Non-mucoid P. Aeruginosa aiming to a safe production of protease and lipase

Abstract

Being an opportunistic pathogen, P. aeruginosa has a limit contribution in biotechnology applications. H 2 O 2 can induce mucoid conversion in P. aeruginosa. Five non-mucoid P. aeruginosa strains were investigated in this study. After applying H 2 O 2 to these non-mucoid strains two strains were converted to be mucoid. The stable non-mucoid strains were investigated for their ability to produce protease and lipase. Agar well diffusion was used for detecting the protease and lipases activity. Additionally Zymogram was used for in gel determination of the protease activity. Spectrophotometrically, proteases and lipases activities were determined using casein and p-nitrophenyl palmitate as substrates respectively. The apparent activity for each enzyme identified as units/ml. Protease activity was 428 units/mL while lipase activity was 21 units/mL. This study proves the ability of isolating stable non-mucoid P. aeruginosa strain able to produce of both protease and lipase. The H 2 O 2 mucoid-non-mucoid test described in this study should be used in the regular P. aeruginosa identification. We recommended using the non-mucoid P. aeruginosa strain isolated in this study in different biotechnological applications.

Full text

103
International Science and Investigation Journal
ISSN: 2251-8576 Vol. 2(5)
Journal homepage: www.isijournal.info
Accepted February 2013
Non-mucoid P. aeruginosa aiming to a safe production of protease and lipase
Amro A. Amara1*, Mohamed A. Hassan1, Ashraf T. Abulhamd2, and
Bakry M. Haroun2
1Protein Research Department, Genetic Engineering and Biotechnology Research Institute
(GEBRI), City of Scientific Research and Technological Applications (SRTA City), New Borg Al-
Arab, P.O. Box 21934, Alexandria, Egypt.
2Botany and Microbiology Department, Faculty of Science (Boys), Al-Azhar University, Cairo,
Egypt.
*Corresponding author: City of Scientific Research and Technological Applications (SRTA
City), New Borg Al-Arab City, P.O. Box 21934, Alexandria, Egypt.
Email: amroamara@web.de
ABSTRACT: Being an opportunistic pathogen, P. aeruginosa has a limit
contribution in biotechnology applications. H2O2 can induce mucoid conversion in P.
aeruginosa. Five non-mucoid P. aeruginosa strains were investigated in this study.
After applying H2O2 to these non-mucoid strains two strains were converted to be
mucoid. The stable non-mucoid strains were investigated for their ability to produce
protease and lipase. Agar well diffusion was used for detecting the protease and
lipases activity. Additionally Zymogram was used for in gel determination of the
protease activity. Spectrophotometrically, proteases and lipases activities were
determined using casein and p-nitrophenyl palmitate as substrates respectively. The
apparent activity for each enzyme identified as units/ml. Protease activity was 428
units/mL while lipase activity was 21 units/mL. This study proves the ability of
isolating stable non-mucoid P. aeruginosa strain able to produce of both protease and
lipase. The H2O2 mucoid-non-mucoid test described in this study should be used in
the regular P. aeruginosa identification. We recommended using the non-mucoid P.
aeruginosa strain isolated in this study in different biotechnological applications.
Keywords: P. aeruginosa, Alginate, H2O2, protease, lipase

