A New Cold-Adapted, Organic Solvent Stable Lipase from Mesophilic Staphylococcus epidermidis AT2

A New Cold-Adapted, Organic Solvent Stable Lipasefrom Mesophilic Staphylococcus epidermidis AT2

Nor Hafizah Ahmad Kamarudin • Raja Noor Zaliha Raja Abd. Rahman •

Mohd Shukuri Mohamad Ali • Thean Chor Leow • Mahiran Basri •

Abu Bakar Salleh

Published online: 29 April 2014

� Springer Science+Business Media New York 2014

Abstract The gene encoding a cold-adapted, organic

solvent stable lipase from a local soil-isolate, mesophilic

Staphylococcus epidermidis AT2 was expressed in a pro-

karyotic system. A two-step purification of AT2 lipase was

achieved using butyl sepharose and DEAE sepharose col-

umn chromatography. The final recovery and purification

fold were 47.09 % and 3.45, respectively. The molecular

mass of the purified lipase was estimated to be 43 kDa.

AT2 lipase was found to be optimally active at pH 8 and

stable at pH 6–9. Interestingly, this enzyme demonstrated

remarkable stability at cold temperature (\30 �C) and

exhibited optimal activity at a temperature of 25 �C. A

significant enhancement of the lipolytic activity was

observed in the presence of Ca2?, Tween 60 and Tween 80.

Phenylmethylsulfonylfluoride, a well known serine inhibi-

tor did not cause complete inhibition of the enzymatic

activity. AT2 lipase exhibited excellent preferences

towards long chain triglycerides and natural oils. The

lipolytic activity was stimulated by dimethylsulfoxide and

diethyl ether, while more than 50 % of its activity was

retained in methanol, ethanol, acetone, toluene, and

n-hexane. Taken together, AT2 lipase revealed highly

attractive biochemical properties especially because of its

stability at low temperature and in organic solvents.

Keywords Staphylococcal lipase � Mesophilic �Purification � Cold-adapted � Organic solvent stable

Abbreviations

A600 Absorbance at 600 nm wavelength

ßME 2-mercaptoethanol

EDTA Ethylenediamineteraacetic acid

DMSO Dimethylsulfoxide

DTT Dithiothreitol

IPTG Isopropyl b-D-1thiogalactopyranoside

OD Optical density

PCR Polymerase chain reaction

PMSF Phenylmethylsulfonylfluoride

PVDF Polyvinylidene fluoride

SDS Sodium dodecyl sulfate polyacrylamide gel

electrophoresis

1 Introduction

Lipase (EC 3.1.1.3) has long been known for its diverse

functions in biocatalysis. The ability of lipase to carry out

hydrolysis and synthesis reactions in aqueous and non

aqueous media, respectively, offers many advantages for

biotechnology exploitation in various industries including

detergents, cosmetics and food industries.

Of many sources of lipases, microbial lipases have

notably attracted the global attention in a very rapid and

exciting rate compared to counterparts from higher

organisms. The enormous labors in exploiting lipases of

N. H. A. Kamarudin � R. N. Z. R. Abd. Rahman (&) �M. S. M. Ali � T. C. Leow � M. Basri � A. B. Salleh

Enzyme and Microbial Technology Research Centre,

Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia

e-mail: [email protected]

N. H. A. Kamarudin � R. N. Z. R. Abd. Rahman �M. S. M. Ali � T. C. Leow � A. B. Salleh

Faculty of Biotechnology and Biomolecular Sciences,

Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia

M. Basri

Faculty of Sciences, Universiti Putra Malaysia, 43400 Serdang,

Selangor, Malaysia

123

Protein J (2014) 33:296–307

DOI 10.1007/s10930-014-9560-3

microbial origin are mainly due to ease in genetic manip-

ulation and economic feasibility [1]. Staphylococcal lipases

(Family I.6), in particular, are relatively unique through

their distinct molecular organization which comprised of

pre-pro-sequence along with their large size (*70 kDa).

The mature active form (40–46 kDa) is generated by pro-

teolytic processing after secretion into the culture medium

[2]. Their unusual molecular properties have led many to

conduct detailed investigation on the molecular and bio-

chemical characteristics as well as the structural architec-

ture and potential biotechnological applications. While the

information is steadily expanding, knowledge on the

structure and function correlation of staphylococcal lipases

at molecular level, however, is still limited. This lack of

information is in part due to the availability of only a single

crystal structure from staphylococcal lipase family that is

derived from S. hyicus lipase [3].

Apart from their importance in biological function,

staphylococcal lipases have emerged as one of the valuable

industrial enzymes worth investigating. Therefore, the

search of staphylococcal lipase with new and attractive

properties remains a continuous pursuit. Lipase exhibiting

organic solvent stable property has been of prime interest

in many studies; driven by its versatility in catalyzing

synthesis reaction in low water environment. Lipases

especially those that can tolerate methanol and ethanol,

have the potential to improve biodiesel production while

others can be employed in food and pharmaceutical

industries by the synthesis of esters and intermediates [4,

5]. Enzymatic reaction in organic media also provides

attractive advantages in chemical reactions and biotech-

nological applications such as enhanced solubility of

hydrophobic substrates, exclusion of microbial contami-

nation and side reactions caused by water [6].

In addition, cold-adapted lipases have been discovered

as valuable enzymes for novel practical applications.

Enzyme reactions at low temperature offer many advan-

tages including reduced energy consumption and preven-

tion from loss of volatile compounds. A broad avenue in

industrial application can be generated by the exploitation

of cold-adapted lipases as additives in detergents for cold-

water washing, additives in food industries and environ-

mental bioremediations [7].

Besides its industrial importance, modern enzyme

studies have also focused on the understanding of the

structure and function relationship. Identification of the key

features that govern specific characteristics allows modifi-

cation of enzymes to circumvent certain limitations hence

producing enzymes with improved properties. This inves-

tigation will also facilitate determination of the structural

patterns from diverse sequences.

