Advances in Microbiology, 2012, 2, 354-357
http://dx.doi.org/10.4236/aim.2012.23043 Published Online September 2012 (http://www.SciRP.org/journal/aim)
Relationship Study between the Alkaline Protease
Production and the Growth Phases of Pseudomonas
aeruginosa Isolated from Patients
Ali Jaffar Saleem
College of Education for Pure Science, Diyala University, Baquba, Iraq
Received July 2, 2012; revised July 31, 2012; accepted August 9, 2012
ABSTRACT
This study was conducted in Diyala University Laboratories collaboration with the Directorate of Diyala Health. Oc-
currence of Pseudomonas aeruginosa was investigated in 161 samples from different clinical sources included Swabs
from wounds, burns, ear, eye and samples from Urine and sputum which were collected from patients. Depending on
the cultural and micro features and biochemical tests 49 isolated items of this bacteria have been diagnosed and all the
isolates showed the proteolytic activity by using skim milk agar through forming clear zone around the growing colo-
nies, and tested the isolates ability of alkaline protease production by quantitative methods, the local isolation P.
aeruginosa AP3 had been selected based on the higher productivity of enzyme comparing to other isolates and
thus it was used in the current study. Studied the relationship between the production of alkaline protease enzymes
and growth phases of P. aeruginosa to determine the time of the enzyme production and the results showed that the
local isolation P. aeruginosa AP3 began production of the enzyme in the later stages of the log phase and increased
production significantly in the stationary phase reaching amaximum after 48 hours as estimated the enzyme activity
159.2 units/ml in the farm leaky and keep the enzyme fully functional almost in the stationary phase.
Keywords: Alkaline Protease; Pseudomonas aeruginosa; Growth Phases; Production
1. Introduction
Pseudomonas aeruginosa is the most important types of
gram-negative pathogenic bacteria to the human, as
characterized by their ability to make different types of
infection intensity ranging between lethal and moderate.
This bacteria is an opportunistic pathogens as they cause
serious infections, especially among cancer patients and
Immunocompromised patients, and also cause these bac-
teria infections in different sites of the body they cause
respiratory tract infection, cystic fibrosis, pneumonia,
urinary tract infection, middle ear infection (Otitis Me-
dia), infection of the eye especially bacterial Keratitis,
endocarditis, meningitis, bacteremia, septicemia, of bones
and joints infection, intestinal infections and skin, burns
and wounds infection especially wounds after operations
[1].
These bacteria cause infections in burn wounds, caus-
ing serious infections in the skin and tissues and may
lead to get septicemia, where the burn wounds, a good
nutrient medium for microorganisms and through it is
colonization and entry into the body and events a series
of infections [2].
The ability of P. aeruginosa in the invasion of tissues
to produce extracellular enzymes and toxins, include
exotoxin-A, hemolysin, exoenzyme-S, urease, elastase
and alkaline protease because they penetrate the body
barriers and damage the host cells and the alkaline prote-
ase which plays an important role in the enabling it to
invade tissues and settled down to comprehensive infec-
tion through the provision of basic food factors for
growth [3], It also serves to provide protection for them
through the inhibition of some components of the im-
mune system of the host, alkaline protease acting to pre-
vent the opsonization by antibodies and complement,
also inhibits neutrophil chemo taxis, also works to reduce
the production of gamma interferon, causing reduced
antiviral activity [4].
Have this enzyme the ability to destroy synthetic pro-
teins such as collagen an d glycoprotein in the tissues [5].
The secretion of this enzyme outside the bacterial cell-by
one step directly from the internal membrane to the ex-
ternal membrane while going through another extracel-
lular proteins secreting by two steps [6].
Proteases are the most important industrial enzymes
that execute a wide variety of functions and have various
C
opyright © 2012 SciRes. AiM

A. J. SALEEM 355
important biotechnological applications [7]. Microorgan-
isms are the most important sources for enzyme produc-
tion which is a big economic return through growth on
the sources of carbon and nitrogen low-cost [8].
