ORIGINAL PAPER

A unique thermostable and organic solvent tolerant lipasefrom newly isolated Aneurinibacillus thermoaerophilusstrain HZ: physical factor studies

Malihe Masomian • Raja Noor Zaliha Raja Abd Rahman •

Abu Bakar Salleh • Mahiran Basri

Received: 26 April 2008 / Accepted: 9 February 2010

� Springer Science+Business Media B.V. 2010

Abstract A newly isolated thermophilic bacterium,

Aneurinibacillus thermoaerophilus strain HZ, from a hot

spring recreational area (Sungai Kelah, Malaysia), showed

an extracellular lipase activity. It was identified based on

16S rRNA sequencing, where phylogenetic analysis

revealed its homology to Aneurinibacillus thermoaerophi-

lus. The strain produced a lipase that was stable in various

organic solvents such as dimethyl sulfoxide, toluene,

p-xylene, and hexane. In order to increase lipase produc-

tion, optimization of physical factors which affected the

growth and lipase production was studied. The optimal

growth was obtained at 50�C and pH 8.0; while the max-

imal lipase production was achieved in the logarithmic

decline phase at 60�C and pH 7.5 with 7% starting inoc-

ulum and 150 rev/min shaking rate for 48 h incubation.

Keywords Aneurinibacillus thermoaerophilus

strain HZ � Thermostable lipase �Optimization of physical factors � Organic solvent tolerant

Introduction

Lipases (triacylglycerol acyl hydrolases (EC 3.1.1.3) are

lipolytic enzymes that catalyse both the hydrolysis and the

synthesis of esters at an oil–water interface (Haba et al.

2000). Many microorganisms, including plants, animals,

bacteria, yeast, and fungi, have been determined to secrete

lipases during their growth in hydrophobic substrates

(Holker et al. 2004). However, only microbial lipases are

commercially significant for their potential use in indus-

tries because of their unique catalytic performance and

versatility of catalyse numerous different reactions. They

are widely diversified in their enzymatic properties and

substrate specificities (Gupta et al. 2004). Lipases are the

choice of biocatalysts as they show unique chemo-, regio-,

enantioselectivites, which enable the production of novel

drugs, agrochemicals and fine products (Gotor-Fernandez

et al. 2006; kamini et al. 2000; Saxena et al. 2003). Many

lipases, due to their ability to perform both hydrolytic and

synthetic reactions, find immense applications in industries

like foods, detergents, pharmaceuticals, leather, textile and

dairy (Saxena et al. 2003; Hasan et al. 2006; Burkert et al.

2004).

Each application requires unique properties with respect

to specificity, stability, temperature, and pH dependence or

ability to catalyse synthetic ester reactions in organic sol-

vents. Meanwhile, thermostable enzymes are particularly

attractive for industrial applications because of their high

activities at the elevated temperatures and stabilities in

organic solvents (Niehaus et al. 1999; Pennisi 1997).

Substrates and products of lipase are often insoluble in

aqueous solutions, and the enzyme is usually insoluble in

organic solvents. Some reactions catalysed by lipase can be

carried out in organic aqueous two-phase media because the

separation of enzyme from substrates or products becomes

M. Masomian � R. N. Z. R. A. Rahman (&)

Enzyme and Microbial Technology Research, Department

of Microbiology, Faculty of Biotechnology and Biomolecular

Science, Universiti Putra Malaysia, 43400 Serdang, Selangor,

Malaysia

e-mail: rnzaliha@biotech.upm.edu.my

A. B. Salleh

Enzyme and Microbial Technology Research, Department

of Biochemistry, Faculty of Biotechnology and Biomolecular

Science, Universiti Putra Malaysia, 43400 Serdang, Selangor,

Malaysia

M. Basri

Enzyme and Microbial Technology Research, Department

of Chemistry, Faculty of Science, Universiti Putra Malaysia,

43400 Serdang, Selangor, Malaysia

123

World J Microbiol Biotechnol

DOI 10.1007/s11274-010-0347-1

easy. However, in general, enzymes are easily inactivated in

the presence of organic solvents, even if they are almost

water-insoluble (Ogino et al. 1999). Accordingly, organic

solvent–stable microbial lipases which are of enormous

biotechnological potential possess the following character-

istics: (1) require no cofactors, (2) remain active in organic

solvents, (3) react on a broad substrate specificity and

(4) exhibit a high enantioselectivity (Bornscheuer 2002).

Pursuant to the above mentioned statements, screening

of thermophilic microorganisms with lipolytic activities

can facilitate the discovery of novel lipases for industrial

purposes that are stable and function optimally at extreme

temperature. Therefore, we conducted an extensive

screening and isolated a new thermophilic lipase-producing

organism, identified as Aneurinibacillus thermoaerophilus

strain HZ that is both organic solvent-tolerant and extre-

mely thermophilic. Here, we report the isolation and

physical optimization of the culture to produce high yield

of thermostable and organic solvent-tolerant lipase.

