This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institution

and sharing with colleagues.

Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third party

websites are prohibited.

In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further information

regarding Elsevier’s archiving and manuscript policies areencouraged to visit:

http://www.elsevier.com/copyright

Author's personal copy

Process Biochemistry 48 (2013) 169–175

Contents lists available at SciVerse ScienceDirect

Process Biochemistry

jo u rn al hom epage: www.elsev ier .com/ locate /procbio

Short communication

A new thermostable and organic solvent-tolerant lipase from Aneurinibacillusthermoaerophilus strain HZ

Malihe Masomiana, Raja Noor Zaliha Raja Abd Rahmana,∗, Abu Bakar Sallehb, Mahiran Basri c

a Enzyme and Microbial Technology Research, Department of Microbiology, Faculty of Biotechnology and Biomolecular Science, Universiti Putra Malaysia, 43400 Serdang, Selangor,Malaysiab Enzyme and Microbial Technology Research, Department of Biochemistry, Faculty of Biotechnology and Biomolecular Science, Universiti Putra Malaysia, 43400 Serdang, Selangor,Malaysiac Enzyme and Microbial Technology Research, Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia

a r t i c l e i n f o

Article history:Received 2 February 2012Received in revised form 17 October 2012Accepted 4 November 2012Available online 14 November 2012

Keywords:Aneurinibacillus thermoaerophilus strain HZThermostable lipaseOrganic solvent tolerantPurificationCharacterisation

a b s t r a c t

A thermostable and organic solvent-tolerant lipase produced by Aneurinibacillus thermoaerophilus strainHZ was purified and characterised. The lipase was purified to apparent homogeneity with two steps: anionexchange chromatography on Q-Sepharose and gel filtration on Sephadex-G75. A final specific activity of43.5 U/mg was obtained with an overall recovery of 19.7% and 15.6 purification fold. The molecular massof the HZ lipase was estimated to be 50 kDa. The optimum pH for the activity of the purified HZ lipasewas 7.0. The stability showed a broad range of pH values between pH 4.0 and 9.0 at 30 ◦C. The purified HZlipase exhibited an optimum temperature of 65 ◦C with a half-life of 3 h and 10 min at 65 ◦C. The activity ofthe purified HZ lipase was stimulated in the presence of Ca2+. Organic solvents such as dimethyl sulfoxide(DMSO), methanol, n-tetradecane and n-hexadecane enhanced the lipase activity. Studies on the effectof oil showed that the lipase preferred natural oil, such as sunflower oil, over synthetic substrates.

© 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Lipases (triacylglycerol ester hydrolases, EC 3.1.1.3) are widelyused in biocatalysis due to their ability to catalyse the hydrolysisof triacylglycerides in aqueous solutions and also synthetic reac-tions in organic media. Lipases must be reasonably thermostableand active in organic solvents if they are to be used in wide rangeof synthetic reactions. Thermostability is important for resistancetowards the chemical modifications caused by the high tempera-tures employed in various industrial lipase-catalysed reactions [1].Lipases constitute the most important group of biocatalysts for thesynthesis of biopolymers and biodiesel, synthesis of fine chemi-cals, the production of enantiopure pharmaceuticals [2], and arealso important in the dairy industry, in detergents and in papermanufacturing [3].

Stability in organic solvents is required since low-water systemsin organic solvents are necessary in order to provide conditions

∗ Corresponding author. Tel.: +60 3 8946 6713; fax: +60 3 8943 0913.E-mail addresses: masomian2000@yahoo.com (M. Masomian),

rnzaliha@biotech.upm.edu.my (R.N.Z.R.A. Rahman), abubakar@biotech.upm.edu.my(A.B. Salleh), mahiran@science.upm.edu.my (M. Basri).

that favour the synthetic reaction over the hydrolytic reaction.Thus, thermostable and organic solvent-tolerant enzymes can offerother advantages for synthetic reactions [1]. Attempts have beenmade to modify the enzymes to enhance their activity and stabil-ity in the presence of organic solvent, however, it would be moreeconomically viable to screen for naturally evolved solvent toler-ant enzymes for application in non-aqueous enzymatic synthesis.There are many reports on the purification and characterisationof microbial lipases but very few lipases have been reported hav-ing both characteristics; thermostable and organic solvent-tolerantsuitable for harsh industrial processes [4–7]. Furthermore, most oforganic solvent-tolerant lipases are stable in either very polar ornon polar organic solvent, but not in both [6,8].

We previously isolated a lipolytic bacterium from a local source,identified as Aneurinibacillus thermoaerophilus strain HZ, whichsecreted a thermostable and organic solvent-tolerant lipase. Asfar as we know, HZ lipase is the only report on lipase fromAneurinibacillus genus. Maximum lipase activity of the crudeenzyme was at 65 ◦C at pH 7.0. The crude lipase was very sta-ble in the presence of various organic solvents. Under optimisedconditions of production, the yield showed an increase up to 1.77-fold compared to the basal medium [9,10]. In this manuscript, thepurification and characterisation of extracellular lipase from theAneurinibacillus thermoaerophilus strain HZ are described.