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1. INTRODUCTION
Pseudomonades a general name referred to a part of a large, heterogeneous and
ubiquitous (Being present everywhere at once) group of microorganisms 1,2. The
treatment of P. aeruginosa infections by strong antibiotic therapy leads to survive of
the patient. This happened usually due to that P. aeruginosa was still in the non-
mucoid form 3. When P. aeruginosa turned to the mucoid form the chance of recovery
decreased 3. The P. aeruginosa mucoid conversion can be easily detected in sputum
because of its ability to produce alginate 4. When P. aeruginosa infects lung, the lung
activate two kinds of defenses the PMNs and antibodies 5. Mathee et al. 6 clearly
proved the role of H2O2 in mucoid conversion of P. aeruginosa in cystic fibrosis and
suggest that mucoid conversion is response to oxygen radical exposure. The big
genetic materials of the P. aeruginosa support the idea of using these microbes in
biotechnological applications. Using genetic techniques P. aeruginosa was studied in
more detail than any other pseudomonad. Amara and Hussain 7 and Hussain and
Amara 8 were highlighted the diversity of P. aerginosa different strains and that each
strain should be studied as a single case 7 ,8. The diversity of P. aeruginosa enables
isolating stable non-mucoid strain(s) which is one aim of this study. Protease, lipase,
uricase and asparaginase are important enzymes produced by P. aeruginosa as well as
many other products like PHA and alginate, which used in many biotechnological
applications 9-17. However, there are increasing interests in P. aeruginosa lipases and
proteases 9, 18. Screening non-mucoid P. aeruginosa for their ability to produce
protease and lipase aiming to establish biotechnological applications is another aim of
this study. H2O2 has been used to screen stable non-mucoid P. aeruginosa strain(s)
able to produce of both lipase and protease aiming to safe biotechnological
production.
2. MATERIALS AND METHODS
2.1. Microbial strains
P. aeruginosa different strains used in this study were isolated from the Egyptian
ecosystem in our Lab., through the past five years from different location and were
screened for their ability to grow and maintain non-mucoid phenotype on LB medium
19.
For confirming the stability of P. aeruginosa non-mucoid phenotype, different strains
were cultivated anaerobically on L-agar (10.0 g of tryptone, 5.0 g of yeast extract, and
5.0 g of NaCl per liter (pH 7.2) and L-agar-nitrate (contain 1% potassium nitrate as an
alternative electron acceptor). Strains which are growing anaerobically on L-agar
nitrate plate at 37oC for 48 h are identified as mucoid 20.
2.2. Study the non-mucoid stability of P. aeruginosa strains
The five non-mucoid P. aeruginosa strains were investigated for their ability to
maintain non-mucoid phenotype after the use of H2O2 (E. coli used as negative

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control). The strains were cultured in static test tube contain 5 mL LB broth culture at
37oC. After four h incubation, 1µL/mL (3%) H2O2 was added to each tube and mixed
briefly. The tubes then left at 37oC for three days. The non-mucoid stable phenotype
was confirmed using L-agar nitrate plates as above.
2.3. Production of Protease
Non-mucoid P. aeruginosa strains were grown on 100 mL flasks contain 20 mL
medium of: 10 g/l meat extract dissolved in tap water. The flasks then were
incubated at shaker incubator (Innova 4230 – New Bruaswick Scientific) at 200
rpm and 37oC for overnight. The cells free supernatant was collected by
centrifugation at 4000 rpm at 4oC and was storage at -20oC for further studies.
2.4. Production of Lipases
Non-mucoid P. aeruginosa strains were grown on 100 mL flasks contain 20 mL
medium: 0.3% (w/v) NH4Cl; 0.3% (w/v) K2HPO4; 0.3% (w/v) KH2PO4; 0.2% (w/v)
MgSO4, olive oil 1% (v/v) and tap water.
The flasks incubated at shaker incubator (Innova 4230 – New Bruaswick Scientific) at
37oC and 200 rpm for overnight. The cells free supernatant collected by centrifugation
at 4000 rpm at 4oC and storage at -20oC for further studies.
2.5. Detection of the proteolytic activity on plates
The different non-mucoid P. aeruginosa strains screened for their ability to produce
protease enzyme using agar well diffusion technique as described by Salem et al. 21
where 0.1 (w/v) gelatin suspended in 100 mL water and autoclaved. After
autoclavation the soluble components added to sterile water agar (16 gm agar/l). The
suspension then stirred gently and distributed in Petri dishes (25 ml/plate). After
complete solidification of the agar on plates, wells were punched out of the agar, b y
using a clean sterile cork borer (6 mm in diameter). The base of each hole was sealed
with a drop of melted sterile water agar (1.5 % (w/v) agar) using sterile Pasteur
pipette. 75 µL of the cell free supernatant of each strain was added to each well and
pre-incubated at -4oC for 30 min and then incubated overnight at 37oC.
2.6. Visualization of the enzyme clear zone
Coomassie blue (0.25%, w/v) dissolved in methanol-acetic acid-water 5:1:4 (v/v/v)
was used for staining the gelatin plates. 10 mL of the staining solution was added to
each plate and incubated in room temperature for 15 min followed by removing the
staining solution from the plate surface and washing gently by distill water. Then the
plates de-stained using destining solution (66 mL methanol, 20 mL acetic acid and
114 mL H2Obidest) for a suitable time 22. Till that the clear zone around each well could
be seen clearly by neck eyes, which an indication about the presence of protease
activity.

