Ann Microbiol (2011) 61:281–290 DOI 10.1007/s13213-010-0135-z

ORIGINAL ARTICLE

Purification and characterization of a novel extracellular carboxylesterase from the moderately halophilic bacterium Thalassobacillus sp. strain DF-E4

Xiao-Yan Lv & Li-Zhong Guo & Lin Song & Qiang Fu & Kun Zhao & Ai-Xia Li & Xiao-Li Luo & Wei-Dong Lu

Received: 17 February 2010 /Accepted: 17 September 2010 /Published online: 1 October 2010 # Springer-Verlag and the University of Milan 2010

Abstract An extracellular carboxylesterase from the moder- inhibited by PMSF, PAO and DEPC, implying that serine, ately halophile Thalassobacillus sp. strain DF-E4 was cysteine and histidine residues at the active site were purified to 8-fold with 11% recovery and specific activity essential for catalysis. of 2,046 U mg−1. The molecular mass of the native enzyme was approximately 49 kDa as determined by analytical Keywords Carboxylesterase . Moderately halophilic ultracentrifugation, while SDS-PAGE analysis showed a bacterium . Purification . Thalassobacillus single protein band corresponding to a molecular mass of 45 kDa, suggesting that the enzyme was a monomer. Among the pNP (p-nitrophenyl) esters tested, p-nitrophenyl butyrate Introduction