Alkaline proteases of microbial origin possess consid-
erable industrial potential due to their biochemical diver-
sity and wide applications in tan nery and food industries,
medicinal formulations, detergents and processes like
waste treatment, silver recovery and resolution of amino
acid mixtures [9,10].
Due to the importance this enzyme and its role in the
pathogenesis of P. aeruginosa as well as the importance
of proteases generally in the fields of pharmaceutical,
medical and industrial, this study was to demonstrate the
relationship the alkaline protease production with growth
phases of local isolation selected of P. aerugi nosa.
2. Material and Methods
Occurrence of P. aeruginosa was investigated in 161
samples from different clinical sources included Swabs
from wounds, burns, ear, eye and samples from Urine
and sputum. Which were collected from patients in Ba’quba
general hospital and Al-Batul hospital for the period
from 1/12/2004 until 3/31/2005 and included samples of
different ages and both sexes.
Swabs and samples were grown on nutrient agar and
blood agar, in cubated aerobic condition s at 37˚C for 24 -
48 hours to investigate P. aeruginosa isolates, and then
conducted diagnostic tests of the isolates according to the
method described by [11,12]. Investigating P. aeruginosa
ability of Proteolytic Activity by using skim milk agar
[13].
Investigate bacterial ability to produce alkaline prote-
ase by using casein broth described by Al-Shehri and
Mostafa [14], have been adopted optimal conditions of
local isolation selected in the alkaline protease produc-
tion which have been identified by Saleem [15], by using
liquid cultures consisting of 0.75% glucose, 1% Tripton,
0.1% CaCl2, 0.01% MgSO4·7H2O, 1.7% K2HPO4 and
0.3% KH2PO4 at primary pH of 8 and 37˚C.
50 ml of medium was taken in a 250 ml conical flask,
the flasks were sterilized in autoclave at 121˚C (15 lb)
for 15 min and after cooling, the flask was inoculated
with 1 × 107 cell/ml. The inoculated flasks were incu-
bated in a shaker incubator at 150 RPM at 37˚C, at the
end of the fermentation period, the culture medium was
centrifuged at 5000 RPM at 4˚C for 15 min to obtain the
crude extract, which served as enzyme source. Then al-
kaline protease was determined by the method of McDon-
ald and Chen, described by [16].
3. Results and Discussion
Depending on the cultural and micro features and bio-
chemical tests 49 isolated items of P. aeruginosa have
been diagnosed, all the iso lates showed the ability to pro-
teolytic activity by u sing skim milk agar through forming
clear zone around the growing colonies.
The local isolation P. aeruginosa AP3 shown in Fig-
ure 1, isolated of burns was selected, because it is the
heaviest of the enzyme production compared with the
other isolates by using the submerged farms and adopted
in the current study.
Follow-up has been the growth curve of local isolation
P. aeruginosa AP3 for 60 hours to know the growth
phases and the start time of alkaline protease production
during this period by counting the living cells and meas-
uring the absorbance at 600 NM wavelength.
Identified the results shown in Figure 2, which shows
the relationship between production and growth phases
estimated by counting the living cells and the Figure 3,
which shows the same relationship estimated by absorp-
tion at wavelength 600 NM, the local isolation selected
interference in the stationary phase after 10 hours, also
was noted the start of alkaline protease production in the
leaky farm after 8 hours of the vaccination, the enzyme
activity was reaching 6.4 units/ml and continued to gra-
dually increase until it reached a maximum after 48 hours,
amounting to 159.2 unit/ml, was also noted the local
isolation P. aeruginosa AP3 intervention Decline phase
after this time, but the enzymatic activity remains rela-
tively high levels was reaching 133.4 unit/ml after 54
hours.
This study shows that the expression of alkaline pro-
tease production start in Logarithmic Phase, and in-
creased significantly in the later stages of the Log phase
and in Stationary phase. Noticeable the stationary phase
elongation of the main points to increase this enzyme
production.
Figure 1. Proteolytic activity of local isolation P. aeruginosa
P3 in skim milk agar. A

A. J. SALEEM 356
0
500000000
1000000000
1500000000
2000000000
2500000000
135791113 15 1719 21 2325 27 2931 33 35 3739 41 4345 47 4951 53 55
Time(hour)
cells count(cell\m l)
57 59
0
20
40
60
80
100
120
140
160
180
E n zym e a ctivi ty(u n it\ml )
Living c el l s countenzymatic activity
Figure 2. Development of alkaline protease production during growth phases local isolation P. aeruginosa AP3 according to
total bacteria count.