Materials and methods

Sample collection and enrichment culture

Samples were collected from hot spring in Sungai Kelah (up

to 100�C), Malaysia. The enrichment of samples was per-

formed at 55�C with shaking (150 rev/min) in a medium

containing peptone (0.5% w/v), yeast extract (0.5% w/v),

NaCl (0.05% w/v), CaCl2 (0.005% w/v), olive oil (1.0% v/v),

pH 8.5 and M2 (tryptone (3.0% w/v), yeast extract (1.0%

w/v), NaCl (0.05% w/v), glucose (0.5% w/v), pH 7.5 for

72 h.

Screening and isolation of lipolytic thermophile

bacteria

Screening for lipase-producing isolates was done qualita-

tively on tributyrin agar plates. Isolates that produced

lipolytic enzyme formed clearing zones around the colo-

nies on the plates. These positive isolates were then tested

for their lipase production on Rhodamine B agar plates and

triolein agar plates at 55�C for 24–48 h. Lipase production

on Rhodamine B agar plate was monitored with UV light at

350 nm. Orange fluorescent halos were shown around the

colonies of lipase producing strains and lipase producers

produced blue zone on triolein agar.

Production medium and growth conditions

A single colony of each lipase producer from a nutrient

agar slant was inoculated in 10 ml tryptone soy broth

medium and incubated at 55�C overnight in water bath

shaker. Two ml were inoculated in 50 ml of production

medium containing yeast extract (0.75% w/v), peptone

(0.75% w/v), olive oil (1.0% v/v), gum arabic (1.0% w/v),

NaCl (0.25% w/v), CaCl2.2H2O (0.001% w/v), pH 7.5 for

72 h at 55�C with shaking.

Lipase assay

Lipase activity was measured by a modified method of

Kwon and Rhee (1986). Ten ml of the culture were taken

out and centrifuged at 10,000g for 10 min at 4�C to sedi-

ment bacteria cell. The supernatant was assayed for lipase

activity. The reaction mixture consisting of 1.0 ml of crude

enzyme, 2.5 ml olive oil emulsion (1:1 ratio of olive oil

and phosphate buffer [K2HPO4 (50 mM), at pH 7.0)] and

0.02 ml of 20 mM CaCl2�2H2O, was incubated for 30 min

with shaking at 200 rev/min at 55�C. Then the reaction was

stopped by adding 1.0 ml of 6 M HCl and 5.0 ml isooc-

tane. The upper layer (4 ml) was pipetted out into a test

tube, and 1.0 ml cupric acetate pyridine [Kupfer(II)-Acetat

monohydrate (5%; pH 6.1), pH adjusted by adding pyri-

dine] was added. The free fatty acids dissolved in isooctane

were determined by measuring the absorbance of isooctane

solution at 715 nm. Each sample was assayed three times

and the average value was taken. One unit (U) of lipase

activity is defined as the rate of fatty acid formation per

min (standard assay condition). Standard deviation was

determined by Microsoft Excel program.

Identification of the bacteria

Aneurinibacillus thermoaerophilus strain HZ was identi-

fied via 16S rDNA sequencing. The genomic DNA was

extracted using DNeasy Tissue Kit (Qiagen, Germany)

according to the manufacture’s instructions and was used

as a template to perform Polymerase Chain Reaction (PCR)

amplification for 16S rDNA identification. The universal

primers were used: 16S-F (50-GAG TTT GAT CCT GGC

TCA G-30) and 16S-R (50-CGG CTA CCT TGT TAC GAC

TT-30). The reaction mixture was initially heated for 4 min

at 94�C, followed by 30 cycles PCR of 94�C 1 min, 58�C

1 min and 72�C 1 min; then, one cycle of 7 min at 72�C

and held at 4�C. The primers amplified a 1,502 bp PCR

product. The amplified products were examined by elec-

trophoresis and extracted by using QIAquick Gel Extrac-

tion Kit (Qiagen, Germany) according to the manufacture’s

instructions. Sequencing of purified product was down by

First Base Laboratories Sdn Bhd (Shah Alam, Selangor,

Malaysia), using BigDye� Terminator v3.1 cycle

sequencing kit chemistry. The complete sequence was

submitted to GenBank at NCBI. DNA homology search

in the GenBank database (http://www.ncbi.nih.gov) was

World J Microbiol Biotechnol

123

performed. A phylogenetic tree was constructed based on

comparison of the 16S rDNA sequence of the isolate with

other strains of Aneurinibacillus spp. All sequences were

aligned with CLUSTALW 1.75 from Biology WorkBench

database at website (http://workbench.sdsc.edu; Thompson

et al. 1994). The tree was constructed in GeneBee service

which involves bootstrap analysis (http://www.genebee.

msn.su). 16S rDNA sequences of other Aneurinibacillus

spp. were obtained from the GenBank database (http://

www.ncbi.nih.gov).

