Biochemical properties and potential applications of an organic solvent-tolerant lipase isolated from Serratia marcescens ECU1010

Process Biochemistry 43 (2008) 626–633 www.elsevier.com/locate/procbio

Biochemical properties and potential applications of an organic solventtolerant lipase isolated from Serratia marcescens ECU1010 Li-Li Zhao a, Jian-He Xu a,*, Jian Zhao a, Jiang Pan a, Zhi-Long Wang b a

Laboratory of Biocatalysis and Bioprocessing, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China b School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China Received 4 December 2007; received in revised form 21 January 2008; accepted 29 January 2008

Abstract An extracellular lipase was purified to homogeneity with a purification factor of 5.5-fold from a bacterial strain Serratia marcescens ECU1010. The purified lipase is a dimer with two homologous subunits, of which the molecular mass is 65 kDa, and the pI is 4.2. The pH and temperature optima were shown to be pH 8.0 and 45 8C, respectively. Among p-nitrophenyl esters of fatty acids with varied chain length, the lipase showed the maximum activity on p-nitrophenyl myristate (C14). The lipase was activated by some surfactants such as Gum Arabic, polyvinyl alcohol (PVA) and Pg350me, but not by Ca2+. The enzyme displayed pretty high stability in many water miscible and immiscible solvents. This is a unique property of the enzyme which makes it extremely suitable for chemo-enzymatic applications in non-aqueous phase organic synthesis including enantiomeric resolution. Several typical chiral compounds were tested for kinetic resolution with this lipase, consequently giving excellent enantioselectivities (E = 83 >100) for glycidyl butyrate (GB), 4-hydroxy-3-methyl-2-(2-propenyl)-2-cyclopenten-1-one acetate (HMPCA), naproxen methyl ester (NME) and trans-3-(40 -methoxyphenyl) glycidic acid methyl ester (MPGM). # 2008 Elsevier Ltd. All rights reserved. Keywords: Lipase; Serratia marcescens; Organic solvent tolerance; Enantiomeric resolution; Chiral compounds; Biochemical properties

1. Introduction Lipases or triacylglycerol hydrolases (E.C. 3.1.1.3) are enzymes that hydrolyze ester bonds of triglycerides at oil–water interface. In recent years, lipases have emerged as key enzymes in swiftly growing biotechnology, owing to their multifarious properties [1–3]. The main application fields of lipase include detergents, dairy, diagnostics, oil and lipid processing, and biotransformation. Recently, special emphasis is lying on the production of chiral chemicals which serve as basic building blocks in the production of pharmaceuticals and agrochemicals [4]. Substrates of lipase are often insoluble or partially soluble in water and thus the use of organic solvents or organic–aqueous solutions is in favor of some reactions. Use of organic solvents also provides many advantages [5], including: (1) relatively high solubility of substrates; (2) relative ease of products recovery in

* Corresponding author. Fax: +86 21 6425 2250. E-mail address: [email protected] (J.-H. Xu). 1359-5113/$ – see front matter # 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2008.01.023

organic phase; (3) possibility of reducing the degree of undesirable substrate and/or product inhibition in organic solvent-water two-phase systems; and (4) ability to shift the reaction equilibrium toward synthetic direction by continuously removing products with organic solvents in biphasic systems. Unfortunately, despite the advantages that biocatalysis in organic solvent-based systems can bring, the catalytic activities of enzymes in these systems are typically much lower than those in aqueous solutions [6]. Furthermore, in an apparent paradox, protein stability is lower in water-miscible solvents than in hydrophobic solvents. The poor stability in hydrophilic solvents represents a problem for the use of lipases in reactions involving the hydrolysis of esters because of the production of alcohol or acid. Therefore, it is anticipated that the solventtolerant enzyme will be applicable and beneficial as catalysts for reactions in the presence of organic solvents. In our laboratory, an extracellular lipase produced by Serratia marcescens ECU1010 was shown to be a potentially useful biocatalyst for the kinetic resolution of trans-3-(40 methoxyphenyl)glycidic acid methyl ester [()-MPGM], as reported previously [7].

