- Research article
- Open Access
Purification and characterization of an extracellular esterase with organic solvent tolerance from a halotolerant isolate, Salimicrobiumsp. LY19
© Xin and Hui-Ying; licensee BioMed Central Ltd. 2013
- Received: 10 March 2013
- Accepted: 29 October 2013
- Published: 10 December 2013
Halotolerant bacteria are excellent sources for selecting novel enzymes. Being intrinsically stable and active under high salinities, enzymes from these prokaryotes have evolved to function optimally under extreme conditions, making them robust biocatalysts with potential applications in harsh industrial processes.
A halotolerant strain LY19 showing lipolytic activity was isolated from saline soil of Yuncheng Salt Lake, China. It was identified as belonging to the genus of Salimicrobium by 16S rRNA gene sequence analysis. The extracellular enzyme was purified to homogeneity with molecular mass of 57 kDa by SDS-PAGE. Substrate specificity test revealed that the enzyme preferred short-chain p-nitrophenyl esters and exhibited maximum activity towards p-nitrophenyl butyrate (p-NPB), indicating an esterase activity. The esterase was highly active and stable over broad temperature (20°C-70°C), pH (7.0-10.0) and NaCl concentration (2.5%-25%) ranges, with an optimum at 50°C, pH 7.0 and 5% NaCl. Significant inhibition of the esterase was shown by ethylenediaminetetraacetic acid (EDTA), phenylmethylsulfonyl fluoride (PMSF) and phenylarsine oxide (PAO), which indicated that it was a metalloenzyme with serine and cysteine residues essential for enzyme activity. Moreover, the esterase displayed high activity and stability in the presence of hydrophobic organic solvents with log Pow ≥ 0.88 than in the absence of an organic solvent or in the presence of hydrophilic solvents.
Results from the present study indicated the novel extracellular esterase from Salimicrobium sp. LY19 exhibited thermostable, alkali-stable, halotolerant and organic solvent-tolerant properties. These features led us to conclude that the esterase may have considerable potential for industrial applications in organic synthesis reactions.
- Organic solvent tolerance
Esterases (EC 18.104.22.168) represent a family of hydrolases that catalyze the hydrolysis and formation of short-chain fatty acid esters. Because of their broad substrate specificity, highly chemo-, regio-, enantio-selectivity and non-aqueous catalytic properties , they have diverse applications in biotechnology which are used as additives in laundry detergents and stereo-specific biocatalysis in pharmaceutical production . However, most industrial processes often require aggressive conditions, which can lead to inactivation of the enzymes. In this sense, novel esterases with better catalytic efficiency and specific properties suitable for special reaction conditions are highly demanded . Extremophiles are bizarre microorganisms that can grow and thrive in extreme environments . Among the extremophiles, halotolerant microorganisms, able to live in saline environments, are good candidates for selecting novel enzymes . Enzymes from these prokaryotes can function optimally under extreme conditions, making them robust biocatalysts with potential applications in harsh industrial processes .
The possibility of using enzymes in organic solvents offers numerous advantages when compared to traditional aqueous enzymology, such as high solubility of hydrophobic substrates and reduced water activity which alters the hydrolytic equilibrium and elimination of microbial contamination . Esterases are widely used as biocatalysis due to their ability to catalyze not only the hydrolysis of triacylglycerides in aqueous solutions, but also enantio-selective synthetic reactions in organic media. Therefore, esterase that remains active and stable in the presence of organic solvents might be very useful for biotechnological applications in which such solvents are used. Since salt tends to greatly reduce water activity like organic solvents, enzymes from halotolerant microorganisms may become the choice for biocatalytic processes performed in low water activity environments . So far, numerous organic solvent-tolerant microbial esterases have been reported ; however, published studies on the enzymatic behavior of esterases from halotolerant bacteria in non-aqueous media are scarce. Recently, screening of lipolytic activity was carried out among microorganisms from Yuncheng Salt Lake, China. In this work, a halotolerant strain LY19 showing lipolytic activity was isolated and identified. Meanwhile, purification and characterization of its extracellular esterase, especially its activity and stability in the presence of organic solvents, were also reported.