Optimal temperature

The effect of temperature of the crude lipase was investi-

gated by assaying at temperatures ranging from 45 to 70�C.

Lipase activity was determined using olive oil as substrate.

Effect of physical factors on bacterial growth and lipase

production

To investigate the influence of physical factors on the

growth and extracellular lipase production by Aneuriniba-

cillus thermoaerophilus strain HZ, an overnight bacterial

inoculum (5%; OD600 = 0.5) was inoculated into basal

medium and incubated for 48 h. Five parameters (medium

volume, pH, temperature, inoculum size and agitation)

were studied for their influences in lipase production and

bacterial growth. Lipase assay was performed after 48 h of

incubation by modified method of Kwon and Rhee (1986)

as described previously. The bacterial growth was esti-

mated by the number of c.f.u. on the nutrient agar plates.

Effect of medium volume

The effect of the medium volume was investigated by

cultivating the bacterium on different volumes of medium

50, 100, 150 and 200 ml in 500-ml blue cap bottles.

Effect of temperature

The ability of A. thermoaerophilus strain HZ to grow and

produce lipase at elevated temperatures (50–70�C) was

studied. Separate cultures were incubated at 50, 55, 60, 65

and 70�C for 48 h with agitation at 150 rev/min.

Effect of pH

The effect of pH was studied by adjusting the media (with

1.0 M of NaOH or HCl) to different pH values from 4 to 9

at 0.5 unit intervals. The media were autoclaved, cooled

and inoculated with an overnight culture of A. thermo-

aerophilus strain HZ.

Effect of inoculum size

The effect of inoculum size (OD600 = 0.5) was investi-

gated by using different inoculum sizes ranging from 1 to

9% at interval of 2% (v/v).

Effect of agitation

The effect of agitation was carried out by cultivating the

bacterium under different agitation rates, 0, 50, 100, 150,

and 200 rev/min.

Organic solvent stability of crude enzyme

A. thermoaerophilus strain HZ was cultured aerobically in

the absence of organic solvents at 55�C for 48 h. The

culture medium was centrifuged at 10,000g for 10 min at

4�C and the supernatant was used as the crude enzyme.

Various organic solvents were tested at the concentration

of 25% (v/v) by addition of 1 ml of organic solvent to 3 ml

of crude enzyme. The reaction mixture was preincubated

for 30 min at 37�C under shaking condition (150 rev/min)

to insure the continuous mixing of the enzyme and solvent.

The enzyme stability was expressed as the remaining

activity assayed according to the method of Kwon and

Rhee (1986) relative to the control value. For control,

phosphate buffer pH 7.0 was added instead of solvent. The

organic solvents used were dimethyl sulfoxide (-1.3),

benzene (2.0), toluene (2.5), 1-octanol (2.9), hexane (3.6),

dodecanol (5.0), decane (5.6), 1-dodecene (6.6), n-te-

tradecane (7.6), hexadecane (8.8).

Statistical analysis

For statistical analysis, a standard deviation for each

experimental result was calculated using the Excel Spread-

sheet available in the Microsoft Excel.

Nucleotide sequence accession number

The nucleotide sequence determined in this study has been

deposited in the Gene Bank database (http://www.

ncbi.nih.gov) under accession number DQ890194.

Results and discussion

Bacterial strain identification and phylogenetic tree

analysis

The 16S rRNA (*1,500 bp) contained several regions of

highly conserved sequence useful for obtaining proper

sequence alignments and on the other hand contained

World J Microbiol Biotechnol

123

sufficient sequence variability in other regions of the

molecule to serve as excellent chronometers. The differ-

ences of sequence in the hypervariable regions reflect the

strain variations. Comparisons of the sequences between

different species suggested a relatively earlier or later time

in which they shared a common ancestor (Woese 1998).

The polymerase chain reaction (PCR) technique was

used to amplify the rRNA gene using universal primers and

it amplified the whole region of the rRNA gene which is

1,502 bp. The amplified product was examined by elec-

trophoresis and then was purified and sequenced (Fig. 1).

Comparison of the 16S rRNA gene of the new isolate

with the GenBank database (http://www.ncbi.nih.gov)

showed that it is closely related to A. thermoaerophilus

strain L420-91, DSM 10154 (X94196) and A. thermo-

aerophilus strain: DSM 10154T (AB112726). The phylo-

genetic tree analysis of new isolate was compared with

other Bacillus spp. sequences and it showed high homology

with A. thermoaerophilus strains such as L420-91, DSM

10154 (X94196), DSM 10154T (AB112726), ATCC 12990,

DSM 10155 (X94197) and Bacillus sp. HC5 (AF252327).