L.-L. Zhao et al. / Process Biochemistry 43 (2008) 626–633

To date, although there were some reports about organic solvent-tolerant lipases, such as lipase from Bacillus species 205y [5] and lipase from Bacillus megaterium CCOC-P2637 [8], no report is available on the organic solvent-tolerant lipase by Serratia strains. In this paper, we report the results on the lipase purification and its properties, especially the enzyme stability against various organic solvents. In addition, the lipase was supposed to be useful for biocatalytic resolution of some chiral compounds based on our previous results on enatioselective hydrolysis of ()-MPGM [7] and ketoprofen [9]. Therefore, further utilization of this lipase for kinetic resolution of more chiral esters was also attempted, affording similar or better enantioselectivities than those reported with other lipases.

2. Materials and methods 2.1. Materials All kinds of p-nitrophenyl esters [10] were prepared from p-nitrophenol and various acids. Naproxen methyl ester [11], trans-3-(40 -methoxyphenyl)glycidic acid methyl ester [()-MPGM] [12], and glycidyl butyrate [13] were also synthesized in our laboratory as described previously. All other chemicals were purchased from various commercial sources and with the highest purity available. DEAE-Toyopearl 650M and Phenyl-Toyopearl 650 M were from Tosoh (Tokyo, Japan). Sephadex G150 was from Pharmacia Fine Chemicals Co. Ultrafiltration membrane system was from Millipore Co. The strain of S. marcescens ECU1010 used in this work was a Gramnegative bacterium stored in our laboratory [7] and currently also deposited at China General Microbiology Collection Center, with an accession number of CGMCC No. 1219. The culture medium was designed according to a reference medium [7]. The culture broth was centrifuged to obtain a clear supernatant, which was used subsequently for enzyme purification.

2.2. Preparation of purified lipase The culture supernatant was concentrated from 1.5 l to 300 ml using an ultrafiltration membrane system (membrane pore size: 30 kDa) after centrifugation of the culture broth at 12,000 rpm for 10 min. Solid powder of ammonium sulfate was slowly added to the concentrated culture supernatant to 35% saturation and then the solution was gently stirred for 1 h. After standing overnight, the resultant precipitate was collected through a micro-filtration membrane (Ø 50 mm). Then the precipitate was dissolved in Buffer A (Tris– HCl, 10 mM, pH 7.5) and applied to a preequilibrated DEAE-Toyopearl 650 M column (Ø 2.5 cm  30 cm), eluted with NaCl gradient from 0.1 to 0.5 M. The fractions with lipase activity were collected, combined, and concentrated to about 1.0 ml by ultrafiltration with Amicon1 Ultra-15 Centrifugal Filter Devices (Millipore). The concentrated solution was added to a Sephadex G150 column (Ø 1.6 cm  100 cm) which has been preequilibrated with the Buffer A. After elution, the fraction possessing the highest activity was pooled, mixed with (NH4)2SO4 up to 2% saturation, and put onto a Phenyl-Toyopearl 650 M column (Ø 0.8 cm  10 cm), already preequilibrated with Buffer A containing (NH4)2SO4 at 2% saturation. The bound protein was eluted from the column with Buffer A, then mixed with lactose, and lyophilized to form a powder. The powder was used for all the following experiments.

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reaction mixture with 30 ml of 100 mM pNPB solution in dimethyl sulfoxide (DMSO) and the variation in absorbance at 405 nm was recorded. One unit of lipase activity was defined as the amount of enzyme releasing 1.0 mmol of pnitrophenol per minute under such conditions. Hydrolytic activities on various p-nitrophenyl esters were measured by a modified lipase assay method. The p-nitrophenyl butyrate was replaced with various p-nitrophenyl esters, respectively. Particularly, for p-nitrophenyl laurate, myristate and palmitate, lipase or blank solution (100 ml) was added to 2.570 ml of 100 mM potassium phosphate buffer (KPB, pH 7.0). After preincubation at 30 8C for 3 min, the reaction was initiated by a quick mixing of the reaction mixture with 300 ml of 10 mM p-nitrophenyl laurate, myristate or palmitate solution in DMSO and the variation in absorbance at 405 nm was recorded.

2.4. Protein concentration and molecular mass determination Protein concentration was determined according to Bradford method [14] using bovine serum albumin as a standard. The molecular mass of the lipase subunit was measured using the method of Laemmli [15] in a 10% (w/v) polyacrylamide slab gel. The molecular mass of the native lipase was measured by gel filtration with TSK-GEL G3000SWXL (30 cm  7.8 mm I.D., 5 mm particles) using FPLC as recorded previously [16]. The reference proteins were: rabbit actin 43 kDa, bovine serum albumin 67 kDa, rabbit phosphorylase b 97 kDa, calmodulin-binding protein 130 kDa, and myosin 200 kDa.

