Characterization of Lipase Producing …. R ESULTS AND D ISCUSSION A. Testing for Lipase Production...

Characterization of Lipase Producing

Rhodococcus sp. from Peninsular Malaysia

Jayesree Nagarajan, Norazah Mohammad Nawawi, and Abdul Latif Ibrahim Institute of BIO-IT Selangor, Universiti Selangor, Malaysia

Email: {jayesree_nagarajan, n_azahmn, alatifbio}@yahoo.com

Abstract—Rhodococcus species is recognized as an excellent

candidate for bioremediation due to its mass storage of

enzymes capacity. However, studies on lipase producing

ability by this actinomycete are far less being explored. Unit

Culture Collection of Institute Bio-IT Selangor preserves

and maintains Rhodococcus isolates which has been isolated

throughout Peninsular Malaysia. In this study, five

Rhodococcus isolates from various environmental niches

were identified for lipase production. The best isolate was

further assessed for lipase producing ability at different

grinding time interval. For better understanding on

behaviorism and adaptations of these isolates, further

characterization based on growth profiles and a few

biochemical tests were performed. Primary goal of this first-

stage study is to introduce new and potential source of lipase

from local resources while understanding the characteristics

of the isolates.

Index Terms—rhodococcus sp., bioremediation, lipase,

growth profile, biochemical tests.

I. INTRODUCTION

Enzyme or better known as protein molecule holds

fundamental tasks of accelerating chemical reactions in

living organisms. Precisely, this molecule serves as basic

tool for energy generation, growth, repair, cell

maintenance and formation of side products in every

biological system [1]. Among diverse available enzymes,

lipases are regarded as most pliable biocatalyst, ideal for

vast commercial applications [2]. Superiority of microbial

lipases is well represented by hiking number of research

articles and denotation as versatile biocatalyst.

Microbial lipases attained great preferability due to its

possibility for bulky production, consistent supply due to

absence in seasonal fluctuation, cheaper and highly stable.

In addition to these, lipase from microbes also exhibit all-

encompassing substrate selectivity, safer chemical

reactions while requiring mild growing conditions mostly.

Advantageously, development in the genetic engineering

also, promises use of recombinant gene technology in

order to increase the mass production of the cell, improve

efficiency of the enzyme either constitutively or by

inducing it and also introducing altered enzyme [3].

It was understood that, only 2% of world’s

microorganisms have been recognized as enzyme source

in which case, bacteria attained higher portion than yeasts

[4]. Diverse numbers of microorganisms have been

identified as supreme source for lipases including

Bacillus sp., Lactobacillus sp., Burkholderia sp. and

Candida sp. [5]. Based on the abundant studies of

bacterial lipases, further deep review revealed that

actinomycete or better known as branched filaments and

rather fragmentary bacteria are scarcely being studied on

enzyme producing ability [6].

Actinomycetes such as Rhodococcus sp. are also

pronounced for lipase producing ability however, detailed

evidences and analysis on this actinomycete are

inadequately explored [7]. This bacterium is popularly

known for its unique feature of producing natural oil

hence, arouses remarkable possibilities on its

triacylglycerol metabolism [8]. Potency of Rhodococcus

sp. in degrading hydrocarbon or crude oil extensively has

become an additional quality as potential source for

lipase enzyme as well [9]. However, the key factor for

this quality lipase, the fat degrading enzyme is far less

being explored.

Unit Culture Collection of Institute Bio-IT Selangor,

Malaysia maintains and preserves Rhodococcus isolates

which has been isolated throughout Peninsular Malaysia.

These isolates have been partially sequenced and further

compared with an established reference strain namely

Rhodococcus sp. 124. In this study, lipase producing

ability of these isolates was assessed. The strains were

further subjected for few characterization tests and

growth profile analysis for better understanding of

lipolytic Rhodococcus sp. behaviorism. This study

introduces new source for lipase enzyme among

diversified number of lipase sources ideal for applications.

