1. factors such as the presence of

1. Introduction

1.1
Proteases and their uses:      

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            Proteases or proteolytic enzymes are
enzymes that are seen to have multiple biological applications in plants and
animals, such as germination, inflammatory processes, complement activation and
many more. They can be divided into two broad subcategories, namely exotoxins and endotoxins.

            For commercial purposes, proteases
are used in leather, food and textile industries. Of both classes of proteases,
endopeptidases have much more significance in industrial use. They have the
following applications:

·       
Processing
of chymosin in cheese preparation

·       
Soy
sauce preparation

·       
Meat
tenderization

·       
As
an alternative to chemical detergents in the leather industry

 

1.2
Isolation from plant sources over microbial sources:

          For industrial purposes, proteases are usually isolated
from microbial sources. But of recent, plant sources have showed promise due to
a variety of factors, such as their broad substrate specificity, allowing for
more flexible substrate preparations for varied plant sources. They also have a
more wide range of permissible temperature, pH and other factors such as the
presence of organic compounds.

            Proteases are seen to be at a high
level especially during and post germination in seeds. This is due to the fact
that these enzymes play an important role in the various biochemical mechanisms
involved in germination and also in the initial stages of development. Of plant
seeds, legume seeds have higher levels of globulin and albumin storage
proteins, which are used for the nourishment of the seedling. Due to the above
factors, the following investigation of comparing the protease content in
multiple samples has been performed.

 

2. Review
of Literature

2.1 In 2013, Ranajit Kumar Shaha and Shyam Sundar Shaha
studied and compared the germinating conditions and protease activity in
multiple leguminous samples including black gram, green gram, etc. They further
performed time course studies for the same and obtained an outline of when best
to perform protease iisolation for maximum yield.

2.2 In 2013, Anupama V., Marimuthu M. and others studied
and performed the partial characterization of proteases from underutilized and
common food legumes. Their method of isolation was seen to be a very simple,
inexpensive procedure for the isolation of the enzyme.

2.3 In 2016, Diego F. Coelho and Elias
Basile Tambourgi performed a study on the use of Azocasein as a substrate for
protease activity determination. Their research provided a reliable procedure
for analyzing the biological activity of proteolytic enzymes.

2.4 In 2014, M. Akhtaruzzaman and Tanjina
Rahman presented the characterization of protease enzymes from seven leguminous
seeds. Their study showed which seeds showed the highest quantity of the
proteolytic enzyme.

 

3. Aim and Objectives:

3.1 Aim

To compare and contrast the
protease content in green gram (Vigna radiata)
and Bengal gram (Cicer arietinum).

 

 

3.2
Objectives

3.2.1 To prepare
a crude enzyme from the sample obtained through straining and precipitation
techniques

3.2.2 To estimate
the protease content in the samples using UV/Spectrophotometry

3.2.3 To compare
the protease content in the samples and identify whichever has the higher
amount present.

 

4. Materials and Methods

4.1 Germination
conditions

          The samples chosen for this
procedure (green gram and Bengal gram) were cleaned thoroughly. They were
surface sterilized with 70% ethanol and repeatedly washed with distilled water.
The seeds were then allowed to germinate in room temperature (28-30°C) in
cycles of 12 hours darkness and 12 hours light for 24 hours. During this phase,
they were wrapped in moistened cloth and kept in a closed vessel during the
dark phases. After 24 hours, they were used for the experiment.

 

4.2
Isolation and preparation of Protease Enzyme (crude extract)

          50g of each of the
samples were weighed and homogenized with pre-chilled acetone. The mixture was
ground finely in a chilled mortar and pestle. The obtained homogenate were
transferred to individual beakers and stored at 4°C.

            The
homogenates were treated with an equal volume of chilled 10mM Tris-HCl buffer
at pH 8, containing 2M NaCl for three hours. Afterwards, the extract mixtures
were filtered through Whatmann’s filter paper and the filtrates were
centrifuged at 10000rpm for 10 minutes at 4°C. The obtained supernatant was the
crude extract. It was collected and stored at 4°C for further treatment.

 

4.3 Assay
of Protease Enzyme

          The assay was made using Azocasein, a
chromomeric substrate in the following manner. 0.25ml of 1% Azocasein, prepared
in 20mM sodium acetate buffer, was mixed with 0.15ml of the enzyme extract. The
mixture was allowed to react for 60 minutes in 37°C. The reaction was stopped
by adding 1.2ml of 10% Trichloro Acetic Acid (TCA). For preparation of the
control, the substrate was treated with 1.2ml of 10% TCA before the addition of
the extract. The contents were incubated for 15 minutes in room temperature
before centrifugation at 3000rpm for 5 minutes.

 

            1.2ml of the supernatant was
transferred to a separate tube and the absorbance was read at 440nm.