The new isolate was identified as Aneurinibacillus ther-

moaerophilus strain HZ. The analysis result indicated that it

is phylogenetically distant from A. thermoaerophilus strain

ATCC 12990, DSM 10155 (X94197). In addition, there

have not yet been any reports on lipase from A. thermo-

aerophilus presented. Phylogenetic relationship of closely

related Aneurinibacillus spp. is shown in Fig. 2.

Effect of different assay temperatures on crude

HZ lipase

On the basis of relatively higher lipase activity detected for

A. thermoaerophilus strain HZ, the effect of temperature on

crude HZ lipase activity was examined from 45 to 70�C. As

shown in Fig. 3, the crude enzyme manifested its maximal

activity at 65�C with olive oil as substrate. Although lipase

activity was strongly dependent on assay temperature, a

further increase in temperature to 65�C did not follow this

trend because, most likely, the flexibility of enzyme

increased and hence loosely bound to substrate. This resul-

ted in decreasing its turnover number. The crude enzyme of

strain HZ was fairly active at higher temperature compared

to lipases from others Bacillus spp. (Sharma et al. 2002;

Li and Zhang 2005), and B. coagulans BTS-3 (0.75 U/ml;

Kumar et al. 2005) which all exhibited maximal activity at

55�C. Kim et al. (2000) reported 0.2 U/ml of crude lipase

activity at 50�C by B. sterarothermophilus L1.

The effect of medium volume on the production

of lipase

The medium volume may have a great effect on the

microorganism growth and enzyme production. While a

large volume contains more oxygen, food and nutrients, the

air space in the container is decreased. The more air space

provides more oxygen that can dissolve slowly in the

medium, so the more bacteria are grown.

The effect of medium volume on lipase production and

bacterial growth by A. thermoaerophilus strain HZ is

shown in Fig. 4. The highest bacterial growth and lipase

production were obtained in 50 ml medium. This volume

with its air space supplied more oxygen for the maximum

lipase production and growth. Lower bacterial growth

occurred in 200 ml production volume with 5% reduction

when compared to the maximum and lipase production

decreased to 50% of the maximum. Lower lipase produc-

tion in 200 ml production volume could be due to the less

air space and consequent poorer aeration.

The highest lipase production in 50 ml was caused

because of the lower volume provided a bigger air space

and also more oxygen that slowly would be dissolved into

the medium. Also, Kumar et al. (2005) reported that higher

lipase production occurred in 50 ml of medium by Bacillus

coagulans BTS-3.

Fig. 1 16S rDNA gene (1,500 bp) of isolate A10 gene amplified via

PCR. Lane 1–3: PCR product of amplified 16S gene. Lane 4 Marker

GeneRuler DNA 1 kb

World J Microbiol Biotechnol

123

The effect of temperature on the production of lipase

The culture was incubated at different temperatures rang-

ing from 50 to 70�C to investigate the optimum

temperature for bacterial growth and lipase production.

Growth and lipase production by A. thermoaerophilus

strain HZ was detected at all temperatures tested (Fig. 5).

Generally, the optimum temperature for lipase production

Fig. 2 Rooted phylogenetic

tree showing the relationship of

A. thermoaerophilus strain HZ

to other Bacillus spp.

Fig. 3 Effect of temperature on crude HZ lipase. Lipase activity was

determined by using olive oil as the substrate with incubation at

various temperatures, i.e.: 45, 50, 55, 60, 65 and 70�C

0

0.05

0.1

0.15

0.2

0.25

0.3

50 100 150 200

Medium volume (mL)

Lip

ase

acti

vity

(U

/mL

)

4.35

4.4

4.45

4.5

4.55

4.6

4.65

4.7

4.75

Log

CF

U/m

L

Lipase activity Colony count

Fig. 4 Effect of medium volume on growth and lipase production by

A. thermoaerophilus strain HZ. Different volumes of production

medium were incubated in 500 ml blue cap bottles at 60�C under 150

rev/min shaking rate for 48 h

World J Microbiol Biotechnol

123

is different from that for optimum growth. The optimum

temperature for growth of A. thermoaerophilus strain HZ

was 50�C, but the optimum temperature for intracellular

lipase production was 60�C.

As the growth temperature increased more than 60�C,

the bacterial growth and lipase production by A. thermo-

aerophilus strain HZ decreased. The low lipase production

at 65�C can have been due to limited amount of total

dissolved oxygen in the medium. Oxygen has a low solu-

bility in aqueous media which further decreases with rising

temperature (Jacques 1977). In addition, increase in tem-

perature more than 60�C in A. thermoaerophilus strain HZ,

may possibly lead to denaturation of enzyme. In case of

extracellular enzymes, temperature influences their secre-

tion, possibility by changing the physical properties of the

cell membrane (Rahman et al. 2005a). Thus, too low and

too high temperatures are not suitable for lipase production

by A. thermoaerophilus strain HZ. The optimum temper-

ature for lipase production of the A. thermoaerophilus

strain HZ was higher than Bacillus coagulans BTS-3 that

exhibited maximum production at 55�C (Kumar et al.