II. MATERIALS AND METHODS

A. Bacteria and Seed Culture Preparation

Five Rhodococcus sp. isolated throughout Peninsular

Malaysia were kindly supplied by Culture Collection

Unit, Institute Bio-IT Selangor. Cultures from glycerol

stock were streaked on nutrient agar plate and incubated

at 30ºC. For seed culture, a loopful of bacteria from

nutrient agar plate was inoculated into 50 ml of nutrient

broth medium and left for shaking in incubator shaker

(Jeio Tech SI-600R, Korea) at 30 ºC at 160 rpm for

overnight. The details of the tested strains were tabulated

in Table I. As a reference strain for positive control

Bacillus subtilis (NCBI accession no: GU191916) from

122014 Engineering and Technology Publishingdoi: 10.12720/jolst.2.1.12-19

Manuscript received March 31, 2014 2014.; revised September 11,

Journal of Life Sciences and Technologies Vol. 2, No. 1, June 2014

the culture collection were compared with the tested

isolates.

TABLE I. RHODOCOCCUS SP. AND ITS ISOLATED LOCATION

Rhodococcus

Strains

Location

R.NAM81 Palm oil Mill effluent, Kuala Selangor

R.NAM319 Palm oil Mill soil, Kuala Selangor

R.NAM350 Palm oil Mill effluent, Kuala Selangor

R.UKMP-5M Petroleum contaminated soil, Port Dickson

R.SeAG1 Oil contaminated soil, Serdang

B. Testing for Lipase Production

1) Production medium

Ten percentage of seed culture was inoculated into

production medium containing 0.1% (w/v) NH2SO4, 0.09%

(w/v) K2HPO4, 0.06% (w/v) KH2PO4, 0.02% (w/v)

MgSO4.7H2O, 0.01% yeast extract and 1% (v/v) olive oil.

2) Preparation of crude lysates

The culture was centrifuged for 15 minutes at 14000

rpm (Tomy MX350, Japan). Upon centrifugation, the

supernatant was discarded. The remaining cell pellet was

washed twice and 2 ml 0.05M potassium phosphate

buffer was added. Cell lysates was prepared by grinding

the cell pellet under liquid nitrogen with a pre-cooled

mortar and pestle for 10 minutes. The lysed sample was

centrifuged again for 15 minutes at 14000 rpm. The

yielded supernatant was used as crude extract for lipase

assay [10].

3) Lipase assay

Lipase activity was determined using titrimetric

method using olive oil as substrate. Emulsified substrate

was prepared using 5% (w/v) olive oil and gum arabic

incubated in water bath for homogenization. 1 ml of

extracted crude was added into 5 ml of emulsified

substrate. The mixture was later on incubated at 30 ºC for

2 hours. The reaction was terminated by addition of 10 ml

of 95% (v/v) ethanol solution into the mixture. Fatty acid

liberated was titrated using 0.05 M NaOH against 2-3

drops phenolphthalein as indicator. One unit of lipase

defined as amount of enzyme required to liberate one

micromole fatty acid under specified assay condition [11]. 4) Empirical study on lipase activity on UHT milk

Ultra-high treatment or UHT processed milk (Dutch

Lady) was used for this analysis. 5 ml of UHT milk was

prepared in a screw capped tube with addition of 2-3

drops of phenolphthalein. Later on, 1 ml of extracted

crude lysates from each isolate was added in the prepared

tubes each. The tubes were incubated at 37 ºC in water

bath and observed for any color changes after 30 minutes

[12].

5) Effect of different grinding time on cell lysates

with best lipase activity

Isolate which demonstrates the best lipase activity was

further cultured for six samples. Each sample was ground

from 10 minutes to 60 minutes with 10 minutes interval.

Lipase activity from each cell lysates was assessed. The

extracted crude enzyme was also subjected for SDS-gel

electrophoresis [13].

