www.sciencejournal.

in

PRODUCTION AND CHARACTERIZATION OF EXTRACELLULAR LIPASES OF STAPHYLOCOCCUS

SP. ISOLATED FROM OIL CONTAMINATED SOIL
Tembhurkar V. R.*, Dama L.B.**, Attarde N. P.*** and Zope P. S.****
* Department of Microbiology, NKSPTs Arts, Science and Commerce College, Badnapur, Jalna (M.S.), India.
**Department of Zoology, D.B.F. Dayanand College of Arts and Science, Solapur, 413002, (M.S.), India.
*** Department of Biotechnology, P. O. Nahata College, Bhusawal, (M.S.), India.
**** Department of Biotechnology, MGMs Institute of Biosciences and Technology, Aurangabad (M.S.), India.
ABSTRACT
Staphylococci sp. was isolated from oil contaminated soil was used for extracellular lipase production. The lipase
production was enhanced by UV mutagenesis. Sodium azide treatment could not improve lipase secretion. Optimum
time and pH for lipase production was found to be 72 hrs and 8 respectively. Media optimization experiments
revealed optimum carbon source was groundnut oil and nitrogen source as meat extract. All the four trace elements
tested (Zinc, Iron, Copper and Calcium) had positive effect with respect to Magnesium on lipase production. Lipase
was working optimally at temperature 30 0C, pH 8 and substrate concentration 15%. Enzyme activity was enhanced
by Zinc, Calcium and Copper at 100ppm concentration; Magnesium and EDTA at 300ppm. Ferrous ions inhibited
lipase action.
KEY WORDS: lipase, sodium azide, Staphylococci sp., UV mutagenesis.
INTRODUCTION
Lipases are serine hydrolases which has uncommon potential of acting at the lipidwater interface. Due to unique
properties of lipases the enzyme has been proved to be useful for wide range of biotechnological applications (Gupta et
al., 2004; Jaeger et al., 1999). Currently bacterial lipases are of great demand because of potential industrial
applications (Sirisha et al., 2010). Different genera of bacteria including Streptomyces spp. are known to produce lipase
but among them Achromobacter spp, Alcaligenes spp, Arthrobacter spp, Pseudomonas spp and Chromobacterium spp
have been well exploited for lipase production (Ghosh et al., 1996). Staphylococci is the another genera shown the
potential of lipase production. Staphylococcal lipases are classified as true lipases (Jaeger et al., 1999; Rosenstein and
Gtz, 2000; Simons et al., 1996).
In most instances lipase production ability of Staphylococci has been related to their pathogenecity. Thus with few
exceptions, there are almost no reports of attempts to purify and characterize lipases synthesized by the coagulasenegative staphylococci which are believed to be nonpathogenic. The Staphylococci whose lipases are been studied till
date are either isolated from medical samples (pathogenic) or does belong to commenssal microfloara of skin (Smeltzer
et al., 1992; Troller and Bozeman, 1970). In the current communication lipase producing Staphylococcus sp. was
isolated from oil and petroleum contaminated soils. Effect of few growth parameters and media components on lipase
production by isolated Staphylococcus sp. was analyzed. And the lipase produced was characterized.
MATERIALS AND METHODS
Isolation of extracellular lipase producing bacteria: Soil samples were collected from few automobile service
stations, petrol pump and oil shops in Aurangabad region. Enrichment of lipase producers done is mineral medium
composed of (g/L); Ammo. dihydrogen orthophosphate 1.0, KCl 0.2, MgSO4 0.2, yeast extract 3.0, pH 7.8. The media
was supplemented with 5% olive oil as sole source of carbon and bromophenol blue indicator (0.1%). Enrichment
carried out till color of the medium changed from dark green to yellow. Individual bacterial cultures were isolated on
nutrient agar plates. All the isolates were screened for extracellular lipase production by spot inoculating on tributyrin
agar (Aaronson, 1970). The isolate showing largest zone of clearance was subjected to biochemical characterization.
For identification the ABIS online software was used. This single culture was used for further studies.
Strain improvement by random mutagenesis: Selected Staphylococcus sp. was subjected to random mutagenesis.
Two mutagens; UV (254nm) and sodium azide (1mg/ml) were used. Mutagen treatment was given up to 50min with
the interval of 5min. Survivors of mutagen treatment were spotted on tributyrin agar to check increased lipase
production.
Lipase assay: Lipase activity was measured according to titrimetric method of Tembhurkar et al. (2012). One unit of
lipase is defined as amount of enzyme required to liberate 1 M of fatty acids per minute under the assay conditions.