1359-5113/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.procbio.2012.11.002

Author's personal copy

170 M. Masomian et al. / Process Biochemistry 48 (2013) 169–175

2. Materials and methods

2.1. Substrates and chemicals

tributyrin, tricaprylin, phenylmethanesulfonyl fluoride, Dithiothreitol and Pep-statin, Coomassie Brilliant Blue R-250, and acrylamide/bis-acrylamide (40% stocksolution – 19:1) were purchased from Sigma, USA. Triolein was purchased fromFluka, Switzerland. Matrices for protein purification were supplied by AmershamBiosciences, Uppsala, Sweden. Pre-stain protein marker was purchased from Fer-mentas, USA. All chemicals used were of analytical reagent grade.

2.2. Microorganism and lipase production

Aneurinibacillus thermoaerophilus strain HZ, which secreted a thermostable andorganic solvent-tolerant lipase, was used in this study. A. thermoaerophilus strainHZ was cultivated in a medium that was previously optimised physically and nutri-tionally for lipase production [9]. Cultivation was conducted at 60 ◦C for 48 h with ashaking rate of 150 rpm.

2.3. Measurement of lipase activity

The cultured supernatant was prepared by removing bacteria cells in the cultureby centrifugation at 10,000 × g for 10 min at 4 ◦C. The cell-free cultured supernatantwas filtered with a 0.45 �m membrane filter and used as crude enzyme. Lipase activ-ity was measured by the modified method of Kwon and Rhee [11]. Olive oil was usedas the substrate. The reaction mixture, consisting of 1.0 ml of crude enzyme, 2.5 mlolive oil emulsion (1:1 ratio of olive oil and phosphate buffer [K2HPO4 (50 mM), atpH 7.0]) and 0.02 ml of 20 mM CaCl2·2H2O, was incubated for 30 min with shak-ing at 200 rpm at 65 ◦C. Then, the reaction was stopped by adding 1.0 ml of 6 N HCland 5.0 ml isooctane. The upper layer (4.0 ml) was pipetted into a test tube and1.0 ml cupric acetate pyridine [copper (II) acetate monohydrate (5%; pH 6.1), pHadjusted by adding pyridine] was added. The free fatty acids dissolved in isooctanewere determined by measuring the O.D. of the isooctane solution at 715 nm. Eachsample was assayed three times and the average value was taken. One unit (U) oflipase activity is defined as the rate of fatty acid formation per min (standard assaycondition).

2.4. Purification of lipase

The enzyme solution was applied to the Q-Sepharose column (1.6 cm × 5.0 cm,5 ml of gel), which was pre-equilibrated with 20 mM glycine–NaOH buffer, pH 9.5.The column was washed with 5× of column bed volume of the equilibration buffer.The lipase bound to the anion exchange gel was eluted at a flow rate of 0.5 ml/minby increasing the concentration of NaCl from 0 to 1 M with a linear gradient using20× of column bed volume of 20 mM glycine–NaOH buffer, pH 9.5 and the samebuffer containing NaCl. The fractions (2.5 ml) were collected and analysed for lipaseactivity. The sample obtained after anion exchange chromatography was loaded ona Sephadex-G75 column (1.6 cm × 65 cm, 80 ml of gel) pre-equilibrated with 2× ofcolumn bed volume of 20 mM glycine–NaOH buffer containing 0.15 M NaCl, pH 9.5.The elution was conducted in the same buffer at a flow rate of 0.25 ml/min. Thefractions (1 ml) that exhibited lipase activity were collected.

The eluted peak was concentrated by 1:1 volume of 100 mg/ml trichloroaceticacid solution, then subjected to SDS-PAGE (10%) under the conditions developed byLaemmli [12] and proteins bands were visualised by Coomassie brilliant blue R-250.The band corresponding to lipase was determined by activity staining of unstainedgel. For this the purified HZ lipase was subjected to SDS-PAGE without boiling (dur-ing sample preparation step) and upon electrophoresis, the gel was washed with20% (v/v) isopropanol for 20 min to remove the SDS and then washed with distilledwater for 10 min. Then, the gel was transferred onto a tributyrin agar plate andincubated at 65 ◦C. The plate was observed every 1 h until the halo zone was visi-ble due to tributyrin hydrolysis on the agar plate. To confirm the homogeneity, thepurified lipase was electrophoresed on Native-PAGE (10%) and the protein band onthe gel was visualised upon Coomassie brilliant blue R-250. The protein content wasassayed by the method of Bradford [13] using bovine serum albumin as the standardprotein.

