Lipases (Triacylglycerol acylhydrolase; EC 184.108.40.206)
are versatile and ubiquitous biocatalysts with a wide range of applications
in food, dairy, detergent and pharmaceutical industries (Gupta et al.
2004a). These enzymes are active at the interface of aqueous and non-aqueous
phases which distinguishes them from esterases (Pandey et al.
1999). The activity of lipases is independent from cofactors and they
do not catalyze side reactions. Moreover, lipases display exquisite chemoselectivity,
regioselectivity and sterioselectivity (Jaeger and Eggert, 2002).
Microbial lipases are commercially important because of their unique properties
and the ease of bulk extracellular production compared to lipases from
other natural sources (Jaeger and Eggert, 2002). Among the lipase producing
bacteria, several species of Bacillus
such as B. subtilis
, B. licheniformis
, B. thermoleovorans
and B. sphaericus
suitable for biotechnological applications (Nthangeni et al.
Rahman et al.
, 2003; Ruiz et al.
, 2003). In addition, alkalophilic
and thermophilic microorganisms have been the focus of many studies for
new sources of lipases that are stable and function optimally at extreme
alkaline pH values and high temperature
s (Nthangeni et al.
Sharma et al.
, 2001). Lipase production is dependent upon
a number of factors including carbon and nitrogen sources, pH, temperature,
aeration and inoculum size (Kim et al.
, 1996; Gupta et al.
2004a). Recently, statistical designs have been employed to optimize enzyme
production and minimize the number of experiments (Krishna et al.
2001; Rathi et al.
, 2002; Gupta et al.
, 2004b). In this
research, we isolated a lipase producing Bacillus
from soil and
partially optimized lipase production using the statistical method of
Taguchi. Identification of the organism was carried out both biochemically
and by 16S rRNA sequencing.
MATERIALS AND METHODS
The study was conducted in 2006 at the Microbiology Department of Shahid
Beheshti University and the National Institute for Genetic Engineering
and Biotechnology, Tehran, Iran.
A number of soil Bacillus
from a previous collection were screened for lipase production (Eftekhar
, 2003). Four isolates were able to produce lipase, one of
which (strain F3) was chosen for the present study. Plasmid pTZ57R/T (Ins
T/A clone, PCR product cloning kit, #k1214, Fermentas) was used as the
cloning vector for 16S rRNA and Escherichia coli
TG1 was used as
the recipient strain for recombinant plasmids.
Screening for lipolytic activity:Lipolytic activity was detected
on TW agar plates containing 1% Tributyrin and 1% Tween 80 (pH 8). Lipase/esterase
production was detected by observing clear zones around isolated colonies
(Akatsuka et al., 2003). Lipase activity was then detected by growth
on Rhodamine B lipase agar at 30°C for 72 h (Kouker and Jaeger, 1987).
Colonies which showed orange fluorescence under UV irradiation indicated
true lipase activity and non-lipolytic bacteria formed pink colonies.
Cloning and sequencing of the 16S rRNA: Plasmid and genomic
DNA were extracted from overnight grown cultures in LB broth using phenol/chloroform
as described before (Sambrook et al., 2001). Amplification of 16S
rRNA was carried out using primer oligonucleotides fD1 (5`-AGA GTT TGA
TCC TGG CTC AG-3`) and rD1 (5`-TAA GGA GGT GAT CCA GC-3`) (Weisburg
et al., 1991) provided by Gen Fanavaran (Tehran, Iran). DNA amplifications
were carried out in 50 µL reaction mixtures containing 5 µL of 10 x PCR
buffer, 2 µL dNTP mixture (10 mM), 1.5 µL MgCl2 (50 mM), 2
µL of each primers (10 pmol µL-1), 1 µL of DNA (5-10 ng) and
0.6 µL Taq DNA polymerase (5 U µL-1). Amplification was performed
in a thermocycler (Techne Flexigene, Model FFG05TUD, Minneapolis, MN,
USA) using the following program. A 5 min denaturation period at 95°C
was followed by 30 cycles each; 1 min at 95°C, 1 min at 50°C and 2 min
at 72°C with a final extension for 10 min at 72°C. All PCR chemicals were
purchased from Cinagen (Tehran, Iran).
The PCR product was purified
from the agarose gel and cloned into plasmid pTZ57R/T in E. coli
TG1 using standard procedures. Recombinant colonies were screened on LB
agar supplemented with ampicillin (100 µg mL-1
), IPTG (0.5
mM) and X-Gal (20 µg mL-1
) as previously described (Sambrook
, 2001). Presence of the cloned 16S rRNA gene in recombinant
plasmids was verified using M13/pUC universal primers (Gen Fanavaran,
Tehran, Iran). The DNA insert was then sequenced using the dideoxy termination
method with ABI automated sequencer at the Research Center for Gastroenterology
and Liver Diseases (Taleghani Hospital, Tehran, Iran) and homology was
analyzed through BLAST (blastn algorithm) at http://www.ncbi.nih.gov/
Selection of salt basal media: M9 medium consisting of 12.8
g Na2HPO4.7H2O, 3 g KH2PO4,
0.5 g NaCl, 2 mM MgSO4.7H2O, 1 g NH4Cl,
2 g glucose and 10 mL olive oil L-1 (pH 7) and G4 medium containing
35 g NH4Cl, 3 g K2HPO4, 1 g KH2PO4,
0.1 g MgSO4.7H2O, 2 g glucose, 0.6 mM MgCl2
and 10 mL olive oil L-1 (pH 7.0) were used (Sambrook et
al., 2001; Gupta et al., 2004b). Two other media, M9+Y and
G4+Y, were prepared by adding 5 g L-1 yeast extract to M9 and
G4. The media were inoculated with 5% seed culture and bacterial growth
and lipolytic activity were measured at 6 h intervals up to 30 h at 30°C.