C. Characterization of the Isolates

1) Growth profile

a) Measurement for optical density (OD600nm)

The cultures were incubated in nutrient broth and

approximately 1ml of samples was withdrawn aseptically

for every 6 hours of intervals including 0 hour. Each

sample was withdrawn in triplicates aseptically. Prior to

quantification, the samples in cuvette were thoroughly

suspended and absorbance readings were obtained at

600nm wavelength against distilled water. The used

cuvettes were originated from Germany

(PLASTIBRAND), a semi-micro disposable cuvette with

the volume capacity of 1.5ml.

b) Measurement for dry cell weight (g/L)

Sterilized micro-centrifuge tubes with the capacity of

1.5 ml were oven-dried at 80 ºC for overnight. Prior to

use, the micro-centrifuge tubes were cooled down in

room temperature and the empty micro-centrifuge tubes

were weighed accordingly (Sartorious TE214S,

Germany). As previous, one ml of samples was pipetted

aseptically into the micro-centrifuge tubes in triplicates

for every interval of 6 hours including 0 hour of

incubation time (Mills & Lee, 1996). Later on, the

samples were subjected for centrifugation for 15 minutes

at 14000 rpm to obtain the cell pellet. Upon

centrifugation, the supernatant was gently discarded and

the cell pellets were re-suspended thoroughly with

autoclaved distilled water. Another three cycles of

centrifugation steps were repeated to eliminate possible

contaminants presence in the final cell pellet. Finally, the

micro-centrifuge tubes containing the washed cell pellets

were oven dried at 80 ºC for 24 hours. The micro-

centrifuge tubes were pre-cooled to room temperature

before being weighed and the readings were tabulates.

The calculated dry cell weight was expressed in (g/L).

2) Biochemical tests

The five isolates were subjected for few biochemical

tests. The chemicals used are shown in Table II.

TABLE II. LIST OF CHEMICAL/REAGENTS INVOLVED FOR EACH

BIOCHEMICAL TESTS

Biochemical Tests Chemical/ Reagents

Gram staining Safranin, iodine, crystal violet,

decolorizing (Merck), Iodine Solution

(PC Laboratory Reagent)

MacConkey MacConkey agar (Pronadisa Conda) Agar-agar (R&M chemicals)

Simon Citrate

utilization

Simon Citrate Agar (Pronadisa Conda),

Agar-agar (R&M chemicals)

Catalase test Hydrogen Peroxide

MR-VP test MR-VP broth (Merck)

Methyl Red (Sigma Aldrich)

Starch Hydrolysis Starch Agar (Pronadisa Conda)

Agar-agar (R&M chemicals) Iodine Solution (PC Laboratory

Reagent)

Kovacs’s indole test Trypthophan Peptone Broth (Oxoid) Methyl Red (Sigma Aldrich)

Antibiotic test Kanaamycin, Gentamicin, Ampicillin,

Tetracycline, Vancomycin (BioBasic, Inc) Antimicrobial susceptibility disks

(Oxoid)

132014 Engineering and Technology Publishing


III. RESULTS AND DISCUSSION

A. Testing for Lipase Production

1) Lipase assay

As shown in Fig. 1, the tested isolates projected lipase

activity from the range of 4.7 U/ml to 9 U/ml. This

outcome emphasizes that Rhodococcus isolates exist as

possible source for lipase production. R.NAM 319

displays highest lipase activity, potent to be most

promising isolate among other isolates. Interestingly, this

isolate even displayed higher enzyme activity compared

to the tested positive culture. R.NAM81, R.NAM350 and

R.SeAG1 on the other hand, exhibited lipase production

almost in similar level. In this assay, lipase hydrolytic

activity was evaluated based on oleic acid (C18), a long

carbon chain [10]. As a hydrolytic enzyme, lipases would

disrupt the major chemical bond of the saturated

hydrocarbons and results into harmless fatty acid droplets.

This mechanism is been influenced by level of lipase

activity (U/ml) occurring on the boundary lipid-water

interface [14].

Figure 1. Titration analysis on tested Rhodococcal isolates

2) Empirical study on lipase activity on UHT milk

The objective of this test is to evaluate efficacy of

Rhodococcal lipase action on milk. UHT milk is known

as long life product; however trace action of lipolysis on

this milk would affect the life span of the product mostly,

6 months without refrigeration [15]. Fig. 2 illustrates the

extent of lipase contamination occurred in the tested UHT

processed milk by different lipase source.

Figure 2. Observation on colour changes on UHT processed milk due to lipase activity.