2.5. Characterisation of the purified lipase

The effect of pH on lipase activity was investigated by emulsifying olive oil invarious buffer systems (pH 4.0–12.0) at a concentration of 50 mM. Then the enzymewas assayed at different pH values at 65 ◦C for 30 min with shaking at 200 rpm. ForpH stability studies, 200 �l of a purified enzyme solution was mixed with 800 �l of50 mM buffer at a specific pH. The mixture was pre-incubated at 30 ◦C for 30 min andthe residual activity was determined. Buffer systems were used at a concentrationof 50 mM: sodium acetate (pH 4.0–6.0), potassium phosphate (pH 6.0–8.0), Tris HCl(pH 8.0–9.0), glycine–NaOH (pH 9.0–11.0) and disodium hydrogen phosphate (pH11.0–12.0). The effect of temperature on lipase activity was studied by assayingthe enzyme reaction mixture at various temperatures in the range of 40–80 ◦C for30 min at pH 7.0, using a phosphate buffer (50 mM). The thermostability of lipase wasdetermined by pre-incubating aliquots of the pure enzyme at various temperatures,

ranging from 60 to 75 ◦C for different time intervals from 0 to 6 h. At each timeinterval, samples were pipetted out and immediately frozen prior to being assayed.

To determine the effect of metal ions and inhibitors on lipase activity, variousmineral salts and inhibitors were tested by treating the purified enzyme with 1 and5 mM of metal ions and metal chelating agents, reducing agents, serine and asparticinhibitors for 30 min at 30 ◦C. The control contained no addition of the test com-pounds. To study the substrate specificity of the enzyme, olive oil was substitutedwith the various triglycerides and natural oils. The oils were emulsified (v/v) in50 mM phosphate buffer (pH 7.0) and assayed with 500 �l of an enzyme solutionat 65 ◦C for 30 min. The stability of purified lipase in the organic solvent was inves-tigated by the method as described by Ogino et al. [14] with slight modification.The purified HZ lipase-containing 50 mM phosphate buffer (pH 7.0) was incubatedwith an organic solvent (25%, v/v) in a vial bottle at 30 ◦C for 30 min with constantshaking at 150 rpm. The remaining lipase activity was measured using the methodproposed by Kwon and Rhee [11] as described above.

3. Results and discussion

3.1. Purification of HZ lipase

Purification steps play an important role in recovering bio-logically active protein with highest purity. In our attempt topurify the HZ lipase, the cell-free supernatant was subjected to ionexchange chromatography on Q-sepharose followed by gel filtra-tion on Sephadex-G75. When the enzyme was loaded onto anionexchange column, the absorbed enzyme was eluted at a 750 mMconcentration of NaCl. This step resulted in increased activity anda recovery rate of 48.7% with 9.3 purification folds. The elutedpeak showed two bands on the SDS-PAGE with sizes of 86 kDaand 50 kDa. The enzyme was further purified through gel filtra-tion chromatography subjected to Sephadex-G75 (Fig. 1A). A singleband was obtained for HZ lipase, which was purified to approxi-mately 15.6-fold with a specific activity of 43.5 U/mg and an overallyield of 19.7% (Table 1). The purity and homogeneity of the elutedpeak was confirmed by SDS-PAGE and Native-PAGE (Fig. 1B and C).Generally, purification methods for different lipases are depend-ent on nonspecific techniques and in some cases using many stepsof purification. The HZ lipase was successfully purified using acombination of anion exchange and gel filtration chromatography.Nawani and Kaur [15] had purified a thermostable lipase fromBacillus sp. using ammonium sulphate precipitation, hydropho-bic interaction chromatography, gel filtration and ion exchangechromatography. Compared with other purified Bacillus lipases, HZlipase showed a higher recovery rate. Lipase from B. stearother-mophilus MC7 was purified to 19.6-fold with 10.2% recovery [16].

The molecular weights of most Bacillus lipases have beenreported to be in the range of 16–69 kDa [16,17]. The molecularweight of the purified enzyme was confirmed by using the modi-fied activity staining method of Singh et al. [18]. The purified lipasewas run on SDS-PAGE and activity staining was conducted usingtributyrin as the substrate. The halo zone on tributyrin agar platewas developed at the band on position around 50 kDa of the purifiedenzyme, which confirmed the size of molecular weight of HZ lipaseand verified that HZ lipase can hydrolyse the ester bond (Fig. 1D).

3.2. Effect of pH on HZ lipase activity and stability

The HZ purified lipase exhibited high hydrolytic activity at pH4.0–10.0 with the maximum activity at pH 7.0 (Fig. 2A). At alkalineconditions, pH 11.0, HZ lipase activity decreased to 34%. At acidicpH (6.0), 93% of relative activity was detected compared to the opti-mum pH. Similarly, Imamura and Kitaura [19] reported that Bacillussp. H-257 lipase had the optimum hydrolytic activity at pH 7.0–9.0.On the other hand, several researchers reported the optimum lipaseactivity in the highly alkaline pH range (8.0–10.6) from Bacillus sp.LBN 4 [20], B. subtilis EH 37 [21] and B. alcalophilus B-M20 [22].

