1.1 Proteases and their uses:
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
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
Soy sauce preparation
As an alternative to chemical detergents
in the leather industry
Isolation from plant sources over microbial sources:
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
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
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 isolation
for maximum yield.
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.
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
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.
Aim and Objectives:
compare and contrast the protease content in green gram (Vigna radiata) and Bengal gram (Cicer
prepare a crude enzyme from the sample obtained through straining and precipitation
estimate the protease content in the samples using UV/Spectrophotometry
compare the protease content in the samples and identify whichever has the
higher amount present.
Materials and Methods
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.
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.
Assay of Protease Enzyme
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.
of the supernatant was transferred to a separate tube and the absorbance was
read at 440nm. Calculations were performed using the following forumula:
Protease activity is denoted in units (U)
Table 5.1: Specific protease activity in
protease activity (U/mg) at different pH