Selection of oil for lipase induction: Seven oils with different
compositions were used for their effects on lipase production. Olive oil,
canola oil, sunflower oil and grape seed oil used at 1% and coconut (1.5%)
were obtained from commercially available sources. Tributyrin used at
2% and glycerol (10%) were purchased from Merck.
Partial optimization of lipase production by the Taguchi experimental
The effect of seven factors (oil, glucose, NH4
yeast extract, inoculum density, pH and agitation) was investigated at
two levels shown in Table 3
. Design Expert 6.0.10 (Stat
Ease Inc., Minneapolis, Minn., USA) was used to generate a set of 8 experimental
trials according to the Taguchi orthogonal L8
). Bacterial growth and lipase production were monitored every 6
h at 30°C for 36 h. All experiments were performed in triplicate and mean
values were used for statistical analysis.
Inoculum preparation: M9 without oil was used to prepare inocula
for selection of salt basal media and G4+Y was used for the rest of the
experiments. Seed cultures were incubated for 18 h at 30°C before use.
Growth estimation and detection of lipase activity: To measure
bacterial growth and lipase production, culture samples (1 mL) were removed
at designated times and were centrifuged at 5000 g for 10 min. Pellets
were resuspended in 1 mL of 0.01 M phosphate buffer (pH 7) and absorbance
was measured at 600 nm (Gupta et al., 2004b). Culture supernatants
were used to determine lipolytic activity using p-nitrophenyl palmitate
substrate (pNPP, Sigma). The substrate was dissolved in 10 mM acetonitrile
followed by adding absolute ethanol and 20 mM Tris-HCl (pH 8) at a ratio
of 1:4:95 (v/v/v), respectively. Substrate hydrolysis was monitored for
5 min at room temperature and the amount of released p-nitrophenol
was determined spectrophotometrically by measuring the increase in absorption
at 405 nm (Model DU-530 Beckman, USA). One unit of lipase was defined
as the amount of enzyme liberating 1 µmol of p-nitrophenol per
minute (Cho et al., 2000).
RESULTS AND DISCUSSION
Among the Bacillus isolates screened for lipase production on
TW agar, one (F3) showed lipolytic zones after 48 h at 30°C. When F3 was
grown on Rhodamine B lipase agar, orange fluorescence was observed under
UV irradiation indicating lipase activity.
Growth and lipase production: Bacillus
F3 grew rapidly in both M9 and G4 media containing yeast
extract (G4+Y and M9+Y). However, lipolytic activity was detected only
in G4+Y (Table 1
). M9+Y supported bacterial growth shown
by high optical density values but no lipolytic activity was observed
in M9 with or without yeast extract. Organic nitrogen sources such as
yeast extract have been shown to play a crucial role on lipase expression
(Gupta et al.
, 2004a; Sharma et al.
, 2001). Maximal lipolytic
activity was shown to be 3 U mL-1
after 24 h of growth in late
logarithmic phase (Table 1
). Medium G4+Y was then chosen
for further studies. Presence of higher concentration of ammonium chloride
(3.5%) in G4 compared to M9 (0.1%) seemed to be another factor responsible
for lipase expression. Lipase production was induced in the presence
of olive oil similar to a number of other reports (Sugihara et al.
1991; Wang et al.
, 1995; Gupta et al.
, 2004a). As a matter
of fact, all long fatty acyl chain triacylglycerol test substrates were
as effective inducers of lipase production as olive oil (Table
). On the other hand, glycerol or tributyrin did not induce lipase
production. Glycerol was previously reported to enhance lipase production
in a thermophilic Bacillus
strain (Gupta et al.
Identification of the lipolytic Bacillus:
characterization of F3 showed it to be a non-motile, catalase positive,
indole and VP negative Gram positive rod with central and elliptical spores.
It produced acid but no gas from glucose, but no acid was produced from
arabinose or mannitol. Growth on anaerobic agar was also detected. Furthermore,
F3 hydrolyzed gelatin and casein but not starch. Metabolization of citrate
and de-amination of phenylalanine did not occur and lecithinase or NO2
were not produced. Growth occurred at temperatures between 20 to 55°C
at pH range 4 to 10 and up to 7% NaCl concentrations. Identification
of F3 by 16S rRNA amplification showed a 1500 bp fragment which was then
cloned in E. coli
TG1. Eight recombinant clones were selected and
their DNA inserts were further amplified using the M13/pUC universal primers.