Vol. 1 No. 1 (2012)

ISSN 23200421(Print); ISSN 2320043X(Online)

2012 DAMA International. All rights reserved.

36

www.sciencejournal.in

Optimization of lipase production condition: Growth conditions and media composition was optimized in separate
experiments. For study of time course and effect of pH, mineral media of composition similar as used for enrichment
step was used. For all the experiments 1% inoculums (24hr old) was used. For time course experiment after every 24hrs
of incubation lipase activity estimated. Optimum pH was determined by adjusting the initial media pH over the range 3
to 10 using 0.1N NaOH and 0.1N HCl. Effect of carbon source was analyzed by supplementing the mineral media with
different carbon sources; coconut oil, safflower oil, mustard oil, groundnut oil, olive oil. Nitrogen source was optimized
by replacing the ammo. dihydrogen orthophosphate and yeast extract in mineral medium with different test nitrogen
sources; Tryptone, yeast extract, meat extract, peptone, beef extract. The media used for analysis of effect of mineral
salts composed of (g/l); Ground nut oil 5.0 ml, meat extract 1.0, KCl 0.2, pH 8. This media was then supplemented with
different mineral salts; MgSO4, MnSO4, CuSO4, ZnSO4, FeSO4, CaSO4.
Characterization of lipase: Effect of temperature, pH, substrate concentration and metal ions of lipase activity was
analyzed. Crude enzyme was used throughout. For temperature optimization reaction mixture was incubated over
temperature range; 30, 40,80 oC. To test effect of pH the buffer in reaction mixture was replaced with buffers of
different pH. Acetate buffer (0.1M) was used for pH in acidic range; 3, 4, 5 and phosphate buffer (0.1M) was used for
pH in neutral range; 6, 7, 8. Effect of increasing substrate concentration on lipase activity was determined at different
olive oil concentrations; 3, 6, 9, 12 and 15%. Lipase activity in presence of different metal ions (Ferrous, Magnesium,
Calcium, Copper and Zinc) at three concentrations (100, 200 and 300 ppm) was analyzed. Additionally effect of EDTA
on lipase activity was also analyzed.
RESULTS
Enrichment and isolation of extracellular lipase producers: Enrichment continued till color of the medium changed
from dark green to yellow to greenish yellow (Fig-1). Individual bacterial cultures were isolated from enrichment broth.
From this 10 isolates were selected based on differences in colony morphology. All the isolates were then subjected to
screening for extracellular lipase production by tributyrin agar plate method. Each of the isolate was capable of
producing extracellular lipase. From these AMSS1 was showing highest zone of clearance; 8mm (Fig-2). This isolate
was identified by its biochemical characteristics (Table-1). The data was analyzed by ABIS online software.
According to the software database the tested isolate were showing 91% similarity with Staphylococcus species.
Parameters for lipase production by this Staphylococcus sp. (Figure 3) were optimized and the lipase was then
characterized.

Figure 1. Enrichment of extracellular lipase producing bacteria. 1 and 2 -No growth, 3 and 4- Yellow color
indicating acidification as a result of lipolysis, 5 and 6- Greenish yellow color due to slow lipolysis.

Figure 2. Screening of extracellular lipase producers

Vol. 1 No. 1 (2012)

ISSN 23200421(Print); ISSN 2320043X(Online)

Figure 3. Staphyloccus sp. used for studies

2012 DAMA International. All rights reserved.

37

www.sciencejournal.in

Table 1. Biochemical Characteristics

Biochemical Test
Grams nature
Morphology
Glucose Fermentation
Fructose Fermentation
Sucrose Fermentation
Lactose Fermentation
Maltose Fermentation

Result
+
Cocci in clusters
+
+
+
+
+

Biochemical Test
Huge & Leifsons Test
Indole production
Methyl red test
Voges proskkauer test
Citrate utilization test
Catalase test
Dehydrogenase test

Result
+
+
-

Strain improvement: The Staphylococcus sp. was subjected to random mutagenesis by UV light and Sodium azide.
As per the exposure time to mutagen dose was increased number of survivors decreased constantly (Fig-4: A & B).
From these survivors eight mutant were randomly selected and sub-cultured. These isolates were screened for enhanced
lipase production by tributyrin agar test. We found that mutants generated by UV exposure for 35-50 seconds were
showing enhanced lipolytic activity. The mutant strain recovered after UV exposure for 50 sec was showing highest
lipase production compared to all other mutants. The zone of clearance for UV 50 strain was 1.5 times larger than that
of wild Staphylococci sp. Sodium azide treatment had no promising effect on lipase production ability rather two
mutants were showing reduction in lipase production. The mutant strain was then used for further studies (Figure 5).

