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

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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

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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

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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.

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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