2.5. Activity staining The activity staining of lipase was carried out according to the reference [17]. After electrophoresis, the gel was soaked in 100 mM Tris–HCl buffer (pH 7.5) containing 0.5% Triton X-100 for 1 h in order to exchange the SDS with Triton X-100. By incubating the gel for 30 min in a 1:1 mixture of Solution A (where 8 mg of 1-naphthyl acetate was dissolved in 2 ml acetone and then mixed with 18 ml of 100 mM Tris–HCl buffer, pH 7.5) and Solution B (20 mg of Fast red TR salt suspended in 20 ml of 100 mM Tris–HCl buffer, pH7.5), the red bands corresponding to the lipase appeared against a transparent background.

2.6. Two-dimensional electrophoresis (2-DE) Two-dimensional electrophoresis was performed as described previously [16].

2.7. Effect of temperature and pH on the lipase activity and stability The powder of the purified lipase was dissolved with Tris–HCl buffer (100 mM, pH 7.5), and 100 ml aliquots (8.8 U/ml, on pNPB) were withdrawn and tested for activity at desired temperatures or pH as described for the lipase assay. For stability tests, 0.5 ml aliquots (25 U/ml, on pNPB) of the above lipase was incubated at the desired temperatures or pH for 1 h. After that the residual activity was tested with the lipase assay method.

2.8. Effect of metal ions and surfactants on the lipase stability After incubating a lipase solution (25 U/ml on pNPB) with various 1 or 10 mM metal ions at 30 8C for 1 h, the lipase activity was measured as the lipase assay, using the Tris–HCl buffer (100 mM, pH 7.5) instead of potassium phosphate as reaction buffer. Incubating a lipase solution (25 U/ml on pNPB) with various surfactants at 30 8C for 1 h, then the lipase activity was measured as the lipase assay.

2.3. Assay of lipase activity 2.9. Effect of various organic solvents on the lipase stability Lipase assay method: lipase was assayed using p-nitrophenyl butyrate ( pNPB) as the substrate. Lipase or blank solution (100 ml) was added to 2.870 ml of 100 mM potassium phosphate buffer (KPB, pH 7.0). After preincubation at 30 8C for 3 min, the reaction was initiated by a quick mixing of the

Effect of various organic solvents at concentrations of 10% (v/v) (0.1 ml of organic solvent plus 0.9 ml of the purified enzyme solution with 30 U on pNPB) and 50% (v/v) (1 ml of organic solvent plus 1 ml of the enzyme solution with

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30 U on pNPB) on the lipase activity was investigated as follows [5]: the reaction mixture was incubated for 24 h at 30 8C with shaking (160 rpm). The remaining activity was assayed under the standard condition and expressed as a percentage of the lipase activity in 100 mM KPB (phosphate buffer, pH 7.5) without any organic solvent. Effect of various organic solvents in neat state on the lipase activity was investigated as below: [18]. The enzyme powder (30 U on pNPB) was suspended in 1.0 ml of the chosen organic solvent and the system was incubated at 30 8C for 24 h with shaking (160 rpm). After that, the enzyme powder was taken out by centrifugation, the residual solvent was evaporated and 1.0 ml of phosphate buffer (100 mM, pH 7.5) was added. The lipase activity was determined according to the lipase assay protocol. The activities were expressed as percentages of that obtained for incubation of the enzyme in 100 mM KPB (phosphate buffer, pH 7.5) without any organic solvent.

2.10. Enantioselective hydrolysis The hydrolysis of methyl mandelate (1a), naproxen methyl ester (2a), and menthol acetate (1b), glycidyl butyrate (2b), 4-hydroxy-3-methyl-2-(2propeny1)-2-cyclopentenon acetate (3b), propylene carbonate (4b) and 4phenyl-[1,3]-dioxolan-2-one (5b) were carried out as follows: to 1.0 ml of 100 mM KPB (phosphate buffer, pH 7.5) with 5% DMSO containing those compounds, was added the purified lipase powder and the mixture was incubated at 30 8C with shaking at 1100 rpm on Thermomixer Compact (Eppendorf, Germany). The reaction mixture was extracted with ethyl acetate (1.0 ml), and the organic layer was dried over anhydrous sodium sulfate for HPLC or GC analysis. The hydrolysis of trans-3-(40 -methoxyphenyl)glycidic acid methyl ester, ()-MPGM (3a), was carried out in toluene-water (100 mM KPB phosphate buffer, pH 7.5) biphasic system (5 ml: 5 ml). After the purified lipase powder was added, the mixture was incubated at 30 8C with shaking at 160 rpm on shaker. Samples of the toluene layer were dried over anhydrous sodium sulfate for HPLC analysis. The conversion (c) and enantiomeric excess (ee) were determined by means of GC or HPLC according to previous reports [7,19–25].

