Protease Production from

Bacillus subtilis
Cayabyab, A. B.; Co, A.K.M.; Cruzado, K. T.; Cu, . G.
Group 2 3 A Biochemistry
General and Industrial Microbiology

Introduction
Proteases
ubiquitous in occurrence
essential for cell growth and differentiation.
are enzymes that break the peptide bond that
joins amino acids together in proteins.
used in dry cleaning, detergents, meat
processing, cheese making, silver recovery from
photographic film, production of digestive and
certain medical treatments of inflammation and
virulent wounds

Introduction
Bacillus subtilis = the preferred source of enzymes
such as protease because of its:
rapid growth
limited space required for their cultivation
ease with which they can be genetically
manipulated

Introduction
Morphological, cultural, and biochemical
characteristics of Bacillus subtilis:
irregular and large colony shape and size
endospore forming
Rods in chains
Gram-positive
motile (due to its peritrichouss flagella)
grows optimally at 28-30 C
Aerobic
positive in Catalase, Oxidase, SCA, Starch
Hydrolysis, VP, and Nitrate reduction tests
negative in MR and Indole tests

Introduction
Objectives of this experiment:
1.) screen and characterize extracellular proteases
from B. subtilis
2.) purify the protease extracted from Bacillus
Subtilis
3.) construct an L-tyrosine curve using the Lowry
assay
4.) quantify the amount of protease produced by B.
subtilis using the Bradford method
5.) determine the effects of pH and temperature with
the use of Lowry reagent and Folin-Ciocalteaus
phenol reagent

Introduction
In the experiment, the Lowry assay was
performed in order to determine free L-tyrosine
residues after the protease digested casein. The
FolinCiocalteu reagent which is a mixture of
tungstates and molybdates works on the
mechanism of oxidationreduction reaction
The method strongly relies on the reduction of the
mixture heteropolyphosphotungsatesmolybdates
by the phenolic compound which results in the
formation of blue coloured chromogen.

Introduction
The phenolic compounds react with Folin
Ciocalteu reagent only under basic conditions
adjusted by sodium carbonate solution. Under
alkaline conditions the divalent copper ion forms a
complex with peptide bonds in which it is reduced
to a monovalent ion.
Monovalent copper ion and the radical groups of
tyrosine, tryptophan, and cysteine react with Folin
reagent to produce an unstable product that
becomes reduced to molybdenum/tungsten blue.
The colour intensity of the formed blue
chromogen can be measured by the absorbance
readings using a spectrophotometer.

Methodology

Enrichment Culture of
Bacillus subtilis

Isolation and Partial

Purification of
Protease and Total
Protein Concentration
Determination

Quantitative
Determination of
Protease Activity at
Different Conditions

Figure 15.1 Process Diagram of Protease Production from Bacillus subtilis

Methodology
Inoculate Bacillus subtilis in an Enriched Culture Medium Containing the following:
Glucose (0.5% w/v), Peptone (0.75% w/v), Salt Solution (5% v/v) containing MgSO4
7H2O (0.5% w/v), KH2PO4 (0.5% w/v), FeSO4 7H2O (0.01% w/v) [7]

Adjust to pH 6-7 using 0.25M NaOH and 0.2M HCl [5]

Incubate for 24 hrs. at 37 oC [5]

Figure 15.2 Enrichment Culture of Bacillus subtilis based on the protocol of [5] and [7].

Methodology
Centrifuge at 10000rpm for
5 mins, Decant supenatant
and redissolve the solids at
0.1M Phosphate buffer at
pH 7

Prepare Dialyzing
membrane using Collodion
and dialyze for 24 hrs.
against same buffer at 4o C.
Collect Dialysate

Centrifuge at 10000 rpm

for 15 mins., collect
supernatant

From supernatant, add

0.30g (NH4)2SO4 crystal
until solid dissolves

Determine Total Protein

Concentration by adding
500 L of dH2O and 2mL
of Bradford Rgt. with 500
L of protease and read at
596 nm.

From the Supernatant, add

3mL 70% (NH4)2SO4

Centrifuge at 10000 rpm

for 5 mins. Collect
supernatant

Analyze using Papain

Calibration Curve, Bradford
Method

Icebath for 5 mins.

Figure 15.3 Isolation and Partial Purification of Protease and Total Protein
Concentration Determination based on the protocol of [2].