In pH stability test, HZ lipase showed very good stability after30 min at pH 4.0–9.0, and the lipase retained 62% of its activity at

Author's personal copy

M. Masomian et al. / Process Biochemistry 48 (2013) 169–175 171

Fig. 1. Purification profile of lipase from A. thermoaerophilus strain HZ. (A) Gel filtration chromatography elution profile of the HZ lipase on Sephadex G-75. The lipase activitywas determined according to the standard method, while the protein profile was monitored by absorption at 280 nm. (B) SDS-PAGE analysis of lipase purified through anionexchange chromatography and gel filtration chromatography. Lane M: pre-stain protein marker; lanes 1, 2 and 3: sample containing non-absorbance proteins from theQ-Sepharose column in the washing step; lane 4: crude enzyme; lane 5: sample containing absorbance proteins from the Q-Sepharose column in the elution step; lane 6:purified HZ lipase from gel filtration. (C) Native-PAGE analysis of purified HZ lipase. Lanes 1 and 2: purified lipase after gel filtration. (D) Lipase activity was detected usinga tributyrin agar plate. The purified HZ lipase was analysed on 10% SDS-PAGE followed by activity staining with tributyrin as a substrate. Lane M: pre-stain protein marker;lane S: the activity band of the purified HZ lipase.

Author's personal copy

172 M. Masomian et al. / Process Biochemistry 48 (2013) 169–175

Table 1Summary of purification of HZ lipase in culture supernatant.

Purification step Sample volume(ml)

Total activity(U)

Total protein(mg)

Specific activity(U/mg) ± SE

Purification(fold)

Yield (%)

Culture supernatant 30.00 16.55 5.953 2.78 ± 0.21 1.00 100Q-Sepharose 15.00 8.06 0.311 25.99 ± 3.34 9.32 48.73Sephadex-G75 16.50 3.26 0.075 43.47 ± 2.59 15.62 19.69

Note: Values are means of three replicates ± SE.

Fig. 2. Effect of pH and temperature on HZ lipase activity and stability. (A) Optimum pH was determined at 65 ◦C and various pH values (4.0–12.0) by a colorimetric method.(B) Stability was analysed after pre-incubating the enzyme in various pH values ranging from pH 4.0 to 12.0 for 30 min at 30 ◦C. (C) For the lipase activity, the enzyme wasassayed at various temperatures ranging from 40 to 80 ◦C at intervals of 5 ◦C for 30 min, using a phosphate buffer (50 mM). (D) For temperature stability, the enzyme waspre-incubated in various temperatures ranging from 60 to 75 ◦C for different incubation times from 0 to 6 h. The residual activity was measured at 65 ◦C. Values are meansof three replicates ± SE.

Author's personal copy

M. Masomian et al. / Process Biochemistry 48 (2013) 169–175 173

pH 10.0 compared to the control (non-treated enzyme; Fig. 2B).Lee et al. [17] reported that both BTID-A and BTID-B lipases from B.thermoleovorans ID-1 were stable in neutral pH range (6.0–8.0) for24 h at room temperature. This phenomenon might be due to theformation of an improper ionic form of the enzyme and resultedin irreversible enzyme activation. The activity of HZ lipase retainedmore than 70% in acidic pH values after 30 min of treatment, whilelipase from Bacillus sp. RSJ-1 was completely inactivated at acidicpH 5.0 [23].

3.3. Effect of temperature on activity and thermostability of HZlipase

The optimum activity of pure HZ lipase was detected at 65 ◦C,which confirmed that the lipase is thermostable (Fig. 2C). Sincemost of the industrial processes involving enzymes operate at tem-peratures above 50 ◦C, the importance of thermostable enzymes isof great significance. HZ lipase was fairly active in between 50 and70 ◦C and the enzyme retained 70% and 62% of its maximum activ-ity at 75 and 45 ◦C, respectively. The optimum lipase activity at50–55 and 60 ◦C was reported from Bacillus sp. RSJ-1[23], B. ther-moleovorans CCR11 [24], Burkholderia sp. C20 [25], Burkholderia sp.HY-10 [26] and B. subtilis EH 37 [21]. The loss of the lipase activityat high temperatures, above 70 ◦C, might be due to the disruptionof hydrogen bonds. This, in turn, may disrupt the conformation ofthe enzyme, which leads to the diminishment of its affinity towardsits substrate. As a result, the enzyme loses its catalytic activity athigh temperatures. However, less activity at lower temperaturescould be due to the optimum temperature needed to trigger the lidopening of the enzyme.

The thermal stability profile of the HZ lipase is shown in Fig. 2D.The lipase was fairly stable at 60 ◦C (>70% residue activity) even upto 4 h of treatment. The half-lives of HZ lipase at 65 and 70 ◦C were3 h 10 min and 1 h 20 min, respectively. The half-lives of the lipasefrom Bacillus sp. strain 42 reported by Rahman et al. [27] was 2 h5 min at 65 ◦C and 45 min at 70 ◦C. Dandavate et al. [28] observedthat the half-life of lipase from Burkholderia multivorans V2 was 50and 10 min at 50 and 60 ◦C, respectively. Although, it was reportedby Park et al. [26] that an extracellular lipase from Burkholderia sp.exhibited maximum activity at 60 ◦C with a half-life of 2 and 0.5 hat 50 and 60 ◦C, respectively, the purified HZ lipase was very stableup to 60 ◦C and lost only 7 and 25% of its original activity after thetreatment at 65 and 70 ◦C for 30 min, respectively. Compared to thelipases reviewed above, the HZ lipase had longer thermostabilitythan other thermostable lipases.