Strain identification and production of extracellular esterase
Results of the esterase purification from Salimicrobium sp. LY19
Total activity (U)
Total protein (mg)
Specific activity (U/mg)
Effect of metal ions and chemical reagents
Effects of metal ions and chemical reagents on esterase activity
Residual activity (% ± SD)
138.9 ± 1.6
97.6 ± 0.8
92.7 ± 1.7
91.8 ± 0.9
80.8 ± 1.8
97.5 ± 1.6
18.4 ± 0.4
97.7 ± 1.3
8.9 ± 0.2
11.1 ± 0.2
92.1 ± 1.1
8.4 ± 0.3
91.3 ± 1.5
Effects of temperature, pH and NaCl concentration
Effect of organic solvents
Activity and stability of the esterase in different organic solvents
Residual activity (%)
100 (3 d)c
81.1 (2 d)
81.4 (1 d)
68.9 (1 d)
18.4 (<1 h)
11.9 (<1 h)
15.5 (<1 h)
17.3 (1 d)
67.9 (4 d)
62.8 (4 d)
87.3 (4 d)
64.1 (>5 d)
65.1 (>5 d)
86.6 (>5 d)
73.9 (>5 d)
119.1 (>5 d)
In recent years, the ability of extremophiles to grow under harsh conditions makes them very attractive for screening of novel enzymes with unusual properties. In this paper, a halotolerant strain LY19 showing lipolytic activity was isolated and identified as belonging to the genus Salimicrobium by 16S rRNA gene sequence analysis (Figure 1). Esterase activity was detected at the mid-exponential phase of bacterial growth and reached a maximum level during the stationary phase. Besides, supplementation of tween-20 as an inducer was required for esterase production. Thus, it was an inducible enzyme secreted into the culture medium. This finding was similar to the lipase from Penicillium sp. DS-39 . Molecular mass of the esterase was estimated to be 57 kDa, which was higher than other halophilic esterases: 45 kDa from Thalassobacillus sp. strain DF-E4  and 50 kDa from Haloarcula marismortui. Substrate specificity test revealed the enzyme was an esterase, as it preferred short-chain p-NP esters (C2 and C4) and had very low ability to hydrolyze long-chain esters. Furthermore, lipolytic activity on Rhodamine B agar plates  showed it could not hydrolyze olive oil. The esterase from halotolerant bacterium Pelagibacterium halotolerans B2T was also reported to show similar substrate specificity . The esterase activity was greatly inhibited by the metal chelator EDTA, indicating hat it was a metalloenzyme. The presence of PMSF (a serine modifier) and PAO (a cysteine modifier) led to the inactivation of the enzyme, which meant that serine and cysteine residues were essential for its catalytic function. Such structural characteristics have not been previously reported for other esterases.
The extracellular esterase can be classified as moderately thermoactive enzyme with optimal activity at 50°C. However, it was worthy noting that the enzyme showed high stability under temperatures below 70°C. After incubation at 80°C for 2 h, about 40% activity still retained. In contrast, other halophilic esterases described previously were inactive under temperatures higher than 70°C [12, 15]. Excellent thermostability may favor its application in processes that lead to inactivation of enzymes with increasing temperature. Optimal pH for the esterase was found to be 7.0, which was similar to the intracellular esterase of Halobacterium sp. NRC-1 exhibiting its maximum activity at pH 7.5 . The esterase showed good stability in the pH range 7.0-10.0, indicating its alkali-stable property. Similarly, a carboxylesterase from Thalassobacillus sp. strain DF-E4 was reported to be active and stable in neutral to alkaline pH range . Alkaline enzymes have received considerable interest because of their tremendous potentiality in industrial processes . Furthermore, the esterase from strain LY19 showed strong tolerance to NaCl. It was highly active and stable in the presence of NaCl concentrations from 2.5 to 25%. This unique property suggested it was a halotolerant enzyme. Similar extreme halotolerance has been observed in other esterases from halophiles [12, 17]. Like most halophlic enzymes which were inactive under low salt concentrations , the esterase activity reduced drastically in the absence of NaCl, indicating it required salt for maintaining enzyme activity.