Four recombinant plasmids with 1500 bp inserts were obtained, one of which
was chosen for sequencing. The sequencing results revealed 99% homology
to Bacillus pumilus
(Genbank accession No. EU285662).
Optimization of bacterial growth and lipase production: In
an effort to optimize bacterial growth and lipase production, the statistical
method of Taguchi was used to design culture conditions with different
media compositions. Statistical designs have been employed to optimize
enzyme production and minimize the number of experiments for a number
of organisms including Bacillus sp. (Krishna et al., 2001;
Rathi et al., 2002; Gupta et al., 2004b; Prakasham et
al., 2005). In order to enhance bacterial growth and lipase production,
seven variables were screened by the Taguchi method using G4+Y medium
and olive oil (Table 3).
||Bacterial growth (OD600)
and lipase activity (U mL-1) in basal media during
30 h incubation at 30°C and 250 rpm shaking
(OD600) and lipase activity (U mL-1) in
the presence of different oils. Cultures were grown for up to
30 h at 30°C and 250 rpm shaker speed
||Measurement of bacterial growth and
lipase activity using the Taguchi orthogonal L8 array
||ANOVA analysis (partial sum of squares)
of bacterial growth for the Taguchi orthogonal array L8
*: The F-value
of 1716.17 implies that the model is significant. **: Values
< 0.1000 for Prob. > F indicate that the model terms are significant
(B, C, E, F and G). Letter(s) B, C, E, F and G correspond to
of bacterial growth for the Taguchi orthogonal array L8
*: The model
F-value of 1716.17 implies that the model is significant. **:
Value of Prob. > F less than 0.1000 indicate model terms are
of experimental factors on bacterial growth and lipase production
obtained by the Taguchi orthogonal array L8
The ANOVA analysis revealed that the inoculum size, glucose
and yeast extract concentrations, pH and agitation significantly enhanced
bacterial growth. On the other hand, oil concentration above 0.5% and
ammonium chloride concentrations above 1.5% were not significant (Table
). The coefficient of determination (R2
) was 0.999 which
was in a reasonable agreement with the predicted R2
and showed a satisfactory adjustment of the quadratic model to the experimental
data. Adequate precision measures the signal to noise ratio, a ratio greater
than 4 is desirable. An adequate precision value of 92.938 indicates an
adequate signal (Table
Contribution of experimental factors on bacterial growth
and lipase production are shown in Table
. Yeast extract at 10% concentration, had the highest contribution
on growth (95.22%) and lipase production (59.99%). In comparison to yeast
extract, the effect of other factors on lipase production was negligible.
In fact, the sum of squares for all other factors showed the Prob. > F-values
greater than 0.1 which are not significant at the 90% confidence interval.
Indeed, lipase was not produced in the absence of yeast extract regardless
of different concentrations of olive oil, glucose, ammonium chloride,
starting inocula, pH or agitation. On the other hand, at least 1.5% NH4
was needed for lipase production in G4+Y medium and higher concentrations
up to 3.5% did not further enhance lipase production.
Lipolytic activity was not detected with high glucose concentrations
and low inoculum density (1%) despite the observed acceptable growth (trial
No. 5, Table
). However, this effect was compensated when high inoculum density
(10%) was used (trial No. 4, Table
). Catabolic repression by glucose has been previously reported for
some lipolytic microorganisms (Gowland et al.
, 1987; Rapp, 1995;
Gupta et al.
The effect of agitation on lipase production varies depending
on the bacterial strain. Optimum lipase production for most Bacillus
species has been reported to occur at lower agitation rates (Gupta
, 2004a). However, production of lipase in Pseudomonas
3SK was maximum at 500 rpm (Lee and Rhee, 1994). It is reasonable
to believe that physical parameters such as pH, temperature and aeration
most probably influence lipase production by modulating bacterial growth
(Gupta et al.
, 2004a). Maximal lipase activity was 4.83±0.22 U
in a medium containing 15 mL olive oil, 2 g glucose, 15
g ammonium chloride and 10 g yeast extract L-1
at pH 8.5 and
low agitation using an initial inoculum of 10 % (trial No. 7, Table
Lipase production in B. pumilus
has been reported
to occur at low levels. Kim et al.
(2002) have reported lipase
yields as low as 0.5 U mL-1
. Maximal lipase production in B.
so far has been reported to be 12.8 U mL-1
, 1991). Our preliminary efforts to produce lipase from B.
F3 led to production of 4.83 U enzyme mL-1
studies are needed to enhance lipase production in this strain. We are
currently in the process of cloning the F3 lipase under a strong promoter
to be able to determine molecular properties of the lipase as well as
increasing enzyme expression and yields for future industrial applications.
We thank Mr. Mohsen Chiani from the Research Center for
Gastroenterology and Liver diseases of Taleghani Hospital for his technical
aid in DNA sequencing. This research was financed by the National Institute
of Genetic Engineering and Biotechnology (Grant 245, NIGEB, Iran). We
also appreciate the assistance of Miss Hoora Ahmadi-Danesh and Mrs. Roya
Razavipour throughout the study.