3.4. Effect of metal ions and inhibitors on HZ lipase activity

The effect of various cations was tested at 1 and 5 mM in 50 mMphosphate buffer at pH 7.0 (Table 2). Among alkaline earth metalions, Mg2+ and Ca2+ were found to have different effect on HZ lipaseactivity. Ca2+ was found to stimulate the HZ lipase activity at 1 mMconcentration up to 46.3% compared to the control, while, Mg2+ hadslightly (1.2%) inhibition effect on HZ lipase activity. Increasing theconcentration of Mg2+ up to 5 mM (30 min) showed 26.2% inhibitioneffect on HZ lipase activity, while a 55% increase in enzyme activ-ity was observed in the presence of 5 mM Ca2+. The Ca2+ ion mightplay a significant role in stabilising the HZ lipase structure thusincreasing the thermostability and enzyme catalysis. In this groupof enzymes, the ions often play a structural rather than a catalyticrole. The ions bind to the enzyme and change the conformation ofthe protein to afford greater stability to the enzyme. According toKim et al. [29], L1 lipase showed an 8–10 ◦C increase in thermosta-bility in the presence of calcium ions. The Ca2+ may stabilise theprotein tertiary structure and, thus, confer thermostability effect[30].

Table 2Effect of various metal ions and inhibitors on the activity of purified lipase.

Metal ions Concentration (mM) Relativeactivitya

(%) ± SE

Control – 100

Na+ 1 80 ± 1.235 68.75 ± 3.10

K+ 1 85 ± 1.585 80 ± 1.23

Mg2+ 1 98.8 ± 2.805 73.8 ± 2.45

Ca2+ 1 146.3 ± 1.405 155 ± 3.33

Mn2+ 1 83.8 ± 0.885 46.3 ± 4.63

Fe3+ 1 30 ± 1.935 NA

Cu2+ 1 53.8 ± 0.885 22.5 ± 1.75

Zn2+ 1 17.5 ± 0.535 NA

PMSF 1 64.9 ± 1.055 41.4 ± 1.05

DTT 1 63.1 ± 2.105 53.2 ± 1.93

EDTA 1 77.5 ± 0.885 73 ± 1.05

�-Mercaptoethanol 1 61.3 ± 0.705 45.9 ± 3.33

Pepstatin 1 94.6 ± 0.705 40.5 ± 1.75

Note: HZ lipase was treated with various metal ions and inhibitors at 30 ◦C for 30 minprior to lipase assay. The activity is expressed as a percentage of the activity relativeto the appropriate control value (with no addition of the test compounds). Valuesare means of three replicates ± SE.

a NA, no activity.

HZ lipase was inhibited in the presence of Cu2+, Fe3+ and Zn2+,known as transition metal ions, losing more than 45% of its activ-ity after treatment at 1 mM concentration. In the presence of 1 and5 mM Mn2+ after 30 min treatment, the lipase activity was inhibitedup to 16.2% and 53.7%, respectively. The inhibition effect by tran-sition metal ions (Zn2+, Fe3+ and Cu2+) on the HZ lipase might bedue to the toxic effect generated by excess amounts of the tran-sition metals. One likely explanation of the effect of metal ions isthe change in the solubility and behaviour of the ionised fatty acidsat interfaces, which changes the catalytic properties of the enzymeitself [31].

Several metal chelating agents, reducing agents, serine andaspartic inhibitors were used to study the inhibition effect and ver-ify the amino acids that play a significant role in enzyme catalysis(Table 2). Among the reducing agents, with 1 and 5 mM DTT, theenzyme activity was retained up to 63.1 and 53.2% after 30 mintreatment, respectively. With �-mercaptoethanol, enzyme activitywas dropped by 38.7% and 54.1% after 30 min, at 1 and 5 mM con-centration, respectively. This suggested the role of SH-groups in thecatalytic mechanism.

Conversely, HZ lipase in the presence of 1 mM EDTA showed22.5% inhibition after 30 min treatment. Increasing the concentra-tion of EDTA up to 5 mM only inhibited 27% of enzyme activity,which indicated that the lipase from A. thermoaerophilus strain HZwas not a metaloenzyme. Similar inhibition (19.1%) was observed

Author's personal copy

174 M. Masomian et al. / Process Biochemistry 48 (2013) 169–175

in thermostable lipase from Geobacillus sp. strain T1 when lipase-treated with EDTA for 30 min [32].