Figure 4. Colonies grown after mutagen treatment, (A) UV treatment, (B) Sodium azide treatment.

Vol. 1 No. 1 (2012)

ISSN 23200421(Print); ISSN 2320043X(Online)

2012 DAMA International. All rights reserved.

38

www.sciencejournal.in

Figure 5. Screening of mutants for improved of lipase production.

Optimization of fermentation condition: Time course of lipase production was tested for 4 days. Lipase production
was highest after 72hrs of incubation. Lipase concentration on third day was 1.83 M / min. Further incubation was
found to be detrimental for lipase activity. Effect of pH on lipase production over the range 3-10 was analyzed. The
results showed that lipase production was highest at pH 8. Lipase activity at this pH was 0.76 M / min. pH above or
below this negatively affected the lipase production (Fig-6). Results of media optimization experiment are presented in
Table-2. Of the five carbon sources tested groundnut oil was found to better stimulant of lipase production compared to
other four oils. Effect of five organic nitrogen sources on lipase production was tested of them meat extract was found
to be better suited. For all the previous experiments the basal media used consist of magnesium. Thus in the experiment
for analyzing trace metal requirements the lipase activity in magnesium containing media was considered as control
value. Accordingly all the four metal ions tested (Zn, Ca, Cu and Fe) were found to have stimulatory effect on lipase
production.

Figure 6. Optimization of time, temperature and media pH

Table 2. Media optimization

EU of Lipase
Carbon Source
(M/min)
Mustard Oil
0.44
Coconut Oil
0.44
Safflower Oil
0.36
Groundnut Oil
0.50
Olive Oil
0.36

Nitrogen Source
Meat extract
Beef Extract
Peptone
Yeast Extract
Tryptone

EU of Lipase
(M/min)
0.50
0.44
0.44
0.36
0.33

Mineral
Salts
Magnesium
Zinc
Iron
Copper
Calcium

EU of Lipase
(M/min)
0.36
0.44
0.44
0.50
0.53

Characterization of lipase: The extracellular lipase produced by Staphylococcus spp. a soil isolate was characterized
for optimum temperature, optimum pH, optimum substrate concentration and cofactor requirement. The optimum
temperature for lipase activity was found to be 30 0C, above this there was consistent fall in activity but suddenly after
500C the lipase activity increased up to 600C and again reduced at 700C. Activity at optimum temperature (30 0C) was
0.66 m/ min (Figure 7). The lipase activity was progressively hampered with increasing acidity. At pH 8 it reached to
maximum value 0.66 m/ min (Figure 8).

Vol. 1 No. 1 (2012)

ISSN 23200421(Print); ISSN 2320043X(Online)

2012 DAMA International. All rights reserved.

39

www.sciencejournal.in

Figure 7. Effect of temperature on lipase activity

Figure 8. Effect of pH on lipase activity

Figure 9. Effect of substrate concentration on lipase activity

Table 3. Effect of metal ions on lipase activity

Metal Ion
Blank
EDTA

Zinc

Calcium

Copper

Magnesium

Ferrous

Vol. 1 No. 1 (2012)

Conc. Of Metal Ion

(in ppm)
No metal ion
100
200
300
100
200
300
100
200
300
100
200
300
100
200
300
100
200
300

ISSN 23200421(Print); ISSN 2320043X(Online)

% Increase in
Lipase Activity
100
133
100
59
185
81
81
122
81
59
326
81
59
81
81
196
81
59
81
2012 DAMA International. All rights reserved.