(C4) was hydrolyzed most effectively, with Km and Vmax values of 0.69 mM and 0.84 μmol min−1 mg−1,respectively, Carboxylesterases (EC 3.1.1.1) and lipases (triacylglycerol and the optimum activity occurred at pH 8.5, 40°C and acylhydrolases, EC 3.1.1.3), are the major two groups within 0.5 M NaCl (w/v). The enzyme activity appeared to be stable the family of carboxylic ester hydrolases (EC 3.1.1.-, http:// over pH 6.0–9.5 and up to 45°C for 1 h. Tests of substrate www.chem.qmul.ac.uk/iubmb/), which catalyze the hydroly- specificity and inhibitor susceptibility revealed it was a sis and the synthesis of fatty acid esters, and are widely serine-type carboxylesterase (EC 3.1.1.1), rather than a distributed in animals and plants as well as microorganisms lipase. None of the divalent cations tested enhanced the (Bornscheuer 2002;Jaegeretal.1999). Microbial carbox- enzyme activity, while most of them had no effect or slightly ylesterases and lipases have been recognized as very useful inhibited the activity. The enzyme activity was strongly biocatalysts since they are usually relatively stable in organic solvents, exhibiting excellent stereoselectivity, and no Xiao-Yan Lv and Li-Zhong Guo contributed equally to this work. cofactors are required (Akoh et al. 2004). Although both X.-Y. Lv : L.-Z. Guo : L. Song : K. Zhao : A.-X. Li : carboxylesterase and lipase catalyze ester bonds and exhibit W.-D. Lu (*) the characteristic of α/β hydrolase fold in three-dimensional Laboratory of Applied Enzymology and Biotechnology, structures, they showed widely differences in substrate College of Life Sciences, Qingdao Agricultural University, ’ specificities (Jaeger et al. 1999). Lipases preferentially Qingdao 266109, People s Republic of China ≥ e-mail: [email protected] hydrolyze long chain esters ( 10 carbon atoms), while carboxylesterases, which are rather unspecific, catalyze the W.-D. Lu short chain aliphatic and aromatic esters (<10 carbon atoms) e-mail: [email protected] (Fojan et al. 2000; Anthonsen et al. 1995). Besides that, Q. Fu carboxylesterases are distinguished from lipases by the lack College of Life Sciences, Ocean University of China, of interfacial activation (Kim et al. 1997). Microbial Qingdao 266003, People’s Republic of China carboxylesterases are currently receiving more and more attention for their potential biotechnological applications X.-L. Luo Bioengineering College, Chongqing University, not only in synthetic chemistry and food processing but Chongqing 400044, People’s Republic of China also in environmental monitoring, biodegradation of 282 Ann Microbiol (2011) 61:281–290 industrial wastewater and agricultural pesticides (Panda p-nitrophenyl hexanoate (pNPH), p-nitrophenyl octanoate and Gowrishankar 2005;Lietal.2008;Wheelocketal. (pNPO), p-nitrophenyl decanoate (pNPD), p-nitrophenyl 2008). laurate (pNPL), p-nitrophenyl myristate (pNPM), p-nitro- Extremophiles dwelling in abnormal environments of phenyl palmitate (pNPP), p-nitrophenyl stearate (pNPS), Fast extreme temperature, pressure, pH and salinity are regarded Blue RR salt, O,O-diethyl O-(4-nitrophenyl) phosphate, horse as being a valuable source of novel biocatalysts (Demirjian radish peroxidase, and glucose oxidase were purchased from et al. 2001; Levisson et al. 2009; van den Burg 2003). The Sigma. Polyoxyethylene sorbitan monolaurate (Tween 20), extremozymes they secrete show high stability and solubil- polyoxyethylene sorbitan monooleate (Tween 80), bovine ity under such extreme environments. As a major group of serum albumin (BSA), and p-nitrophenol were purchased extremophiles, moderately halophiles have received grow- from Sangon (Shanghai, China). All other chemicals were of ing attention for their potential applications as a source of analytical grade. salt-adapted enzymes (Ventosa et al. 1998). Moderately halophilic bacteria can grow over a wide range of salinity Strain, growth condition and production of enzyme varied from seawater to highly concentrated brines. The extracellular enzymes they produce show higher catalytic Strain DF-E4 was screened and isolated from the solar activity under a wide range of salt concentrations, especially salterns of Jiaozhou Bay located at the coast of Yellow Sea, at the hypersaline condition that may denature or inactivate China. The lipolytic or esterolytic enzyme it produced on most of the homologous enzymes from non-halophiles, and DSMZ-755 medium (1.71 M NaCl) agar plates containing play an important role in degrading organic pollutants of 1% Tween 80 (v/v), was detected by the formation of saline wastewaters (Oren 2002). opaque halo around the colonies after incubating at 37°C Although a large number of esterases/lipases from non- for 5–7 days. The morphological, biochemical and physi- halophilic bacteria have been reported, only a very few ological characterization of the strain was carried out have so far been studied on those from moderately according to Liu et al. (2005). The 16S rRNA gene of halophiles or extremely halophiles (Sánchez-Porro et al. strain DF-E4 was amplified by polymerase chain reaction 2003; Bhatnagar et al. 2005; Müller-Santos et al. 2009). from genomic DNA using the general bacterial primers 27f Enzymatic properties of lipolytic/esterolytic enzymes from and 1492R. The obtained 16S rRNA gene sequence was halophilic bacteria or archaea had been obtained based on aligned with its closely related neighbor sequences retrieved the results from the crude enzymes (Amoozegar et al. 2008; from GenBank and a phylogenetic tree was constructed as we Boutaiba et al. 2006; Martín et al. 2003; Ozcan et al. 2009; have described before (Lu et al. 2006). Cells were routinely Rohban et al. 2009). Rao et al. (2009) reported that a grown at 37°C in DSMZ-755 medium [http://www.dsmz.de/ halophilic esterolytic enzyme from archeaon Haloarcula microorganisms/] consisting of (g/l): casamino acids, 7.5; marismortui was heterologously overexpressed in Escherichia yeast extract 1.0; Na3-citrate, 3.0; NaCl, 100; MgSO4•7H2O, coli, and its physical states in different salt concentration 20; KCl, 2.0. The pH was adjusted to 7.5 prior to autoclaving. solution were determined from the biophysical point of view. About 3×107 CFU (colony-forming unit) of strain DF-E4 was Hitherto, an electrophoretically pure preparation of inoculated into a 250-ml Erlenmeyer flask with 50 ml of esterase from moderately halophile has not been medium and grown