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2.7. Zymogram assay
Zymogram assay was used to detect the protease enzyme activity in-gel. The
supernatant of the three non-mucoid strains were conducted simply on SDS-PAGE as
described by Sambrook et al. 19. The distilled water in 12% separating gel was
replaced by 1% (w/v) gelatin powder soluble in universal buffer pH 8 23.
After running gel at 80 v the SDS-PAGE gel was stripped off from the gel plate and
soaked in 1% (v/v) triton X100 for 2 h with changing the solution every 1 h to remove
the SDS content aiming to protease refolding. The gel then washed three times with
tape water and soaked in universal buffer (pH 8) for 2 h at 37ºC. The gel then stained
with amido black stain (0.1% amido black in methanol-acetic acid-water 30:10:60
[v/v/v]) for 5-20 minutes and washed with tape water three times. The appearance of
clear bands means the presence of protease activity.
2.8. Preparation of L-tyrosine standard curve
1.1 mM L-tyrosine was dissolved in 100 mL deionized water by heating gently
(without boiling). After dissolving of the L-tyrosine the standard curve generated by
reading the absorbency for 0, 12.5, 25, 50, 100, 200, 250 and 500 µL from L-tyrosine
solution completed to 1 mL by adding deionized water at 280 nm. The relation among
the absorbency and the mM L-tyrosine then plotted as y/x line plot.
2.9. Preparation of Casein-Universal buffer for different pH enzyme activity
Universal buffer was prepared according to Britton and Robinson 23, which consists of
40 mM H3PO4 and 40 mM acetic acid. The pH was adjusted to 7.3-7.4 using 0.2 M
NaOH.
0.325 mg casein weighted and dissolved in 50 mL of Universal buffer. The mixture
dissolved by heating gently to 80-90oC without boiling. The mixture used
immediately or incubated for short time at 4oC [not longer than 12 h].
2.10. Protease assay
The proteolytic activity of the P. aeruginosa supernatant was determined following
the method described by Salem et al. 21 with modification where 25 µL of each
supernatant (contain the crude enzyme) added to the 575 µL of the Casein-Universal
buffer solutions (pH 7). The enzymes-substrate mixture incubated at 37oC in water
bath for 10 min. After the incubation period, the enzyme reaction stopped by adding
600 µL of 10% trichloroacetic acid (TCA). The mixture allowed to stand at room
temperature for 15 min then centrifuged at 10000 rpm/min for 10 min. The
absorbance was determined spectrophotometrically at 280 nm (PerkinElmer-UV/VIS
Spectrometer Lambda), their tyrosine content derived from the tyrosine standard
curve, and the enzyme activity calculated as units/ml.