2005). However, thermostable lipase from Geobacillus sp.

TW1 was reported to exhibit higher temperature of above

65�C (Li and Zhang 2005).

The effect of pH on the production of lipase

As microbial metabolism usually changes the pH of the

medium, some form of pH control is necessary in culture

systems.

Production medium of various pH values, ranging from

pH 4.0 to pH 9.0 were tested to determine the optimum pH

for lipase production and bacterial growth. As shown in

Fig. 6, very low lipase production was detected at acidic

(pH4.0 and 5.0) and alkaline conditions (pH 9.0). Maxi-

mum lipase production by A. thermoaerophilus strain HZ

was obtained at pH of 7.5 (0.26 U/ml) while maximum

bacterial growth was observed at pH 8.0. At pH 7.5, the

bacterial growth was almost the same with pH 8.0 but the

lipase production at pH 8.0 was only 71% of the maximum.

In acidic media from pH 4.0–6.5, bacterial growth was the

same but lipase production detected at pH 6.0 was only

70% of the maximum. The bacterial growth at alkaline pH

values (8.5 and 9.0) was lower compared with that in acidic

media and in pH 9.0 growth was greatly reduced.

The decrease in pH might have been due to the pro-

duction of acidic metabolites (Moon and Parulekar 1991).

In view of the close relationship between lipase synthesis

and use of the medium compounds, pH variation during

fermentation may reflect the kinetic changes in the lipase

production, such as the onset of production.

The effect of agitation on the production of lipase

Agitation has variable effect on lipase production by dif-

ferent organisms. It has been indicated as a key factor

influencing lipolytic enzyme production by mesophilic

microorganism (Sharma et al. 2002; Chen et al. 1999). The

relevance of this variable is even more remarkable when

thermophilic strains are considered, due to the low solu-

bility of gases at the high temperatures utilized throughout

the cultures. Although themophilic bacteria have adapted

to survive at low gas concentrations in their natural habi-

tats, rather high values of specific oxygen up-take rate have

0

0.05

0.1

0.15

0.2

0.25

50 55 60 65 70

Temperature (°C)

Lip

ase

acti

vity

(U

/mL

)

2

2.5

3

3.5

4

4.5

5

5.5

6

Log

CF

U/m

L

Lipase activityColony count

Fig. 5 Effect of temperature on growth and lipase production by

A. thermoaerophilus strain HZ. Cultures contain 50 ml of production

medium were incubated in different temperatures in water bath shaker

under 150 rev/min shaking rate for 48 h

0

0.05

0.1

0.15

0.2

0.25

0.3

4 5 6 6.5 7 7.5 8 8.5 9

pH

Lip

ase

acti

vity

(U

/mL

)

2

2.5

3

3.5

4

4.5

5

5.5

Log

CF

U/m

L

Lipase activityColony count

Fig. 6 Effect of pH on lipase production and growth by A.thermoaerophilus strain HZ. Cultures with different pHs from 4 to

9 at 0.5 unit intervals were incubated at 60�C in water bath shaker at

60�C under 150 rev/min shaking rate for 48 h

World J Microbiol Biotechnol

123

been reported in some cases (Belo et al. 2000). The use of

pressurized or gas lift fermentors has been proposed as

means to provide an adequate oxygen concentration in the

culture medium. The latter is also useful to minimise shear

stress, to which some extreme thermophiles appear to be

extremely sensitive (Belo et al. 2000; Holst et al. 1997).

The effect of agitation on bacterial growth and lipase

production was studied under static condition, 50, 100, 150

and 200 rev/min. As demonstrated in Fig. 7, the highest

lipase production was obtained when agitated at 150 rev/

min. The static condition to 50 rev/min shaking enhanced

bacterial growth by A. thermoaerophilus strain HZ while

lipase production was only 11% and 13% of the maximum,

respectively. Shaking at 100 rev/min supported HZ lipase

production at 77% compared to that at 150 rev/min, but its

bacterial growth was 84% at static condition and decreased

to 83% at 150 rev/min. Higher shaking rate reduces the

bacterial growth because during respiration, hydrogen

atoms may be combined with oxygen forming hydrogen

peroxide which is lethal to the cell. Too much aeration

facilitates hydrogen peroxide production to the certain

level where enzyme that is produced by the bacterial is not

enough to breakdown the hydrogen peroxide and thus leads

to cell death (Lee 2002).

Chander et al. (1980) opined that increased enzyme

secretion by Aspergillus wentii in liquid media when agi-

tated may be due to the greater availability of dissolved

oxygen for its growth and lipase production. Kumar et al.

