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Protease Production From Bacillus Subtilis
Protease Production From Bacillus Subtilis
Protease Production From Bacillus Subtilis
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
Quantitative
Determination of
Protease Activity at
Different Conditions
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]
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
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
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
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
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
Read at 750 nm
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
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
100
0.003543
75 deg. Celsius at pH 7
0.00102
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 /
6
pH
10
12
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
Conclusion
References
[1] Blainsky, A. et. Al. (2013) Application and Analysis of the Folin Ciocalteu Method for the Determination of the
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
[4] Johnson, M. (2012) Protein Quantitation. MATER METHODS 2012;2:115
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
protease from Bacillus Sp isolated from soil sample. Journal of Microbiology and Biotechnology Research,
Scholars Research Library. p. 164-167
Retrieved: 10/26/14
http://scholarsresearchlibrary.com/JMB-vol2-iss1/JMB-2012-2-1-163-168.pdf