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2.11. Detection of the lipolytic activity on plates
Chromogenic substrate plates were prepared by using phenol red (0.01%) along with
2% (v/v) olive oil as substrate, 2 (v/v) % Arabic gum, 10 mM CaCl2 and 2% (v/v)
agar. The pH was adjusted to 7.3–7.4 by using 0.1 N NaOH. The phenol red then
added and mixed well. 25 mL of the mixture was distributed in each plate. After
complete solidification of the agar on plates, wells were punched out of the agar, b y
using a clean sterile cork borer (8 mm in diameter). The base of each hole was sealed
with a drop of melted sterile water agar (15 g agar per liter H2O) using sterile Pasteur
pipette. 75μl of the cell free supernatant of each strain was added to each well and
pre-incubated at -4oC for 30 min and then the plates were incubated overnight at
37oC. The clear zone around each well could be seen clearly by necked eye, which
indicate the presence of lipase activity.
2.12. Lipase assays
Extracellular lipase activity was assayed according to a method described by Salem et
al. 21. The substrate mixture was prepared by adding 200 µL Tween-20 to 40 mg p-
nitrophenyl palmitate dissolved in 10 mL DMSO. The crude enzyme activity was
determined by adding 500 µL from the substrate mixture to 500 µL from 50 mM Tris
HCl, pH 8.0. The enzyme reaction was started by adding 500 µL from the supernatant
which contain the crude enzymes. The buffer and the supernatant each kept worm at
30oC during the assay. The lipase activity was determined by the rate of p-nitrophenol
production (pNP) which measured at 405 nm spectrophotometerically (PerkinElmer-
UV/VIS Spectrometer Lambda). The increase in absorbance against times is
measured against different time interval [Till constant absorbance]. The pNP
extinction coefficient under the conditions described was 14500 L mol cm-1.
One unit (U) was defined as the amount of enzyme catalyzing the liberation of 1 pmol
p-nitrophenol/min at 30°C under the given experimental conditions 24.
3. RESULTS
3.1. Detection of mucoid and non-mucoid strains
Screening for mucoid and non-mucoid P. aeruginosa strains was conducted b y
cultivation different strains on LB agar at 37ºC for 48 h. Strains, show mucoid growth
are neglected while other, which grow normally are subjected for further
confirmation. The phenotype of the mucoid can be distinguished by necked eye due
the slimy feature of the colonies as in figure 1. Five strains show stable non-mucoid
growth on LB agar plats were subjected to further investigation and conformation.
H2O2 used for forcing mucoid conversion in P. aeruginosa strains. After subjecting
different P. aeruginosa strains to H2O2 as above the strains were reinvestigated for the
IR stability regarding to the non-mucoid phenotype by cultivation them anaerobically
in L-agar-nitrate plates at 37oC for overnight cultivation. The true non-mucoid strains
were unable to grow in L-agar-nitrate medium anaerobically while only mucoid
strains can grow. Table 1 show that, two strains have been lost their stability and
changed to mucoid.

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Table 1: P. aeruginosa non-mucoid stability test using H2O2 and their ability to produce
lipase and protease
Lipase
activity
Protease
activity
Phenotype
Strain
No.
+H2O2
a
- H2O2
a
37ºC 37ºC
-ve -ve Mucoid Non mucoid 1
-ve -ve Mucoid Non mucoid 2
+ve +ve Non mucoid Non mucoid 3
-ve -ve Non mucoid Non mucoid 4
-ve -ve Non mucoid Non mucoid 5
a-/+ mean with or without
Figure 1: Mucoid [1] and non-mucoid [2] phenotype of P. aeruginosa strains cultivated
on LB medium
3.2. Detection of the proteolytic activity on plates
The stable three non-mucoid P. aeruginosa were further investigated for their ability
to produce proteases. The proteolytic activity of each strain was determined on
gelatin agar plate. The clear zones around the bacterial growth visualize clear after
coomassie blue staining method as in figure 2.
This technique gives preliminary fast idea about the ability of different P. aeruginosa
strain to produce proteases. The proteolytic activities of the enzymes appear as a clear
zoon around the well which indicates the presence of protyoletic activity in the
supernatant. One strain out of the three stable non-mucoid strains is able to produce
stable amount of protease.

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The methods is simple, fast, cheap and give a preliminarily sign about the presence of
extracellular proteases activities
3.3. Protease activity
Protease enzyme activity of the non-mucoid P. aeruginosa which is show cleare zone
in agar diffusion method was determined spectrophotometricaly. The enzyme assay
was conducted at 37oC using casein as substrate as described above. The apparent
enzyme activity of the protease was 428 Unit/ml.
Figure 2: Protease assay
3.3.1. Zymogram
The zymogram experiment for screening the presence of protease activity in the three
non-mucoid strains was performed. Only one strain was able to degrade the gelatin in
the gel and gave a clear band as shown in Figure 3.
Figure 3: Zymogram gel show the activity of P. aeruginosa strains. Only one strain gives
positive result (clear band).

