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American Journal of  
Life Science and Innovation (AJLSI)

Characterisation Studies on Lipases of  Brevibacterium, Bacillus and Pseudomonas Spp  
Produced Under Sub-Merged Fermentation of  Different Carbon Sources

Omolade, O.A1, Orji, F.A.2*, Agu, G.C1, Adebajo, L.O1

Volume 1 Issue 2, Year 2022
ISSN: 2833-1397 (Online)

DOI: https://doi.org/10.54536/ajlsi.v1i2.303
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Article Information ABSTRACT

Received: June 01, 2022

Accepted: August 13, 2022

Published: August 16, 2022

Lipases are a class of  hydrolytic enzymes that catalyze the hydrolysis of  insoluble triacylglyc-
erol to glycerol, acylglycerols, and free fatty acids. The present study is aimed at identifying 
Lipase producing bacteria isolated from soil determining the optimal conditions (tempera-
tures, pH, and metal ion concentrations) of  Lipases produced through submerged fermen-
tation by bacteria of  different three (3) genera. The different bacterial isolates with good hy-
per-producing potentials for Lipases were identified by Polymerase Chain Reactions (PCR). 
The effect of  temperature on cellulase activity was determined by estimating the lipase activ-
ity at pH 9.0 within a temperature range of  (300C-600C). In order to determine the behaviour 
of  the enzymes within some metallic ions, the reaction of  the enzyme and gum Arabica/
olive oil mixture  was allowed to proceed at 500C with duplicate test tubes containing 50mM 
CaCl2 (Ca2+). MgSO4 (Mg 2+), Nacl(Na+), Kcl (K+). The identifies of  the lipase produc-
ing bacteria were identified as Brevibacterium brevis strains Hk 544, Pseudomonas aeruginosa 
strain WES, Bacillus megaterium strain WH13, and Bacillus subtilis strain BS 01 for Isolate H, 
A, B and F., respectively. Optimum temperatures for the activities of  Brevibacterium brevis 
strains Hk 544, Pseudomonas aeruginosa strain WES, Bacillus megaterium strain WH13, and 
Bacillus subtilis strain were determined to be 50oC, 50oC , 45oC and 45oC, respectively. Op-
timum pH for the activities of  Brevibacterium brevis strains Hk 544, Pseudomonas aeruginosa 
strain WES, Bacillus megaterium strain WH13, and Bacillus subtilis strain were determined 
to be alkaline (8.0 to 9.0). The observation that sodium and potassium ions at 50mm con-
centration enhanced the activity of  some of  the lipases under this investigation showed that 
sodium and potassium are likely to be co-factors for the performance of  these lipases.

Keywords

Brevibacterium, Bacillus, 
Pseudomonas, Polymerase Chain 
Reactions

1 Department of  Microbiology, Faculty of  Science, Olabisi Onabanjo University, Ago-Iwoye, Ogun State, Nigeria.
2 Department of  Biotechnology, Federal Institute of  Industrial Research, Oshodi, PMB 20123, Ikeja, Lagos-Nigeria.
* Corresponding author’s e-mail: orjifa@yahoo.com

INTRODUCTION 
Lipases are ubiquitously produced by the plants 
(Belguith et al. 2009); animals (Carriere et al. 1994); and 
microorganisms (Ramesh et al., 2013). Microbial lipases 
are the preferred potent source due to several industrial 
potentials (Hasan et al., 2006). Lipases are becoming more 
and more popular in the field of  biotechnology, as they 
have received great interest in industrial applications 
because of  their properties (Almeida et al., 2019) The 
uniqueness and its ability to benefit from a wide range 
of  reaction materials and its high stability towards 
temperature and acidity function and organic solvents, 
as most industrial processes are carried out at a high 
temperature and therefore enzymes with high stability 
(Sahu & Martin, 2011). Lipases are considered to be 
the third biggest enzymes group following proteases 
and amylases, based on total sales volume. Because of  
its extensive range of  applications lipase production is a 
billion dollar (Jaeger et al., 1998). Lipases (Glycerol ester 
hydrolases E.C. 3.1.1.3) are much-demanded enzymes with 
significant commercial applications in industries. Lipases 
stimulate the hydrolysis of  triacylglycerol to glycerol and 
free fatty acids. A real lipase will cleave emulsified esters 
of  glycerin and lengthy chain fatty acids such as triolen 
and tripalmitin (Gayathri et al., 2013). Many applications 
of  lipases include specialty organic syntheses, hydrolysis 
of  fats and oils, modification of  fats, flavor enhancement 
in food processing, resolution of  racemic mixtures, and 

chemical analyses (Afaf  et al., 2020). Microbial lipases 
detoxify and degrade the oil effluents as one by innovative 
technologies (Shart & Elkhalil, 2020).
The present study is aimed at identifying Lipase 
producing bacteria isolated from soil, determining the 
optimal conditions (temperatures, pH, and metal ion 
concentrations) of  Lipases produced through submerged 
fermentation by bacteria of  different three (3) genera.

