin ring generally and catalyzes a redox reaction between H202 as an electron acceptor and many kinds of substrates by means of oxygen liberation from HzOz (Brill, 1996).

1. Isolation and Purification
of Peroxidase from
soyabean
by
Pooja Walke

2. Abstract
• Plants and/or plant food wastes have been given much less attention or
evendisregarded. In some instances, however, oxidative enzymes from residual
plant tissueshave been shown to effectively degrade recalcitrant pollutants.
Soybean seed coat peroxidase (SBP) is an inexpensive oxidoreductive enzyme
and could be potentially used to oxidize/polymerize various organic pollutants of
the industrial and petrochemical wastes. The catalytic properties of SBP are
retained under a wide range of pH and at elevated temperatures. In the present
study, the biocatalytic properties of SBP were estimated. The enzyme exhibited
the highest activity and stability at pH 6.0 and retained over 75% of the
maximum activity for 12 hours. The activity of SBP was found to be 2.5 times
higher at an elevated temperature of 65°C compared to the activity at room
temperature. The activity is retained over 95% for 30 min at 75°C. The pH and
temperature of the reaction mixture showed significant influence on SBP
activity. SBP is fairly active in organic solvents and exhibited the optimal activity
in the presence of 20% (v/v) acetone. Increasing the organic solvent content
resulted in a reduction in SBP activity. Molecular weight of peroxidase was
measured 46kg/dalton and the protein was estimated 1.813 by the biuret test.

3. Introduction
• Peroxidase (EC. 1.11.1.7), an oxidoreductase, has iron porphyrin ring generally and catalyzes
a redox reaction between H202 as an electron acceptor and many kinds of substrates by
means of oxygen liberation from HzOz (Brill, 1996). Peroxidase is usually intracellular, as are
the other oxidoreductases (Reed, 1975). The enzyme occurs in plants like radish, soybean
(Ambreen et aI., 2000), tomato (Zia et aI., 2001), potato, turnip, carrot, wheat, pear, apricot,
banana, dates (Reed, 1975), strawberry (Jen et al., 1980) and horseradish (Rehman et al.,
1999). The separation and isolation of peroxidase from leukocyte by Agner (1943) assured
its presence in animal tissue as well. It has also been found in spleen, lungs, mammary and
thyroid glands, bone marrow and intestine (Harris and Loew, 1996).
• Reed (1975) purified this enzyme from Streptococcus faecalis.Peroxidase is a heat stable
enzyme, having wide range of applications in health sciences as a diagnostic tool (Kwak et
al., 1995). It is preferred for preparing Enzyme conjugated antibodies due to it's high
specificity towards certain substances, and hence widely used in Enzyme Linked
Immunosorbant Assays and other sensitive analytical techniques (Barnes et aI.1993). The
oxidative properties of peroxidase have been employed usefully in assay systems to detect
metabolites by the formation of their respective oxidases (Eisenstaedt, 1939; Trinder,
1969). Regardless of its wide use in quality control laboratories of food, pharmaceutical
industry and in enzymological research purposes, no appreciable work has so far been done
in Pakistan to produce commercial peroxidase. Peroxidase and it's kits especially for glucose
estimation are therefore, being imported at a very high cost involving a huge amount of
foreign exchange. Thus there is a need to commercialize and standardize these enzymes
and kits depending upon our own sources and technology to reduce burden on our
economy. The present project was, therefore, designed to purify the enzyme peroxidase
from soybean seeds and to standardize the conditions for glucose estimation from/in
serum.

4. Preparation of Crude Extract:
• Soy bean seeds (100 g) were soaked overnight in distilled water and then
blended with 400 mL distilled water for 15 min. It was centrifuged at 10,000
rpm tor 15 min at 4°C and the supernatant was passed through filter paper
(Ambreen et al., 2000; Zia, 2002). It was heated in water bath at 65°C for 3
min to inactivate catalase present in the extract and cooled promptly by
placing it in ice bucket for 10 min. (Zia, 2002). This extract was subjected to
enzyme assay and protein estimation.

5. Purification of Peroxidase:
• 2.1 Ammonium sulfate precipitation
• The enzyme extract was subjected to (NH4) ZS04 precipitation by the method of Evans (1968) for
salting out of enzyme.
•
• 2.2 Dialysis
• The presence of inhibitors in the crude extract had been removed by the precipitation followed
by. The separation of molecules across the semi permeable membrane is driven by. The
concentration differential between the solution on either side of the membrane and is
constrained by the size (molecular weight) of the molecules relative to the size of the pores within
the membrane.
• For dialysis of peroxidase and many proteins a molecular weight cut off 12000 -14000 is suitable.
• Preparation of dialysis tubing
•
• Cut the tubing into pieces of convenient length (10-20cm)
• Boil the tubing for 10 minutes in a large volume of 2% (w/v) sodium bicarbonate and 1mm EDTA
(pH-8)
• Rinse the tubing thoroughly in distilled water
• Boil the tubing for 10 minute in 1mm EDTA (pH-8)
• Allow the tubing to cool and then store it at 4oC.
• Be sure that the tubing is always submerged.