(2005) reported 1.16 U/ml of lipase production by B. co-

agulans BTS-3 under 170 rev/min agitation of culture

medium. In contrast, Alford and Smith (1965) reported that

Geotrichum candidum cultured without shaking produced

more lipase than with shaking. This maybe due to

liberation of a proteolytic enzyme by G. candidum which

resulted in hydrolysis of lipase (Lawrence et al. 1967).

Rahman et al. (2006) found significant lipase production by

Pseudomonas sp. strain S5 under static condition. In con-

clusion, the general increase in lipase production with

agitation rate is probably due to improved oxygenation of

the medium.

The effect of inoculum size on the production of lipase

The total dissolved oxygen can be affected by using dif-

ferent inoculum size and hence influence the lipase

production.

Inoculating production medium with various inoculum

sizes of A. thermoaerophilus strain HZ affected the bac-

terial growth and lipase production. As observed in Fig. 8,

enzyme production and bacterial growth increased with the

increase of inoculum size with an optimum at 7% (v/v;

0.24 U/ml) after which the level of enzyme production and

bacterial count both decreased. At low inoculum size, the

nutrient and oxygen levels were sufficient for the growth of

bacteria and, therefore, enhanced lipase production.

A. thermoaerophilus strain HZ only produces high lipase

at the end of the stationary phase (data not shown). Lipase

production and bacterial growth were decreased to 84 and

83% when 9% inoculum size was applied to the medium,

respectively. This may be because the nutrients begin to be

used up by the large population of the bacterium before it is

physiologically ready to start producing lipase. On the

other hand, the low lipase production by the 1% (v/v)

0

0.05

0.1

0.15

0.2

0.25

0.3

0 50 100 150 200

Agitation (rpm)

Lip

ase

acti

vity

(U

/mL

)

2

2.5

3

3.5

4

4.5

5

5.5

6

Log

CF

U/m

L

Lipase activityColony count

Fig. 7 Effect of agitation on lipase production and growth by A.thermoaerophilus strain HZ. Cultures contain 50 ml of production

medium with initial pH 7.5 were incubated at 60�C with different

shaking rate at 60�C for 48 h

0

0.05

0.1

0.15

0.2

0.25

0.3

1 3 5 7 9

Inoculum size (%)

Lip

ase

acti

vity

(U

/mL

)

2

2.5

3

3.5

4

4.5

5

5.5

Log

CF

U/m

L

Lipase activityColony count

Fig. 8 Effect of inoculum size on lipase production and growth by

A. thermoaerophilus strain HZ. Cultures contain 50 ml of production

medium were inoculated with different percentage of inoculum size

and incubated at 60�C under 150 rev/min shaking rate for 48 h

World J Microbiol Biotechnol

123

inoculum is probably due to insufficient bacterium to

produce much enzyme. ul-Haq et al. (2002) stated that the

optimum population of microorganism will produce the

high amount of enzyme. However, high inoculum sizes

might not necessarily give higher enzyme yield or cell

growth. In addition, greater inoculum size can lead to an

impaired nutrient supply and thereby decreasing bacterial

growth and production capacity (Kopp 1987). Previous

studies have identified different optimum inoculum sizes

1% (v/v; Kambourova et al. 2003), 3% (v/v; Gupta et al.

2007), 6% (v/v; Rahman et al. 2005b) and 10% (v/v; Jasvir

et al. 1999) for lipase production of different enzymes. So,

there is no specific inoculum size for maximum lipase

production.

Organic solvent stability of crude enzyme

Substrates of the lipase are often insoluble in aqueous

solutions, and organic solvents or organic-aqueous two-

phase media are favorable for these reactions. However,

enzymes are generally not stable in the presence of organic

solvents and are apt to denature (Ogino et al. 1994). It is

well known that effect of organic solvents on lipase activity

differs from lipase to lipase (Sugihara et al. 1991).

Recently many efforts have been made to discover a new

organic solvent tolerant lipase that could create new

applications.

Organic solvents of various log P values from -1.3 to

8.8 were chosen to study the effect of solvent on the

hydrolysis of oil. Log P value, the partition coefficient

between water and 1-octanol, is often used as an indicator

of solvent polarity. Table 1 shows the crude lipase from

A. thermoaerophilus strain HZ that was stable in most of

the organic solvents with log P values of -1.3 to 3.6 and

the enzyme activity was more than 90% of that in the

absence of an organic solvent. The higher lipase activity

was exhibited by HZ lipase in present of dimethyl sulf-

oxide (Log P -1.3). In addition, toluene (Log P 2.5),

p-xylene (log P 3.1), and hexane (Log P 3.6) slightly

enhanced the crude lipase activity of A. thermoaerophilus

strain HZ. However, the HZ lipase exhibited less than 40%

decrease of its activity in the presence of dodecanol (log P

5.0), n-tetradecane (log P 7.6) and hexadecane (log P 8.8).

