lipase-producing bacterium and its enzyme · pdf filethese lipase-producing bacteria ......
Post on 24-Mar-2018
220 Views
Preview:
TRANSCRIPT
Lipase-Producing Bacterium and its Enzyme
Characterization
Patcha Boonmahome
Graduate School/Department of Microbiology/Faculty of Science/Khon Kaen University, Khon Kaen, Thailand
hellokitty_preaw@hotmail.com
Wiyada Mongkolthanaruk Department of Microbiology/Faculty of Science/Khon Kaen University, Khon Kaen, Thailand
wiymon@kku.ac.th
Abstract—Recently, renewable energy is very important use
for industrial development, transport because it is
environmental friendly and can reduce high cost of
imported fossil fuel. The evolution of biodiesel production
has been reported in many researchers. Transesterification
process which is mainly reaction for biodiesel is required
catalysts such as acid, base catalyst or biocatalyst. In this
study, 134 isolates were selected from soil contaminated
with oil in Khon Kaen region by using culture medium
containing 1% olive oil and 0.0001% rhodamine B with
incubation at 30°C for 48 h. These lipase-producing bacteria
were also determined their activities on agar medium with 1%
tributyrin. The isolate NA37 showed high lipase activity
(190 mU/ml) detecting with p-nitrophenyl palmitate as a
substrate. Cooking-palm oil was contributed lipase
production of the NA37 more than other oils. The optimum
temperature and pH for lipase production is 30°C and pH 9,
resulting in lipase activity of 481 mU/ml.
Index Terms—Bacterial lipase, rhodamine B, tributyrin
I. INTRODUCTION
Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3)
can hydrolyze ester bond of long chain fatty acid which is
mainly component of oil. The sources of lipase enzyme
are generally found in nature such as plants, animals,
yeast, fungi and bacteria, for example, Candida rugosa [1]
Thermomyces lanuginosus [2] Fusarium oxysporum f. sp.
lini [3] Candida antarctica [4] Rhizopus oryzae [5]
Lactobacillus spp. [6] Bacillus stearothermophilus L1 [7]
Burkholderia sp. C20 [8]. Bacterial lipases are important
enzymes applications in various industries, because of
friendly for environment, non-toxic and no harmful
residues [9]. For instant, there are widely uses in dairy
industry and pharmaceutical industry [3], detergent and
surfactant [9], taste or flavor industry [10], agricultural
industry, chemical, cosmetic and perfume [9]. Especially,
they are applied for biodiesel productions such as lipase
enzyme from Acinetobacter venetianus RAG-1 could
produce biodiesel using transesterification process [11].
Advantages of lipase enzyme for biodiesel production are
catalysis in mild reactions, using less energy and easy
Manuscript received October 1, 2013; revised December 10, 2013.
recovery glycerol from biodiesel [12]. There are many
types of oil for biodiesel production, e.g. olive oil, palm
oil, soybean oil and sunflower oil. Ha et al. [13] reported
that immobilized Candida antarctica gave biodiesel from
reaction of soybean oil with methanol. Ban et al. [5] used
immobilized cell of Rhizopus oryzae to produce biodiesel
from soybean oil with methanol.
II. MATERIALS AND METHODS
A. Screening, Isolation and Growth Conditions
Lipase-producing bacteria were isolated from soil
contaminated with cooking oil in Khon Kaen region.
Two gram of soil sample were added into YOC medium
(yeast extract 1 g, olive oil 2.5 ml, CaCl2 10 g in distilled
water 1 L, pH 8), YM medium and nutrient broth. The
culture was incubated at 30°C for 48 h on a rotary shaker.
Lipase-producing bacteria were selected by spread plate
technique on rhodamine B agar plate (rhodamine B
0.0001 % (w/v) in YOC medium). Plates were incubated
at 30°C for 48 h. The lipase production was determined
by observation of pink-orange colony under UV 350 nm
and confirmed on nutrient agar with 1% tributyrin to
observe a high clear zone around colony.
B. Lipase Activity Assay
Lipase activity was assayed using p-nitrophenly
palmitate (p-NPP) as a substrate. The 30 mg of p-NPP
was added into 10 ml of 2-propanol and mixed with 90
ml of 5 mM phosphate buffer (pH 8) containing 207 mg
of sodium deoxycholate (NaDOC) and 100 mg of gum
arabic. The 100 µl of crude enzyme were added in 2ml of
the reaction mixture and then incubated at 55°C for 15
min. The 2.9 ml of 2 M sodium carbonate (211.8 mg of
sodium carbonate in distilled water 1 L) were added to
stop the reaction after incubation. The lipase reaction was
measured absorbance by spectrophotometer at
wavelength of 410 nm. One Unit of lipase activity is
defined as an enzyme releasing 1µmol of free p-
nitrophenol per minute [3].