Owing to the overwhelming, non-stop progression in

lipase biotechnology, discovery of novel staphylococcal

lipase genes is of great important for better fundamental

understanding and industrial exploitation. Previously, the

gene encoding a lipase from a newly soil-isolated meso-

philic Staphylococcus epidermidis AT2 (AT2 lipase) has

been sequenced, cloned and expressed in E. coli system [8].

Herein, the purification and biochemical characteristics of

the purified staphylococcal lipase are described. It is worth

emphasized that very few lipases have been reported to

exhibit both cold-adapted and organic solvent stable

properties. In fact, this is by far, the first report on purified

recombinant staphylococcal lipase demonstrating such

unique and potentially marketable properties. Furthermore,

literature on cold-adapted lipases produced by mesophilic

bacterium is relatively few, hence, it is hoped that these

findings will expand the knowledge of lipases particularly

of those produced by Staphylococcus sp.

2 Materials and Methods

2.1 Bacterial Strains, Plasmid and Media

Staphylococcus epidermidis AT2 was isolated from con-

taminated soil at a car service area in Port Dickson,

Malaysia. The lipase gene was identified, sequenced and

cloned in pTrcHis2-TOPO expression vector (Invitrogen,

UK) [8]. E. coli Tuner (DE3) pLacI (Novagen, UK) was

used as expression host. The bacterial culture was culti-

vated in Luria–Bertani medium (10 g tryptone, 5 g yeast

extract, 5 g NaCl in 1 L at pH 7, Difco, USA), supple-

mented with 50 lg/mL of ampicillin (Sigma, USA).

2.2 Expression of AT2 Lipase Gene

The mature active form of AT2 lipase containing 390 amino

acids was cloned in pTrcHis2/AT2 expression vector in

E. coli Tuner (DE3) pLacI (complete open reading frame

(ORF) was submitted to GenBank and assigned with

Accession No. EU814893). E. coli Tuner (DE3) pLacI har-

boring recombinant pTrcHis2/AT2 was grown in 10 mL LB

broth overnight at 37 �C and subsequently sub-culture in

200 mL of LB broth for 3–4 h until OD at A600 = 0.5 for

isopropyl b-D-Thiogalactopyranoside (IPTG) induction.

Cells were induced with 60 lM of IPTG and further incu-

bated for 12–16 h. The induced culture was further incubated

for 12 h and harvested by centrifugation (10,0009g, 10 min,

and 4 �C). The supernatant was discarded, while pellet

(intracellular fraction) was used to obtain the soluble protein.

2.3 Purification of AT2 Lipase

The cell pellet of 600 mL culture was resuspended with

buffer comprising of 20 mM Tris–HCl and 1 M

Stable Lipase from Mesophilic Staphylococcus epidermidis 297

123

ammonium sulfate (pH 8) and lysed by sonication (Branson

Sonifier 250, Branson, USA). The lysed cells were centri-

fuged for 10,0009g, 10 min, 4 �C. The cleared crude cell

lysate was loaded into the first hydrophobic interaction

chromatography column (HIC); butyl sepharose 4 fast flow

(GE Healthcare, UK) which was equilibrated with the same

buffer at a flow rate of 1.0 mL/min. The target proteins

were eluted at 50 % of salt-free elution buffer (20 mM

Tris–HCl pH 8) by a descending linear gradient (20 column

volume, CV). The fractions containing active protein were

pooled and subjected to dialysis/buffer exchange against

20 mM Tris–HCl (pH 9). The dialyzed sample was loaded

into a second ion exchange chromatography column (IEX),

DEAE Sepharose Fast Flow (GE Healthcare, UK) at a flow

rate of 0.5 mL/min. The active, target protein was eluted at

approximately 10 % of elution buffer (20 CV of 0–0.2 M

NaCl linear gradient). For each purification step, fractions

were examined for lipase activity and monitored by SDS-

PAGE analysis.

2.4 Lipase Activity Assay

Lipase activity assay was conducted according to Kwon

and Rhee method (1986) [9] with slight modification. Olive

oil (C18:1) was used as substrate. The substrate was pre-

pared by mixing equal volume of olive oil and buffer at 1:1

ratio. Reaction mixture containing 1 mL of enzyme,

2.5 mL of substrate emulsion, was incubated in a water

bath shaker for 30 min at 25 �C with vigorous agitation,

200 rpm. After 30 min of incubation, the enzyme reaction

was terminated by adding 1 mL of 6 N HCl and the lib-

erated fatty acids were dissolved in 5 mL of isooctane. The

mixture was vigorously vortexed for 30–60 s and two

layers formed. Four ml of the upper layer was drawn out

into test tubes containing 1 mL of 5 % (w/v) copper acetate

pyridine pH 6.1 and mixed vigorously by vortexing. The

absorbance reading was taken at 715 nm and isooctane was

used as the blank. Enzyme activity was determined based

on oleic acid standard curve. One unit of activity is defined

as the rate of free fatty acids released in 1 min.

2.5 Protein Determination (Bradford Assay)

Protein concentration was determined by Bradford method

using bovine serum albumin (BSA) as a standard. The

absorbance reading was taken at 595 nm.

2.6 Electrophoresis (SDS-PAGE)

SDS-PAGE was carried out as described by Laemmli [10]

using 6 % of stacking gel and 12 % of separating gel. The

molecular weight of protein was estimated by comparison

with a broad range of protein molecular weight marker

(116–14.4 kDa; Thermo, UK).

2.7 Activity Staining and N-terminal Sequencing

Lipase activity staining was performed according to the

method outlined by Sommer et al. [11]. Sample was pre-

pared in non-denaturing condition. Following SDS-PAGE,

the unstained gel was immersed in 20 % (v/v) isopropanol

for 30 min on a rotary shaker, washed two to three times

with ultrapure water, and further overlaid on a tributyrin

agar (TB agar) for 30 min. For N-terminal sequencing,

sample was separated by SDS-PAGE and the gel was

electrophoretically transferred to PVDF membrane. The

target band was excised and subjected to Edman Degra-

dation by Applied Biosytems, Procise Sequencer (Life

Technologies, USA).

