PresentationOn

Partial purification of protease enzyme from a bacterial isolate obtained from Cattle Hide

PresentationOn

Partial purification of protease enzyme from a bacterial isolate obtained from Cattle Hide

Under Guidance of:

Ms. Payal Mehtani

Assistant Professor

Dept. of Biotechnology

Presented By:

Pooja Soni

M.Sc. BTE (SEM III)

• 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.

• 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

SALTING

OUT

DIALYSIS

TOTAL PROTEIN ESTIMATION

S.No. Reagent B S1 S2 S3 S4 S5 T

1. Std. Protein

BSA(mg/ml)

--- 0.1 0.2 0.3 0.4 0.5 ---

2. Test solution(ml) --- --- --- --- --- --- 1.0

3. Water(ml) 0.5 0.4 0.3 0.2 0.1 --- ---

4. Alkaline

solution(ml)

5.0 5.0 5.0 5.0 5.0 5.0 5.0

Incubated for 10 mins. at room temperature

5. Folin

Ciocalteau(ml)

0.5 0.5 0.5 0.5 0.5 0.5 0.5

Incubated for 30 mins. at room temperature

SDS

PAGE

ZYMOGRAPHY

Process same as SDS-PAGE except these few changes i.e.- No SDS. No β-Mercaptoethanol Add Gelatin as a substrateZYMOGRAM (Gel) Treatment and staining: -

y = 0.778x + 0.012

0

0.10.20.3

0.40.5

0.60.7

0.80.9

0 0.2 0.4 0.6 0.8 1 1.2

Abso

rban

ce(n

m)

BSA Concentration(mg/ml)

Y-Values

Linear (Y-Values)

Total Protein:

Crude Sample 60% Saturation Sample Crude Sample 60% Saturation Sample

20% Saturation Sample 40% Saturation Sample 20% Saturation Sample 40% Saturation Sample

DIALYSIS

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 % SDS-

polyacrylamide 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.