MATERIALS AND METHODS
Molecular identification of  isolates
DNA extraction
DNA was extracted using the protocol stated by Fuguri et 
al., (2015). Briefly, Single colonies grown on medium were 
transferred to 1.5 ml of  liquid medium and cultures were 
grown on a shaker for 48 h at 28 ºC. After this period, 
cultures were centrifuged at 4600g for 5 min. The resulting 
pellets were re-suspended in 520 μl of  TE buffer (10) 
mMTris-HCl, 1mM EDTA, pH 8.0). Fifteen microliters 
of  20% SDS and 3 μl of  Proteinase K (20 mg/ml) were 
then added. The mixture was incubated for 1 hour at 37 
ºC, then 100 μl of  5 M NaCl and 80 μL of  a 10% CTAB 
solution in 0.7 M NaCl were added and vortexed.  The 
suspension was incubated for 10 min at 65 ºC and kept on 
ice for 15 min.  An equal volume of  chloroform: isoamyl 
alcohol (24:1) was added, followed by incubation on ice 
for 5 min and centrifugation at 7200g for 20 min. The 
aqueous phase was then transferred to a new tube and 

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isopropanol (1: 0.6) was added and DNA precipitated at 
–20 ºC for 16 h. DNA was collected by centrifugation at 
13000g for 10 min, washed with 500μl of  70% ethanol, 
air-dried at room temperature for approximately three 
hours and finally dissolved in 50μl of  TE buffer.

Polymerase chain reaction
PCR sequencing preparation cocktail consisted of  10 µl 
of  5x GoTaq colourless reaction, 3 µl of  25mM MgCl2, 
1 µl of  10 mM of  dNTPs mix, 1 µl of  10 pmol each 
27F 5’- AGA GTT TGA TCM TGG CTC AG-3’ and 
- 1525R, 5′-AAGGAGGTGATCCAGCC-3′ primers and 
0.3units of  Taq DNA polymerase (Promega, USA) made 
up to 42 µl with sterile distilled water 8μl DNA template. 
PCR was carried out in a GeneAmp 9700 PCR System 
Thermalcycler (Applied Biosystem Inc., USA) with a Pcr 
profile consisting of  an initial denaturation at 94°C for 
5 min; followed by a 30 cycles consisting of  94°C for 30 
s, 50°C for 60s and 72°C for 1 minute 30 seconds; and a 
final termination at 72°C for 10 mins. And chill at 4oC.
GEL (Hassan et al., 2016; Gunajit et al., 2017).

Integrity of  DNA
The integrity of  the amplified about 1.5Mb gene fragment 
was checked on a 1% Agarose gel ran to confirm 
amplification.  The buffer (1XTAE buffer) was prepared 
and subsequently used to prepare 1.5% agarose gel. The 
suspension was boiled in a microwave for 5 minutes. The 
molten agarose was allowed to cool to 60°C and stained 
with 3µl of  0.5 g/ml ethidium bromide (which absorbs 
invisible UV light and transmits the energy as visible 
orange light). A comb was inserted into the slots of  the 
casting tray and the molten agarose was poured into the 
tray. The gel was allowed to solidify for 20 minutes to 
form the wells. The 1XTAE buffer was poured into the 
gel tank to barely submerge the gel. Two microliter (2 
l) of  10X blue gel loading dye (which gives colour and 
density to the samples to make it easy to load into the 
wells and monitor the progress of  the gel) was added to 
4µl of  each PCR product and loaded into the wells after 
the 100bp DNA ladder was loaded into well 1. The gel 
was electrophoresed at 120V for 45 minutes visualized 
by ultraviolet trans-illumination and photographed. The 
sizes of  the PCR products were estimated by comparison 
with the mobility of  a 100bp molecular weight ladder that 
was ran alongside experimental samples in the gel.

Purification of  Amplified Product
After gel integrity, the amplified fragments were ethanol 
purified in order to remove the PCR reagents. Briefly, 7.6 
µl of  Na acetate 3M and 240 µl of  95% ethanol were 
added to each about 40µl PCR amplified product in a new 
sterile 1.5 µl tube eppendorf, thoroughly by vortexing 
and keep at -20°C for at least 30 min. Centrifugation for 
mix 10 min at 13000 g and 4°C followed by removal of  
supernatant (invert tube on trash once) after which the 
pellet were washed by adding 150 µl of  70% ethanol and 
mix then centrifuge for 15 min at 7500 g and 4°C. Again 

remove all supernatant (invert tube on trash) and invert 
tube on paper tissue and let it dry in the fume hood at 
room temperature for 10-15 min. then re-suspend with 
20 µl of  sterile distilled water and kept in -20oC prior 
to sequencing. The purified fragment was checked on a 
1.5% Agarose gel ran on a voltage of  110V for about 
1hr as previous, to confirm the presence of  the purified 
product and quantified using  a nano-drop of  model 2000 
from thermo scientific.

Plate 1: Agarose gel electrophoresis indicating the 
positive amplification of  the bacteria isolate’s samples 
using ITS universal primers (Band 1 is the marker, band 
2, 3, 4 and 4 represents Brevibacterium brevis strains Hk 544, 
Pseudomonas aeruginosa strain WES, Bacillus megaterium strain 
WH13, and Bacillus subtilis respectively.