On the other hand, pepstatin at 1 mM concentration showedno significant effect on the lipase activity of HZ lipase. However,at in higher concentration the lipase activity was inhibited up to59.5%. Leow et al. [32] found that T1 lipase was strongly inhibitedby pepstatin. The inhibition profile showed that HZ lipase activitywas inhibited by 58.6% under the treatment with phenyl methylsulfonyl fluoride (PMSF) (5 mM), which indicated that HZ lipasebelonged to the serine group. Partial inactivation of the HZ lipaseby PMSF might be caused by modification of an essential serineresidue that plays a key role in the catalytic mechanism [33,34].

3.5. Effect of substrate specificity

HZ lipase demonstrated wide substrate specificity towardstriglycerides from C2 to C18 with the highest affinity to triolein.It was found that HZ lipase was more selective towards long car-bon chain natural oils, such as olive oil, sunflower oil, soybean oil,corn oil and rice bran oil (data not shown). Since oils and fats aremixed triglycerides, strict parallelism with the hydrolysis of triglyc-eride consisting of only one type of fatty acid cannot be expected.However, preference for the oils with a relatively high content oflinoleic and/or oleic acid compares well with the lipase that hadhigh activity towards triolein. This provides good potential for HZlipase as a catalyst in industrial applications.

The hydrolysis of palm oil (solid lipid) was low, which contained50% saturated fatty acids as compared to other natural oils. How-ever, the hydrolysis of solid lipid was more difficult compared toother liquid lipids due to limited interaction between the enzymeand substrate. In addition, lower hydrolysis was observed withcoconut oil because it contains a high amount of short-chain fattyacids, 47% of lauric acid (C12:0) and 18% of myristic acid (C14:0).This shows that HZ lipase is highly specific towards the long chainfatty acids. The carbon chain length selection of HZ lipase wasprobably due to the hydrophobic entity of longer chain fatty acids,which might have favoured a stronger hydrophobic interactionbetween the substrate and helix lid of the enzyme. In agreement,Leow et al. [32] reported that T1 mature lipase showed prefer-ence to olive oil as compared to coconut oil. Moreover, HZ lipase isdifferent from other thermostable lipases with respect to its pref-erence for triolein as a substrate compared to other shorter carbonchain triglycerides. BTL2 lipase from B. stearothermophilus MC 7[16] showed high activity towards tributyrin (C4), whereas the P1lipase from B. stearothermophilus [35] preferred tricaprylin (C8) assubstrate.

3.6. Effect of organic solvent stability on HZ lipase

Treatment of the purified HZ lipase with various organic sol-vents for 30 min demonstrated that the enzyme was stable in all theorganic solvents tested (Table 3). The purified enzyme was very sta-ble in water-miscible solvents with a low log P value. High stabilitywas observed in the presence of dimethylsulfoxide and methanolwith a ∼45% increase in their activities compared to the control(without solvent). The results were in agreement with Sulong et al.[36] which stated that B. sphaericus 205y lipase showed high sta-bility in the presence of DMSO and methanol. In addition, HZ lipaseretained activities of 84% and 61% in 1-propanol and propyl acetatecompared to the control (without solvent).

The purified lipase showed moderate activity in the organicsolvents used with log P values between 2 and 3.1. The relativeactivity of HZ lipase in organic solvents such as benzene, tolueneand p-xylene were 85, 92 and 77%, respectively. Ogino et al. [37]reported very low lipase activity in the presence of toluene and

Table 3Stability of purified lipase in the presence of various organic solvents.

Solvent Log P Relativeactivity (%) ± SE

Control – 100DMSO −1.3 144 ± 2.34Methanol −0.76 146 ± 2.731-Propanol 0.28 84 ± 3.65Propyl acetate 1.2 61 ± 0.68Benzene 2 85 ± 1.21Toluene 2.5 92 ± 0.84p-Xylene 3.1 77 ± 2.951-Decanol 4 48 ± 2.51Dodecanol 5 25 ± 1.22n-Dodecane 6.6 86 ± 5.33n-Tetradecane 7.6 124 ± 1.64n-Hexadecane 8.8 106 ± 2.58

Note: The enzyme and organic solvent were mixed in a 3:1 ratio, and the mixture wasincubated at 30 ◦C with shaking at 150 rpm for 30 min, followed by lipase activityassay. Activities of HZ lipase in the presence of various organic solvents are shownas values relative to those in the absence of an organic solvent. Values are means ofthree replicates ± SE.

p-xylene at a final concentration of 25% (v/v) form P. aeruginosaLST-03 lipase.

Non-polar solvents are often less harmful to enzymes. PurifiedHZ lipase in water-immiscible organic solvents showed variableresults. The relative activity of the enzyme was enhanced in thepresence of both n-tetradecane (24%) and n-hexadecane (6%). Rah-man et al. [38] reported that n-tetradecane and n-hexadecaneenhanced the relative activity of lipase S5 from P. aeruginosa S5after 30 min of incubation at 37 ◦C. On the other hand, in the pres-ence of n-dodecane, 1-decanol and dodecanol, 14, 52 and 75% lossof activity was observed, respectively.