2.8 Effect of Temperature on Lipase Activity

and Stability

Optimal temperature for AT2 lipase was determined by

varying assay temperatures ranging from 15 to 50 �C at

5 �C intervals. Lipolytic activities at different temperature

were assayed for 30 min. For thermal stability/half-life

study, purified enzyme was pre-incubated at three different

temperatures (20, 25 and 30 �C) for 300 min in which, at

every 30 min intervals, sample was removed and the

lipolytic activity was determined at 25 �C. Sample at 0 min

(t = 0) was regarded as the control of the experiment

(100 %).

2.9 Effect of pH on Lipase Activity and Stability

The optimal pH of purified AT2 lipase was determined by

preparing substrate emulsion (olive oil) in 50 mM buffer of

different pH ranging from pH 4 to 12 at 1:1 ratio (olive oil

and buffer), and assayed at 25 �C for 30 min. Lipolytic

activity in 50 mM Tris–HCl (pH 8) was further selected as

the control reaction for stability study. To determine the pH

stability profile of AT2 lipase, purified enzyme was pre-

incubated in buffer of tested pHs for 30 min followed by

lipase activity assay at 25 �C, 30 min. Buffer systems used

were sodium acetate (pH 4–6), phosphate (pH 6–8), Tris–

HCl (pH 8–9), Glycine/NaOH (pH 9–11) and sodium

hydrogen phosphate, Na2HPO4/NaOH (pH 11–12).

2.10 Effects of Metal Ions on Lipase Activity

The effects of additional metal ions were investigated by

pre-incubating the purified enzyme with metal ions at two

concentrations (1 mM and 5 mM) for 30 min at 20 �C and

subsequently subjected to lipase assay. The metal ions used

298 N. H. A. Kamarudin et al.

123

were: K?, Na?, Mg2?, Ca2?, Mn2?, Cu2?, Co2?, Zn2? and

Fe3?. Control reaction corresponded to the non-treated

enzyme (without incubation with metal ions) and regarded

as 100 %.

2.11 Effect of Surfactants on Lipase Activity

To assimilate the effect of surfactants on AT2 lipase, the

enzyme was treated with 0.1 % and 0.5 % (v/v) of Tween

20, 40, 60, 80, Triton X-100, and SDS for 30 min at 20 �C

prior to lipase assay. Surfactant-free enzyme served as the

control reaction (100 %) and the remaining activities of the

treated enzymes were determined in comparison to the

control of the experiment.

2.12 Effect of Inhibitors on Lipase Activity

The influence of inhibitors, chelating agents and reducing

agents (phenylmethylsulfoxide (PMSF), pefabloc, pepsta-

tin A, ethylenediaminetetraacetic acid (EDTA), dithio-

threitol (DTT) and b-mercaptoethanol (bME)) on the

purified enzyme were investigated at two different con-

centrations (1 and 5 mM). AT2 lipase was pre-treated with

the respective inhibitors for 30 min prior to lipase assay.

Relative activities were measured against the lipolytic

activity of the non-treated sample which considered as

100 %.

2.13 Effect of Substrates on Lipase Activity

Triglycerides and natural oils were exploited to study the

effect of different carbon chain length substrates on AT2

lipase activity. Substrates were prepared by mixing buffer

(50 mM Tris–HCl pH 8) and triglycerides/oils at 1:1 ratio.

Reaction mixtures containing different type of substrates

were assayed at 25 �C for 30 min. Substrates used were

triacetin (C2:0), tributyrin (C4:0), triolein (C18:1), coconut

oil (C12:0), palm oil (C16:0), palm kernel oil (C16:0),

olive oil (C18:1), rice bran oil (C18:1), soy oil (C18:2),

corn oil (C18:2), sunflower oil (C18:2). Lipolytic activities

of AT2 lipase at different substrates were calculated in

relative to that obtained in olive oil which served as control

(100 %).

2.14 Effect of Organic Solvents on Lipase Activity

Fifteen organic solvents with different log P were tested on

AT2 lipase stability; DMSO (-1.3), methanol (-0.76),

acetonitrile (-0.33), ethanol (-0.24), acetone (-0.24),

1-propanol (0.28), ethyl acetate (0.68), diethyl ether (0.85),

chloroform (2.0), benzene (2.0), toluene (2.5), octanol

(2.9), xylene (3.1), n-hexane (3.5) and n-heptane (4.0).

Purified enzyme was pre-incubated with 25 % (v/v) of

organic solvent for 30 min at 20 �C with vigorous shaking

(150 rpm) in a water bath shaker. The treated enzymes, as

well as the organic solvent-free enzyme (which served as

the control of the experiment), were assayed for their

lipolytic activities. Residual activities were measured in

relative to the control reaction which was regarded as

100 %.

3 Results

3.1 Expression of AT2 Lipase

A lipase gene from Staphylococcus epidermidis AT2

(termed AT2 lipase) was previously sequenced and

expressed in E. coli system [8]. The deduced gene which

contained a single ORF comprising of 643 amino acids was

deposited in GenBank with accession number EU814893.

The nucleotide sequence of AT2 lipase is organized as a

pre-pro sequence, and this molecular organization is sim-

ilarly found in other deduced staphylococcal lipase genes.