In the presence of decanol (Log P 4.0) and decane (log P

5.6), slight loss of activity was observed.

Hun et al. (2003) reported that 80% activity of lipase

from B. sphaericus strain 205y was lost in the presence of

dimethyl sulfoxide. In contrast, the most activity of the

crude HZ lipase was noticed in the presence of hydrophilic

solvents (-1.3 \ log P \ 3.6). However, the HZ lipase

activity was observed to be decreased by water-immiscible

organic solvents with log P [ 4. No lipase activity was

detected from lipase of B. sphaericus strain 205y in the

presence of hexadecane, which has a high log P value of

8.8 (Hun et al. 2003). From the results of this study, no

specific styles of lipase activities with respect to the

polarity of organic solvents were seen. Some organic sol-

vent tolerant lipases, including the HZ lipase, still retain

their activity even if few molecules of water remain; pre-

sumably just sufficient to stabilize the conformation of the

active site. Therefore, their stability in various organic

solvents may be different from other organic solvent tol-

erant lipase.

Acknowledgment This work was supported by the Sciencefund

Grant No. 02-01-04-SF0212 from the Malaysian Ministry of Science/

Technology and Innovation (MOSTI).

References

Alford JA, Smith JL (1965) Production of microbial lipase for the

study of triglyceride structure. J Am Oil Chem Soc 42(12):1038–

1040

Belo I, Pinheiro R, Mota M (2000) Response of the thermophile

Thermus sp. RQ-1 to hyperbaric air in batch and fed-batch

cultivation. Appl Microbiol Biotechnol 53:517–524

Bornscheuer UT (2002) Microbial carboxyl esterase: classification,

properties and application in biocatalysis. FEMS Microbiol Rev

26:73–81

Burkert JFM, Maugeri F, Rodrigues MI (2004) Optimization of

extracellular lipase production by Geotrichum sp. using factorial

design. Bioresour Technol 91(1):77–84

Chander H, Batish VK, Sannabhadti SS, Srinivasan RA (1980)

Factors affecting lipase production in Aspergillus wentii. J Food

Sci 45:598–600

Chen JY, Wen CM, Chen TL (1999) Effect of oxygen transfer on

lipase production by Acinetobacter radoresistens. Biotechnol

Bioeng 62:311–316

Gotor-Fernandez V, Brieva R, Gotor V (2006) Lipases: useful

biocatalysts for the preparation of pharmaceuticals. J Mol Catal

B Enzym 40(3–4):111–120

Table 1 Effect of organic solvents on stability of the crude lipase

from A. thermoaerophilus strain HZ .25% (v/v) of organic solvents

were added to the cell-free supernatant and incubated at 65�C with

200 rev/min shaking for 30 min

Organic solvent Log P Remaining lipase

activity (%)

SD

Dimethyl sulfoxide -1.3 123 0.005

Benzene 2.0 93.8 0.004

Toluene 2.5 106.9 0.01

p-Xylene 3.1 101.01 0.02

Hexane 3.6 106.4 0.03

Decanol 4.0 83.9 0.02

Dodecanol 5.0 61.1 0

Decane 5.6 91.2 0.03

n-Tetradecane 7.6 64.9 0.04

Hexadecane 8.8 72.9 0.06

No solvent – 100 0.04

World J Microbiol Biotechnol

123

Gupta R, Gupta N, Rathi P (2004) Bacterial lipases: an overview of

production, purification and biochemical properties. Appl

Microbiol Biotechnol 64(6):763–781

Gupta N, Sahai V, Gupta R (2007) Alkaline lipase from a novel strain

Burkholderia multivorans: statistical medium optimization and

production in a bioreactor. Process Biochem 42(4):518–526

Haba E, Bresco O, Ferrer C, Marque’s A, Busquets M, Manresa A

(2000) Isolation of lipase-secreting bacteria by deploying used

frying oil as selective substrate. Enzyme Microb Technol

26(1):40–44

Hasan F, Shah AA, Hameed A (2006) Industrial applications of

microbial lipases. Enzyme Microb Technol 39(2):235–251

Holker U, Hofer M, Lenz J (2004) Biotechnological advantages of

laboratoryscale solid-state fermentation with fungi. Appl Micro-

biol Biotechnol 64:175–186

Holst O, Manelius A, Krahe M, Markl H, Raven N, Sharp R (1997)

Thermophiles and fermentation technology. Comp Biochem

Physiol 118(3):415–422

Hun CJ, Rahman RNZA, Salleh AB, Basri M (2003) A newly isolated

organic solvent tolerant Bacillus sphaericus 205y producing

organic solvent-stable lipase. Biochem Eng J 15(2):147–151

Jacques R (1977) Culture media. In: Moss MO, Smith JE (eds)