on a reciprocal shaker (200 strokes per obtained. To investigate the lipolytic/esterolytic enzymes min) at 37°C. Growth in the medium was monitored by from moderately halophiles, we screened 155 strains of measuring optical density at 600 nm (OD600) against the fresh moderately halophilic bacteria isolated from the brine medium. After incubation for 48 h, cells were harvested by and sediment of solar saltern on agar plates for centrifugation at 10,600 g at 4°C for 15 min, and the cell-free extracellular lipolytic activity toward Tween 80 (Dong supernatant was used as crude enzyme for further purification. et al. 2009). In this paper, an extracellular esterase from moderately halophile strain DF-E4 was purified and its Preparation of cell fractions enzymatic properties were characterized. To determine the localization of the lipolytic activity, the culture broth was separated into extracellular (E), intracel- Materials and methods lular (I) and membrane-bound (M) fractions, and the lipolytic activity in each fraction was tested. The shake Chemicals and reagents flask cultures incubated at 37°C for 48 h were collected and centrifuged at 10,600 g for 15 min. The supernatant phase DEAE-Sephadex A-25 and Sephadex G-75 were purchased was recovered and kept as the extracellular fraction. The from Amersham Biosciences (Uppsala, Sweden). p-nitro- cell pellet was washed twice with 0.15 M NaCl (w/v) phenyl acetate (pNPA), p-nitrophenyl butyrate (pNPB), solution, suspended in 50 mM Tris-HCl buffer (pH 8.5), Ann Microbiol (2011) 61:281–290 283 and then disrupted by a Xinzhi® cell disruption system Step 2 (Ion exchange chromatography): the samples (Ningbo, China) for 10 min, followed by centrifugation at resulting from Step 1 were applied onto a 10,600g for 10 min. The resulting supernatant was collected DEAE-Sephadex A-25 column (1.6 cm×50 cm) and kept as the intracellular fraction, while the pellet was previously equilibrated with 50 mM Tris-HCl resuspended in 50 mM Tris-HCl buffer (pH 8.5) containing buffer (pH 8.0). The column was washed with 0.2% non-ionic detergent Triton X-100 and mixed for 100 ml of the same buffer, and eluted with 100 ml 20 min at room temperature using a magnetic stir plate. of 50 mM Tris-HCl buffer (pH 8.5) containing Then, they were centrifuged and kept as the membrane- 0.5 M NaCl, then followed with a linear gradient bound fraction. All the cell fractions mentioned above were of NaCl (0–0.5 M) of the same buffer containing stored at 4°C. The activity was measured in all fractions for 0.1% Triton X-100 at a flow rate of 1 mL/min. the lipolytic activity. The esterolytic activity was assayed and the active fractions were pooled and dialyzed twice against Esterase assay 50 mM Tris-HCl buffer (pH 8.0). Step 3 (Gel filtration chromatography): the active frac- Unless otherwise stated, esterase activity was determined tions obtained from Step 2 were concentrated and spectrophotometrically by measuring the amount of p- dialyzed against 50 mM Tris-HCl buffer (pH 8.5). nitrophenol released using p-NPB as the substrate. The The dialyzed solution was then applied to a assay mixture consisted of 1.9 ml of 50 mM Tris-HCl Sephadex G-75 column (3 cm×40 cm) equilibrat- buffer (pH 8.0), 0.05 ml of 40 mM pNPB in acetonitrile. ed with the same buffer. The column was eluted After preincubation for 5 min at 40°C, the reaction was with the same buffer at a flow rate of 12 mL/h. initiated by adding 0.05 ml of the purified enzyme solution The active fractions showing esterase activity were to the reaction mixture and incubated for 10 min at 40°C. The pooled and concentrated by ultrafiltration with increase in absorbance due to formation of p-nitrophenol centrifugal filters with 10-kDa cutoffs (Millipore), (pNP) was measured against a blank with thermally then dialyzed against 50 mM Tris-HCl buffer inactivated enzyme solution (100°C, 10 min), and (pH 8.5). continuously monitored at 400 nm. The concentration of liberated pNP was calculated with the molar extinction coefficients at different pHs, which were determined Protein determination before according to the standard solutions of pNP. The effect of nonenzymatic hydrolysis of substrates was taken The protein concentration was determined by the method of into consideration and its value was subtracted from the Bradford (1976), using bovine serum albumin (Sangon, final measured result. One unit of enzyme activity (U) was China) as the standard. defined as the amount of enzyme required to release 1 μmol of p-nitrophenol per min under the assay Optimum pH and pH stability conditions. The specific activity is expressed in the units of enzyme activity per milligram of protein. The effect of pH on the enzyme activity towards pNPB was studied at 40°C over a pH range from 6.0 to 10.0 by using 50 mM Tris-HCl buffer with different pH values. Analysis Purification of enzyme of the effect of various pH levels on enzyme stability was carried out by incubating the enzyme at room temperature All purification steps were carried out at room temperature, for 1 h with 50 mM of the following buffers: sodium unless otherwise specified. acetate (pH 4.0 to 5.5), sodium phosphate (pH 6.0 to 7.0), Step 1 (Ultrafiltration): 1,000 ml of the cell-free culture Tris-HCl buffer (pH 7.0 to 9.5), or sodium carbonate supernatant was concentrated to 200 ml by an (pH 10-11), and then the residual activity was measured. ultrafiltration hollow fiber cartridges (Amersham The molar extinction coefficients of p-nitrophenol at pH 6, Biosciences) and a membrane with a nominal 6.5, 7.0, 7.5, 8, 8.5, 9, 9.5 and 10.0 were determined to be molecular mass cutoff of 10 kDa. Then, 200 ml of 2,414.3, 5,516.8, 10,783, 15,759, 18,961, 20,189, 20,517, −1 −1 50 mM Tris-HCl buffer (pH 8.5) was added and 20,696, and 20,762 cm M , respectively. again concentrated to 200 ml, and this step was repeated 3 times. Finally, the retentate was Optimum temperature and thermostability lyophilized and dissolved in 50 mM Tris-HCl buffer (pH 8.5), and dialyzed against the same The temperature optimum was determined at pH 8.5 in a buffer overnight. range of 20–80°C using pNPB as substrate. To assay the 284 Ann Microbiol (2011) 61:281–290 enzyme thermal stability, the purified enzyme was incu- Kinetic study bated in 50 mM Tris-HCl (pH 8.5) at different temper- atures (20–80°C) for 1 h and immediately placed on ice. The Michaelis-Menten constant (Km) and the apparent

