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Isolation of fungal protease

Isolation, Purification and Characterization of Fungal Protease
Syeda Mahnoor, Erum Hanif and Shafaq Aiyaz Hassan
Department of Biotechnology, University of Karachi.
Email : syedamahnoorkhushbakth@gmail.com
An Alkaline Protease producing source was isolated from Aspergillus niger from lemon
sample on PDA plates and enzyme production was observed at room temperature using
Protease growth media as nutritional medium and incubation duration was of about a
week, broth was filtered and ammonium sulphate precipitation and after that centrifugation was done to obtain crude enzyme. Later on the crude enzyme was dissolved in Tris
HCl and checked through kunitz Assay after that to estimate enzyme’s activity Bradford
Method was used which gives fruitful results. For crude enzyme purification dialysis of
enzyme was done against Tris HCl at low temperature for few hours, and after that
DEAE Cellulose Ion Exchange chromatography was performed to obtain purified enzyme fractions (unbound proteins and bound proteins are obtained via washing of column
by different salts concentrations). After having a purified Enzyme sample another step
Aspergillus niger taken from lemon
Transferred on PDA plate, incubated at 37◦C for 24 hrs.
Transfer loopful of spores in Protease growth medium and
incubate it for 7 days at room temperature
Filter the broth
Ammonium sulphate precipitation on ice bath
comes which includes SDS-PAGE electrophoresis of enzyme samples and then enzyme
Centrifuge @low temperature & high speed for 15 min.
characterization takes place at different pH and temperature to identify the most suitable
Separate precipitates and dissolve in Tris HCl buffer
pH and temperature at which enzyme shows best activity.
Crude enzyme obtained
Protease (also known as peptidase or proteinase) is an enzyme that performs prote-
Bradford Assay performed for total protein estimation
olysis i.e.
Protein catabolism by hydrolysis of peptide bonds. Proteases has
evolved multiple of times, various classes of proteases can perform same function
through several different catalytic functions. Proteases clearly do a lot more than
digest your meals; they act on substrates that are fundamental to a raft of physio-
Casein Agar Plate showing activity of Fungal Protease
Dialysis of crude enzyme against Tris HCl @ low temp. for 3 hours
DEAE- Cellulose ion exchange chromatography
Collection of unbound protein fractions
logical processes, from ovulation to programmed cell death or apoptosis. Proteases
Treat column with several concentrations of salts
clearly do a lot more than digest your meals; they act on substrates that are funda-
Protein fractions obtained
Protease from fungal source is isolated and purified and gives
mental to a raft of physiological processes, from ovulation to programmed cell
SDS-PAGE of crude enzyme ppt., dialyzed sample and protein fractions from column
death or apoptosis. Proteases from fungi induce inflammatory responses by altering
far more better results as compare to that of microbial protease
the permeability of epithelial barrier and by induction of proinflammatory cyto-
Purified enzyme obtain
kines through protease-activated receptors. Many fungal allergens possess proteo-
lytic activity that appears to be essential in eliciting Th2 responses. Allergenic fun-
Treatment of purified enzyme with different salt concentrations
gal proteases can act as adjuvants, potentiating responses to other allergens. Prote-
Identification of suitable concentration for protease
olytic enzymes from fungi contribute to inflammation through interactions with the
Treatment of purified enzyme with different pH
kinin system as well as the coagulation and fibrinolytic cascades. Their effect on
the host protease-antiprotease balance results from activation of endogenous proteases and degradation of protease inhibitors. Recent studies of the role of fungi in
human health point to the growing importance of proteases not only as pathogenic
we have screened in beginning from Bacillus specie.
To identify Suitable pH at which enzyme shows best activity
Treatment of purified enzyme with different surfactants
To check it’s activity
agents in fungal infections but also in asthma, allergy, and damp building related
illnesses. Proteolytic enzymes from fungi are widely used in biotechnology, mainly
in food, leather, and detergent industries, in ecological bioremediation processes
and to produce therapeutic peptides ]. Fungal proteases are active over a wide pH
range (pH 4 to 11) and exhibit broad substrate specificity (Rao et al 1998). One of
the first known representatives of proteases was proteinase K, an alkaline enzyme
from Engyodontium album also known as Tritirachium album
(a). https://en.wikipedia.org/wiki/Protease
(b). http://www.worthington-biochem.com/disp/default.html
Isolated fungal protease undergoes in purification following several procedures
(c). https://www.biotecharticles.com/Biology-Article/General-Inroduction-on-
such as Dialysis, Ion exchange chromatography and SDS-PAGE and at the end
it gives purified enzyme which later on characterized on several criteria i.e. pH,
(d). https://www.nature.com/horizon/proteases/background/searching.html
temperature and reaction with various surfactants (tween 20, triton X, SDS etc.)