ISOLATION, EXTRACTION, PURIFICATION AND CHARACTERIZATION OF ALKALINE PROTEASE FROM NEUROSPORA CRASSA AND ENZYME ASSAY

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OBSERVATION:


Estimation of purified enzyme:

S.No

Vol.

of std.(BSA)

Vol. of

D/W

Concentration of Purifier Enzyme

 in µg/ml

Vol. of Alkaline copper sulphate reagent

Incubation at room

temperature

Vol of

 Fc Reagent

Boiling at

1000C in water bath

O.D values at 660nm

Blank

0.0ml

1.0ml

00 µg

5ml

10min

0.5ml

10min

0.00

1.

0.2ml

0.8ml

20 µg

5ml

10min

0.5ml

10min

0.20

2.

0.4ml

0.6ml

40 µg

5ml

10min

0.5ml

10min

0.40

3.

0.6ml

0.4ml

60 µg

5ml

10min

0.5ml

10min

0.60

4.

0.8ml

0.2ml

80 µg

5ml

10min

0.5ml

10min

0.80

5.

1.0ml

0.0ml

100 µg

5ml

10min

0.5ml

10min

1.00

6.

Unknown

0.5 ml

0.5ml

60 µg

5ml

10min

0.5ml

10min

0.60

Observation:


ESTIMATION OF PURIFIED ENZYME

Graph:


RESULT: In 0.5 ml of purified enzyme solution the concentration of enzyme is 60µg which is taken as unknown sample.

VII. OPTIMIZATION:
The activity of enzyme is influenced by various factors includes pH, temperature and Substrate.

EFFECT OF pH: The rate of enzymatic reaction depends on pH of medium. The enzymatic activity is maximum at a particular pH called optimum pH.

Reagents: enzyme, Phosphate Buffer, 0.5% BSA, TCA, FCR, 5% sodium hydroxide

Procedure: 1.0 ml of phosphate buffer of different pH 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0 and 12.0 was taken in nine test tubes. 1ml of 0.5%BSA was added to all the test tubes followed by the addition of 1ml of enzyme.All the test tubes were incubated at 370C for 10min. After incubation, add 2.0 ml of 10% TCA was added and then the contents of the tubes were centrifuged at 10,000rpm for 10min. To the 0.5ml of the supernatant, 2ml of 5% sodium hydroxide and 0.5 ml of FCR were added and incubated at 370 C for 10 min. After incubation, the enzyme activity was measured at 660nm.

Sr. No

Buffer of Various PH

Vol of BSA 0.5%

Vol of Enzyme

Incubation at 370C

Vol of TCA

Vol of NaoH

Incubation For 10min at 370C

Centrifuge for 10min at 10000

OD At 660nm

1

4ml

1ml

1ml

10min

2ml

2ml

10min

0.5

0.04

2

5ml

1ml

1ml

10min

2ml

2ml

10min

0.5

0.09

3

6ml

1ml

1ml

10min

2ml

2ml

10min

0.5


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