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3.4. Detection of the extracellular lipase activity on plates
The stable three non-mucoid P. aeruginosa were further investigated for their ability
to produce lipases. The supernatants which contain activities give a clear zone around
the phenol red plate. Only one strain out of the three stable non-mucoid strains is able
to produce lipase as shown in Figure 4. The positive strain gives positive protease
activity.
The methods is simple, fast and cheap and give a preliminarily indication about the
presence of stable extracellular lipase activity.
Figure 4: Lipase assay
3.4.1. Lipase activity
The increase in absorbance at 405 nm which is a result of liberation of pNP were
determined spectrophotometrically (PerkinElmer-UV/VIS Spectrometer Lambda) as
units/ml. The apparent calculated lipase activity for P. aeruginosa was 21 units/ml.
DISCUSSION
P. aeruginosa is an opportunistic [taking immediate advantage, often unethically, of
any circumstance of possible benefit] pathogen, normally did not cause problem to the
healthy persons but can cause virulence diseases for immunocompromised patients. P.
aeruginosa has a big genomic structure, which enable it to show a diverse ability of
environmental adaptation. One of the strangest phenomena in P. aeruginosa is its
ability to mutate themselves to overcome cretin conditions. Reports show that P.
aeruginosa can mutat nalB gene which lead to overexpression of outer membrane
protein OprM (49 KDa) and increase its resistance to quinolones, cephems, penams,
moropenem, tetracyclibe, chloramphenicol and erythromycin 25. Moreover P.
aeruginosa during lung infection and because of the elevation of H2O2 as one of the
lung defense mechanism can be change from non-mucoid to mucoid 6. The ability of
P. aeruginosa to mutat themselves put a big task regarding its control. Moreover P.
aeruginosa strains have a big diversity as reported by Amara and Hussain 7 and
Hussain and Amara 8. Hussain and Amara 8 prove that P. aeruginosa must be

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examined as Case-by-Case study and there is no general role for controlling P.
aeruginosa 8. The big diversity of P. aeruginosa give the possibility to isolate safe
and stable non-mucoid P. aeruginosa strains which could be used in biotechnological
applications. Out of many strains isolated in our Lab., five strains were show stability
regarding to their non-mucoid phenotype were subjected for further investigation.
H2O2, which cause mucoid conversion in P. aeruginosa, was used to study the non-
mucoid stability of these five strains. Three strains were stable while the other two
were converted to mucoid. These results prove the fact that H2O2 mediate mucoid
conversion in P. aeruginosa. While our aim is to identify a P. aeruginosa strain able
to be used in biotechnological applications, the three stable non-mucoid strains were
investigated for their ability to produce protease and lipase enzymes. Simple detection
method was used where agar well diffusion method was used for screening protease
and lipase activities. For protease assay gelatin was the substrate and the plat simply
stained using coomassie blue staining method. One strain was able to produce
protease. Moreover zymogram was used for in gel determination of the protease
activity. The same strategy using agar well diffusion method was used with olive oil
as substrate in presence of phenol red. The same P. aeruginosa strain was able to
produce lipase. Spectrophotometrically, proteases and lipases activities were
determined using casein and p-nitrophenyl palmitate as substrates respectively. The
apparent enzyme activity for each enzyme was identified as units/ml. In this study we
succeeded to analysis the stability of the non-mucoid P. aeruginosa strains, re-prove
the role of H2O2 in P. aeruginosa mucoid conversion, establish a method for analysis
the stability of non-mucoid P. aeruginosa and isolate stable non-mucoid P.
aeruginosa able to produce both of protease and lipase. We recommended using our
strain in biotechnological applications for production of protease and lipase, including
the H2O2 mucoid-non-mucoid test described in this study can be used in the regular P.
aeruginosa identification. The strategies used in this study can lead to isolate a safe
strains of P. aeruginosa could produce different kind of biotechnological products
aiming to industrial applications.
ACKNOWLEDGMENT
We kindly thanks and appreciate the Academy of Scientific Research and Technology
for the grant kindly provided to M.Sc. Mohamed A. Hassan which support this study.
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