Methodology
Table 15.1 Papain Calibration Curve, Bradford Method [2]
Test
1
Tube
Papain
0

1mg/m
l std.
(L)
dH2O 1000
(L)

10

20

40

80

160

320

640

990

980

960

920

840

680

360

+2mL Bradford Rgt.

Read at 596 nm

Figure 15.4 Quantitative Determination of Protease Activity at Different pH

based on protocol of [7]

Incubate 0.5 mL of
enzyme in 2.5 mL of 0.6
w/v Casein at 37 oC in
the following 0.01M
buffers: Gly/HCl (pH 3),
Tris-HCl (pH 7.2),
Gly/NaOH (pH 10)

After 30 mins.
Stop the reaction
by adding 10%
Trichloroacetic
acid

Analyze using L-Tyr

Standard Calibration
curve. Determine
protease activity from
amount of Free L-Tyr.
Construct Absorbance
vs. pH plot.

Centrifuge at 10000 rpm

for 5 mins. Collect 2900
L Supernatant and add
1000 L Lowry Rgt. and
100 L Folin-Ciocalteu's
Phenol Rgt. Read at 750
nm

Figure 15.5 Quantitative Determination of Protease Activity at Different

Temperatures based on protocol of [7]

Incubate 0.5 mL of
enzyme in 2.5 mL of 0.6
w/v Casein in pH 7.2 TrisHCl buffer at the
following Temperatures:
17 oC, 24 oC, 37 oC, 75 oC

After 30 mins. Stop the

reaction by adding 10%
Trichloroacetic acid

Analyze using L-Tyr

Standard Calibration
curve. Determine
protease activity from
amount of Free L-Tyr.
Construct Absorbance
vs. Temperature plot.

Centrifuge at 10000 rpm

for 5 mins. Collect 2900
L Supernatant and add
1000 L Lowry Rgt. and
100 L Folin-Ciocalteu's
Phenol Rgt. Read at 750
nm

Methodology
Table 15.2 L-Tyrosine Calibration Curve, Lowry Protocol [6]
Test
Tube
L-Tyr - 1
mg/mL
std. (L)

10

20

40

80

160

dH2O
(L)

2900

1890

1880

1860

1820

1740

+ 1000 L Lowry Reagent

+100 L Folin-Ciocalteus Phenol Reagent

Read at 750 nm

Results and Discussion

Papain Standard Curve for Total Protein
Determination via Bradford Method
0.7
y = 0.0009x + 0.0135
R = 0.9824

Abgorbance at 596 nm

0.6
0.5
0.4

Series1

0.3

Linear (Series1)

0.2
0.1
0
0

100

200

300

400

500

600

700

Papain in g

Figure 15.6 Papain Standard Curve for Total Protein Determination via Bradford Method

Results and Discussion

-Figure 15.6 shows the standard curve used for the
determination of total protein concentration via
Bradford Assay.

-Using the equation of the line, this yielded to a total

protein concentration of 40.88 g/mL.
-Papain was used as a standard to minimize deviation of
the protease from the standard, since the binding of
Coomassie Brilliant Blue G-250 dye may vary from
protein to protein. [4]

Results and Discussion

L-Tyrosine Standard Curve via Lowry Assay
1.2

Aborbance at 750 nm

y = 0.012x + 0.1289
R = 0.9567

0.8

0.6

Series1
Linear (Series1)

0.4

0.2

0
0

20

40

60

80

L-Tyr in g

Figure 15.5 L-Tyrosine Standard Curve via Lowry Assay

100

Results and Discussion

-Figure 15.5 shows the L-Tyr standard curve used for the
determination of free L-Tyr after incubation of protease
in casein.
-Centrifugation was done to sediment the undigested
casein substrate and protease enzyme, and to isolate LTyr freed as a result of proteolysis.

-Phenolic compounds, in the context of the experiment,

the free L-Tyr reduce Folin-Ciocalteu Reagent (F-C Rgt.)
at Basic conditions producing a Molybdenum Blue
Chromophore [1] measurable at 750 nm wavelength
(max of reduced F-C Rgt.) [3].

Results and Discussion

The purpose of determining free tyrosine residues
was to find out whether the protease was actually
present in solution and to determine the amount of
casein it can digest at a certain condition.
The amount of casein digested was directly
proportional to the free tyrosine residues present in
the sample which was, in respect, directly
proportional to its absorbance at 750 nm, hence we
can relate that to the activity of the protease under
certain pH and temperatures.