Industrial application of microbiology. Surrey University Press,

London, pp 27–58

Jasvir S, Gill N, Devasahayam G, Sahoo DK (1999) Studies on

alkaline protease produced by Bacillus sp.NG312. Appl Bio-

chem Biotechnol 76(1):57–63

Kambourova M, Kirilova N, Mandeva R, Derekova A (2003)

Purification and properties of thermostable lipase from a

thermophilic Bacillus stearothermophilus MC 7. J Mol Catal B

Enzym 22:307–313

Kamini NR, Fujii T, Kurosu T, Lefuji H (2000) Production,

purification and characterization of an extracellular lipase from

the yeast, Cryptococcus sp. S-2. Process Biochem 36(4):317–324

Kim MH, Kim HK, Lee JK, Park SY, Oh TK (2000) Thermostable

lipase of Bacillus sterarothermophilus: high-level production,

purification and calcium-dependent thermostability. Biosci Bio-

technol Biochem 64(2):280–286

Kopp B (1987) Long-term alkaloid production by immobilized cells

of Claviceps purpurea. Meth Enzymol 136:317–329

Kumar S, Kikon K, Upadhyay A, Kanwar SS, Gupta R (2005)

Production, purification, and characterization of lipase from

thermophilic and alkaliphilic Bacillus coagulans BTS-3. Protein

Expr Purif 41:38–44

Kwon DY, Rhee JS (1986) A simple and rapid colorimetric method

for determination of free fatty acids for lipase assay. J Am Oil

Chem Soc 63(1):89–92

Lawrence RC, Fryer TF, Reiter B (1967) The production and

characterization of lipases from a micrococcus and a pseudo-

monad. J Gen Microbiol 48(3):401–418

Lee PG (2002) Production, purification, characterization and

expression of the organic solvent tolerant protease gene. Ph.D.

thesis, Universiti Putra Malaysia, Malaysia

Li H, Zhang X (2005) Characterization of thermostable lipase from

thermophilic Geobacillus sp. TW1. Protein Expr Purif 42:153–159

Moon SH, Parulekar SJ (1991) A parametric study of protease

production in batch and fed-batch culture of Bacillus firmus.

Biotechnol Bioeng 37(5):467–483

Niehaus F, Bertoldo C, Kahler M, Antranikian G (1999) Extremo-

philes as a source of novel enzymes for industrial application.

Appl Microbiol Biotechnol 51:711–729

Ogino H, Miyamoto K, Ishikawa H (1994) Organic solvent-tolerant

bacterium which secretes organic-solvent-stable lipolytic

enzyme. Appl Environ Microbiol 60:3884–3886

Ogino H, Miyamoto K, Yasuda M, Ishimi K, Ishikawa H (1999)

Growth of organic solvent-tolerant Pseudomonas aeruginosaLST-03 in the presence of various organic solvents and

production of lipolytic enzyme in the presence of cyclohexane.

Biochem Eng J 4:1–6

Pennisi E (1997) Biotechnology: in industry, extremophiles begin to

make their mark. Science 276:705–706

Rahman RNZA, Geok LP, Basri M, Salleh AB (2005a) Physical

factors affecting the production of organic solvent-tolerant

protease by Pseudomonas aeroginosa strain K. Bioresource

Technol 96(4):429–436

Rahman RNZRA, Baharum SN, Basri M, Salleh AB (2005b) High-

yield purification of an organic solvent-tolerant lipase from

Pseudomonas sp. strain S5. Anal Biochem 341:267–274

Rahman RNZRA, Baharum SN, Salleh AB, Basri M (2006) S5 lipase:

an organic solvent tolerant enzyme. J Microbiol 44(6):583–590Saxena RK, Davidson WS, Sheoran A, Giri B (2003) Purification and

charactrization of an alkaline thermostable lipase from Asper-gillus carneus. Process Biochem 39(2):239–247

Sharma R, Soni SK, Vohra RM, Gupta LK, Gupta JK (2002)

Purification and characterization of a thermostable alkaline

lipase from a new thermophilic Bacillus sp. RSJ-1. Process

Biochem 37(10):1075–1084

Sugihara A, Tani T, Tominaga Y (1991) Purification and character-

ization of a novel thermostable lipase from Bacillus sp.

J Biochem 109(2):211–215

Thompson JD, Higgins DG, Gibson TJ (1994) CLUST AL W:

important the sensitivity of progressive multiple sequence

alignment through sequence weighting, position-specific gap

penalties and weight matrix choice. Nucleic Acids Res

22(22):4673–4680

ul-Haq I, Idrees S, Rajoka MI (2002) Production of lipases by

Rhizopus oligosporous by solid-state fermentation. Process

Biochem 37(6):637–641

Woese C (1998) The universal ancestor. Proc Natl Acad Sci USA

95(12):6854–6859

World J Microbiol Biotechnol

123