Protease enzyme

Presented by:
Amna Ahmed
Kawther Hussain
Haneen habeeb
Proteases are enzymes that break down proteins into
smaller peptides or amino acids. They are important in a
variety of biological processes, including digestion, protein
turnover, and signaling. There are many different types of
proteases, each with specific substrate preferences and
physiological roles.
Function in vivo
• They are required for the blood coagulation process.
• Protease enzymes are involved in the cell division, growth, apoptosis
and migration.
• Protein recycling and transport across membranes.
• They are involved in the activation of precursor proteins.
• Proteases provide immune support and regulate the process of
tumour growth, inflammation, etc.
• They may help in wound healing and muscle soreness.
Source of protease enzyme

1-prokaryotes
2-fungi
3-plants
4-animals
 Source of Proteases
1-The use of plants as a source of proteases is governed by several
factors such as the availability of land for cultivation and the
suitability of climatic conditions for growth.
production of proteases from plants is a time-consuming process.
Papain, bromelain, keratinase, and ficin represent proteases of plant
origin.
 The most familiar proteases of animal origin are pancreatic
trypsin, chymotrypsin, pepsin, and rennin. These are prepared in
pure form in bulk quantities. However, their production depends on
the availability of livestock for slaughter, which in turn is governed
by political and agricultural policies
Proteases are present in all forms of life, they are produced by
microorganisms, various plants and animals. Among them bacterial
proteases secure most important place due to their enormous
industrial applications. They represent one of the largest groups of
industrial enzymes and find application in detergents, leather, food,
pharmaceutical and textile industries as well as silver recovery and
bioremediation processes. The largest application of protease is in
laundry detergents and leather industry, where they remove protein
based stains from clothes and dehairing purpose, respectively .
 Microorganisms represent an excellent source of enzymes owing to their
broad biochemical diversity and their susceptibility to genetic manipulation.
Due to these proteases from microbial sources are preferred over the
enzymes derived from plant and animals. In addition, they possess
almost all characteristics desired for their biotechnological applications.
Among microorganisms, Bacteria and fungi are the most prominent one.
However, compared to the bacterial protease, fungal proteases are active
over a wide pH range (pH 4 to 11) and exhibit broad substrate specificity, for
instance Aspergillus oryzae produces the three proteases; acid, neutral and
alkaline. However, they have a lower reaction rate and not as good as heat
tolerance than do the bacterial enzymes. Among bacteria, Bacillus spp is
attractive industrial tools for a source of proteases
Mechanism of action
Peptidases or proteases are a group of proteolytic enzymes that
hydrolyze large protein molecules and break them down into short
proteins. The enzyme begins by breaking down the long protein chain
by hydrolyzing the peptide bonds that hold the amino acids together in
the protein-forming peptide chain. Peptidase is an enzyme that aids
digestion, which breaks down proteins in the stomach and intestines
into their essential compounds - amino acids.
Proteolytic enzymes catalyse the hydrolysis of peptide bonds. Catalysis
facilitates the nucleophilic attack of an activated water molecule on the
peptide bond.
intracellular and extracellular enzyme
Intracellular proteases are important for various cellular and
metabolic processes, such as and differentiation, protein turnover,
maturation of enzymes and hormones and maintenance.
Extracellular proteases are important for the hydrolysis of proteins
in cell-free environments and enable the cell to absorb and utilize
hydrolytic products. At the same time, these extracellular proteases
have also been commercially exploited to assist protein
degradation in various industrial processes
Classification of Proteases
Proteases are classified based on:
chemical nature of the active site
the reaction they catalyse
and their structure and composition
catalytic site on the substrate
.
 catalytic site on the substrate
1- Exoproteases act at the end of the polypeptide chain.
Exoproteases are further classified into
A - amino peptidases which act at the free N-terminus of the
polypeptide substrate
B- carboxypeptidases proteases which act at the free free C-terminal
of the polypeptide chain.
 endoproteases preferably act at the inner region of the
polypeptide chain
endoproteases are also classified based on their side chain
specificity and functional group present in characteristic active site
 classification based on their active site

(i)Serine proteases (EC. 3.4.21): Serine proteases are proteases having

a serine group (-OH) in their active site.
(ii) Cysteine proteases (EC 3.4.22) : The activity of these depends on
the presence of an intact-SH group in their active site.
(iii) Metalloproteases(EC 3.4.24): These depend on the presence of
more of less tightly bound divalent cations for their activity.
(iv) Aspartic proteases(EC 3.4.23): Acid proteases contain one or
more side chain carboxyl groups in their active site. Commonly
known as acid protease
 Classification based on PH
1- Acidic proteases: Acid proteases are proteases which are active in
the pH ranges of 2-6.
2- Neutral proteases: Neutral proteases are proteases which are
active at neutral, weakly alkaline or weakly acidic pH.
3- Alkaline proteases: Alkaline protease can be defined as the
protease those are active in alkaline range of pH (8-12).
 Purification and isolation methods
Chromatography: This is a common method for purifying proteins, including
proteases. There are several types of chromatography that can be used, including ion
exchange chromatography, size exclusion chromatography, and affinity
chromatography.
Precipitation: Proteases can be purified by precipitating them out of solution using a
variety of methods, including salt precipitation and ammonium sulfate precipitation.

Gel filtration: Proteases can be separated based on their size using gel filtration
chromatography.
 Purification and isolation methods
Dialysis: Proteases can be purified by dialysis against a buffer to
remove impurities.
Ultracentrifugation: Proteases can be purified by centrifugation at high
speeds to separate them from other proteins and impurities.
It is important to carefully optimize the purification and isolation
conditions to ensure that the protease is pure and active. This may
involve testing a range of different conditions and methods to find the
most effective approach.
 Enzyme extraction and purification from S aureus

1- Enzyme extraction and purification protease enzyme was extracted

from the selected local isolate S aureus AG10 after developing the
isolate in optimal conditions for enzyme production and harvesting
cells using centrifugation at a speed of 6000 rpm for 15 minutes.
Purify the enzyme by performing the following
1- precipitation with ammonium sulfate
2-Precipitation with organic solvents (acetone)
3-Ion exchange chromatography
 3-Ion exchange chromatography
1- 5 ml of the concentrated extract obtained from previous steps was added to the ion
exchanger column.
2- the column was washed with 0.005 molar Tris with a pH of 7.5 at a flow rate of 0.5
ml/min.
3- The permeable solution was collected from the column and recovered using Tris
buffer with a pH of 7.5 and a linear salt gradient of sodium chloride.
The protein in the transmissible fractions was monitored by reading the absorbance at
a wavelength of 280 nm. The activity was also measured, and the fractions containing
the enzymatic activity were collected and concentrated by means of dialysis tubes
with sucrose for the subsequent purification steps.
 References
Bacterial Protease Enzyme: Safe and Good Alternative for Industrial
and Commercial Use Temam Abrar Hamza
Thank you