Sequencing
The amplified fragments were sequenced using a Genetic 
Analyzer 3130xl sequencer from Applied Biosystems 
using manufacturers’ manual while the sequencing kit used 
was that of  Big Dye terminator V3.1 cycle sequencing kit. 
Bio- Edit software and MEGA 6 were used for all genetic 
analysis.

Lipase production under different carbon source 
media
The determination of  optimum carbon Lipase production 
medium hydrolytic activity of  isolated bacteria lipase 
was done on composed of  (g/L): peptone, 10; NaCl, 
5; CaCl2.2H2O, 0.1; Trybutyrin selective agent, 10 mL 
(v/v). (NH¬4)2 SO4(1.4g), K2HPO4 (2.0g), CaCl2 (2.0g), 
MgSO4.7H2O (0.3g), peptone (7.5g), FeSO4 (5.0g), MnSO4 
(1.6g), ZnSO4 (1.4g). In addition different Erlenmeyer 
flasks containing the above chemical compounds 
amended using different carbon sources such as Tobacco 
seed oil (5ml/100ml of  production medium), Tobacco 
seed oil (10ml /100ml of  production medium), Olive 
oil (5ml/100ml of  production medium), Olive oil  (10ml 
/100ml of  production medium). The Erlenmeyer flasks 
were loaded at the shaker incubator at 150RPM for 5days.
there after 5 days of  incubation; the Lipase activities were 
determined using NaOH titration method as previously 

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described above.

Characterization of  Enzyme Based on Stability
The purified fraction showing highest specific activity was 
characterized by varying the parameters that influence 
enzyme activity.

Effect of  pH on Activity and Stability of  Lipase
This was determined by emulsifying 25ml of  olive oil 
with 75ml of  7% gum Arabic for 10mins. The reaction 
mixture containing 5ml of  olive oil emulsion, 2ml of  
0.1M phosphate buffer (pH 7.0) Gum Arabica 1% (w/v) 
as substrate suspended in various buffer systems: 0.1M 
sodium acetate buffer (pH 6.0 - 7.0): 0.1 M sodium 
phosphate buffer (6.0-7.0); tris-HCl buffer (8.0) and 
a glycerine- NaOH buffer (pH 9.0-). The pH stability 
studies was performed by pre-incubating the purified 
enzyme without substrate in pH values ranging from 
4.0-10.0 at 50 0C for 2 h and subsequent analysis was 
determination under standard assay carried out (Okoli et 
al., 2017).
Effect of  Temperature on Activity and Stability of  Lipase 
The effect of  temperature on Lipase activity was 
determined by estimating the lipase activity at pH 
9.0 within a temperature range of  (300C-600C) for 2h 
using gum Arabica and olive oil mixture as substrate. 
The thermal stability of  lipase was determined by pre-
incubating the purified enzyme preparation (pH 9.0) 
at different temperatures (300C - 600C) for 2 h without 
substrate. The residual cellulase activity was determined 
under standard assay conditions (Sharma et al., 2018; 
Okoli et al., 2017). 

Effect of  Metal Ions on Enzyme Activity
The reaction of  the enzyme and gum Arabica/olive oil 
mixture  was allowed to proceed at 500C with duplicate 
test tubes containing 50mM CaCl2 (Ca2+). MgSO4 (Mg 2+),  
Nacl(Na+), Kcl (K+). Test tubes were labeled according 
to the above mentioned salts. This was determined by 
emulsifying 25ml of  olive oil with 75ml of  7% gum 
Arabic for 10mins. The reaction mixture containing 5ml 

of  olive oil emulsion, 2ml of  0.1M phosphate buffer (pH 
7.0), Gum Arabica 1% (w/v) as substrate suspended in 
0.1M phosphate buffer, In addition. Into each of  the 
tubes 50mm of  the various metal ions (5ml) were added, 
incubated at 45OC, and thereafter titrated against 0.1N 
NaOH using Phenolphthalein indicator.

Determination of  Lipase activities 
Lipase screening assay was carried out using olive oil 
emulsion prepared by emulsifying 25ml of  olive oil with 
75ml of  7% gum Arabic for 10mins.
Titration: 2-3 drops of  phenolphthalein indicator were 
added to the reaction mixture and the liberated free fatty 
acids were titrated with 0.5N NaOH to the end point of  
pink color at pH 10.0 (Macedo et al., 1997; Lopes et al., 
2011; Mendes et al., 2011; Bhavani, et al., 2012; Nagarajan 
et al., 2014; Ullah et al., 2015). Lipase activity was calculated 
as micromoles of  free fatty acids formed from olive oil 
per ml of  lipase enzyme as given by the equation:
Activity = (VS – VB). N. 1000
                                 S
Where, VS is the volume of  0,05M NaOH solution 
consumed by the enzyme _substrate cocktail (ml); VB 
is the volume of  0.05M NaOH solution consumed in 
the titration by the substrate (control) cocktail; N is the 
molar strength of  the NaOH solution used for titration 
(0.05M); S is the volume of  substrate cocktail solution. 
One unit of  lipase enzyme is defined as the amount of  
enzyme required to liberate 1µmol of  fatty acids from 
triglycerides (Okoli et al., 2019).

