Isolation and purification of peroxidase from soyabean

Isolation and Purification of Peroxidase from soyabean

by

Pooja Walke

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.

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.

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.

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.

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:

•

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).

Observations and Graphs • Molecular weight of peroxidase enzyme: 46 kg/Dalton

• Biuret test: 1.813 optical density at 540 nm

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

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

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 .

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