0
0.5
1
1.5
2
2.5
3
1 357 9111315171921232527293133353739414345474951535
Time(hour)
Absorp atio n at 600nm
55759
0
20
40
60
80
100
120
140
160
180
E n z yme acti vi ty (u i n t\ ml)
Absorpation enz y m atic ac ti vit y
Figure 3. Development of alkaline protease production during growth phases local isolation P. aeruginosa AP3 according to
absorption.
Indicated many of the scientific literature that the pro-
duction of protease happens at the end of Log phase and
the beginning of Stationary phase, as noted Oreilly and
Day the protease produ ction is happening in th e end Log
phase and the beginning of Stationary phase and keep the
enzyme active in the Enzyme production medium for a
period of not less than 12 hours of the entry Stationary
phase [17], and this was confirmed by several studies
that the extracellular enzymes produced in the beginning
of Stationary phase [18]. In another study noted Hors-
burhg et al., the genes cloned which responsible for the
protease production mainly occur in the period post-Log
phase [19], and gene las R control to stimulate the pro-
duction of virulence factors through its role in the proc-
ess of elastase gene las B cloning and protease gene las A
and Tox A [20].
In the local studies indicated Al-Tai that the highest
specific activity of the protease product from Aeromonas
hydrophila occurred at the end of Log phase and the be-
ginning of Stationary phase [21], and Indicated Saud that
the protease produced from Staphylococcus aureus be-
gins at the end of Log phase and during Stationary phase
[22].
REFERENCES
[1] M. Pollack, “Principles and Practice of Infectious Dis-
eases,” 5th Edition, Churchill Livingstone, New York,
2000.
[2] J. A. Sleigh and M. C. Timbury, “Notes on Medical Bac-
teriology,” 5th Edition, Churchill Livingstone, Singa-
pore, 1998.
[3] K. A. Kernacki, J. A. Hobden, L. D. Hazlett, R. Fridman
and R. S. Berk, “In Vivo Bacterial Protease Production
during Pseudomonas aeruginosa Corneal Infection,” In-
vestigative Ophthalmology & Visual Science, Vol. 36, No.
7, 1995, pp. 1371-1378.

A. J. SALEEM 357
[4] C
. Van Delden, E. C. Pesci, J. P. Pearson and B. H.
Iglewski, “Starvation Selection Restores Elastase and Rha-
mnolipid Production in a Pseudomonas aeruginosa Quo-
rum-Sensing Mutant,” Infection and Immunity, Vol. 66,
No. 9, 1998, pp. 4478-4502.
[5] K. Sakata, H. Yajima, K. Tanka, Y. Sakamoto, K. Ya-
mamoto, A. Yoshida and Y. Dohi, “Erythromycin Inhibits
the Production of Elastase by Pseudomonas aeruginosa
with Affecting Its Proliferation in Vitro,” The American
Review of Respiratory Disease, Vol. 148, No. 1, 1993, pp.
1061-1065. doi:10.1164/ajrccm/148.4_Pt_1.1061
[6] J. L. Kadurugamuwa and T. J. Beveridge, “Virulence
Factors Released from P. aeruginosa in Associated with
Member Vesicles during Normal Growth and Exposure
Gentamicin Novel Mechanism of Enzyme Secretion,”
Journal of Bacteriology, Vol. 177, No. 14, 1995, p p. 3998-
4008.
[7] F. N. Mohen, D. Dileep and D. Deepthi, “Potential Ap-
plication of Protease Isolated from Pseudomon aerugi-
nosa PD100,” Electronic Journal of Biotechnology, Vol.
8, No. 2, 2005, pp. 197-203.
[8] K. Kalaiarasi and P. U. Sunitha, “Optimization of Alka-
line Protease Production from Pseudomonas fluorescens
Isolated from Meat Waste Contaminated Soil,” African
Journal of Biotechnology, Vol. 8, No. 24, 2009, pp. 7035-
7041.