RESULTS AND DISCUSIONS 
The identifies of  the lipase producing bacteria were 
identified as Brevibacterium brevis strains H k 544, 
Pseudomonas aeruginosa strain WES, Bacillus megaterium strain 
WH13, and Bacillus subtilis strain BS 01 for Isolate H, A, 
B and F., respectively (Table 1). These bacterial isolates 
had their genomes blasted on the blast software of  the 
NCCI, and similarities between 99-100% at different 
accession numbers were recorded (Table 1.)The bands of  
the nucleotide were also arranged in get as shown in plate.

Table 1: Summary of  Molecular Identity of  some selected lipase hyper-producing strains of  Bacteria
S/N Isolate codes Identity % Simi-larity Accession Num-ber
1 H Brevibacillus brevis strain HK 544 99.44 CPO42161.1
2 A Pseudomonas aeruginosa WES2 99.93 MN960116.1
3 B Bacillus  megaterium strain WH13 100 Mn372086.1
4 F Bacillus subtilis BS 01 100 MT372489.1

This current investigations that observed different strain 
of  Brevibacterium brevis, Pseudomonas aeruginosa strain WES, 
Bacillus megaterium strain WH13, and Bacillus subtilis strain 
BS 01 as hyper –producing bacteria is also tandem with 
other reports by other workers at different parts of  the 
globe. Ertuğrul et al., (2007) reported the use of  hyper – 
producing strain of  Bacillus from lipase production. Kiran 
et al. (2008) also documented the use of  Pseudomonas for 
sub nerved production of  extra- cellular lipase.

This report on this study is also in tandem with periods 
study by Bradoo et al. (1999) which also documented 
lipase- hyper producing strain of  Bacillus species resident 
in soil.      
In independent related studies, Ambu et al., (2010) 
reported the presence of  extra-cellular lipase producing 
Acinetobacter Junii in the soil of  south Korea. Bompensieri 
et al. (1996) had also reported the isolation of  Lipse-
Producing bacterial such as Acinetobacter from different 

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environmental sources.
Thus, one concludes that the observation of  Bacillus, 
Pseudomonas, Acinetobacter, Brevibacterium strains in this 
study as Lipases hyper-Producing is within the pattern 
of  previous observation in this subject matter. The 
physiology and genetics of  enzyme expressions in 
microorganisms show that enzyme production potentials 
in bacteria, and fungi lie on genes and equally regulated 
by genes. This could be extended to mean that any 
microorganisms irrespective of  genes or species can 
produce any enzyme provided it has the genes to express 
such enzymes. In a horizontal gene transfer within a wild 
environment, bacterial and fungal strains can transmit 
genes from one genus to closely related genus.
In addition, under a conventional condition, bacterial 
and fungal isolates that do not have to express lipase 
production, could along their co-existence with lipase-
producing bacterial and fungal isolates acquire the 
genes for lipase production either on their genome/
chromosome of  outside the chromosome (in this case, it 
is said that the microorganism has acquired plasmids for 
such enzyme productions).

The lipases isolated under different conduction from 
Isolate H (Brevibacterium brevis), Isolate A (Pseudomonas 
aeruginosa), Isolate B (Bacillus megaterium), and Isolate F 
(Bacillus subtilis) were subjected to temperature stability 
studies (Figure 1). 
Brevibacterium brevis at temperatures of  250, 350, 400, 540, 
and 600 on a centigrade scale showed a lipase activity of  
8.0, 9.0, 7.0, 10.0, 22, and Units of  lipase respectively 
(Figure 1). This showed that lipase of  Brevibacterium brevis 
has an optimum temperature of  500c. This implies that 
industrial application using this lipase of  Brevibacterium 
brevis strain HK 544 must be maintained at 50oC to 
achieve the best desired hydrolysis.
Furthermore in a related study, Luz et al. (2021) reported 
Pseudomonas fluorescens isolated from water, bryophytes and 
soil showed different tolerance pattern to temperature. 
Luz et al. (2021) showed that S. marcescens lipase had an 
optimum temperature at 35oC while Pseudomonas fluorescens 
lipase had optional temperature at temperature of  55oC.
Pandey et al. (2016) working on lipase of  Penicillum 
fungus discovered that temperature of  250C. Kojima 
et al. (1994) observed that similar strain of  Pseudomonas 

Figure 1: pH stability of  the lipase enzymes from Brevibacillus brevis strain HK 544 (ISOLATE H) Pseudomonas aeruginosa 
WES2 (ISOLATE A), Bacillus megaterium strain WH13 (ISOLATE B) and Bacillus subtilis BS 01 (ISOLATE F)