The mature active form of AT2 lipase which translated to

390 amino acids was cloned and expressed in pTrcHis2-

TOPO vector in E. coli TOP10. The expression system is

designed for direct insertion of PCR product into the

plasmid vector via 30 thymidine (T) overhangs and requires

no ligase and post PCR procedures. The resulting lipolytic

activity obtained at its optimum condition (16 h, at 0.6 mM

of IPTG) was 5.58 U/mL which corresponded to 18-fold

increase in the activity compared to its wild type [8]. In the

present study, the expression of soluble AT2 lipase was

improved when E. coli Tuner (DE3) pLacI was used as

host. The lipolytic activity increased by 4.5-fold under its

optimized condition; 60 lM of IPTG, 12 h of post induc-

tion time yielding an optimal activity of 25.20 U/mL (data

not shown). Owing to its higher activity, E. coli Tuner

(DE3) pLacI harboring recombinant pTrcHis2/AT2 was

further used for the purpose of purification and character-

ization studies.

3.2 Purification of AT2 Lipase

Isolating a desired protein from a complex mixture requires

the employment of an ideal purification step. In the attempt

to purify AT2 lipase, the crude lysate was loaded into

hydrophobic interaction chromatography (HIC) butyl

sepharose fast flow column followed by ion exchange

chromatography (IEX) on DEAE Sepharose Fast Flow

exchanger. The efficiency of each purification protocol was

evaluated by measuring the total protein and specific

activity as presented in Table 1. HIC displayed a good

capturing strategy for the purification of AT2 lipase as

denoted by 92.47 % of recovery and almost a onefold


123

increment of the purification fold. A bulk of impurities in

the cell-free supernatant was removed during sample

binding, leaving few proteins to tightly bind to the matrix.

Two separated broad peaks were detected during the elu-

tion fractionation. The second peak which eluted at

approximately 0.5 M of ammonium sulfate was identified

as the target protein through lipase activity assay and SDS-

PAGE analysis. Following HIC, the accomplishment of

IEX as the final polishing step was evidenced by marked

improvement in purity with 3.45 purification fold and a

final recovery of 47.09 % over the crude enzyme. A single

sharp peak was obtained at approximately 0.05 M NaCl of

the linear gradient and the fractions were further assayed

for confirmation.

To assimilate the purity level of AT2 lipase at every

stage of purification, SDS-PAGE, followed by activity

staining were conducted. Figure 1a denotes the SDS-PAGE

analysis for each steps of purification. The impurities in the

crude enzyme were removed by almost 70 % through HIC

as clearly observed in Lane 3. Meanwhile, a pure, single

band was detected on the gel after the final step of purifi-

cation (Lane 5). The molecular weight (MW) of the puri-

fied product was estimated to be 43 kDa, which agreed

with the calculated MW of AT2 lipase based on its primary

sequence analysis.

Besides the quantitative lipase measurement, activity

staining was performed to qualitatively ensure that the target

protein is biologically active. A clearing zone (halo) sur-

rounding the target protein band was observed when the gel

was placed on tributyrin agar for 30 min at 25 �C. Tributyrin

agar is one of the selective media for lipase detection where

tributyrin (C4:0) serves as the substrate. The active protein

hydrolyzed the substrates surrounding it resulting in the

formation of clearing zone. This signified and confirmed that

the purified product was a lipase (Fig. 1b).

The purity level of AT2 lipase was further determined

by Native PAGE analysis. Somewhat unexpected, no dis-

tinct band was observed on the gel, instead, a low mobility

band and smearing were detected (data not shown). Such

behavior is deduced to be related to the tendency of protein

to form aggregates or oligomer [12, 13]. The oligomeric

state of the enzyme was then determined by analytical ultra

centrifugation (AUC) based on sedimentation velocity

(SV) analysis. This method is used to probe the aggregation

behavior of proteins in solution by physical separation of

molecular species according to variation in mass and shape

Fig. 1 Analysis of the purified

product. a SDS-PAGE analysis

of AT2 lipase at different

purification steps. Lane 1

Standard protein marker, Lane 2

crude enzyme, Lane 3 AT2

lipase after hydrophobic

interaction chromatography

(HIC), Lane 4 pooled fractions

after dialysis, Lane 5 purified

AT2 lipase after ion exchange

chromatography (IEX).

b Activity staining of AT2

lipase on Tributyrin agar. Lane

1 purified AT2 lipase (clearing

zone was observed after 30 min

of incubation at 25 �C); Lane 2

Standard protein marker

Table 1 Flow sheet of AT2

lipase purification

a One Unit of activity

corresponds to the rate of free

fatty acids released in 1 min

using olive oil emulsion as

substrate

Step Volume

(mL)

Activitya

(U/mL)

Protein

(mg/mL)

Total

Activity

(U)

Specific

Activity

(U/mg)

Yield

(%)

Fold

Crude 30 36.81 0.88 1,104.30 41.83 100 1

Hydrophobic interaction

chromatography (butyl

sepharose fast flow)

28 36.47 0.45 1,021.16 81.04 92.47 1.94

Ion exchange chromatography

(DEAE sepharose fast flow)

12 43.34 0.30 520.08 144.47 47.09 3.45


123

[14]. Through this analysis, a dominant single peak with a

molecular mass of approximately 42.1 kDa was obtained,

suggesting the occurrence of a single protein species in the

solution thereby, reflecting the existence AT2 lipase in a

monomeric form. No corresponding peaks for any oligo-

mers were detected (data not shown).

3.3 Effect of Temperature on Lipase Activity

and Stability

Effect of temperature on AT2 lipase activity and stability

were investigated and presented in Fig. 2. Interestingly,

high lipolytic activity was discovered at low temperatures,

15 and 20 �C with 23.89 and 26.55 U/mL, respectively,

while AT2 lipase achieved its optimum activity of 28.01

U/mL at 25 �C. At elevated temperature (30 �C and

above), the activity started to decline gradually and a total

wipe out of activity was observed at 45 and 50 �C.