The residual activities were then assayed under the maximal velocity (Vmax) of the enzyme were determined standard assay conditions by taking the activity at 40°C using various concentrations of p-nitrophenyl butyrate as 100%. ranging from 0.1 to 2 mM under the standard conditions. The program GraphPad prism 5.02 (GraphPad Software, Optimum NaCl concentration and osmostability San Diego, CA, USA) was used for hyberbolic Wtting of the substrate saturation data and for estimating the Km and

The effect of NaCl concentration on esterase activity was Vmax values. also measured in the presence of NaCl ranging from 0 to 2.56 M. To study the effect of NaCl on the enzyme stability, Molecular weight estimation the enzyme was incubated with NaCl in the range of 0–3.41 M at 25°C for 1 h and the residual activity was measured under The molecular weight (MW) of the native enzyme was the standard assay conditions. estimated by analytical ultracentrifugation as described by Measurements were corrected for autohydrolysis of the Stuer et al. (1986), and horse radish peroxidase (3.6 S) and substrate during the process of determining the optimal glucose oxidase (7.9 S) were used as the internal markers. reactive condition. The percentage residual activity was Alternatively, the MW of the protein was also estimated by determined under the standard assay by comparison with sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified enzyme without incubation. (SDS–PAGE) with 12% separating gel under reducing condition described by Sambrook et al. (1989), followed Effects of various additives on the activity of enzyme by staining with Coomassie Brilliant Blue R-250.

For determination of the effects of different additives on Non-denaturing PAGE and zymography enzyme activity, various metal ions (Ca2+,Mg2+,Cu2+,Zn2+, Fe2+,Co2+,Mn2+,Hg2+,Fe3+,Na+,orK+), surfactants (SDS, Native PAGE was performed according to standard techni- Triton X-100, Tween 20, and Tween 80), inhibitors [β- ques (Sambrook et al. 1989) with 8% separating gel and 5% mercaptoethanol (2-ME), ethylenediaminetetraacetic acid stacking gel. SDS and DTT were omitted from the stacking (EDTA), phenylmethylsulfonyl fluoride (PMSF), diethyl and separating gel, and the enzyme applied to the gel was p-nitrophenyl phosphate (E600), phenylarsine oxide (PAO) not boiled. The zymogram staining for the detection of and diethyl pyrocarbonate (DEPC)] and water-miscible esterolytic activity was performed according to Reddy et al. organic solvents [methanol, ethanol, acetonitrile, dimethyl (1970). sulfoxide (DMSO), and dimethyl formamide (DMF)] were incubated with equal volume of the purified enzyme Other assays solutionat25°Cfor1h,containingonlyonekindof additive at a time and the residual enzyme activity was Proteolytic activity was detected by clearing band of casein measured under the standard assay condition. All metals hydrolysis as described by Tesch et al. (1996). To determine ions were used in the chloride form except for Mn2+ and whether LIP4 has a lipase activity, rhodamine B/trioleoyl- Zn2+, which were assayed in the sulfate form. The final glycerol plate assay (Kouker and Jaeger 1987 and pH-stat concentration of the additive was as follows: 1 mM metal method (Isobe and Wakao 1996) were employed. Activities ion, 1 mM or 5 mM inhibitor, 0.2% surfactant, and 10% presented represent the average of measurements conducted (v/v) organic solvent. in triplicate.

Substrate specificity Results and discussion The substrate specificity of the purified enzyme was studied by a spectrophotometric assay with various p-nitrophenyl esters of Screening and identification of Thalassobacillus sp. strain fattyacid(C2-C18, at a final concentration of 10 mM). 0.1% DF-E4 Triton X-100 was added in the case of p-NPO, while 2% 2-propanol was included when p-NPP was used as We screened various moderately halophilic bacteria show- substrate in order to solubilize the substrate. Data were ing high lipolytic activity on agar plates towards Tweens expressed as a percentage of the maximal activity obtained from solar soltern of Jiaozhou Bay, China (Dong et al. with pNPB (100% activity). 2009). Strain DF-E4, which exhibited relatively high Ann Microbiol (2011) 61:281–290 285 hydrolytic activity toward Tween 80, was chosen for further Most of the microbial esterases are usually considered as study. It is a motile, aerobic spore-forming rod. Indole, inducible enzymes, their production being promoted by