fluorescens has an optimal temperature of  550C.
In addition, Lipase isolated from Isolate A (Pseudomonas 
aeruginosa WES 2) should lipase activities of  10.0 units 12.0 
14.0, 19.0 30.0, and 13.0 units, at temperatures of  250C, 
350C, 400C, 450C, and 600C respectively (Figure 1). This 
implies that 50oC remain the best /optimum temperature 
that can active the highest level of  hydrolysis during the 
application of  two other workers in Brazil (Chandra et 
al. 2020) in an extensive review reported that Lipases of  
Chromobacterium viscosum, Aspergillus niger, Rhizopus species 
and Pseudomonas nitroaceducans has an optimal activities 
at lower temperatures between 35OC – 40OC. This is in 
contrast with this current study as lipase of  Pseudomonas 
aeruginosa has optimal activity a temperature of  50O on a 
centigrade scale.
This current work is in agreement with the report of  
Kiran et al. (2008) which reported lipase of  marine strain 
of  Pseudomonas aeruginosa   having an optimal temperature 

of  40-500C.
Lipase of  bacterial isolate B (molecularly identified as 
Bacillus megaterium strain WH 13) at temperature of  (25oC, 
350C, 40oC, 45oC, 50oC and 60oC showed Lipase activities 
of  5.0, 6.0, 9.1, 20.0, 8.0, and 5.0 units, respectively 
(Figure 1). Statistical analyses using ANOVA (analyses of  
variance) showed that among the four (4) Lipases from 
four (4) different bacterial isolates in this study, Lipase 
of  Bacillus megaterium had the least response in terms of  
Lipase activities. It is also recorded that the optimum 
temperature for Lipase isolated from Bacillus megaterium 
was observed at 450C. In another study on Lipase of  
Bacillus species isolated from the hot spring of  had an 
optimal temperature of  60oC. 
Thermal stability of  Lipase of  Bacillus megaterium strain 
WH 13 at 40oC could be also explained by the presence 
of  polyamines in the general protein structure. The 
increased number of  hydrogen bonds, salt bridges and 

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relatively high amount of  thermo-tolerant amino acids 
may contribute to the thermal stability of  this Lipase at 
40oC. The result of  this current study is in contrast with 
the work of  Brune & Gotz (1992), which reported that 
Lipase of  Pseudomonas sp KWA-56 showed an optimum 
temperature of  60oC and remain stable at 600C.
In terms of  ergonometric, it has become very important 
to develop novel enzymes that remain active and stable 
temperature ranges. This enzyme being most active at 
40oC implies a lot of  cost optimization implication as 
little heat energy is required to achieve enzyme- substrate 
catalysis during industrial applications (Brune & Gotz, 
1992).
In terms of  bio-energetics, Lipase of  Bacillus megaterium 
at 40oC means that little/ relatively low amount of  heat 
energy is required to achieve bund breaking in substrates 
in order to reduce the activation energy, create an 
alternative pathway and establish catalysis products (Fatty 
acids in this case). Studies showed that the Lipase of  
Bacillus subtilis (isolate F) at temperatures of  25oc, 35oc, 
40oc, 45oc, 50oc, and 60oc showed Lipase activities of  6.0, 
16.0, 8.0, 20.0, 4.0, and 4.0 units, respectively. In this case, 
it is deduced that the Lipase of  Bacillus subtilis had two 
temperature optima (35oC and 45oC). 
This implies that industrial applications of  this Bacillus 
subtilis Lipase is best at 35oC and 45oC in order to achieve 
the best hydrolysis desired. Bakir & Metin (2017) identified 
a thermophilic Bacillus species from the hot springs of  
Ayidin, Turkey and reported in a similar fashion to this 
work that the thermophilic Bacillus sp strain remained 
most active at temperature of  45oC – 50oC. 
Other similar independent studies also had previously 
confirmed that Bacillus coagulans Lipases have optimum 
temperature ranges of  45oC – 55oC (Fojan, 2000; Lima 
et al., 2004; Sulong et al., 2006). The pH responses of  the 
Lipase invested from Pseudomonas aeruginosa (Isolate A) 
showed that pH values of  6.0, 7.0, 8.0, and 9.0,  showed 
Lipase activities of  6.0, 9.0, 13.0 and 5.6 units statistical 
analysis using ANOVA showed that there are significant 

differences in the units of  activities from pH 6.0 to pH 
9.0. Inductively, the Lipase of  this Pseudomonas aeruginosa 
strain WES 2 (Isolate A) has alkaline pH optima and 
applications must be maintained within alkaline ranges to 
ensure optimal hydrolysis by the enzyme. This observation 
is in tandem with similar studies in Japan where Yoshitaka 
et al. (1982) reported the presence of  alkaline Lipase from 
different strain of  Gram-negative Alcaligenes species.
Relatively recently, Liew et al. (2015) reported the 
isolation of  alkaline Lipase from Burkholderia cepacia after 
optimization using submerged fermentation technology. 
The result obtained in this study is further agreement with 
the work of  Gupta et al. (2009) which reported isolation 
of  alkaline Lipase from Burkholderia sp C20.
Furthermore, Liu et al. (2006) and Sharma et al. (2002) 
independently observed at different location that strain 
of  Arthrobacter sp had Lipases with optimum activity at 
pH of  HP8.0 – 9.0.
In addition, the work of  Gupta et al. (2004) contradicts 
this current study. Gupta et al. (2009) reported that some 
bacterial Lipase showed optimum activities at neutral 
pH (7.0). The pH responses of  the three (3) Lipase 
investigations were studies and documented in Figure 2.
Lipase harvested from isolate H (Brevibacillus brevis strain 
Hk 544) at pH values of  6.0, 7.0, 8.0, and 9.0 showed Lipase 
activities of  5.0, 6.0, 5.0, and 23.0 units, respectively. This 
implies that the Lipase of  Brevibacillus brevis strain HK 544 
is an alkaline Lipase and all application of  the enzyme 
must be carried out using pH 8.0. (Figure 2) Similarly in 
Pradesh, India Bora & Bora (2012) reported the isolation 
of  alkaline Lipase from thermophilic Bacillus species 
resident in soil. The study in strong  agreement with 
the report of  Rathi et al (2001) which observed strains 
of  Bacillus species with Lipase having optimal activities 
beyond 17.5 units of  activity at pH of  8.5 (alkaline).
The Lipase harvested from Bacillus megaterium WH 13 
(isolate B) at pH values of  6.0, 7.0, 8.0, and 9.0 had 
activities of  5.6, 4.0, 7.0 at 10.0 units, respectively. This 
showed that the Lipase from WH 13 strain of  Bacillus 