Results and Discussion

Table 15.3 Protease Activity
Protease Activity
(Units/mL)
pH 10 at 37 deg. Celsius

0.003543

75 deg. Celsius at pH 7

0.00102

Table 15.3 shows the protease activity in Units/mL, where 1 unit is

equal to mol Free L-Tyr per minute incubated. [6] Only free L-Tyr at
pH 10 at 37 deg. Celsius, and at 75 deg. Celsius at pH 7 were
computed because at other pH and Temperatures, free L-Tyr fell below
the sensitivity of the Lowry Assay (10-1000 g/mL) [4] and thus, the
amount of free L-Tyr in pH 3 and 7 as well as at 17 oC, 24 oC and 37
oC, is less than 10 g (refer to Computations).

Results and Discussion

Computations for Protease activity in Units/ mL enzyme:
Amount of L-Tyr in pH 10 (37 oC ) derived from the equation of the line (g): 38.52 g

38.52
1
1

30 .
2
181.19

= 3.5432 103 /

Amount of L-Tyr 75 oC (pH 7) derived from the equation of the line (g): 11.09 g

11.09
1
1

30 .
2
181.19

= 1.020 103 /

Effect of pH to the Protease Activity

0.6
0.5
0.4
0.3
0.2
0.1
0
0

6
pH

10

Figure 15.7 Effect of pH to the

Protease Activity

12

Effect of Temperature in the

Protease Activity
0.3
Absorbance at 750 nm

Absorbance at 750 nm

0.7

0.25
0.2
0.15
0.1
0.05
0
0

20
40
60
Temperature in Degrees Celsius

80

Figure 15.8 Effect of Temperature in the

Protease Activity

Results and Discussion

-Figure 15.7 and 15.8 shows the effect of pH and
Temperature to the protease activity.
-Since Free L-Tyr cannot be quantified, as it fell below
the sensitivity of the assay, the plot used absorbances at
750 nm instead, to correlate the data.
-It had been found out that the isolated protease was
optimal at alkaline conditions (pH 10) and at high
temperatures (75 deg. Celsius). Thus, the protease was
highly thermo-stable and stable at alkaline conditions.

Results and Discussion

Possible source of error:
-Dialysis might not have been effective enough in
removing Ammonium sulfate salt used in the
Purification. This may lead to chemical interferences
with Folin-Ciocalteus Rgt. That might have affected the
absorbance during the assay.
-The centrifugation was done at Room temperature
whereas, ideally, it should be carried out at 4 deg.
Celsius.

Documentation (put pics here)

Conclusion

- Protease Isolated in Bacillus subtilis is

stable at high temperature and pH.

References
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Total Phenolic Content from Limonium Brasiliense L. Molecules 2013, 18, 6852-6865;
doi:10.3390/molecules18066852
Retrieved: 11/6/14
http://www.mdpi.com/1420-3049/18/6/6852
[2] Crisostomo, A.C., Daya, M.L., de Guia, R.M., Farrow, F.L., & Gabona, M.G. (2010). Laboratory Manual in
General Biochemistry. Quezon City: C & E Publishing, Inc.
[3] Hayworth, D. (2014). Chemistry of Protein Assays. ThermoScientific Pierce Protein Biology Products.
Retrieved on: 11/6/14
http://www.piercenet.com/method/chemistry-protein-assays
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Retrieved on: 11/6/14
http://www.labome.com/method/Protein-Quantitation.html
[5] Santiago, M.R., Santiago, L.A. (2014). Protease Production from Bacillus subtilis. Laboratory Manual in
General and Industrial Microbiology - Basic Microbiology Technique and Applications to Industrial Microbiology.
Department of Biochemistry, Faculty of Pharmacy, University of Santo Tomas
[6] Sigma-Aldrich Co. LLC. (2014). Universal Protease Activity Assay: Casein as a Substrate
Retrieved on: 11/6/14
http://www.sigmaaldrich.com/life-science/learning-center/life-science-video/universal-protease.html
[7] Soundra Josephine, Ramya V S, Neelam Devi et.al. (2012) Isolation, production and characterization of
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Scholars Research Library. p. 164-167
Retrieved: 10/26/14
http://scholarsresearchlibrary.com/JMB-vol2-iss1/JMB-2012-2-1-163-168.pdf