Figure 2b denotes the stability and half-life study of

AT2 lipase at three different temperatures, 20, 25 and

30 �C. Excellent stability was discovered at 20 �C where

almost no reduction of activity was observed throughout

the total incubation period. At its optimal temperature,

25 �C, the enzymatic activity retained to more than 80 %

up to 270 min. Further increased in incubation time caused

a sharp fall in lipolytic activity by 56 % as viewed at

300 min. The half-life of AT2 lipase at 25 �C was esti-

mated to be at 300 min. In contrast, higher temperature

(30 �C) demonstrated greater deleterious effect on AT2

lipase activity. Prolonged incubation up to 240 min dis-

played a decline in relative activity by 52 % which can be

anticipated as its half life at 30 �C (t1/2 = 240 min).

3.4 Effect of pH on Lipase Activity and Stability

As shown in Fig. 3a, AT2 lipase exhibited high lipolytic

activity at pH 7 to pH 9 with maximum activity at pH 8

(23.06 U/mL), thereby indicating it as an alkaline lipase.

The hydrolytic activities of AT2 lipase at pH 7 and pH 9

were 8.77 and 19.80 U/mL, respectively. A complete

deactivation of AT2 lipase activity was observed at both

highly alkaline and acidic pH (pH 4–5 and pH 11–12).

Stability of AT2 lipase at various pHs was also monitored

by pre-incubating the purified enzyme at pH 4 to pH 12 for

30 min prior to lipase assay. Figure 3b indicates that this

enzyme is stable at a broad range of pH (pH 6–9) and most

stable at neutral pH with 86 % of relative activity. At

Fig. 2 Effect of temperature on AT2 lipase (a) activity (b) stability.

Optimal temperature was determined by varying the assay temper-

atures. Stability study was investigated by pre-incubating AT2 lipase

at different temperature for 30 min. Relative activities were measured

against activity of the non-incubated sample (t = 0). Symbols

represent the incubation temperature at; 20 �C (diamond), 25 �C

(filled square), and 30 �C (filled triangle). Relative activities are

represented by mean value ± standard deviations (n = 3)

Fig. 3 Effect of pH on AT2 lipase (a) activity (b) stability. Optimal

pH was determined by varying the pH of the substrates. Lipolytic

activity at pH 8 (Tris–HCl) was further selected as the experimental

control (100 %). In stability study, purified enzyme was pre-incubated

for 30 min at the tested pHs and assayed at pH 8, 25 �C, for 30 min.

Relative activities were calculated against the activity of the non-

incubated sample. Buffer systems used are as follows: (filled

diamond) sodium acetate (pH 4–6); (filled square) sodium phosphate

(pH 6–8); (filled triangle) Tris–Cl (pH 8–9); (x) glycine-OH (pH

9–11); (filled circle) sodium hydrogen phosphate (pH 11–12). Error

bars represent standard deviations of means (n = 3)


123

slightly acidic environment (pH 6), 70 % of the residual

activity was retained while at pH 8 and 9, the activity

remained up to 52 and 58 %, respectively. AT2 lipase was

very unstable at extremely acidic and alkaline pH (pH 4–5

and pH 10–12).

3.5 Effect of Metal Ions on Lipase Activity

Metal ions are known to play important roles in enzyme

catalysis. Data in Table 2 shows the influence of metal ions

on AT2 lipase activity. Group I metal ions (Na? and K?)

retained the activity for more that 50 % at 1 mM, however,

the inhibitory effect was greater at 5 mM as observed

through the loss of activities by 84 and 53 %, respectively.

No inhibition of the activity was observed when the

enzyme was treated with 1 mM of Mg2? and Ca2?. In fact,

Ca2? ion was found to significantly boost up the activity of

AT2 lipase to 145 %. Addition of Mn2?, Co2? and Cu2? at

1 mM concentration neither caused any impairment nor

activation to the catalytic activity of AT2 lipase. At higher

concentration of Mn2? and Co2? (5 mM), the activity was

fairly retained by 50–75 %. Conversely, in the presence of

5 mM Cu2?, the lipolytic activity was greatly impaired by

a reduction of 95 %. Heavy metal ions such as Zn2? and

Fe3? at 5 mM concentration, showed severe impediment of

the lipolytic activity with inhibition by more than 90 %.

3.6 Effect of Inhibitors on Lipase Activity

Several inhibitors and chelating agents were tested on AT2

lipase activity (Table 2). EDTA, a well-known chelating

agent, showed a strong detrimental effect on AT2 lipase

activity. Almost 97 % of the activities were diminished

when treated with 1 and 5 mM of EDTA. Pepstatin A, an

aspartyl protease inhibitor, showed strong inhibition on

lipolytic activity of AT2 lipase. Low concentration of this

inhibitor was enough to cause total disruption of the

enzyme activity as denoted by 2 % of the relative activity

at 1 mM of pepstatin A. Consequently, no activity was

detected at higher concentration of pepstatin A (5 mM).

Surprisingly, PMSF and pefabloc which are serine inhibi-

tors do not completely inhibit the lipolytic activity of AT2

lipase. The enzymatic activity was suppressed by only 33

and 61 % at 5 mM of PMSF and pefabloc, respectively. No

significant reduction of lipolytic activity was observed

when AT2 lipase was treated with reducing agents; DTT

and ßME at both concentrations.

3.7 Effect of Surfactants on Lipase Activity

Lipase is also known as surface active protein due to its

nature property in hydrolyzing lipids at water–lipid inter-

face [15]. Surfactants reduce the surface tension of the

microemulsion and prevent the substrate from separating

into two layers. Several surfactants including ionic and

nonionic, were tested on AT2 lipase activity and the results

are depicted in Table 2. Tween 60 and Tween 80 stimu-

lated the activity to more than twofold at 0.1 % (v/v).