Upon degradation of the milk and excessive yield of

free fatty acid, the colour of UHT milk changes from pink

(alkaline) to colourless (acidic). This phenomenon

indicates the presence high amount of free fatty acid thus,

the milk turns acidic. As per theory, in case spoilage

although the lipase producing microbe has been destroyed

yet the activity of lipase still remains determining the life

span of the dairy product. As the milk portion has been

degraded to free fatty acid, it tends to develop bitter –off

flavors by free fatty acid yielded [16]. Therefore, this first

stage outcome proves that the extracted crude lipase

practically applicable in food products. 3) Effects of different grinding time on cell lysates

with best lipase activity

Apart from analysis on bacteria growth conditions and

nutrients, another essential aspect influencing the enzyme

analysis would be the grinding time required. Being the

most potential lipase producer, demonstration in Fig 3

shows analysis on crude lysates of R.NAM391 obtained

at different grinding time. Rhodococcus sp. evidently has

been proven to own rigid cell envelope, where the

internal osmotic pressure in the microorganism cytoplasm

regarded as another resistance to disintegrate cell wall

from the intracellular content [17]. Hence, evaluation on

level of grinding for high operable quality of lipase

enzyme esteemed to be necessary.

Figure 3. Effect of grinding time on lipase activity of R.NAM319

Cryogenic grinding persistently earned great protein

content when Rhodococcus cell membrane is disrupted. It

was understood that constant non-stop force from

grinding will overcome barrier like “capsular slime”

properties produced by this bacteria. Convincingly,

structure of lipase enzyme made of α/β hydrolase folds, is

also recognised as a strong robust catalyst with greater

capacity to withstand harsh conditions [18]. Hence,

possibilities for denaturation or damage on this specified

protein during consistent grinding can be least expected. Extracted protein content proportionally increases by

grinding time for R.NAM319. Therefore, lipase activity

noticeably increases as time grows, indicating increasing

level amount of hydrolysis end product from prolong

lysis. In agreement to the previous finding, extracted cell

lysates resulted in visible protein bands for every 10

minutes of interval grinding. Due to the increasing

protein recovery for each grinding intervals, darker

protein bands were observed from 1st lane to 6

th lane as

shown in Fig 4.



Figure 4. Protein separation from R.NAM 319 cell lysates at different grinding time. Lane 1: 10 min; Lane 2: 20 min; Lane 3: 30 min, Lane 4:

40 min; Lane 5: 50 min; Lane 6: 60 min.

B. Characterization of the Isolates

1) Growth profile

Growth pattern of the five potential isolates were

assessed for 144 hours in nutrient broth as depicted in Fig

5 (a) and (b). Microbial growth is termed as increase in

cell size and cell mass due to the microbe metabolism and

sustainability over a range of environmental condition.

As per theory, a growth curve comprises four different

phases including lag, exponential, stationary and death

phase. The growth curve of the bacteria was assessed

based on optical density (OD600nm) and dry cell weight

(g/L) as shown in Fig 5 (a) and (b). From the analyzed

data, each five isolates exhibited different period of each

phases. There is no significant lag phase shown by these

isolates. From the plotted graph, the lag phases of the

strains are not evidently apparent. Prior to inoculation in

fresh medium, the bacteria cells were grown on solid

medium initially. Hence, upon transferring the cells from

nutrient-less medium into fresh nutrient rich medium, the

cells easily adapted the new environment requiring

duration of less than 6 hours. Therefore, a shorter lag

phase was depicted by the isolates.

Figure 5(a). Optical density (OD600nm) measurement of the lipolytic Rhodococcus strains in nutrient broth

The exponential phase is period of time where cell

undergo rapid division and proliferation. In general the

viewed graph, show an evident exponential phase during

24 hours of incubation with OD600 ranging from 0.7 to1.2

and approximate weight of 1 g/L. Meanwhile, R.UKMP-

5M and R.SeAG1 displayed increasing proliferation until

30 hours of incubation. R.NAM 319 on the other hand,

exhibited slower cell division and concentration, as seen

increasing trend was observed until 72 hours of

incubation in both graph.