[9] R. Gupta, Q. K. Beg and P. Lorenz, “Bacterial Alkaline
Proteases: Molecular Approaches and Industrial Applica-
tion,” Applied Microbiology and Biotechnology, Vol. 59,
No. 1, 2002, pp. 15-32. doi:10.1007/s00253-002-0975-y
[10] D. Agarwal, P. Patidar, T. Banerjee and S. Patil, “Produc-
tion of Alkaline Protease by Penicillium sp. under SSF
Conditions and Its Application to Soy Protein Hydroly-
sis,” Process Bioc hemistry , Vol. 39, No. 8, 2004, pp. 977-
981. doi:10.1016/S0032-9592(03)00212-7
[11] J. G. Collee, B. P. Marmion, A. G. Fraser and A. Sim-
mons, “Mackie and McCartne Practical Medical Micro-
biology,” 40th Edition, Churchill Livingstone, New York,
1996.
[12] J. G. Holt, N. R. Kreig, P. H. A. Sheath, T. T. Staley and
S. T. Williams, “Bergey’s Manual of Determinative Bac-
teriology,” 9th Edition, Williams and Wilkns, Philadel-
phia, 1994.
[13] R. Cruickshank, J. P. Duguid, B. P. Marmion and R. H. A.
Swain, “Medical Microbiology,” 12th Edition, Vol. 2.
Churchill Livingstone, London, 1975.
[14] M. A. Al-Shhri and S. Y. Mostafa, “Production and Some
Properties of Protease Produced by Bacillus Licheni-
formis Isolated from Tihamet Aseer, Saudi Arabia,” In-
ternational Journal of Biological Sciences, Vol. 7, No. 9,
2004, pp. 1631-1635.
[15] A. J. Saleem, “Biochemical Study of Alkaline Protease
Produced from Pseudomonas aeruginosa Isolated from
Patients,” M.Sc. Thesis, College of Education for Pure
Science, University of Diyala, Baquba, 2007.
[16] Ikram-ul-Haq, H. Mukhtar, Z. Ali and N. Riaz, “Protease
Biosynthesis by Mutant Strain of Penicillium griseoro-
seum and Cheese Formation,” International Journal of
Biological Sciences, Vol. 7, No. 9, 2004, pp. 1473-1476.
[17] T. Oreilly and D. F. Day, “Effect of Cultural conditions
on Protease Production by Aeromonas hydrophilia,” Ap-
plied and Environmental Microbiology, Vol. 45, No. 3,
1983, pp. 1132-1135.
[18] A. L. Cheung, K. J. Eberhardt, E. Chung, et al., “Dimin-
ished Virulence of sar-/agr-mutant of Staphylococcus
aureus in the Rabbit Model of Endocarditis,” Journal of
Clinical Investigation, Vol. 94, No. 5, 1994, pp. 1815-
1822. doi:10.1172/JCI117530
[19] M. J. Horsburgh, J. L. Aish and I. J. White, “Sigma B
Modulates Virulence Determinant Expression and Stress
Resistance: Characterization of a Functional rsb V Strain
Derived from Staphylococcus aureus 8325-4,” Journal of
Bacteriology, Vol. 184, No. 19, 2002, pp. 5457-5467.
doi:10.1128/JB.184.19.5457-5467.2002
[20] M. J. Gambello, S. Kaye and B. H. Laglewski, “Las R of
P. aeruginosa Is a Transcriptional Activators of the Alka-
line Protease Gene (apr) and an Enhancer of Exotoxin-A
Expression,” I nfection and Immunity, Vol. 6 1, No. 4, 1993,
pp. 1180-1184.
[21] N. M. I. Al-Tai, “Biochemical Study of Protease Enzymes
Produce from the Locally Isolated Aeromonas hydro-
phila,” Ph.D. Thesis, College of Science, University of
Baghdad, 2005.
[22] A. M. Saud, “Biochemical Study of Protease Enzymes
Produce from Staphylococcus aureus Isolated Locally,”
M.Sc. Thesis, College of Science, 2005.