High activity and stability of enzyme in organic solvents is an essential prerequisite for applications in organic synthesis . Effect of organic solvents on the esterase from Salimicrobium sp. LY19 was shown in Table 3. Significant esterase inactivation in the presence of hydrophilic organic solvents, such as methanol, acetonitrile, ethanol and acetone, was observed, which maybe due to the stripping-off of crucial bound-water monolayer from the enzyme molecule essential for its activity . Although esterases are diverse in their sensitivity to solvents, there is a tendency for hydrophilic solvents to cause more significant enzyme inactivation than hydrophobic solvents . Interestingly, the esterase activity increased greatly in the presence of isooctane. This activation could be explained that organic solvent molecules could interact with hydrophobic amino acid residues present in the lid that covers the catalytic site of the enzyme, thereby maintaining the esterase in its open conformation and conducting to catalyze . Besides, in the presence of hydrophobic organic solvents, the half-lives of the esterase were much longer than in the absence of the solvents or in the presence of hydrophilic solvents. Together these results indicated that, in contrast to the organic solvent stability of some lipases , which had no relationship with the polarity of the organic solvents, the stability of the esterase from strain LY9 was probably dependent on the polarity of the solvents, which increased only in non-polar organic solvents with higher log Pow values. Similar findings were also observed in some halophilic lipases, which showed high tolerance towards non-polar hydrophobic solvents with significant instability in polar solvents [11, 22].
In the present investigation, a halotolerant strain Salimicrobium sp. LY19 producing extracellular esterase was isolated and identified. The esterase was purified to homogeneity with molecular mass of 57 kDa. It was a novel metalloenzyme with serine and cysteine residues essential for enzyme catalysis. Also, considering its thermostable, alkali-stable, halotolerant, and organic solvent-tolerant properties, the esterase might be potentially useful for future applications in biotechnological processes under harsh conditions.
Strain isolation, identification and esterase production
The strain LY19 was isolated from the saline soil of Yuncheng, China. Production of esterase was performed in the complex medium (CM) containing (g/l): casein peptone 7.5; yeast extract 10.0; sodium citrate 3.0; MgSO4•7H2O 20.0; KCl 2.0; FeSO4•7H2O 0.01; NaCl 40.0 and pH 7.0. Morphological, physiological and biochemical characteristics of strain LY19 were studied either on CM agar plate (2% agar, w/v) or in CM broth plus 4% NaCl. 16S rRNA gene was amplified using the general bacterial primers 8 F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACCTTGTTACGACTT-3′), and has been deposited to GenBank with the accession number HQ683737. The strain LY19 was deposited at China Center of Industrial Culture Collection with the accession number CICC 10492. The strain LY19 was incubated aerobically in CM broth supplemented with 1% (v/v) tween-20 at 37°C for 48 h with shaking. After centrifugation at 6,000 g for 15 min, cell-free supernatant was used for esterase purification.
The culture supernatant was treated with solid ammonium sulphate to 60% saturation and stirred overnight at 4°C. The precipitate collected by centrifugation was dissolved in buffer A (20 mM Tris–HCl containing 5% NaCl, pH 7.0). After dialysis against buffer A overnight, the sample was applied to a DEAE- Cellulose column (2.5 cm × 30 cm). The column was eluted with a linear gradient of 0–1 M NaCl in Tris–HCl buffer at a flow rate of 0.6 ml/min. Active fractions showing esterase activity were pooled and concentrated by freeze-drying. The resulting concentrate was dissolved in buffer A, and then loaded on a Sephacryl S-100 gel filtration column (1.6 cm × 60 cm). The bound protein was eluted with buffer A at a flow rate of 0.2 ml/min. Active fractions were pooled and used for further characterization. The molecular mass of the purified enzyme was estimated using the same column, which was calibrated previously with bovine serum albumin (BSA) (67 kDa), ovalbumin (43 kDa), bovine carbonic anhydrase (29 kDa) and cytochrome C (12.4 kDa). Blue Dextran was used to determine the void volume of the column. Protein concentration was determined by the method of Bradford , using bovine serum albumin as standard. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to determine the purity and molecular mass of the esterase on 12% (w/v) polyacrylamide gel . After electrophoresis, the gel was stained with Coomassie Brilliant Blue R-250.
Enzyme activity assay
The esterase activity was determined using p-NPB (p-nitrophenyl butyrate) as substrate. 0.4 ml of substrate solution (10 mM dissolved in 2-propanol) was mixed with 3.6 ml of Tris–HCl buffer (20 mM, pH 7.0) containing 5.8% NaCl. The enzymatic assay was initiated by adding the enzyme solution (0.2 ml) to the reaction mixture and incubated at 50°C for 10 min. The amount of p-nitrophenol (p-NP) released was measured at 410 nm against a blank. One unit (U) of esterase activity was defined as the amount of enzyme liberating 1 μmol of p-NP per minute under the assay conditions. The specific activity was expressed as the units of enzyme activity per milligram of protein.