Moderate inhibition on AT2 lipase activity was observed in

the presence of 0.1 % (v/v) of Tween 20 and Tween 40

with 32 and 10 % of the relative activity, respectively. A

dramatic fall in hydrolytic activity was monitored when the

enzyme was treated with 0.5 % (v/v) of Tween 20, 40, 60

and 80. Treatment with Triton X-100 and SDS, on the other

hand, showed a strong deleterious effect on AT2 lipase

activity. Suppression of activity by 67 and 87 % was

detected upon addition of 0.1 % (v/v) of the surfactant,

Table 2 Effects of metal ions, inhibitors, chelating and reducing

agents, surfactants on AT2 lipase activity

Characterization Characteristics

Relative activity (%)

Concentration 1 mM 5 mM

Control 100 100

Metal ionsa

Na? 66.8 16.5

K? 88.8 47.1

Mg2? 100.1 73.4

Ca2? 145.9 64.8

Mn2? 106.9 76.4

Cu2? 93.3 5.0

Co2? 93.8 52.8

Zn2? 5.7 4.9

Fe3? 16.0 6.0

Inhibitors/chelating agents and reducing agentsa

DTT 124.2 127.2

b-mercaptoethanol 119.8 94.9

PMSF 89.6 67.5

Pefabloc 127.6 39.8

Pepstatin A 2.2 0.7

EDTA 2.8 3.0

Surfactantsb

Concentration 0.1 % (v/v) 0.5 % (v/v)

Tween 20 73.4 41.5

Tween 40 107.5 37.8

Tween 60 245.2 72.3

Tween 80 288.4 32.5

Triton X-100 36.6 21.9

SDS 13.5 9.1

a Purified AT2 lipase was treated with 1 and 5 mM of metal ions,

inhibitors, chelating agents and reducing agents for 30 min prior to

lipase assayb Purified AT2 lipase was treated with 0.1 % (v/v) and 0.5 % (v/v) of

surfactants for 30 min prior to lipase assay


123

respectively. Meanwhile, at higher concentration of the

surfactants, greater inhibition on AT2 lipase activity was

observed.

3.8 Effect of Substrates on Lipase Activity

Substrate selectivity and specificity are unique among

enzymes. AT2 lipase demonstrated excellent preference

towards long-chain triglycerides (C12-C18), suggesting

that this enzyme is a true lipase (Fig. 4). A remarkable

enhancement of the hydrolytic activity was monitored in

triolein and all natural oils. Its hydrolytic activity was

enhanced by more than 100 % in triolein (C18:0), coconut

oil (C12:0), rice bran oil (C18:1), soy oil (C18:2) and

sunflower oil (C18:2). An increment to almost 300 % of

the activity was observed when corn oil was used as the

substrate. Palm oil, a C16 substrate, improved the lipolytic

activity by 53 % whereas palm kernel olein and coconut

oil, both composed of 12-carbon chain, enhanced the

activity 24 and 124 %, respectively. In contrast, only

2–4 % of the relative lipolytic activity was detected in

substrates comprising of short chain triglycerides, namely,

triacetin (C2) and tributyrin (C4).

3.9 Effect of Organic Solvents on Lipase Activity

Organic solvents are commonly classified in terms of log

P value; where log P is the partition coefficient of organic

solvents in 1-octanol/water mixture [16]. In general, AT2

lipase was fairly stable in both polar and non-polar solvents

(Fig. 5). DMSO which exhibited the lowest log P value

(-1.3), displayed a marked enhancement on AT2 lipase

activity by 40 % increment. More than 50 % of AT2 lipase

activity remained in solvents with log P \ 2, namely,

methanol, ethanol, acetone and diethyl ether. Conversely,

1-propanol and ethyl acetate displayed deleterious effects

on AT2 lipase. A complete wipe out of the enzymatic

activity in these organic solvents reflected their high tox-

icity. Meanwhile in non-polar organic solvents (log P [ 1),

toluene and n-hexane retained the lipolytic activity by 91

and 71 %, respectively. In contrast, other non-polar organic

solvents, namely benzene, octanol, xylene and n-heptane

displayed strong inhibitions on AT2 lipase by repressing

the activity to over 50 %. Chloroform was manifestly

regarded as the most toxic hydrophobic organic solvent to

AT2 lipase in which no lipolytic activity was detected upon

treatment.

4 Discussion

A lipase gene from a local soil-isolate, Staphylococcus

epidermidis AT2 was expressed, purified and character-

ized. In comparison to our previous report, the expression

of soluble protein was improved by 4.5-fold when

expressed in E. coli Tuner (DE3) pLacI. A suitable com-

bination of expression vector and host strain is essential for

transcription and translation of target protein hence

Fig. 4 Effect of various substrates at different chain length on AT2

lipase activity. Substrates were prepared in the form of emulsion in

50 mM Tris–HCl pH 8 at ratio (1:1). Lipolytic activity of each treated

enzyme was measured in relative to the activity obtained in reaction

containing olive oil as the substrate and regarded as control (100 %).

Error bars represent standard deviation of means (n = 3)

Fig. 5 Effect of organic solvents on AT2 lipase activity. Purified

enzyme was pre-treated with 25 % (v/v) of tested organic solvents for

30 min with vigorous shaking (200 rpm) in a water bath shaker

followed by lipase assay at 25 �C for 30 min. Lipolytic activity in

aqueous solution (without organic solvent) served as the control

reaction (100 %). Error bars represent standard deviation of mean

(n = 3)


123

producing soluble protein at high level. AT2 lipase was

further purified by two-step column chromatography; HIC

butyl sepharose and IEX DEAE sepharose. In many cases

of lipase purification, HIC was successfully employed as a

sole or part of the purification strategies. The strong

binding affinity of AT2 lipase towards the matrix may in

part due to the occurrence of approximately 21.3 % of total

hydrophobic residues (leucine, isoleucine, valine, phenyl-

alanine) in the protein; which interacted with the hydro-

phobic 4-linear alkyl chain ligand (butyl sepharose) besides

being aided with high ionic strength in the mobile phase.

