Soybean And Their Partial Optimization

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02 Nov 2017

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Vivek Kumar, Arif Kamal Khan, Pallavi Chaudhary

School of Biosciences and Technology, VIT University, Vellore-632014, Tamil Nadu, India

[email protected]; +91-94-44047684

Abstract

Peroxidase enzyme was extracted from soybean seeds and isolated by ammonium sulfate precipitation technique followed by purification by dialysis and gel filtration chromatography. The crude enzyme was subjected to ammonium sulfate precipitation technique for partial purification and the activity and specific activity were noted. After ion exchange chromatography using DEAE-cellulose, maximum activity was noted. This fraction was then applied to sephadex G-75 column and after elution, the activity and specific activity was determined.

Key Words: Soybean seeds, peroxidase purification, ion exchange chromatography, gel filtration.

Introduction:

Peroxidases are a large family of enzymes that typically catalyze a reaction of the form:

ROOR' + electron donor (2 e-) + 2H+ → ROH + R'OH

The optimal substrate for most of these enzymes is hydrogen peroxide, but others are more active with organic hydroperoxides such as lipid peroxides. Peroxidases can contain a heme cofactor in their active sites, or alternately redox-active cytosine or seleno-cysteine residues.

It was first discovered by Hugo Theorell in Horse radish roots. Later it was found that peroxidase enzymes are found throughout the plant kingdom. Peroxidase is usually intracellular, as are the other oxido-reductases. Peroxidase is a heat stable enzyme, having wide range of applications in health sciences as a diagnostic tool. It is used to prepare enzyme conjugated antibodies and used widely in ELISA and other sensitive detection assays. The oxidative properties of peroxidase have been used successfully in assay systems to detect metabolites by the formation of their respective oxidases. Though it is present in abundant amounts, extraction and purification techniques add up to the cost of the final product. Therefore, optimization of the processing techniques of peroxidises is essential.

Materials and methods:

Sample:

1. Soya bean seeds

2. Garlic

Chemicals used:

TMB, tris-HCl, EDTA, Comassie blue-G250, phosphoric acid, ethanol, ammonium sulphate, citric acid, sodium citrate dihydrate, H2O2 from Hi Media Chemicals, Mumbai and Sigma chemicals, Bangalore.

Methodology:

Preparation of crude extract: 100gm of each sample i.e peeled garlic bulbs & soya bean seeds were weighed, and frozen with liquid N2. With the help of mortar-pestle, the frozen tissues are then homogenized in the presence of 230ml tris-HCl buffer (pH 7.5) containing 2mM EDTA. Ultrasonication treatment was given to the homogenized samples. The homogenized mixture is then centrifuged at 14000rpm at 4°C for 20 min. The supernatant is stored at 4°C and the pellet was discarded. Supernatant containing peroxidase is then further purified and assayed.

Determination of proteins: Bradford method (Bradford, M. 1976) was followed for the quantification of protein in crude extract. Bovine serum albumin was used as the standard. Absorbance was recorded at 595nm by UV-Vis Spectrophotometer.

Ammonium sulphate precipitation: Crude extract was precipitated with ammonium sulphate (30%, 50% & 80% saturation). It was then centrifuged at 14000rpm for 10min. The pellet was dissolved in 0.01M phosphate buffer (pH 7) for further analysis.

Dialysis:

1 ml of each precipitated sample (30%, 50% & 80%) was dialyzed overnight in a beaker containing solvent (pH-7) and the crude protein was stored at 4°C.

After dialysis the volume of the sample was found to be 2ml.

Results and Discussion:

Standard curve for BSA by Bradford assay was obtained. As per the standard curve, the amount of protein per mL for varied concentrations of crude extract was estimated.

BSA concentration

Figure 1- BSA standard curve

Concentration analysis of test proteins:

Sample

Volume(µl)

Water(µl)

Bradford Reagent (µl)

OD of Ginger

O.D of Soyabean

Ginger

soyabean

0-30%

20

180

800

0.234

0.122

0-50%

20

180

800

0.540

0.445

0-80%

20

180

800

0.923

1.03

Following Ammonium sulfate precipitation and dialysis of the crude extract, an enzyme assay was performed using TMB as a chromogenic substrate. From the previous results we know that 50µL of 10X TMB was used with 25µL of crude enzyme and 50 µL of H2O2 to give color production. Thus, the results obtained showed color change at OD 655nm.

3 samples were produced for 3 different fractions as per the given data:

30% fraction - 25 µL crude enzyme+ 50 µL 10X TMB + 50 µL H2O2

50% fraction - 25 µL crude enzyme+ 50 µL 10X TMB + 50 µL H2O2

80% fraction - 25 µL crude enzyme+ 50 µL 10X TMB + 50 µL H2O2

Activity analysis of enzyme:

Sample fraction

Volume(µl)

TMB+H2O2 (Ul) (10X)

INCUBATE FOR 20 Minute

O.D at 655

Ginger

Soyabean

For Ginger

For Soyabean

0-30%

25

50+50

0.123

0.013

0-50%

25

50 + 50

0.212

0.342

0-80%

25

50 + 50

0.333

0.459

Concentration of protein was found more in 80% fraction of both the sample, in which concentration in soyabean sample was high.

After incubation, 80% fraction was having higher concentration of peroxidase which act on H2O2 & TMB & show blue color production.

The Effect of Various Temperatures on Peroxidase Activity

Temperature 4°C 15°C 25°C 43°C 55°C 70°C 100°C

O.D: 0.106 0.177 0.251 0.312 0.289, 0.164 0

SDS-PAGE:

Conclusion:

As the concentration of protein was maximum in 80% fraction as well as the enzyme activity on substrate was higher in the same. Thus, on loading this fraction on SDS-PAGE bands of different molecular weights were observed showing the presence of different protein containing our desired enzyme i.e peroxidase.



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