40

www.sciencejournal.in

Similar trend was seen in substrate concentration optimization experiment. Substrate concentration was increased from
3% to 15% and with increasing substrate concentration the lipase activity was also increased steadily (Figure 9). To
find cofactor dependence the lipase activity was determined in presence of EDTA. And it was seen that at 300 ppm
EDTA reduced the lipase activity to 59% of normal value. This shows that enzyme requires cofactor for its activity.
Effect of five metal ions at three concentrations was tested on lipase activity. Zinc was stimulant when at 100ppm
concentration and suppressed lipase activity when concentration increased further. Similar was the observation in case
of calcium, but increasing concentration of calcium was inversely related with lipase activity. Copper at 100ppm
concentration increased lipase activity more than three fold. In case of magnesium 300ppm was required to stimulate
lipase activity. Ferrous rather was found to be inhibitor of lipase activity.
DISCUSSION
Joseph et al., (2006) studied lipase production by Staphylococcus epidermidis. The lipase exhibited optimum activity
within 72 h in batch fermentation. Presence of metals in the media had an inhibitory effect. The optimum temperature
and pH for enzyme activity was 200 C and 7.0 respectively. Vadehra and Harmon (2008) reported that optimum pH for
lipase production by Staphylococcus aureus is within pH 7.59.0. According to Esakkiraj et al. (2010) suitable nitrogen
source for lipase production by Staphylococcus epidermidis was meat extract. And trace element for maximum lipase
production was ZnSO4 followed by MgSO4. Partial characterization of the crude lipase revealed that pH 7 and a
temperature of 50C gave optimal lipase activity.
ACKNOWLEDGEMENT
All the authors are grateful to MGMs Institute of Biosciences and Technology, Aurangabad (MS), India, for providing
all the funding and laboratory facility throughout the research work.
REFERENCES
Aaronson S. (1970). Isolation of lipolytic microorganisms. Experimental microbial ecology. Academic Press, New
York US. 94-95.
Esakkiraj P., Rajkumarbharathi M., Palavesam A., Immanuel G. (2010). Lipase production by Staphylococcus
epidermidis CMST-Pi 1 isolated from the gut of shrimp Penaeus indicus. Ann. Microbiol. 60(1):37-42.
Ghosh P. K., Saxena R. K., Gupta R., Yadav R. P., Davidson S. (1996). Microbial lipases: production and
applications. Sci. Progress. 79(2): 119-157.
Gupta R., Gupta N. and Rathi P. (2004). Bacterial lipases: an overview of production, purification and biochemical
properties. Appl. Microbiol. Biotechnol. 64:763-781.
Jaeger K. E., Dijkstra B. W. and Reetz M. T. (1999). Bacterial Biocatalysts: Molecular biology, three-dimensional
structures and biotechnological applications of lipases. Annu. Rev. Microbiol. 53:315-351.
Joseph B., Ramteke P.W. and Kumar P.A. (2006). Studies on the enhanced production of extracellular lipase by
Staphylococcus epidermidis. J. Gen. Appl. Microbiol. 52(6):315-20.
Rathi P., Goswami V.K., Sahai V., Gupta R. (2002). Statistical medium optimization and production of a
hyperthermostable lipase from Burkholderia cepacia in a bioreactor. J. App. Microbiol. 93:930-936.
Rosenstein R. and Gtz F. (2000). Staphylococcal lipases: Biochemical and molecular characterization. Biochimie.
82(11):1005-1014.
Simons J. W. F. A., Adams H., Cox R. C., Dekker N., Gotz F, Slotboom A. J., Verheij H. M. (1996). The lipase
from Staphylococcus aureus, expression in Escherichia coli, large-scale purification and comparison of substrate
specificity to Staphylococcus hyicus lipase. Eur. J. Blochein. 242:760-769.
Sirisha E., Rajasekar N. and Narasu M. L. (2010). Isolation and Optimization of Lipase Producing Bacteria from Oil
Contaminated Soils. Adv. Bio. Res. 4(5):249-252.
Smeltzer M. S., Hart M. E. and Iandolo J. J. (1992). Quantitative spectrophotometric assay for Staphylococcal
lipase. App. Env. Microbiol. 58(9): 2815-2819.
Tembhurkar V. R., Kulkarni M. B. and Peshwe S. A. (2012). Optimization of Lipase Production by Pseudomonas
spp. in submerged batch process in shake flask culture. Sci. Res. Rep. 2(1):46-50.
Troller J. A. and Bozeman M. A. (1970). Isolation and characterization of a Staphylococcal lipase. App. Microbiol.
20(3): 480-484.
Vadehra D. V., Harmon L. G. (2008). Factors affecting production of Staphylococcal lipase. J. App. Microbiol.32(2):
147-150.

Vol. 1 No. 1 (2012)

ISSN 23200421(Print); ISSN 2320043X(Online)

2012 DAMA International. All rights reserved.

41