IEX was employed based on its powerful capability in

separating biomolecules with minor difference in charge

properties. A pure, single band of AT2 lipase was observed

through SDS-PAGE analysis after DEAE Sepharose chro-

matography. Evidently, IEX has been widely used in many

purification strategies. S. haemolyticus lipase was purified

using a series of IEX chromatography techniques. DEAE

Sepharose CL-6B, CM Sepharose CL-6B, and Resource Q

column were applied respectively in the attempt to obtain

highly purified lipase [18]. Sakinc et al. (2007) [19]

reported on the use of DEAE Sepharose CL-6B as a final

polishing strategy in purification of S. saprophyticus lipase.

AT2 lipase was found prone to aggregation as observed

in Native PAGE analysis. Smears on Native PAGE are

often associated with incomplete solubility of protein while

low mobility band corresponds to oligomer [12, 13]. Fur-

ther investigation on the oligomeric state of AT2 lipase by

AUC showed that this enzyme existed in monomeric form

(data not shown). Staphylococcal lipases have been

claimed to exhibit a complex structure and its tendency to

form aggregates which is driven by the hydrophobic force

from high content of hydrophobic residues [20].

Temperature is one of the most crucial factors in

mediating enzyme-catalyzed reaction rates. It is therefore a

primary prerequisite to determine the optimal temperature

of an enzyme for its best function. Interestingly, in this

study, S. epidermidis AT2 - a mesophile, produced a cold-

adapted lipase that exhibited optimal activity at a temper-

ature of 25 �C. In fact, the enzyme was catalytically active

and stable at temperature below 30 �C, while little or no

activity was detected at higher temperature. It is therefore

clearly evidenced the heat-sensitivity of AT2 lipase, a

distinguishing feature of a cold-loving enzyme. Cold-active

enzymes are commonly produced by psychrophiles as part

of their adaptation and survival strategies in cold climate.

There are very few reports on mesophilic microorganisms

producing cold-adapted enzymes. Of the limited examples,

Cai et al. [21] reported on the production of two cold-

adapted lipases (Lipase-A and Lipase-B) from Geotrichum

sp. SYBC WU-3, a mesophile. Recently, a cold-adapted

lipase from Streptomyces strain which exhibited stability at

temperature below 25 �C was reported [22]. It has been

generally accepted that the stability of enzyme at cold

temperature is governed by the nature of protein structures

and conformations. Conformational flexibility remains the

dominating theory in cold adaptation however the issue on

either local or global flexibility is still under argument [23].

Enzymes adopt specific strategies to become catalytically

active in the extreme environment such as through reduced

number of disulphide bridge and large proportion of sur-

face-exposed charged residues [24], decreased in proline

content [25], and increased number of glycine, methionine

and loops [26].

AT2 lipase was identified as an alkaline lipase where it

exhibited its optimal activity at pH 8. Alkaline lipases are

important in detergent industries. Most staphylococcal

lipases share similar preference towards alkaline pH (pH

8.5) including S. haemolyticus lipase and S. simulans lipase

[18, 27]. In contrast, S. aureus NCTC8530 lipase favored

slightly acidic pH (pH 6.5) [28]. In terms of stability, this

enzyme was found stable at a broad range of pH (pH 6–9).

Similarly, lipase from S. saprophyticus, showed stability at

pH 6–9; below or above this range, the activity was greatly

inactivated [19]. pH is important in maintaining a proper

protonation form of active site of an enzyme as the site

composed of charge residues which involve in substrate

binding. Changes in pH will also lead to disruption of ionic

bonds that hold the enzyme’s tertiary structure.

Nature chooses metals for their biological function. For

instance, Mg2? and Ca2?, both divalent charged metal

ions, are involved in substrate activation and electrostatic

stabilization of enzyme [29].Ca2? was found to strongly

stimulate the activity of AT2 lipase. Enhancement of the

lipolytic activity by Ca2? has been observed in many

staphylococcal lipases as well as other bacterial lipases. In

S. hyicus lipase crystal structure, the role of Ca2? has been

proposed to be involved in stabilizing the three-dimen-

sional structure rather than directly involved in enzyme

catalysis [3]. Primary sequence comparison between AT2

lipase and S. hyicus lipase revealed that the calcium

binding site was highly conserved thereby similar reason

might explain the functional role of Ca2? in AT2 lipase.

Meanwhile, the toxic effect of Zn2? and Fe3? on AT2

lipase might be generated by excess amount of these heavy

metals.

A complete inhibition of AT2 lipase activity was

observed when treated with EDTA. This total deleterious

consequence reflected the essential role of metal ions in

enzyme structure and catalysis. Hence, AT2 lipase can be

classified as a metal-dependent enzyme. Such characteristic

is highly in line to the activation of AT2 lipase in the

presence of metal ion, particularly, Ca2? as described

earlier. Many lipases are inhibited by this heavy metal ion

scavenger for their similar dependency towards Ca2? such

as that in S. saprophyticus and S. warneri lipase 2 [19, 30].


123

Besides EDTA, pepstatin A also demonstrated severe

impairment on AT2 lipase. The probable reason for the

pronounced inhibitory effect of pepstatin A may be

attributed by its potent action on aspartate residues par-

ticularly the catalytic aspartate and the aspartate residues

involve in Ca2? binding site. Insensitivity of AT2 lipase

towards PMSF was found to be another interesting finding.

Lipase which active site composed of catalytic serine is

theoretically sensitive towards serine inhibitors. These

inhibitors bind covalently at the active serine and cause

inactivation. However, notably, with very few exceptions,

some lipases demonstrated resistancy towards inactivation

by PMSF such as that observed in S. hyicus lipase [31]. In

the case of palmitoyl protein thioesterase (PPTI), the

occupancy of PMSF in the catalytic region was restricted

by the narrow substrate-binding cleft [32]. Presumably, low

sensitivity AT2 lipase towards PMSF and pefabloc may be

explained by the limited accessibility of aromatic sulfonyl

fluoride to the active site. Lacking of cysteine residues

which possibly involved in disulphide bond formation, this

enzyme was not inhibited by reducing agents, DTT and

ßME.