6. SDS Page:
• Standard proteins and purified enzymes were incubated in 10 % SDS and in
sample buffer for 2 h at 37°C. Staining was performed in staining solution.
Stain was removed and replaced by by destaining solution with frequent
changes. After destaining, gels were stored in 2 % acetic acid.
• 1] 1x SDS gel loading buffer:
• 2] 1x Triss glycine electrophoresis buffer:
• Stacking gel: (5%)
• Resolving Gel: (10%)
• Staining solution:
• Destaining solution:
•

7. Enzyme Assay and Protein
Estimation:
• Peroxidase assay was carried out in a reaction mixture containing 46 mL
phosphate buffer of pH 6, 0.320 mL of H202 (Substrate) and 0.850 mL
guaiacol (chromogen). The absorbance of the coloured complex was read
on spectrophotometer at 470 nm wavelength after 3 min. of reaction
interval (Ambreen et al., 2000). Protein contents of the enzyme extract at all
steps were measured by biuret method (Gornall et aI., 1949).

8. Observations and Graphs
• Molecular weight of peroxidase enzyme: 46 kg/Dalton
• Biuret test: 1.813 optical density at 540 nm

9. Activity of enzyme at
different pH
Sr. no pH Activity
1 4 0.20
2 5 0.30
3 6 0.47
4 7 0.27
5 8 0.20

10. Activity of enzyme at different
temperature:
Sr. no Temerature Activity
1 20 0.12
2 40 0.24
3 60 0.35
4 80 0.20

11. Result and Discussion
• To purify the enzyme, the crude extract was subjected to 50-85% saturation with
(NH4)S04 to remove unwanted proteins. The results of our findings are
presented in Table 2 which shows 3.58 fold, purification. This is the most
commonly used reagent for salting out of proteins because its high solubility
permits the achievement of solution with high ionic strength (Voet et aI., 1999).
Evans (1968) standardized the saturation values of (NH4hS04 as 50-85%, while
Rehman et aI., (1999) purified peroxidase from different seeds with 35-90%
precipitation. The finding of Ambreen et aI., (2000) was 1.21 fold purification for
soybean peroxidase using the same (85%) saturationvalues where as Rehman et
al., (1999) reported the degree of purification as 46 kg/Dalton soyabean
• peroxidase. This difference may be due to grade of salt used for purification or
the concentration of salt and duration of saturation for purification.
• Effect of pH on activity:
• Relative activity is defined as the rate of H2O2 consumption at a particular pH
normalized with respect to the highest rate [20]. Experiments were performed
using different buffer solutions to maintain the identical pH conditions in the pH
ranging from 4 to 8. Maximum activity was at pH 6.0 .

12. References:
• Amisha Kamal, J.K. and Digambar Behere, V., “Steady-state and picosecond
timeresolved fluorescence studies on native and Apo seed coat soybean
peroxidase,” Biochem. Biophys. Res. Commun. 289, 27 (2001).
• Bódalo, A., Gómez, J.L., Gómez E., Hidalgo, A.M., Gómez, M., Yelo, A.M.,
“Removal of 4-chlorophenol by soybean peroxidase and hydrogen peroxide in a
discontinuous tank reactor,” Desalination 195, 51 (2006).
• Cooper, V.A., Nicell, J.A., “Removal of phenols from a foundry wastewater using
horseradish peroxidise,” Water Res. 30, 954 (1996).
• Chance, B., Maehly, A.C., “Enzymatic Assay of peroxidase (EC 1.11.1.7) from
Soybean,” Methods in Enzymology. 2, 773 (1955).
• David, J.S., Agric. Res. 44, 23 (1996).
• Estela da Silva, M., Teixeira Franco, T., “Purification of soybean peroxidase
(Glycine max) by metal affinity partitioning in aqueous two-phase systems,” J.
Chromatogr. B. 743, 287 (2000).
• Fatima, A., Husain, Q., Hasan Khanb R., “A peroxidase from bitter gourd
(Momordica charantia) with enhanced stability against organic solvent and
detergent: A comparison with horseradish peroxidise,” J. Mol. Catal. B:
Enzymatic. 47, 66 (2007).