Figure 2: pH stability of  the lipase enzymes from Brevibacillus brevis strain HK 544 (ISOLATE H) Pseudomonas aeruginosa 
WES2 (ISOLATE A), Bacillus   megaterium strain WH13 (ISOLATE B) and Bacillus subtilis BS 01 (ISOLATE F).

megaterium is an alkaline type of  Lipase with pH 9.0 as 
optimum pH for optimal activity or hydrolysis.
Rasmey et al. (2017) took a different position and reported 
that Pseudomonas monteili 2403 showed optimum Lipase 

activities at pH 6.0 which is moderately acidic. Other 
scholars such as Qamsari et al. (2011), Sooch and Kaulder 
(2013) had independently reported that different species 
and strains of  Bacillus and Pseudomonas spp expressed 

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Lipases that had optimum activities at pH ranges of  6.0 
– 6.5.
In addition, Lipase harvested from Bacillus subtilis (isolate 
F), at pH values of  6.0, 7.0, 8.0, 9.0 showed a lipolytic 
activity of  6.0, 7.0 9.0 and 15.0, respectively (Figure 2). 
This equally showed alkaline optimum pH, and could be 
used at pH 9.0 for the best hydrolytic performance.
This observation in the current investigation, is in 
agreement with previous study by Prasasty et al. (2016) 
which documented that Bacillus species and Pseudomonas 
alcaligenes had Lipases with optimum activities of  1.0 x 
104 U/mg and 0.8 x 104 U/mg, respectively (Prasaty et 
al. (2016.).
However, Tang and Xia (2005). showed that Bacillus 
coagulans ZJU strain works optimally at 7.0 – 10.0 pH 
values and activity remained depressed at acidic pH 

ranges. In further characterization based on reaction with 
heavy metals from alkalis and alkaline earth metals in the 
periodic group, the performance as regards activities of  
the three (3) Lipases have been documented as in figure 3.
Lipase of  Brevibacterium brevis HK 544 got higher lipolytic 
activity of  20 units at 50mM concentration of  sodium 
and at 50MmM, concentrations of  Potassium (K), 
Calcium (Ca), and magnesium (Mg) the lipase harvested 
from Brevibacterium brevis stain HK 544 got repressed to 
6.0, 8.0 and 8.0 units, respectively (Figure 3).
The observation that sodium ion at 50mm concentration 
enhanced the activity of  lipase of  Brevibacterium showed 
that sodium is likely to be a co-factor for the performance 
of  this lipase from B. brevis.
The lipase of  Pseudomonas aeruginosa  (Isolate A), showed 
a repressed enzyme activity of  6.0/12.0 and 9.0 units at 

Figure 3: Metallic ion stability of  the lipase enzymes from Brevibacillus brevis strain HK 544 (ISOLATE H) Pseudomonas 
aeruginosa WES2 (ISOLATE A), Bacillus   megaterium strain WH13 (ISOLATE B) and Bacillus subtilis BS 01 (ISOLATE 
F)
50mM concentration of  Sodium (Na+) Potassium (K+), 
and Calcium (Ca2+) ions, while at 50mM concentration of  
magnesium (Mg2+) had an enhanced activity of  18.0 units 
of  lipase activity. The essence of  this characterization has 
identified magnesium as possible co-factor for the activity 
of  Pseudomonas aeruginosa Wes2 strain.
In a related pattern, the lipase of  Bacillus subtilis BS 01 
(isolate f) had a strong increase in activity on exposure 
of  substrate and enzyme under 50mM concentration of  
sodium ion (12.0 units) while the activities of  the Bacillus 
subtilis enzyme /lipase at 50mm concentrations of  K+, 
Ca2+ and Mg2+ had repressed enzyme units of  4.0, 8.0, 
and 8.0 units (figure 3).
The Lipase of  Bacillus megaterium strain WH13 (Isolate 
B,) showed complete repression on exposure with 
50mm concentrations of  sodium (Na+), potassium (K+), 
Calcium(Ca2+), and Magnesium (Mg2+) as 8.0, 6.0, 4.0, and 
7.0 Units of  Lipase activities lose recorded respectively  
(Figure 3).
CONCLUSION 
The Properties of  Lipases from Brevibacterium brevis 
strains Hk 544, Pseudomonas aeruginosa strain WES, Bacillus 
megaterium strain WH13, and Bacillus subtilis strain BS 
01have been tracked and industrial application of  any of  
the Lipases can now be achieved under the scientifically 
proven-conditions as observed in this study. The study is 
expected to utilize the produced Lipases for production of  

Biodiesel. However, at this point, the aspect of  Biodiesel 
production using the Lipases cannot be established as a 
result of  limitation in funds. 