H2S, methyl red and Voges–Proskauer tests are negative. their substrates (Donaghy and Mckay 1992; Isobe and Acid is produced from trehalose, D-fructose, D-mannose, Wakao 1996; Lloyd et al. 1971). When Tween 80 was D-glucose, and maltose. Colonies are uniformly round, omitted from the growth medium, the lipolytic activity from circular, regular, convex and cream-coloured on DSMZ-755 strain DF-E4 could still be detected from fractions E, I and medium, growing in a wide range (0.5–18%, w/v) of salt M, indicating that the extracellular esterase is constitutively concentrations with optimum growth at 5–10% (w/v) NaCl. produced irrespective of the presence of inducer (Tween No growth in the absence of NaCl. Phylogenetic analysis 80). The release of intracellular lipolytic enzyme due to the based on 16S rRNA gene sequence comparisons revealed that cell lysis could be neglected, because most of the cells strain DF-E4 (accession number in GenBank: GU138121) fell formed spores at this stage (data not shown), thus the within the branch encompassing members of the genus production of extracellular lipolytic enzyme was closely Thalassobacillus and was most closely related to Thalasso- related to the cell growth. bacillus devorans G-19T (99.5% 16S rRNA gene sequence similarity) (Fig. 1). But oxidase test of strain DF-E4 is Enzyme purification positive, which is different from that of Thalassobacillus devorans G-19T, indicating that they were not the same The results of the purification procedure are summarized in strain. Thus, strain DF-E4 was named as Thalassobacillus Table 1. By this purification procedure, the esterase was sp. strain DF-E4. purified 8-fold over the crude enzyme, with an overall recovery of 11%, and had a specific activity of 2,046 U mg−1 Localization and production of lipolytic enzyme from strain towards pNPB. Although the first step, ultrafiltration, did DF-E4 not increase the specific activity, the salt concentration in the supernatant efficiently decreased. We also noticed that As shown in Fig. 2, the lipolytic activity was found the enzyme had strong non-specific adsorption to hollow predominantly in fraction E, while only minor activity was fiber, which led to the decrease of the purification yield also measured in fractions M and I. These results strongly (from 100 to 44.89%) after ultrafiltration. The second step, suggested that the activity is localized at the culture through a DEAE column, was the most efficient in isolating supernatant. The production of extracellular lipolytic enzyme the esterase, as the specific activity after this step increased during the growth of strain DF-E4 was also determined 7.75-fold relative to the crude exact. Because there were (Fig. 2). The pNPB-hydrolysing activity in the culture strong hydrophobic interactions between the lipolytic brothwithoutTween80couldnotbedetectedattheearly enzyme and the gel matrix, various detergents were assayed exponential phase, and then increasedslowlyinthelate and 0.1% Triton X-100 proved to be the most effective in exponential phase from 0.74 U/ml at 12 h to 4.92 U/ml at desorbing the enzyme. The DEAE-Sephadex A-25 step 24 h, reaching the maximum activity of 7.28 U/ml in the removed most of the contaminating protein before the middle stationary phase (40 h). The lipolytic activity in the esterase was eluted with 0.1% Triton X-100. The third step, culture broth remained constant at least 10 h. After that with a Sephadex G-75 column, purified the enzyme to time, the activity decreased from 7.24 U/ml at 50 h to homogeneity and slight increased the specific activity. The 4.63 U/ml at 70 h. purified lipolytic enzyme from strain DF-E4, named as

Fig. 1 Neighbor-joining DF-E4 stain (GU138121) tree based on 16S rRNA gene 82 99 Thalassobacillus devorans G19.1 (AJ717299) sequences showing the positions 96 T of strain DF-E4 and other relat- Thalassobacillus cyri HS286 (FM864226) T ed taxa. Numbers at nodes are 97 Thalassobacillus hwangdonensis AD-1 (EU817571) percentage bootstrap values Halobacillus mangrovi MS10T (DQ888316) 99 based on 1,000 replications; Halobacillus yeomjeoni MSS-402T (AY881246) 70 only values greater than 50% are T 66 Halobacillus seohaensis ISL-50 (EF612763) shown. Bar 0.02 substitutions 72 89 Halobacillus salsuginis JSM078133T (FJ456889) per nucleotide position Sediminibacillus halophilus EN8dT (AM905297) 96 Lentibacillus salis BH113T (AY762976) Bacillus subtilis ATCC 6633 (AB018486) Salsuginibacillus kocurii CH9dT (AM492160) Alicyclobacillus acidocaldarius DSM 446T (X60742)

0.02 286 Ann Microbiol (2011) 61:281–290

activity decreased to 17% of its original activity after incubation at acetate buffer (pH 5.0) for 1 h.