C. Optimization of Conditions for Lipase Production
1) Determination of a suitable carbon source
Journal of Life Sciences and Technologies Vol. 1, No. 4, December 2013
2013 Engineering and Technology Publishing 196doi: 10.12720/jolst.1.4.196-200
There were various kinds of oils used in this
experiment, such as olive oil, soybean oil, used cooking
(palm) oil, and palm oil. These oils might induce lipase
production. The bacterial isolates were inoculated into
nutrient broth containing each kind of oil. The cultures
were incubated at 30°C for 48 h and shaken at 125
rpm/min. The cell and supernatant were separated by
centrifugation at 5000 x g for 15 min, at 4°C. The
supernatant as a crude enzyme were determined the lipase
activity at 410 nm using spectrophotometer.
2) Optimum temperature
The optimization of temperature was determined for
lipase production by inoculating bacteria into nutrient
broth containing appropriate 1% of oil (in experiment
C.1.), incubating at 30°C, 35°C and 50°C. After 48 h, the
cultures were centrifuged at 5,000 x g for 15 min, at 4°C;
and then the supernatant was determined the lipase
activity using spectrophotometer at 410 nm.
3) Optimum pH
The bacteria were grown into nutrient broth containing
appropriate 1% of oil (in experiment C.1.); the pH of the
media was adjusted to pH 4, 5, 6, 7, 8, 9 and 10. These
media were incubated at appropriate temperature (in
experiment C.2.) for 48 h. The supernatant was harvested
by centrifugation at 5,000 x g for 15 min, at 4°C and
determined the lipase activity using spectrophotometer at
410 nm
D. Identification of Bacterial Strains
The DNA template was extracted using “Genomic
DNA mini kit (blood/culture cell)” (Geneaid Biotech Ltd.,
Taiwan) and amplified using the two primers, 20F (5’-
GAG TTT GAT CCT GGC TCA G-3’) and 1500R (5’-
GTT ACC TTG TTA CGA CTT-3’) [14]. PCR
amplification was programmed to carry out an initial
denaturation step at 94°C for 3 min, 25 cycles of
denaturation at 94°C for 1 min, annealing at 50°C for 1
min and elongation at 72° for 2 min, followed by a final
amplification step at 72°C for 3 min. The PCR product
was analyzed by agarose gel electrophoresis and purified
with a QIAquick®PCR purification kit (QIAGEN GmbH,
Hilden, Germany). The nucleotide sequence obtain from
all primers were assembled using Cap contig assembly
program, an accessory application in BioEdit (Biological
sequence alignment editor) Program
(http://mbio.ncsu.edu/BioEdit/BioEdit.html). The
identification of phylogenetic neighbors was initially
carried out by the BLASTN [15]. PCR amplification step,
sequencing, and phylogenetic tree were analyzed by
National Center for Genetic Engineering and
Biotechnology (Biotec).
III. RESULTS AND DISCUSSION
A. Screening Lipase-Producing Bacteria
Total of 134 isolates were isolated from contaminated-
oil soil samples in Khon Kaen region by showing pink-
orange fluorescent colonies under UV wavelength (350
nm) on agar plates containing 1% olive oil and 0.0001%
rhodamine B (Fig. 1). This appearance is caused by a
complex formation between cationic rhodamine B and
uranyl fatty acid ion [16]. The mechanism may be the
generation of excited dimmers of rhodamine B which
fluoresce at longer wavelengths than the exited monomer
[17]. All 134 positive isolates were confirmed lipase
production by duplication on 1% tributyrin agar plate.
After incubation for 48 h at 30°C, the plates were
checked a clear zone around colony. The most effective
lipase-producing bacterium was isolate NA37, which
gave the largest clear zone occurring by hydrolysis ester
bond of triglyceride from lipase enzyme.
B. The Optimum Conditions of Lipase Production
The isolate NA37 was investigated the lipase activity
by growing in nutrient broth with 1% olive oil for 48 h at
30°C. This isolate showed a high activity of 190 mU/ml
lipase activity more than other isolates; therefore, this
isolate was used to determine the optimum conditions for
lipase production. As oil can be a good inducer to
promote lipase production during cell growing in
cultivation. The various kinds of oil were determined for
suitable oil in lipase production of the isolate NA37.