The hydrolytic activity of AT2 lipase was remarkably

enhanced in the presence of 1 % (v/v) of Tween 60 and

Tween 80. Both surfactants were likely to stabilize the

substrate emulsion and trigger the lid displacement of AT2

lipase through hydrophobic interaction between the sur-

factant and hydrophobic residues, giving access for more

substrates to interact at the active site. The latter was

deduced as surfactants are known to resemble lipase sub-

strate [33]. However, at higher concentration of the sur-

factants (0.5 % (v/v), a dramatic fall in the lipolytic

activity of AT2 lipase was detected. Such decline in

activity may be caused by treatment of surfactants near or

above the critical micellar concentration (CMC). At this

point, surfactants were in saturation and restricting the

absorption of lipase at the oil/water interface resulting in

enzyme deactivation [34]. Ionic surfactant, SDS, showed a

total inhibition on AT2 lipase activity. Deleterious effect

by SDS may be attributed by the formation of additional

hydrophobic interactions which resulted in complete

unfolding of the tertiary structure and altered surface

hydrophobicity [34].

AT2 lipase showed strong preferential specificity for

higher chain length triglycerides and natural oils. Long

chain substrates which are less water soluble seem to be

essential in stimulating the lipase-substrate interaction

through the formation of oil/water interface between the

substrates and enzyme. A greater accessibility for the

substrate to enter the active site was triggered by strong

hydrophobic interaction between the lid and substrate.

Chain length selectivity of staphylococcal lipases differs

from one another. S. aureus lipase was reported to favor

short chain triacylglycerols but poorly hydrolyzed medium

and long chain triglycerides as well as phospholipids [28].

S. warneri lipase 2 was capable of hydrolyzing short chain

substrates and phospholipids [30].

Stability of enzyme in organic solvents is regarded as an

extra-valuable property for various biotechnology applica-

tions. AT2 lipase was found stable in both hydrophilic and

hydrophobic organic solvents. The enzyme displayed sta-

bility not only in methanol, ethanol and acetone but the

lipolytic activity was also enhanced in the presence of

DMSO and diethyl ether. Stimulation of activity in DMSO

has been observed to associate with glycine-rich enzyme

[35]. Classically, solvents with log P \ 2 are not favorable

for biocatalysis due to their inactivation effects towards

biocatalysts [16]. Therefore, the capability of AT2 lipase to

tolerate solvents such as methanol and ethanol is highly

important for industrial applications especially in biodiesel

production. Exception was observed on 1-propanol and ethyl

acetate whereby these solvents showed complete inhibition

to the enzymatic activity. The notion on stripping water

molecules off the protein surface by hydrophilic organic

solvents may explain the inactivation effect by these two

solvents. Optimal amount of water molecules is of necessity

to lubricate the enzyme for increased activation [36].

Purified AT2 lipase displayed variable results when

treated with water-immiscible organic solvents. The enzyme

was catalytically active in toluene and n-hexane mixture.

Hydrophobic organic solvents have been regarded as more

superior and favorable in the enzyme-solvent interaction

mainly by maintaining the solvation shell of the protein [37].

Furthermore, in most of the transesterification reactions for

biosynthesis of esters, hexane is the most favoured solvent.

Hence AT2 lipase is also a good candidate for the biocatal-

ysis of esters in food and flavouring industries, cosmetics,

and pharmaceuticals manufacturing. In contrast, benzene,

octanol, xylene, n-heptane and chloroform inhibited the

enzymatic activity of AT2 lipase. A plausible explanation for

the instability of AT2 lipase in these solvents is in their

involvement in penetrating and attacking the active centre of

lipase which is hydrophobic in nature.

Few staphylococcal lipases (wild-type) have been

claimed as organic solvent stable enzymes. For instance,

extracellular lipase from S. caseolyticus EX-17 displayed

enhancement in activity by 1.5-fold in the presence of n-

hexane [38]. Stability of S. saprophyticus M36 lipase in

organic solvents was achieved in 25 % (v/v) of toluene,

benzene, p-xylene and n-hexane in which residual activity

remained in the range of 80-90 % [39]. Crude lipase from

S. epidermidis CMST-Pi 1 lipase conserved its activity in

hexadecane, tetradecane and dodecane [40]. Clearly, AT2

lipase is unique by having stability in both polar and non

polar organic solvents compared to other staphylococcal

lipases.


123

Stability of lipase in organic solvents is deduced to

correlate with the distribution of surface charged resi-

dues and surface property of the enzyme [35]. In

agreement, Ogino et al. [41] reported that the surface

residues of the molecule were important for the solvent

stable nature of PST-01 protease. There is however

relatively few published studies on the mechanism of

enzyme adaptation towards organic solvents thereby

limiting our understanding on this subject matter. No

clear correlation and structural pattern have yet been

observed as different enzymes adopt different adapta-

tion strategies. A detailed investigation on the 3D

structure will permit better understanding on the struc-

ture and function relationship.

5 Conclusion

AT2 lipase displays new and exciting biochemical prop-

erties exclusively as a cold-adapted, organic solvent stable

enzyme produced by a mesophilic bacterium. To the best

of our knowledge, no staphylococcal lipase exhibiting both

cold-adapted and organic solvent stability properties has

been reported so far. This finding may serve as useful

information for further exploration on the structural basis

and commercial application in modern biotechnology such

as in cold water washing, food industries and production of

intermediates.

Acknowledgments We thank Prof Dr. Andrew H.-J Wang and Dr.

Lee Cheng-Chung from Institute of Biological Chemistry, Academia

Sinica, Taiwan for AUC analysis. This project was supported by

Ministry of Science and Technology, Malaysia [02-01-04 SF 1024].

Conflict of interest The authors declare that there is no conflict of

interest.

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Documents

A New Cold-Adapted, Organic Solvent Stable Lipase from Mesophilic Staphylococcus epidermidis AT2