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.
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.

 RESULT:
The protease activity was found for
Fungal Protease_______________
Bacterial Protease ___________

 Observation and Calculation

Conc. Of Vol. Vol. Total Vol. of Incubation Vol. of Incubation OD at

BSA of of vol. alk. Cu @ RT (min) folins @ RT (min) 640
(mg/ml) stock D/W (ml) reagent ciocalteu nm
(ml) (ml) (ml) reagent
(ml)
100 0.2 0.8
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     1       3        15       0.5        30
Unknown

Enzyme Activity
Test Enzym PO4 buffe BS Incubatio 10% Centrifugatio 1 ml of Incubatio O
tube e ext r A n TC n supernatan n D
(ml) A t + 2 ml
Blan 1 1 ml 0 10 min 2 ml (0.5N
k NaOH) +
3000 rpm for 0.5 ml 30 min @
Test 1 1 ml 0.5 10 min 2 ml 10 min Folins RT
reagent