The stability of the HZ lipase in aqueous-organic mixture sug-gested the resistance of this enzyme towards denaturation byorganic solvents. A possible explanation is that the outer part of HZlipase is more hydrophilic and, therefore, it binds to essential watermore tightly, thereby leaving the biocatalyst in an active state inorganic solvents. This explanation was in agreement with Klibanov[39], who demonstrated that the pancreatic lipase appears to bindthe necessary water tightly to these enzyme molecules, leaving itto be catalytically active in organic solvents. However, differentorganic solvents showed different tolerance profiles of HZ lipase. Itis well known that the effect of organic solvents on enzyme activitydiffers from lipase to lipase. There was no clear correlation betweenthe solubility of an organic solvent in water and the stability oflipase in its presence [14].

4. Conclusion

Aneurinibacillus thermoaerophilus strain HZ is a source of novellipase that has high specific activity with high purification fold,good thermostability and high solvent tolerance that makes theenzyme very attractive for biotechnological applications. An effec-tive method for purification of HZ lipase to obtain high recoverywith fewer steps was developed. As far as we know this is thefirst report in characterising the extracellular lipase from theAneurinibacillus thermoaerophilus strain HZ and it has unique prop-erties which can fulfil various industrial applications.

Acknowledgment

This work was supported by Malaysian Ministry of Sci-ence/Technology and Innovation (Sciencefund: 02-01-04-SF1024).

Author's personal copy

M. Masomian et al. / Process Biochemistry 48 (2013) 169–175 175

References

[1] Lima GMV, Kreiger N, Mitchell AD, Baratti CJ, Filippisde I, Fontana DJ. Evaluationof the potential for the use in biocatalysis of a lipase from a wild strain of Bacillusmegaterium. J Mol Catal B: Enzym 2004;31:53–61.

[2] Jaeger KE, Eggert T. Lipases for biotechnology. Curr Opin Biotechnol2002;13:390–7.

[3] Miroliaei M, Nemat-Gorgani M. Effect of organic solvents on stability and activ-ity of two related alcohol dehydrogenases: a comparative study. Int J BiochemCell Biol 2002;34:169–75.

[4] Deive FJ, Costas M, Longo MA. Production of a thermostable extracellular lipaseby Kluyveromyces marxianus. Biotechnol Lett 2003;25:1403–6.

[5] Eltaweel MA, Rahman RNZRA, Salleh AB, Basri M. An organic solvent-stablelipase from Bacillus sp. strain 42. Ann Microbiol 2005;55:187–92.

[6] Peng R, Lin J, Wei D. Purification and characterization of an organic solvent-tolerant lipase from Pseudomonas aeruginosa CS-2. Appl Biochem Biotechnol2010;162:733–43.

[7] Yan J, Yang J, Xu L, Yan Y. Gene cloning, overexpression and characterizationof a novel organic solvent tolerant and thermostable lipase from Galactomycesgeotrichum Y05. J Mol Catal B: Enzym 2007;49:28–35.

[8] Universiti Putra Malaysia. Novel lipase gene from Bacillus sphaericus 205y.Patent PI20040958 MY.

[9] Rahman RNZA, Masomian M, Salleh A, Basri M. A new thermostable lipase byAneurinibacillus thermoaerophilus strain HZ: nutritional studies. Ann Microbiol2009;59:133–9.

[10] Masomian M, Rahman RNZRA, Salleh AB, Basri M. A unique thermostableand organic solvent tolerant lipase from newly isolated Aneurinibacillus ther-moaerophilus strain HZ: physical factor studies. World J Microbiol Biotechnol2010;26:1693–701.

[11] Kwon DY, Rhee JS. A simple and rapid colorimetric method for determinationof free fatty acid for lipase assay. J Am Oil Chem Soc 1986;63:69–92.

[12] Laemmli UK. Cleavage of structure protein during assembly of the head ofbacteriophage T4. Nature 1970;277:680–5.

[13] Bradford MM. A rapid and sensitive method for the quantitation of microorgan-ism quantities of protein utilizing the principles of protein–dye binding. AnalBiochem 1976;72:248–54.

[14] Ogino H, Nakagawa S, Shinya K, Muto T, Fujimura N, Yasuda M, et al. Purificationand characterization of organic solvent-stable lipase from organic solvent-tolerant Pseudomonas aeruginosa LST-3. J Biosci Bioeng 2000;89:451–7.

[15] Nawani N, Kaur J. Studies on lipolytic isoenzymes from a thermophilic Bacillussp.: production, purification and biochemical characterization. Enzyme MicrobTechnol 2007;40:881–7.

[16] Kambourova M, Kirilova N, Mandeva R, Derekova A. Purification and propertiesof thermostable lipase from a thermophilic Bacillus stearothermophilus MC 7. JMol Catal B: Enzym 2003;22:307–13.

[17] Lee DW, Kim HW, Lee KW, Kim BC, Choe EA, Lee HS, et al. Purification andcharacterization of two distinct thermostable lipases from the gram positivethermophilic bacterium Bacillus thermoleovorans ID-1. Enzyme Microb Technol2001;25:363–72.