Figure 1. Lipase activity on Rhodamine B agar plate. A, without UV determination; B and C, determination under UV 350 nm. All plates were incubated at 30ºC for 48 hours.
The results showed that the isolate NA37 grew well in
all media (data not shown) and gave high lipase activity
in medium containing palm oil, particular in used cooking
(palm) oil as shown lipase activity at 361 mU/ml (Fig. 2).
Palm oil is a good raw material for biodiesel production,
as the plant grows widely in many areas of Asia and it is
A B C
Journal of Life Sciences and Technologies Vol. 1, No. 4, December 2013
2013 Engineering and Technology Publishing 197
used in food cooking so the waste oil or used cooking oil
can be re-used, increasing the value of the product.
The optimum temperature and pH for lipase
productions were determined by inoculating the isolate
NA37 into nutrient broth with 1% used cooking (palm)
oil. After incubation for 48 h, the results showed the
highest activity at 30°C of cultivation, resulting in 255
mU/ml (Fig. 3). There was no activity at 50°C as no
growth was observed (data not shown). The initial pH of
media was 9, showing high activity of 481 mU/ml (Fig.
4). This enzyme might be highly active in alkaline
condition which is a suitable condition for biodiesel
production. However, the lipase enzyme should be
investigated optimum temperature and pH for lipase
activity including stability of the enzyme. These data will
develop the process of biodiesel using enzyme as a
biocatalyst.
Figure 2. Lipase activity of the isolate NA37 produced from nutrient broth with 1% of each olive oil, soybean oil, used cooking (palm) oil and palm oil. The cultures were incubated at 30°C for 48 h and the activity was performed.
Figure 3. Lipase activity of the isolate NA37 in various temperatures of cultivation. The bacterium grew in nutrient broth with 1% used cooking (palm) oil and was incubated for 48 h.
Figure 4. Lipase activity of the isolate NA37 in medium adjusted pH in the range of 4-10. The bacterium grew in nutrient broth with 1% used
cooking (palm) oil and was incubated at 30°C for 48 h.
0
100
200
300
400
olive oil soybean oil used
cooking
(palm) oil
palm oil
Lip
ase
act
ivit
y (
mU
/ml)
Substrate
Journal of Life Sciences and Technologies Vol. 1, No. 4, December 2013
2013 Engineering and Technology Publishing 198
C. Identification of the Isolate NA37 Based on 16S
rDNA
The isolate NA37 was characterized the morphology as
a gram-negative, rod shape bacterium. The 16S rDNA
gene sequence was compared with the database and it
gave 99.93% identity with Pseudomonas auruginosa. The
phylogenic tree showed the relationship between P.
aeruginosa LMG1242 and the isolate NA37 in the same
group with 100% bootstrap (Fig. 5). Therefore, the isolate
NA37 was identified as P. aeruginosa NA37.
Figure 5. Phylogenetic tree of the isolate NA37. The accession number of each strain is given after the name.
IV. RESULTS AND DISCUSSION
In this study, we screened lipase-producing bacteria
from soil sample contaminated with cooking oil in areas
of Khon Kaen region. The totals of 134 isolates were
observed the clear zone on nutrient agar with 1%
tributyrin and the pink-orange fluorescent colonies on
rhodamine B agar. The isolate NA37 was an effective
lipase-producing bacterium, giving lipase activity at 190
mU/ml using p-nitrophenyl palmitate as a substrate. The
lipase production of the isolate NA37 had the optimum
temperature and pH at 30°C and pH of 9, respectively.
The raw material was used cooking (palm) oil for lipase
production of the isolate NA37, resulting in 481 mU/ml
of lipase activity for the optimum cultivation. The isolate
NA37 was identified to be Pseudomonas aeruginosa. In
the future, we will characterize factors of enzyme and
application of lipase from the isolate NA37 for biodiesel
production.
ACKNOWLEDGMENT
This work was supported by Cluster biofuels of a
national university of research projects, Khon Kaen
University. This also was partially supported by the
Protein and Proteomics Research Center for Commercial
and Industrial Purposes (ProCCI), Khon Kaen University,
Thailand.
REFERENCES
[1] G. Kouker and K. Jaeger, “Specific and sensitive plate assay for
bacterial lipases” Applied and Environmental Microbiology, vol. 53, pp. 211-213, 1987.
[2] N. Dizge and B. Keskinler, “Enzymatic production of biodiesel
from canola oil using immobilized lipase,” Biomass and Bioenergy, vol. 32, pp. 1274-1278, 2008.