To determine the substrate specificity of the esterase, p-nitrophenyl (p-NP) esters with different chain lengths (acetate, C2; butyrate, C4; hexanoate, C6; octanoate, C8; decanoate, C10; laurate, C12; myristate, C14; palmitate, C16) were added to the reaction mixture with the final concentration of 1 mM, respectively, and then the released amount of p-nitrophenol was measured at 410 nm. Data were expressed as the percentage of the observed maximal activity obtained with p-NPB ester (C4).
Effects of metal ions and chemical reagents
Effects of different metal ions and chemical reagents [ethylenediaminetetraacetic acid (EDTA), phenylmethylsulfonyl fluoride (PMSF), phenylarsine oxide (PAO), diethyl pyrocarbonate (DEPC), β-mercaptoethanol] on the esterase activity were examined by pre-incubating the enzyme with them at 30°C for 1 h, respectively, and then residual activity was determined under the standard assay conditions. Esterase activity in the absence of any additives was taken as 100%.
Effects of temperature, pH and NaCl concentration on esterase activity and stability
The temperature optimum of the purified esterase was determined under temperatures from 20°C to 90°C. To assess its thermostability, the enzyme was pre-incubated at different temperatures for 2 h and then residual activity was measured using p-NPB method as described above. Effect of pH on the esterase activity was measured over a pH range of 5.0-10.0. The buffers (20 mM) used were as follows: sodium acetate (pH 4.0-5.5), sodium phosphate (pH 6.0-7.5), Tris–HCl (pH 8.0-9.0) and glycine-NaOH (pH 9.5-10.0). The pH stability was examined by pre-incubating the esterase under different pH at 50°C for 2 h, and residual activity was measured as described above. Effect of NaCl was tested by measuring the esterase activity in the reaction mixture containing different NaCl concentrations (0-25%). To determine its salt stability, the esterase was pre-incubated in Tris–HCl buffer (20 mM, pH 7.0) containing various NaCl concentrations at 50°C for 2 h. The residual activity was measured using the standard assay.
Effect of organic solvents on esterase activity and stability
The effect of organic solvents with different Log Pow values at 20% (v/v) concentration on the purified esterase was determined by incubating the enzyme solution with different organic solvents at 30°C with shaking, respectively. At different time intervals, aliquots were withdrawn and residual activity was measured under the standard conditions. If residual activity was more than 50% after 5 d, half-life was taken as “>5 d”. While activity was less than 50% after 1 h, half-life was taken as “<1 h”.
This work was financially supported by National Natural Science Foundation of China (grants no. 31300002), Natural Science Fund of Shanxi Province (grants no. 2011021031-4) and PhD Start-up Foundation of Yuncheng University (grants no. YQ-2011043).
- Bornscheuer UT: Microbial carboxyl esterase: classification, properties and application in biocatalysis. FEMS Microbiol Rev. 2001, 26: 73-81.View ArticleGoogle Scholar
- Panda T, Gowrishankar BS: Production and applications of esterases. Appl Microbiol Biotechnol. 2005, 67: 160-169. 10.1007/s00253-004-1840-y.View ArticleGoogle Scholar
- Rao L, Zhao X, Pan F, Xue Y, Ma Y, Lu JR: Solution behavior and activity of a halophilic esterase under high salt concentration. PLoS ONE. 2009, 4: e6980-10.1371/journal.pone.0006980.View ArticleGoogle Scholar
- Gomez J, Steiner W: The biocatalytic potential of extremophiles and extremozymes, extremophiles and extremozymes. Food Technol Biotechnol. 2004, 2: 223-235.Google Scholar
- Van den Burg B: Extremophiles as a source for novel enzymes. Curr Opin Microbiol. 2003, 6: 213-218. 10.1016/S1369-5274(03)00060-2.View ArticleGoogle Scholar
- Margesin R, Schinner F: Potential of halotolerant and halophilic microorganisms for biotechnology. Extremophiles. 2011, 5: 73-83.View ArticleGoogle Scholar