Figure 5(b). Dry cell weight (g/L) of lipolytic Rhodococcus strains in nutrient broth

Several opinions do not correlate optical density and

dry cell weight; however, there are several factors could

be possible reasons for fluctuation. Factors such as

inconsistent amount of taken sample in per ml, or the

sample might not be dried completely during the drying

process in oven might influence the reading of dry cell

weight (g/L). Additionally, total volume taken for each

analysis (1ml) could be too small for analysis, as the dry

matter of the cell is easy to lose in high temperature

hence, results in variation in triplicate readings. Other

than that, prolong incubation could have caused the

occurrence of cell lysis. Another possible reason also

could be due to insufficient centrifugation speed to

collect the cell debris. Therefore, the triplicates reading of

cell mass differ among each other. Subsequent phase or

stationary phase is referred as the non-dividing resting

state and increased resistance of provided environment

mainly due to nutrient depletion [19].

The view graphs comparably depicted consistent

reading until 96 hours of incubation, meanwhile, a slight

deterioration in biomass concentration while a drastic

drop of cell number was noted after 96 hours. This shows

that the cell are approaching death phase and the

bacterium clearly loses its ability to reproduce and dies

eventually.

Although optical density reading does not differentiate

the active and dead cells, however, measurement of dry

cell weights estimates effectively the weight of viable

cells [20]. Relationship between optical densities against

cell mass is regarded to be specific to the microorganism

species. This might be the reason fluctuation of growth

curve can be observed from graph of dry cell weight (Fig.

3b) than graph optical density (Fig. 3a). Another key

factor that contributes the independency of cell biomass



and optical density is the robust wavelength [21]. Various

range of pigment profile displayed by the cell in the

medium lead to diverse absorption spectra. Hence,

suspension samples with similar absorption ability results

in reduced deviation during optical density analysis.

Figure 6. Morphology of Rhodococcus colonies (a) R.NAM81, (b) R.NAM319, (c) R.NAM350, (d) R.UKMP-5M, (e) R.SeAG1, (f)

Positive culture.

2) Biochemical test

a) Gram staining

Cell envelope of Rhodococcus cell is being capsulated

with large branch chain lipid known as mycolic acids.

Additionally, this bacterium is well-denoted for its tough

arabinogalactan (AG) cell wall polysaccharide anchoring

the formed mycolic acids [22]. This tough and rigid

layers cell envelope retains the crystal violet dye stained

initially during this test though stages of decolorizing and

washing has been done subsequently. Thus, the stained

cells appear to be purple under microscope.

Figure 7. Gram staining observation under microscope 4 x100

b) Mac conkey agar test

As both selective and differential medium,

composition of Mac Conkey agar includes mixture of bile

salt and crystal violet which inhibits growth of gram

positive bacteria. In agreement to gram staining result,

the streaked bacteria could not tolerate bile salts and

crystal violet in the agar, thus do not exhibits any growth

on the agar. This confirms that the streaked strains belong

to gram positive category.

c) Simmon citrate agar test

This differential medium is composed of sodium

citrate as the sole carbon source and ammonium ion as

the only nitrogen source. Bromothymol blue indicator has

been incorporated in the agar, where the medium remains

green for pH <7 but changes to blue when pH >7.6.

Therefore, upon utilization of citrate carbon source for

energy, an alkaline by- product formed will initiate colour

changes from green to blue. In accordance to Table III,

few lipolytic Rhodococcus isolates namely R.NAM81,

R.NAM319 and R.SeAG1 changes colour to blue,

indicating their ability to utilize citrate as the carbon

source, though few other Rhodococcus isolates resulted

no changes. This indicates although same species, their

adaptability can differs according to the metabolism of

the strain [23].

Figure 8. Observation on simmon citrate agar

d) Catalase test

In this test, the ability of tested microorganism to

produce catalase enzyme were tested. From this test, the

specimen can be known either living in aerobic or

anaerobic condition. Biologically, hydrogen peroxide is

the by- product of respiration and in case of accumulation,

it endangers the cell. Therefore, to prevent cell damages,

any cell that uses oxygen or lives in presence of oxygen,

produces catalase enzyme to breakdown hydrogen

peroxide into water and oxygen. From Fig. 9, it was clear

that the lipolytic Rhodococcus isolates are aerobic

bacteria meanwhile; the tested positive control is an

anaerobic microorganism.