Effect of temperature on enzyme activity and stability

As shown in Fig. 4b, LIP4 was very active between 30 and 50°C with an optimal temperature of 40°C, with about 32 and 71% activity at 20 and 30°C, respectively. LIP4 was stable at temperature below 45°C and lost about 36% of its activity when incubated for 60 min at 50°C, while the enzyme was completely inactivated at 80°C. The half-life of the enzyme was 103 min at 50°C, 36 min at 55°C, and 9 min at 60°C without any additive. In addition, the Fig. 2 Cell growth (■) and extracellular (fraction E, ♦), intracellular purified esterase was stable for at least 4 weeks when stored (fraction I, □), and membrane-bound (fraction M, ▲) lipolytic activity in 50 mM Tris-HCl buffer (pH 8) at 4°C (data not shown). of Thalassobacillus sp. stain DF-E4 grown in DSMZ-755 medium containing 10% NaCl (without Tween 80) after incubation at 37°C for Effect of NaCl concentration on enzyme activity 48 h and stability

LIP4, showed a single protein band on a SDS-PAGE gel The enzyme activity was also measured in the presence of (Fig. 3a) and a single activity band on a native-PAGE gel different NaCl concentrations (Fig. 4c). Interestingly, after zymogram activity staining (Fig. 3b). although the salt (NaCl) limit for strain DF-E4 was only 3 M NaCl, the enzyme was quite stable up to 4 M NaCl and Molecular mass retained 90% of the original activity even after 12 h incubation. In the absence of NaCl, LIP4 still showed The apparent molecular mass of the esterase, calculated by relatively high activity, indicating it was a halotolerant SDS-PAGE, was 45 kDa, while the native molecular mass enzyme not a halophilic one. LIP4 displayed extreme of the esterase was estimated to be 49 kDa by analytical stability at different NaCl concentrations even up to 4 M ultracentrifugation. Those results suggested that the enzyme NaCl, and retained almost all its original activity after was a monomer, consisting of a single polypeptide chain incubation for 1 h (Fig. 4c). These features are unique and with 45 kDa. make it a very attractive enzyme in biotechnological implication with salt concentration between 0.5–1.71 M. It Effect of pH on enzyme activity and stability is known that extracellular hydrolytic enzymes produced by extremely halophilic bacteria usually need high-salt con- Unlike many other esterases with a low pH-activity centration for stability and optimal activity, and their optimum (around pH 6), LIP4 is optimally active at alkaline activity was lost at such salinity (Bonete et al. 1996), while pH (Fig. 4a). The enzyme was active in the pH range from the activity of enzymes from non-halophilic bacteria was 6 to 10, exhibiting high activity at alkaline pH level, with obviously inhibited to varying degrees. an optimal pH of 8.5 on pNPB. The enzyme retained over 77% of its maximum activity at pH 8, but only 18% at Substrate specificity pH 6.0. The activity at higher pH values (pH >10) was not tested because of spontaneous hydrolysis of pNPB (Tesch Although the enzyme could hydrolyze a wide range of p-NP et al. 1996). LIP4 had a broad pH stability in the three esters from C2 to C10 (Table 2), the highest activity was − different buffers covering the pH range of 6–10, but the observed with pNPB (2,046 U mg 1). The activity declines

Table 1 Purification of carboxylesterase from the Step Volumn Total protein Total activity Specific activity Yield Purification a −1 culture supernatant of strain (ml) (mg) (U) (U mg ) (%) fold DF-E4 Culture supernatant 1,000 28.55 7,200 252 100 1.00 Ultrafiltration 200 12.57 3,232 257 44.89 1.02 DEAE-Sepharose A-25 5 0.76 1,485 1,954 20.63 7.75 a Lipolytic activity was measured Sephadex G-75 3 0.39 798 2,046 11.08 8.11 using pNPB as the substrate Ann Microbiol (2011) 61:281–290 287

M 1 2 1 2 a b

Fig. 3 SDS-PAGE (a) and non-denaturing PAGE (b) of the purified esterase from Thalassobacillus sp. stain DF-E4. a M molecular weight markers, lane 1 crude extracts after ultrafiltration, lane 2 the purified enzyme. The masses of the molecular weight markers were: β- galactosidase (116 kDa), bovine serum albumin (66.2 kDa), ovalbu- min (45 kDa), lactate degydrogenase (35 kDa), REase Bsp981 (25 kDa), β-lactoglobulin (18.4 kDa) and lysozyme (14.4 kDa). b lane 1 the extracellular esterase activity in supernatant after ultrafil- tration, lane 2 the esterase activity of the purified enzyme