REFERENCES
Almeida JM., Martini V, Iulek J, Alnoch R, Moure V, 

Müller-Santos M and Krieger N 2019. Biochemical 
characterization and application of  a new lipase and 
its cognate foldase obtained from a metagenomic 
library derived from fat-contaminated soil. International 
journal of  biological macromolecules, 137: 442-454.

Ambu, P., Noth, M.J, Kim, D-H, Seo, J-S, Hur, B-K, and 
Kyeong, H.M.(2011). Screening and optimization of  
extracellular Lipases by Acinetobacter species isolated 
from oil contaminated soil in South Korea, African 
Journal of  biotechnology, 10(20):4147-4156.

Brune, A.K.; Gotz, F. (1992). Degradation of  lipids by 
bacterial lipases. In Winkelmen G(ed) Microbial 
degradation of  natural products, VCH, Weinhein, pp 
243-266. 

Bora, L., and Bora, M. (2012). Optimization of  
extracellular thermophilic highly alkaline Lipase from 
thermophilic Bacillus species isolated from hotspring 
of  Arunachal Pradesh, India, 30-34.

Bakir, Z.B., and Metin, K. (2017). Production and 
characterization of  an alkaline Lipase from 
thermophilic Anoxybacillus sp. HBB16, Chem 

https://journals.e-palli.com/home/index.php/ajlsi


Pa
ge

 
7

https://journals.e-palli.com/home/index.php/ajlsi

Am. J. Life Sci. Innov. 1(2) 1-8, 2022

Biochem. Eng. , 31(3): 303-317.
Belguith H, Fattouch S, Jridi T, Ben Hamida J.(2009) 

Immuno-purification and characterization of  a rape 
(Brassica napus L.) seedling lipase. Afr J Biotechnol., 
12(21): 3224-3234.

Chandra, p.,   Nespa, E., Singh, R., and Arora, P.K. (2020). 
Microbial Lipases and their Industrial applications: a 
comprehensive review, Microbial cell factories, 19: 169-
210.

Carriere F, Thirstrup K, Hjorth S.(1994) Cloning of  the 
classical guinea pig pancreatic lipase and comparison 
with the lipase related protein 2. FEBS Lett., 388: 63-
68

Ertuğrul, S., Dönmez, G., Takaç, S., Isolation of  lipase 
producing Bacillus sp. from olive mill wastewater and 
improving its enzyme activity, J. Hazard. Mater. 149 
(2007) 720.

Fojan, P. (2000). What distinguishes an esterase from a 
lipase: A novel structural approach, Biochimie 82: 1033-
1034.

Fugiri, I.,  M. Atigui, M. Ziadi, S. Arroum and T. 
Khorchani. 2015. Biochemical and molecular 
identification of  lactic acid bacteria isolated from 
camel milk in Tunisia. Emir. J. of  Food and Agric., 27 
(9):716-720.

Gupta, R.; Gupta, N.; Rathi, P. (2004). Bacterial lipases: an 
overview of  production, Purification and Biochemical 
properties. Appl Microbiol Biotechnol 64: 763-781. 

Gunajit G., Sudupta, S.B., Assma P.,  Robin C.B.,  
Madhumita B. (2017).Identification and functional 
properties of  dominant lactic acid bacteria isolated 
from Kahudi, a traditional rapeseed fermented food 
product of  Assam, India Journal of  Ethnic Foods, 4 (3): 
2-13

Gayathri, V.R., Perumal, P., Mathew, L.P. and Prakash, B. 
(2013) Screening and Molecular Characterization of  
Extracellular Lipase Producing Bacillus Species from 
Coconut Oil Mill Soil. International Journal of  Science and 
Technology, 2: 502-509.

Hassan, ZMR, Efat, B.A.M., Magdoub, M.M.I., Tawfik, 
N.F., Sadek, Z.I.M, Mabrouk, A.M.N. (2016). 
Molecular Identification of  Lactic Acid Bacteria 
isolated from dairy products, International Journal of  
Biology, Pharmacy and Allied Sciences, 5(12): 3221-3230.

Hasan F, Shah AA, Hameed A. (2006). Industrial 
applications of  microbial lipases. Enzyme Microb 
Technol; 39: 235-251. Shart, A. and Elkhalil, E. (2020) 
Biochemical Characterization of  Lipase Produced by 
Bacillus spp. Isolated from Soil and Oil Effluent. Advances in 
Enzyme Research: 8, 39-48. 