Figure 9. Catalase test on microscope slide

e) MR-VP test

MR-VP broth is a differential medium, which

differentiate specimen based on acid or acetylmethyl



production. The broth composed of peptone, buffers,

glucose or dextrose. In the process of converting the

dextrose or glucose to pyruvate, the cultured strain uses

mixed acidic pathway which yields acidic end such as

lactic acid, formic acid and acetic. Pertaining Table III, it

was evident that all the cultured strains do not produce

acidic product in their metabolism, thus conversion of

dextrose to pyruvate do not occur in these bacteria

metabolism.

TABLE III. RESULTS OF DIFFERENT BIOCHEMICAL TESTS CONDUCTED ON EACH RHODOCOCCUS ISOLATE

Isolate Biochemical Test

Gram

staining

Mac

Conkey

Simon

Citrate

Catalase

test

MR-VP test Starch

hydrolysis

Kovacs

indole

R.NAM81 + - + + - - -

R.NAM319 + - + + - - -

R.NAM350 + - - + - - -

R.UKMP-5M + - - + - - -

R.SeAG1 + - + + - - -

Positive control + - - - - - - “+” indicates positive changes occurred during the test; “-” indicates no changes have been detected during the test. Positive control= Bacillus subtilis

f) Starch hydrolysis

This test is used to differentiate the ability of specimen

to produce enzyme known as α-amylase or amino-1,6-

glucosidase. Starch is a macro nutrient which is hard to

be absorbed into the cell. Hence, in order to breakdown

starch into smaller subunits, the specimen produces

exoenzymes to initiate breakdown. A clearing around the

streaked colony after addition of iodine on the plate

indicates starch has been hydrolysed. However, none of

the streaked strains could produce exoenzymes

effectively thus, no changes seen on starch agar.

g) Kovac’s indole test

This test reveals ability of a specimen to degrade the

amino acid tryptophanane produce indole as the end

product. This is the chemical reaction involved:

Tryptophan+water=indole + pyruvic acid +ammonia

In presence of indole after the cultivation, top of the

broth will form a “cherry-red” ring upon addition of

Kovac’s reagent. As shown in the equation, formation of

indole is the result of reductive deamination from

tryptophan via the intermediate molecule indole pyruvic

acid. However, the cultivated isolates resulted in negative

observation, indicating these strains are not able to

breakdown tryptophan amino acids [24].

h) Antibiotic resistance test

Disk diffusion method was applied to test the

susceptibility of the bacteria on few antibiotics on

Mueller Hinton agar plate. Table IV represents the mean

of inhibition zone diameter measurement in mm for

tested antibiotics discs.

This test would determine the most effective antibiotic

for treatment against particular type of microbe infection.

Size of inhibition zone represents degree of resistance,

and can be predicted for resistance mechanism and

resistance gene involved. Rhodococcus sp. relevantly, is

known as uncommon opportunistic pathogen in human

especially for immunocompromised patient [25].

Formation of an inhibition zone cannot be automatically

concluded as susceptible yet; the diameters of zone need

to be compared to standard zone inhibition chart [26].

Table V are the subsequent interpretations of Table IV.

TABLE IV. RESULTS FOR ANTIBIOTIC RESISTANCE TEST ON CHOSEN LIPOLYTIC RHODOCOCCUS STRAINS

Isolate Mean of inhibition zone (mm)

Tet (30μg) Vanco (30μg) Gent (10μg) Kana (30 μg) Amp (10μg)

R.NAM81 25.17a ± 0.76 18.33b ± 0.76 13.17c ± 1.04 14.83d ± 0.29 13.67e ± 0.76

R.NAM319 20.83a ± 0.29 15.5c ± 0.5 12d ± 0.86 15.67 b ± 0.76 7.33e ± 0.58

R.NAM350 16 ± 0.5 13.83 ± 0.29 13.33 ± 0.58 14.17 ± 0.29 9 ± 0.5

R.UKMP-5M 27.5 ± 0.86 24.83 ± 0.76 13.83 ± 0.29 19.5 ± 0.5 17.83± 0.29

R.SeAG1 22.83 ± 0.29 25.67 ± 0.58 24.17 ± 0.29 20.67 ± 0.58 25.67 ± 0.58

+ve control 17.17 ± 0.76 15.5 ± 0.5 12.5 ± 0.5 16.7 ± 0.58 9.33 ± 0.76

TABLE V. INTERPRETATION ON ZONE OF INHIBITION AS PER THE STANDARD CHART

Isolate Mean of inhibition zone (mm)