with longer chain-length, reaching 78% activity with pNPH and 46% with pNPO, respectively. No activity was detected on pNP ester substrates with an acyl chain length greater than C12. This phenomenon was consistant with those of other esterases. The purified enzyme also hydrolyzes various aliphatic and aromatic carboxylesters, as shown by zymo- gram staining with α-napthyl acetate and β-napthyl acetate. Substrates for protease and lipase were not hydrolyzed Fig. 4 Effects of pH, temperature, and NaCl concentration on activity by the purified enzyme, so the purified enzyme is an and stability of LIP4. a Effects of pH on activity (□) and stability esterase, rather than a protease or a lipase. Although it (filled symbols) of LIP4. For the pH stability, the enzyme was had been reported that some esterases also showed preincubated at room temperature for 1 h in the following buffer: acetate buffer (filled diamand,pH4–5.5), sodium phosphate buffer proteolytic activity at the same time (Frenette et al. (filled square,pH6–7), Tris-HCl buffer (filled triangle,pH7–9.5) and 1986;Salmassietal.1992), a negative result was obtained sodium carbonate buffer (filled circle,pH10–11). Relative activity is by casein digestion assay for LIP4, indicating that the expressed as percentage of the maximum activity, while residual enzyme had no proteolytic activity. activity was calculated by taking the activity at pH 8.5 as 100%. b Effect of temperature on activity (♦) and stability (■) of LIP4. Relative activity is expressed as percentage of the maximum activity, while Kinetic parameters residual activity was calculated by taking the activity at 40°C as 100%. c Effect of NaCl concentration on activity (♦) and stability (■) A kinetic analysis was conducted using pNPB, and of LIP4. Relative activity is expressed as percentage of the maximum activity (50 mM Tris-HCl, pH 8.5, 3% NaCl, 40°C), while residual kinetic parameters, such as Km (0.69±0.07 mM), activity was calculated by taking the activity at 3% NaCl (50 mM −1 −1 Vmax(0.84±0.03 μmol min mg ), were calculated from Tris-HCl, pH 8.5, 40°C) as 100% the measured activity. 288 Ann Microbiol (2011) 61:281–290

Table 2 Hydrolyzing activity of LIP4 toward p-NP esters enzyme activity was not affected or slightly inhibited by 2+ 2+ 2+ 2+ 2+ 2+ Substrates Relative activity (%)a Mg ,Ca ,Mn ,Co ,Hg and Zn , and the residual activity decreased to 23, 51 and 76%, respectively, in the p-NP acetate (C2) 89±1.7 presence of Fe3+,Fe2+, and Cu2+, while metal chelator like p-NP butyrate (C4) 100 EDTA had no influence on the activity, suggesting that no p-NP hexanoate (C6) 78±1.9 metal ions are needed. Furthermore, low concentration + + p-NP octanoate (C8) 46±1.1 (1 mM or 10 mM) of Na or K had no obvious effects on + p-NP decanoate (C10) 14±1.2 the esterase activity, whereas high concentration of Na or + p-NP laurate (C12) ND K (500 mM) stimulated the activity by over 12%. p-NP myristate (C14) ND Incubation with detergents, such as SDS and Triton X- p-NP palmitate (C16) ND 100, slightly enhanced the enzyme activity, while Tween 20 p-NP stearate (C18) ND and Tween 80 had no effect on the activity. Trioleoylglycerol b ND The effects of some known enzyme inhibitors on the Casein ND esterase activity are summarized in Table 4. The enzyme activity was not affected by the addition of β-mercaptoethanol, ND Not detectable which implied that the enzyme either did not have a The specific activity toward p-nitrophenyl butyrate (C4) corresponding to disulfide bonds, or if present, the disulfide bonds were −1 2,046.15 U mg was defined as 100%. The data represent the mean not essential for activity, whereas the activity was values of three experiments completely inhibited by PMSF, a serine protease inhib- b Substrate specificity toward olive oil was performed as described by Tesch et al. (1996) itor, suggesting that the active-site serine residue is necessary for catalysis. In addition, the enzyme activity was also strongly inhibited by DEPC, a histidine Effect of various additives on enzyme activity modifier, and PAO, a cysteine modifier, respectively, indicating that the enzyme inactivation could be attribut- Table 3 showed the effects of various metal ions and ed to the modification of histidine residue (Wragg et al. detergents on the esterase activity. Of the divalent cations 1997) and cysteine residue. The catalytic activity was also tested, none of them stimulated the activity of enzyme, The strongly inhibited in the presence of diethyl p-nitrophenyl phosphate (E600). On the basis of the broad specificity to Table 3 Effects of various metal ions and detergents on LIP4 p-nitrophenyl esters ranging from C2 to C10, sensitivity to PMSF and diethyl p-nitrophenyl phosphate, and the Reagents Concentration Residual activity (%) criterion by Oosterbaan and Janz (1965), we classified the enzyme as B-type esterase or carboxylesterase. Control 100 The effects of water-miscible organic solvents on the Ca2+ 1 mM 97±0.8 activity were also investigated (Table 5). LIP4 was highly Mg2+ 1 mM 98±0.7 stable in 10% ethanol, methanol, acetonitrile, and DMF, Cu2+ 1 mM 76±1.1 whereas the activity decreased to 55.9% of its original Zn2+ 1 mM 92±0.9 activity in the presence of DMSO. Fe2+ 1 mM 51±1.3 Co2+ 1 mM 95±2.1 Mn2+ 1 mM 93±1.4 Hg2+ 1 mM 95±1.7 Table 4 Effects of protein inhibitors on LIP4 Fe3+ 1 mM 23±2.1 Inhibitors Residual activity (%)a Na+ 1 mM 99±1.6 500 mM 116±1.3 1mM 5mM K+ 1 mM 99±1.1 500 mM 112±1.4 EDTA 99±1.3 34.6±1.5 SDS 0.2% (w/v) 108±1.8 PMSF 0 0 Triton X-100 0.2% (w/v) 120±2.3 2-ME 109±1.9 145±1.6 Tween 20 0.2% (w/v) 98±1.6 DEPC 14±0.8 8±1.1 Tween 80 0.2% (w/v) 99±1.5 PAO 39±1.2 10±1.7 E600 16±1.4 0 a Residual activity was determined under the standard assay conditions and expressed as the percentage of the control value (with no addition). The a The activity of enzyme in the absence of protein inhibitor was taken as data represent the mean values of three experiments the 100% level. The data represent the mean values of three experiments Ann Microbiol (2011) 61:281–290 289