- Sellek GA, Chaudhuri JB: Biocatalysis in organic media using enzymes from extemophiles. Enzyme Microb Technol. 1999, 25: 471-482. 10.1016/S0141-0229(99)00075-7.View ArticleGoogle Scholar
- Marhuenda-Egea FC, Bonete MJ: Extreme halophilic enzymes in organic solvents. Curr Opin Biotechnol. 2002, 13: 385-389. 10.1016/S0958-1669(02)00338-5.View ArticleGoogle Scholar
- Doukyu N, Ogino H: Organic solvent tolerant enzymes. Biochem Eng J. 2010, 48: 270-282. 10.1016/j.bej.2009.09.009.View ArticleGoogle Scholar
- Larsen H: Halophilic and halotolerant microorganisms-an overview and historical perspective. FEMS Microbiol Rev. 1986, 39: 3-7. 10.1111/j.1574-6968.1986.tb01835.x.View ArticleGoogle Scholar
- Dheeman DS, Antony-Babu S, Frias JM, Henehan GTM: Purification and characterization of an extracellular lipase from a novel strain Penicillium sp. DS-39 (DSM 23773). J Mol Catal B-Enzym. 2011, 72: 256-262. 10.1016/j.molcatb.2011.06.013.View ArticleGoogle Scholar
- Lv XY, Guo LZ, Song L, Fu Q, Zhao K, Li AX, Luo XL, Lu WD: Purification and characterization of a novel extracellular carboxylesterase from the moderately halophilic bacterium Thalassobacillus sp. strain DF-E4. Ann Microbiol. 2010, 61: 281-290.View ArticleGoogle Scholar
- Kouker G, Jaeger KE: Specific and sensitive plate assay for bacterial lipase. Appl Environ Microbiol. 1987, 53: 211-213.Google Scholar
- Jiang X, Huo Y, Cheng H, Zhang X, Zhu X, Wu M: Cloning, expression and characterization of a halotolerant esterase from a marine bacterium Pelagibacterium halotolerans B2T. Extremophiles. 2012, 16: 427-435. 10.1007/s00792-012-0442-3.View ArticleGoogle Scholar
- Ozcan B, Ozyilmaz G, Cokmus C, Caliskan M: Characterization of extracellular esterase and lipase activities from five halophilic archaeal strains. J Ind Microbiol Biotech. 2009, 36: 105-110. 10.1007/s10295-008-0477-8.View ArticleGoogle Scholar
- Chakraborty S, Khopade A, Biao R, Jian W, Liu XY, Mahadik K, Chopade B, Zhang LX, Kokare C: Characterization and stability studies on surfactant, detergent and oxidant stable α-amylase from marine haloalkaliphilic Saccharopolyspora sp. A9. J Mol Catal B-Enzym. 2011, 68: 52-58. 10.1016/j.molcatb.2010.09.009.View ArticleGoogle Scholar
- Camacho RM, Mateos-Díaz JC, Diaz-Montaño DM, González-Reynoso O, Córdova J: Carboxyl ester hydrolases production and growth of a halophilic archaeon, Halobacterium sp. NRC-1. Extremophiles. 2010, 14: 99-106. 10.1007/s00792-009-0291-x.View ArticleGoogle Scholar
- Madern D, Ebel C, Zaccai G: Halophilic adaptation of enzymes. Extremophiles. 2000, 4: 91-98. 10.1007/s007920050142.View ArticleGoogle Scholar
- Ogino H, Ishikawa H: Enzymes which are stable in the presence of organic solvents. J Biosci Bioeng. 2001, 91: 109-116.View ArticleGoogle Scholar
- Rúa L, Díaz-Mauriño T, Fernández VM, Otero C, Ballesteros A: Purification and characterization of two distinct lipases from Candida cylindracea. Biochim Biophys Acta. 1993, 1156: 181-189. 10.1016/0304-4165(93)90134-T.View ArticleGoogle Scholar
- Ji Q, Xiao S, He B, Liu X: Purification and characterization of an organic solvent-tolerant lipase from Pseudomonas aeruginosa LX1 and its application for biodiesel production. J Mol Catal B-Enzym. 2010, 66: 264-269. 10.1016/j.molcatb.2010.06.001.View ArticleGoogle Scholar
- Lima VMG, Krieger N, Mitchell DA, Fontana JD: Activity and stability of a crude lipase from Penicillium aurantiogriseum in aqueous media and organic solvents. Biochem Eng J. 2004, 18: 65-71. 10.1016/S1369-703X(03)00165-7.View ArticleGoogle Scholar
- Bradford MM: A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976, 72: 248-254. 10.1016/0003-2697(76)90527-3.View ArticleGoogle Scholar
- Laemmli UK: Cleavage of structural proteins during the assembly of the head of becteriophage T4. Nature. 1970, 227: 680-685. 10.1038/227680a0.View ArticleGoogle Scholar
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