Presentation

On
Partial purification of protease enzyme from a
bacterial isolate obtained from Cattle Hide
Under Guidance of:
Ms. Payal Mehtani
Assistant
Professor

Presented By:
Pooja Soni

M.Sc. BTE (SEM II

 Protein is one of the three major food groups needed for proper
nutrition. Proteolytic enzymes or proteinases are the group of
enzymes whose catalytic function is to hydrolyze (breakdown)
proteins.
Proteolytic enzymes are ubiquitous in occurrence, found in all
living organisms, and are essential for cell growth and
differentiation.
Renewed interest in the study of these enzymes is recognition of
their role not only in cellular metabolic processes but also in
industrial community.
Proteases represent one of the three largest groups of industrial
enzymes.

 Plant Protease

i. Papain
ii. Bromelain
iii. Keratinases
Animal Protease
i. Trypsin
ii. Chymotrypsin
iii. Pepsin
iv. Rennin
Microbial Protease

 Crude extraction of protease from CH-4.

Salting out of protease from crude extract.
Desalting of the extract by DIALYSIS.
Confirmation of protease production by SDS-PAGE.
Confirmation of protease activity by ZYMOGRAPHY
using gelatin as substrate.

PROTEASE PRODUCTION & CRUDE

EXTRACTION

S
A
L
T
I
N
G
O
U
T

D
I
A
L
Y
S
I
S

TOTAL PROTEIN
ESTIMATION
S.No. Reagent
B
1.
Std.
Protein ---

S1
0.1

S2
0.2

S3
0.3

S4
0.4

S5
0.5

T
---

2.

BSA(mg/ml)
Test

---

---

---

---

---

---

1.0

3.
4.

solution(ml)
Water(ml)
Alkaline

0.5
5.0

0.4
5.0

0.3
5.0

0.2
5.0

0.1
5.0

--5.0

--5.0

solution(ml)
Incubated for 10 mins. at room
temperature
5.
Folin

0.5

0.5

0.5

0.5

0.5

0.5

0.5

Ciocalteau(ml
)
Incubated for 30 mins. at room

S
D
S
P
A
G
E

Process same as SDS-PAGE except these few changes i.e.-

Z
Y
M
O
G
R
A
P
H
Y

No SDS.
No -Mercaptoethanol
Add Gelatin
as aTreatment
substrate and staining: ZYMOGRAM
(Gel)

Total Protein:

DIALYSIS
Sample

Crude Sample

20% Saturation Sample

Saturation Sample

60% Saturation

40%

SDS-PAGE
Lane 1

Lane 2

Lane 3

Lane 4

Lane 5

Lane 1 Crude Sample; Lane 2 20% saturation sample; Lane 3 40% saturation sample;
Lane 4 60% saturation sample; Lane 5 PPMWM(Prestained protein molecular marker)
B.Genei

ZYMOGRAPHY
Lane 1

Lane 2

Lane 3

Lane 4

Lane 1 Crude sample; Lane 2 20% saturation; Lane 3 40% saturation;

Lane 4 60% saturation

Among all samples obtained after ammonium sulphate precipitation, highest

protein content was obtained in pellet at 40% saturation (P40). Samples
(crude, 20%, 40%, and 60%) were dialyzed in 50 mM K2HPO4 buffer (pH
7.5) through semi permeable membrane, shade of the colour of samples
changed. When these sample and marker (PPMWM (Prestained protein
molecular marker) B.Genei) were electrophoresed on 7.5 % SDSpolyacrylamide gel. The band of 40% sample equal to the band of marker, so
the molecular weight of 40% sample may be 14178 Da., and when these
samples were electrophoresed for zymography a band of hydrolysis on a
7.5% gelatin zymogram were observed. 40% sample showed higher
hydrolysis and 20% & 60% sample showed lower hydrolysis.