Table 5 Effects of organic solvents on LIP4 Boutaiba S, Bhatnagar T, Hacene H, Mitchell DA, Baratti JC (2006) Preliminary characterization of a lipolytic activity from an Organic solvents Concentration (%, v/v) Residual activity (%)a extremely halophilic archaeon, Natronococcus sp. J Mol Catal B Enzym 41:21–26 Acetonitrile 10 82.8±1.2 Bradford MM (1976) A rapid and sensitive method for the Ethanol 10 75.9±0..5 quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254 Methanol 10 80.7±1.4 Demirjian DC, Morís-Varas F, Cassidy CS (2001) Enzymes from DMSO 10 55.9±0.8 extremophiles. Curr Opin Chem Biol 5:144–151 DMF 10 74±1.1 Donaghy J, Mckay AM (1992) Extracellular carboxylesterase activity of Fusarium graminearum. Appl Microbiol Biotechnol 37 a Equal volume of the purified enzyme was incubated with each of 20% (6):742–744 organic solvent dissolved in 50 mM Tris-HCl (pH 8.5, 3% NaCl) for 1 h at Dong W, Guo LZ, Li CC, Wang L, Lu WD (2009) Isolation and 25°C. Remaining activity was determined under the standard assay characterization of a moderately halophilic bacterium high- conditions and expressed as the percent of the control value (with no producing esterase. Microbiology (in Chinese) 36:479–483 addition of organic solvent). The data represent the mean values of three Fojan P, Jonson PH, Petersen MT, Petersen SB (2000) What experiments distinguishes an esterase from a lipase: a novel structural approach. Biochimie 82:1033–1041 Frenette G, Dubé JY, Tremblay RR (1986) Proteolytic activity of arginine esterase from dog seminal plasma towards actin and Conclusion other structural proteins. Int J Biochem 18:697–703 Isobe K, Wakao N (1996) Tween-hydrolyzing esterase produced by In this study, LIP4, a novel carboxylesterase from Thalasso- acidiphilium sp. J Gen Appl Microbiol 42:285–295 bacillus sp. strain DF-E4, was characterized. This is the first Jaeger KE, Dijkstra BW, Reetz MT (1999) Bacterial biocatalysts: Molecular biology, three-dimensional structures and biotechno- report on purification and characterization of a carboxyles- logical applications of lipases. Annu Rev Microbiol 53:315–351 terase from moderately halophilic bacterium. Our study Kim KK, Song HK, Shin DH, Hwang KY (1997) Crystal structure of demonstrated that LIP4 is a novel, halo- and alkali-tolerant carboxylesterase from Pseudomonas fluorescens,anα/β hydro- – carboxylesterase and can be purified by the combination of lase with broad substrate specificity. Structure 5:1571 1584 Kouker G, Jaeger KE (1987) Specific and sensitive plate assay for ultrafiltration, anion exchange chromatography, and gel bacterial lipases. Appl Environ Microbiol 53:211–213 permeation chromatography. The purified enzyme is a Levisson M, van der Oost J, Kengen SWM (2009) Carboxylic ester monomer, with a molecular mass of 45 kD. Furthermore, it hydrolases from hyperthermophiles. Extremophiles 13:567–581 is very stable at different NaCl concentrations (0–3.4 M) and Li G, Wang K, Liu YH (2008) Molecular cloning and characterization – of a novel pyrethroid-hydrolyzing esterase originating from the active in a broad range of pH (6.0 10.0) and in hypersaline Metagenome. Microb Cell Factories 7:38–48 conditions. In addition, LIP4 also exhibited activity in Liu WY, Zeng J, Wang L, Dou YT, Yang SS (2005) Halobacillus solutions including 10% organic solvents. dabanensis sp. nov. and Halobacillus aidingensis sp. nov., isolated from salt lakes in Xinjiang, China. 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