[18] Singh R, Gupta N, Goswami VK, Gupta R. A simple activity staining protocol forlipases and esterases. Appl Microbiol Biotechnol 2006;70:679–82.

[19] Imamura S, Kitaura S. Purification and characterization of a monoacylglyc-erol lipase from the moderately thermophilic Bacillus sp. H-257. J Biochem2000;127:419–25.

[20] Bora L, Kalita M. Production and optimization of thermostable lipase from athermophilic Bacillus sp. LBN 4. Internet J Microbiol 2007:4.

[21] Ahmed EH, Raghavendra T, Madamwar D. A thermostable alkaline lipase froma local isolate Bacillus subtilis EH 37: characterization, partial purification, andapplication in organic synthesis. Appl Biochem Biotechnol 2010;160:2102–13.

[22] Ghanem EH, Al-Sayed HA, Saleh KM. An alkalophilic thermostable lipase pro-duced by a new isolate of Bacillus alcalophilus. World J Microbiol Biotechnol2000;16:459–64.

[23] Sharma R, Soni SK, Vohra RM, Gupta LK, Gupta JK. Purification and character-ization of a thermostable alkaline lipase from a new thermophilic Bacillus sp.RSJ-1. Process Biochem 2002;37:1075–84.

[24] Castro-Ochoa CDL, Gomez RC, Alfaro VG, Ros OR. Screening, purification andcharacterization of the thermoalkalophilic lipase produced by Bacillus ther-moleovorans CCR11. Enzyme Microb Technol 2005;37:648–54.

[25] Liu CH, Lu WB, Chang JS. Optimizing lipase production of Burkholderia sp. byresponse surface methodology. Process Biochem 2006;41:1940–4.

[26] Park DS, Oh HW, Heo SY, Jeong WJ, Shin DH, Bae KS, et al. Characterization of anextracellular lipase in Burkholderia sp. HY-10 isolated from a longicorn beetle.J Microbiol 2007;45:409–17.

[27] Rahman RNZRA, Hamid THTA, Eltaweel MA, Basri M, Salleh AB. Overexpress-ion and characterization of strep-tagged thermostable organic solvent-stablelipase from Bacillus sp. strain 42. J Biotechnol 2008;136:290–344.

[28] Dandavate V, Jinjala J, Keharia H, Madamwar D. Production, partial purifica-tion and characterization of organic solvent tolerant lipase from Burkholderiamultivorans V2 and its application for ester synthesis. Bioresour Technol2009;100:3374–81.

[29] Kim MH, Kim HK, Lee JK, Park SY, Oh TK. Thermostable lipase ofBacillus stearothermophilus: high-level production, purification and calcium-dependent thermo stability. Biosci Biotechnol Biochem 2000;64:280–6.

[30] Noble MEM, Cleasby A, Johnson LN, Egmond MR, Frenken LGJ. The crystalstructure of triacylglycerol lipase from Pseudomonas glumae reveals a partiallyredundant catalytic aspartate. FEBS Lett 1993;331:123–8.

[31] Lesuisse E, Schanck K, Colson C. Purification and preliminary characterizationof the extracellular lipase of Bacillus subtilis 168, an extreamly basic pH-tolerantenzyme. Eur J Biochem 1993;216:155–60.

[32] Leow TC, Rahman RNZRA, Basri M, Salleh AB. High level expression of ther-mostable lipase from Geobacillus sp. strain T1. Biosci Biotechnol Biochem2004;68:96–103.

[33] Arpigny JL, Jaeger KE. Bacterial lipolytic enzymes: classification and properties.Biochem J 1999;343:177–83.

[34] Jaeger K-E, Dijkstra BW, Reetz MT. Bacterial biocatalysts: molecular biology,three-dimensional structures, and biotechnological applications of lipases.Annu Rev Microbiol 1999;53:315–51.

[35] Sinchaikul S, Sookkheo B, Phutrakul S, Pan F-M, Chen S-T. Optimization ofa thermostable lipase from Bacillus stearothermophilus P1: overexpression,purification, and characterization. Protein Expr Purif 2001;22:388–98.

[36] Sulong MR, Zaliha Raja Abd Rahman RN, Salleh AB, Basri M. A novel organicsolvent tolerant lipase from Bacillus sphaericus 205y: extracellular expressionof a novel OST-lipase gene. Protein Expr Purif 2006;49:190–5.

[37] Ogino H, Mimitsuka T, Muto T, Matsumura M, Yasuda M, Ishimi K, et al. Cloning,expression, and characterization of a lipolytic enzyme gene (lip8) from Pseu-domonas aeruginosa LST-3. J Mol Microbiol Biotechnol 2004;7:212–23.

[38] Rahman RNZRA, Baharum SN, Basri M, Salleh AB. High-yield purification of anorganic solvent-tolerant lipase from Pseudomonas sp. strain S5. Anal Biochem2005;341:267–74.

[39] Klibanov AM. Enzymatic reactions in organic media. Protein engineering: appli-cation in science, medicine and industry. London: Academic Press; 1986. p.341–9.