Jaeger, K.E., Dijkstra, B.W. and Reetz, M.T. (1998) 
Bacterial Biocatalysts: Molecular Biology Three 
Dimensional Structures and Biotechnological 
Applications of  Lipases. Annual Review of  Microbiology, 
53: 315-351.

Kiran, S.G.; Shanmughapriya, S.; Jayalakshmi, J.; Selvin, J.; 
Gandhimathi, R.; Sivaramakrishnan, S.; Arunkumar, 
M.; Thangavelu, T.; Natrajaseenivasan, K. (2008). 

Optimization of  extracellular psychrophilic lipase 
produced by marine Pseudomonas sp. (MSIO57). 
Bioprocess Biosyst Eng 31: 483-492.

Kiran, S.G.; Shanmughapriya, S.; Jayalakshmi, J.; Selvin, J.; 
Gandhimathi, R.; Sivaramakrishnan, S.; Arunkumar, 
M.; Thangavelu, T.; Natrajaseenivasan, K. (2008). 
Optimization of  extracellular psychrophilic lipase 
produced by marine Pseudomonas sp. (MSIO57). 
Bioprocess Biosyst Eng 31: 483-492.

Lima, V. M. G., Krieger, N., Mitchell, D. A., Baratti, J. 
C.,Filippis, I., Fontana, J. D. (2004) .  Evaluation of  
the potential for use in biocatalysis of  a lipase from 
a wild strain of  Bacillus megaterium, J. Mol. Catal. B: 
Enzym. 31: 53-57.

Lima, V. M. G., Krieger, N., Mitchell, D. A., Baratti, J. 
C.,Filippis, I., Fontana, J. D., Evaluation of  the 
potential for use in biocatalysis of  a lipase from a wild 
strain of  Bacillus megaterium, J. Mol. Catal. B: Enzym. 31 
(2004) 53.

Luz, B.D., SArrouh, B., Bicas, L.S., and Lofrano, R.C.Z. 
(2021). Lipase production by microrganisms isolated 
from Serra de Ouro Branco State Park, Annals of  
Academy of  Brazillian Scientists, 93(3): 1-12.

Okoli, C.B., Orji, F.A., and Lawal, A.K. (2019). Isolation, 
Screening, and Optimization of  Bacillus spp for 
lipolytic enzyme production, Journal of  Industrial 
Research and Technology,7 (1): 112-129.

Pandey, N.,  Dhaka, K.,  Jain, R.,  and Pandey, A. (2016). 
Temperature dependent Lipase production from cold 
pH tolerant species of  Penicillium, Mycosphere, 7(10): 
1533-1545.

Ramsey, A-H., Aboseidah, A.A., Gaber, S., and Mahran 
F. (2017). Characterization and optimization of  
Lipase activity produced by Pseudomonas monteilii 2403-
ky120354 isolated from ground beef  , African Journal 
of  Biotechnology, 16 (2): 96-105

Ramesh S, Kumar R, Devi A, Balakrishnan, K.(2014). 
Isolation of  a lipase producing bacteria for enzyme 
synthesis in shake flask cultivation. Int J Curr Microbiol 
App Sci.; 3(3): 712-719.

Riaz M, Shah AA, Hameed A., Hasan F (2010). 
Characterization of  lipase produced by Bacillus sp. 
FH5 in immobilized and free state. Ann Microbiol.; 60: 
169-175.

Rathi, P.; Saxena, R.K.; Gupta, R. (2001). A novel 
alkaline lipase from Burkholderia cepacia for detergent 
formulation. Process Biochem 37: 187-192.

Sulong, M. R., Abdul Rahman, R. N. Z. R., Salleh, A. B., 
Basri, M. (2006). A novel organic solvent tolerant 
lipase from Bacillus sphaericus 205y: extracellular 
expression of  a novel OST-lipase gene, Protein Expr. 
Purif. 49: 190-195.

Sahu G and Martin M . (2011). Optimization of  growth 
conditions for the production of  extracellular lipase 
by bacterial strains from dairy industry effluents. 
Biotechnol Bioinf  Bioeng, 1(3): 305-31.

Sharma, A., Meena, K. R., and Kanwar, S. S. (2018).
Molecular characterization and bioinformatics studies 

https://journals.e-palli.com/home/index.php/ajlsi


Pa
ge

 
8

https://journals.e-palli.com/home/index.php/ajlsi

Am. J. Life Sci. Innov. 1(2) 1-8, 2022

of  a lipase from Bacillus thermoamylovorans BHK67. 
Int. J. Biol. Macromol. 107, 2131–2140. doi: 10.1016/j.
ijbiomac.2017.10.092

Sulong, M. R., Abdul Rahman, R. N. Z. R., Salleh, A. 
B., Basri, M., A novel organic solvent tolerant lipase 
from Bacillus sphaericus 205y: extracellular expression 

of  a novel OST-lipase gene, Protein Expr. Purif. 49 
(2006) 190.

Veerapagu M, Sankara D.R., Narayanan A, Ponmurugan 
K., Jeya K.R. (2013). Screening selection identification 
production and optimization of  bacterial lipase from 
oil spilled soil. Asian J Pharm Clin Res. 6: 62- 67.

https://journals.e-palli.com/home/index.php/ajlsi