Tet (30μg) Vanco (30μg) Gent (10μg) Kana (30 μg) Amp (10μg)

R.NAM81 S S I I I

R.NAM319 S S R I R

R.NAM350 I I I I R

R.UKMP-5M S S I S S

R.SeAG1 S S S S S

+ve control I I I I R

S= susceptible, I= Intermediate; R= resistant



It was understood that the formed inhibition zone

largely depends on several factors such as degree of

susceptibility of bacteria and rate of growth of the

inoculum. Evident to few studies of antibacterial

activities on Rhodococcus sp., the obtained outcome was

in agreement to the reported results. As an example,

generally most of Rhodococcus sp. is reported to be

susceptible to vancomycin [25]. There is also a report

stated that Rhodococcus equi potrays moderate

susceptibility to ampicillin, tetracycline and gentamicin

[24]. Meanwhile in current finding, susceptibility of

different Rhodococcus isolates seems to be different for

each tested antibiotics, suggesting variable anti-microbial

properties displayed by different Rhodococcus isolates.

IV. CONCLUSION

This initial stage of study confirms that locally isolated

Rhodococcus isolates are potent to be lipase producers.

As discussed, R.NAM 391 being the most promising

isolate, reveal that as grinding time of the cell increases,

lipase activity of the cell lysates would increase. Since

the tested isolates are tentatively characterized,

biochemical tests resembles that the tested isolates

resemble characteristics of Rhodococcus sp. as per

analysed in literature. This initial stage of study serves as

an introductory study on locally available isolates, which

has not been fully exploited for lipase producing

capacities.

ACKNOWLEDGMENT

The authors would like to express gratitude to Ministry

of Science, Technology and Innovation (MOSTI),

Malaysia (3090104000(G)) and the Selangor State

Government, Malaysia for the financial assistance. We

would like to acknowledge Institute Bio-IT, Universiti

Selangor, for providing us necessary lab facilities

throughout this investigation.

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Jayesree Nagarajan is a postgraduate student in Institute Bio-IT Selangor,

Universiti Selangor, Malaysia. She obtained

her Bachelor of Science (Hons) Biotechnology from University Tunku Abdul

Rahman in 2011. Her majoring was on mycology and lipase studies during her

degree. Her current project highlights on

Rhodococcal lipases and its practical applications in industry under the supervision

of Prof. (E) Dato’ Dr. Abdul Latif Ibrahim. Recently she has published

a full length research article entitled “Rhodococcus UKMP-5M, an endogenous lipase producing from actinomycete from Peninsular

Malaysia” under Biologia, Springer publication.



Norazah Mohammad Nawawi is a senior lecturer in Universiti

Selangor. Earlier she pursued her Masters in Universiti Kebangsaan

Malaysia and currently continuing her PhD in Universiti Putra Malaysia.

Her expertise field is on Microbiology and Molecular biology. Her current project focuses on Rhodococcus sp. and phenol hydroxylase

enzyme.

Prof. (E) Dato’ Dr. Abdul Latif Ibrahim is a senior professor in

Universiti Selangor since 2003. He started his career as a Veterinary

Assistant at the Veterinary Office in Selangor. Later on, he pursues a

Bachelor Degree in Veterinary Medicine at the East Pakistan. He furthered his studies at the University of Hawaii for an Msc in

microbiology. And then he successfully graduated with a PhD from

University of California USA. He also developed a vaccine for Newcastle disease during his service at Universiti Putra Malaysia. He

received Personality Academic Award from Ministry of Education in

2012.



Characterization of Lipase Producing …. R ESULTS AND D ISCUSSION A. Testing for Lipase Production...

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