[3] T. Hoshino, T. Sasaki, Y. Watanabe, T. Nagasawa, and T. Yamane,
“Purification and some characteristics of extracellular lipase from fusarium oxysporum f. sp. Lini,” Bioscience Biotechnology and
Biochemistry. vol. 56, pp. 660-664, 1992.
[4] L. Goujard, P. Villeneuve, B. Barea, J. Lecomte, M. Pina, and S. Claude, “A spectrophotometric transesterification-based assay for
lipases in organic solvent,” Analytical Biochemistry. vol. 385, pp.
161–167, 2008. [5] K. Ban, M. Kaieda, T. Matsumoto, A. Kondo, and H. Fukuda,
“Whole cell biocatalyst for biodiesel fuel production utilizing
rhizopus oryzae cells immobilized within biomass support particles,” Biochemical Engineering Journal. vol. 8, pp. 39–43,
2001.
[6] B. Padmapriya, T. Rajeswari, E. Noushida, D. Sethupalan, and C. Venil, “Production of lipase enzyme from Lactobacillus spp. and
its application in the degradation of meat,” World Applied
Sciences Journal, vol. 12, no. 10, pp. 1798-1802, 2011. [7] S. Hwang, K. Lee, J. Park, B. Min, S. Haam, and I. Ahn, et al.
“Stability analysis of bacillus stearothermophilus L1
lipaseimmobilized on surface-modified silica gels,” Biochemical Engineering Journal, vol. 17, pp. 85-89, 2004.
[8] D. Tran, C. Chen, and J. Chang, “Immobilization of burkholderia
sp. lipase on a ferric silica nanocomposite for biodiesel production,” Journal of Biotechnology, vol. 158, pp. 112–119,
2012.
Journal of Life Sciences and Technologies Vol. 1, No. 4, December 2013
2013 Engineering and Technology Publishing 199
[9] F. Hasan, A. Sanh, and A. Hameed, “Industrial applications of microbial lipases,” Enzyme and Microbial Technology, vol. 39, pp.
235–251, 2005.
[10] L. C. Ochoa, C. R. Gomez, G. V. Alfaro, and R. Ros, “Screening, purification and characterization of the thermoalkalophilic lipase
produced by bacillus thermoleovorans CCR11,” Enzyme and
Microbial Technology, vol. 37, pp. 648–654, 2005. [11] S. A. Ericks and C. R. Rita, “Transesterification activity of a novel
lipase from acinetobacter venetianus RAG-1,” Antonie van
Leeuwenhoek, vol. 94, pp. 621–625, 2008. [12] A. Bajaj, P. Lohan, P. Jha, and R. Mehrotra, “Biodiesel production
through lipase catalyzed transesterification,” Journal of Molecular
Catalysis B: Enzymatic, vol. 62, pp. 9-14, 2010. [13] S. Ha, M. Lan, S. Lee, S. Hwang, and Y. Koo, “Lipase-catalyzed
biodiesel production from soybean oil in ionic liquids,” Enzyme
and Microbial Technology, vol. 41, pp. 480-483, 2007. [14] J. Brosius, T. J. Dull, D. D. Sleeter, and H. F. Noller, “Gene
organization and primary structure of a ribosomal RNA operon
from escherichia coli,” Journal of Molecular Biology, vol. 148, pp. 107-127, 1981.
[15] S. F. Altschul, T. L. Madden, A. A. Schaeffer, J. Zhang, et al.,
“Gapped blast and PSI-BLAST: A new generation of protein database search programs,” Nucleic Acids Res. vol. 25, pp. 3389-
3402, 1997.
[16] D. R. Mackenzie, R. T. Blohm, M. E. Auxier, and C. A. Luther, “Rapid colorimetric micromethod for free fatty acids,” The
Journal of Lipid Research. vol. 8, pp. 589-597, 1967.
[17] G. Kouker and K. E. Jaeger, “Specific and sensitive plate assay for bacterial lipases,” Applied and Environmental Microbiology, vol.
53, pp. 211-213, 1987.
Patcha Boonmahome is a master student at faculty
of science, department of microbiology, Khon Kaen
University. She works on bacterial lipases for biodiesel applications. Also, the area of research is
development of enzyme technology.
Wiyada Mongkolthanaruk graduated from Sheffield University in
molecular biology and biotechnology. The research areas are
endophytic bacteria and their applications, bacteria enzyme for
biotechnology, e.g. laccase, lipase. Also, she is interested in bioactive
compounds for plant-microbe interactions.
Journal of Life Sciences and Technologies Vol. 1, No. 4, December 2013
2013 Engineering and Technology Publishing 200
top related