LAB MANUAL
Production And
Estimation Of Protease Activity Through Solid State Fermentation
Aim :
To produce and
estimate protease produced by Aspergillus
niger and Bacillus sp. using
wheat bran and dried skimmed milk respectively by solid state fermentation.
Introduction :
Proteases
are the most important industrial enzymes and comprise about 25% of commercial
enzymes in the world.
It refers to a group of enzymes whose catalytic function is to hydrolyze
(breakdown) peptide bonds of proteins. They are also called proteolytic enzymes
or proteinases. Proteases differ in their ability to hydrolyze various peptide
bonds. Ex : fungal protease, pepsin, trypsin, chymotrypsin, papain, bromelain,
and subtilisin.
The mycelium of
many fungi store sufficient amount of nutrient proteins required for their
growth. The enzyme protease becomes active during growth and breakdown of
stored proteins into peptides and amino acids which can be used by the fungi
for its metabolic and energy requirements. The amount of amino acids released
gives the measure of activity of the enzyme protease in the organism.
Proteases are
divided into four major groups according to the character of their catalytic active site and
conditions of action: serine proteinases, cysteine (thiol) proteinases,
aspartic proteinases, and metalloproteinases.
Common protease
producing microbes are Aspergillus sp., Aeronomas sp., Alcaligenes sp., Bacillus
sp., Staphylococcus sp. and Pseudomonas sp.
The present uses of
the protease in industrial applications are as detergents, cosmetics, leather
processing, solubilising agent, in food industry as softening agent and as well
as pharmaceutical industry to treat
digestive ailments.
Principle
:
Sterile wheat and skimmed
milk are inoculated with A. niger and Bacillus respectively. The
fungal cultures are incubated for a week and the enzyme is extracted using
phosphate buffer. When protease enzyme acted upon protein, the peptide bonds
were cleaved resulting in the liberation of free amino acids. First the
proteins are pre-treated with copper ion in alkaline solution and then the amino acids in the treated
sample reduce the phosphomolybdate and phosphotungstate
acid present in the Folin ciocalteu reagent. The reagent reacts with phenolic
and non-phenolic substances and reduce them to heteropolymolybdenum by the
copper-catalyzed oxidation of aromatic acids and finally produces blue coloured
complex that can be detected spectrophotometrically at 660nm.
.
Requirements:
Bovine Serum albumin, skimmed milk, wheat bran,
wheat bran media, phosphate buffer, folin ciocalteu reagent, mortar &
pestle, NaOH, CuSO4, volumetric flask, pipettes and test tubes.
Reagents:
·
Phosphate Buffer: 58.9ml of 0.1M KH2PO4
and 61.1ml of 0.1M Na2HPO4.
·
Bovine Serum Albumin: 1% Bovine serum albumin
(1g of BSA in 100ml of distilled water) used for enzyme activity.
·
Standard Stock: 50mg of BSA was dissolved in
100ml of distilled water to give a concentration of 500mg/ml.
·
Folin ciocalteu reagent: the commercially
available reagent is diluted in the ration of 1:2 with water.
Procedure:
Preparation of wheat bran media:
|
Peptone
|
1g
|
|
CaCl2
|
100mg
|
|
NaNO3
|
1g
|
|
K2HPO4
|
100mg
|
|
MgSO4
|
100mg
|
Dissolve the above ingredients in 100ml of
distilled water and autoclave. Take 10g each of wheat bran and skimmed milk
powder in separate flask and sterilize.The medium is cooled. 10ml of wheat bran
media is added aseptically in to the flask containing wheat bran and skimmed
milk using sterile pipette.
Inoculation
and Incubation:
A small amount of A. niger and Bacillus
sp. is inoculated in the flask containing wheat bran and skimmed milk powder
respectively. After incubating for a week enzymes are extracted from the medium
using phosphate buffer.
Preparation
of Standard Graph:
i.
Different aliquots of standard protein
solution (500mg/ml)
were pipetted out in to 5 test tubes (100-500mg/ml).
ii.
Ranging from 0.2ml to 1.0ml with a total
volume of 1ml using distilled water. 3ml of alkaline copper reagent was added
and the tubes were incubated at room temperature for 15mins.
iii.
0.5ml of folin ciocalteu reagent was added
and the tubes were incubated at room temperature for 30 mins.
iv.
The absorbance was read at 640nm.
v.
A standard graph was plotted by taking
absorbance on Y- axis and protein concentration on X- axis.
Extraction
and estimation of protein:
Extraction:
For fungi:
1g of fermented substrate is taken and 10ml of
phosphate buffer is added homogenised with mortar and pestle. Centrifuge at
3000rpm for 15 minutes. Take the supernatant for enzymatic activity.
For Bacteria:
Directly centrifuge 1ml of culture and homogenised
in 10ml of phosphate buffer and
centrifuge at 1000rpm for 10-15 minutes. Take the supernatant.
Estimation
i.
1g of mycelial mat is homogenised with 10 ml
of ice cold phosphate buffer solution.
ii.
The homogenate is centrifuge at 3000rpm for
10 mins.
iii.
1ml phosphate buffer is taken in to 3 test
tubes with 1 kept as blank.
iv.
Add 0.5ml of substrate solution (1% BSA) to
each of the tubes, allow to stand for 10 minutes at room temperature.
v.
Add 2ml of TCA
vi.
Centifuge at 3000 rpm for 10mins.
vii.
Transfer 1ml of supernatant to fresh tubes.
viii.
Add 2ml of 0.5N NaOH, 0.5ml of CuSO4 and
0.5ml of Folin’s reagent.
ix.
Incubate at room temperature for 30mins.
x.
Measure the absorbance at 640nm.
The protease activity was found for
Fungal Protease_______________
Bacterial Protease
___________
|
Conc.
Of BSA
(mg/ml)
|
Vol.
of stock (ml)
|
Vol.
of D/W
(ml)
|
Total
vol. (ml)
|
Vol.
of alk. Cu reagent
(ml)
|
Incubation
@
RT (min)
|
Vol.
of folins ciocalteu reagent
(ml)
|
Incubation
@
RT (min)
|
OD
at 640 nm
|
|||||||||||||||
|
100
|
0.2
|
0.8
|
1
|
3
|
15
|
0.5
|
30
|
|
|||||||||||||||
|
200
|
0.4
|
0.6
|
|
||||||||||||||||||||
|
300
|
0.6
|
0.4
|
|
||||||||||||||||||||
|
400
|
0.8
|
0.2
|
|
||||||||||||||||||||
|
500
|
1.0
|
0.0
|
|
||||||||||||||||||||
|
Blank
|
0.0
|
1.0
|
|
||||||||||||||||||||
|
Unknown
|
|
|
|
Enzyme Activity :
|
Test tube
|
Enzyme ext (ml)
|
PO4 buffer
|
BSA
|
Incubation
|
10%
TCA
|
Centrifugation
|
1 ml of supernatant + 2 ml (0.5N NaOH) + 0.5 ml
Folins reagent
|
Incubation
|
OD
|
|
Blank
|
1
|
1 ml
|
0
|
10 min
|
2 ml
|
3000 rpm for 10 min
|
30 min @ RT
|
|
|
|
Test
|
1
|
1 ml
|
0.5
|
10 min
|
2 ml
|
|
References :
www.nature.com/protocolexchange/protocols/267
No comments:
Post a Comment