3. Results and discussion 3.1. Purification and properties of the lipase The extracellular lipase produced and secreted by the bacterium S. marcescens ECU1010 was purified about 5.5-fold to homogeneity with an overall yield of 15.8%, by a five-step procedure as summarized in Table 1. Activity staining (Fig. 1a) and Coomassie Brilliant Blue staining (Fig. 1b) revealed the presence of a single protein with a molecular weight of 65 kDa, whereas molecular mass of the native lipase was 140 kDa, as determined by a TSK gel column. The two-dimensional electrophoresis (2-DE) indicated only one single spot on the gel (data not shown). Therefore, it was inferred that the lipase is a dimer with two homologous subunits.

Fig. 1. SDS-PAGE of purified lipase. Gels were first activity stained followed by staining with Coomassie Brilliant Blue. (a) Native staining; (b) Coomassie Brilliant Blue staining. Lane 1: purified lipase plus MW; Lane 2: purified lipase. MW: molecular weight standard (94 kDa, phosphorylase b; 67 kDa, bovine serum albumin; 43 kDa, ovalbumin; 30 kDa, carbonic anhydrase; 20.1 kDa, trypsin inhibitor; 14.4, a-lactalbumin).

The molecular mass of 65 kDa is a relatively high value among other known lipases from bacteria [26]. Yet, S. marcescens strains are known to produce lipases that have relatively large molecular masses, ranging from 52 to 65 kDa. The molecular mass of S. marcescens ECU1010 lipase agrees closely with previous results of 64.8–64.9 kDa reported for the S. marcescens lipases from S. marcescens SM6 [27], S. marcescens ES-2 [26], and S. marcescens Sr41 8000 [28]. The lipase’s pI was about 4.2, as measured by 2-DE. It is similar to that reported from S. marcescens Sr41 8000 (pI 4.5) [28] and lower than that from S. marcescens SM6 (pI 5.8) [27]. The optimal activity of S. marcescens ECU1010 lipase was observed at pH 8.0, and it was stable within pH 6–9. The optimal temperature of this lipase was 45 8C, and the lipase retained 71% activity after being incubated at 50 8C for 1 h (data not shown). These performances are quite similar to those of S. marcescens Sr41 8000 lipase [28]. 3.2. Substrate specificity to various p-nitrophenyl esters Substrate specificity of the lipase was investigated with various p-nitrophenyl esters. The esters of various fatty acids with a moderate to long chain (NC > 6) served as good

Table 1 Purification of an extracellular lipase from S. marcescens ECU1010 Purification step

Total activity (U)

Total protein (mg)

Specific activity (U/mg)

Yield (%)

Purification fold

Crude enzyme Ultrafiltration (NH4)2SO4 fractionation DEAE-Toyopearl Sephadex G-150 Phenyl-Toyopearl

33, 900 31, 723 17, 315 14, 513 5, 515 5, 353

374 293 151 19.8 13.9 10.8

90 109 115 273 397 497

100 93.6 51.1 42.8 16.3 15.8

1 1.2 1.3 3.0 4.4 5.5

The enzyme was purified from 1.5 l of culture broth. The activity was assayed using p-nitrophenyl butyrate as substrate.

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Table 2 Effect of various metal ions on activity of the lipase from S. marcescens ECU1010 Inorganic salts

None FeSO4 FeCl3 CoCl2 MgSO4 ZnSO4 MnCl2 CaCl2 CuSO4 NiCl2 NaCl KCl EDTA

Fig. 2. Fatty acid specificity of the lipase from S. marcescens ECU1010. The activity towards p-nitrophenyl butyrate was taken as 100%. All assays were performed in duplicates.

Relative activity (%) a 1 mM

10 mM

100 31.0  4.9 22.0  8.5 93.0  4.0 90.0  6.4 44.0  4.5 73.0  1.1 95.7  0.1 85.7  0.4 86.0  3.4 98.0  5.9 101.0  7.7 19.6  0.5

100 14.0  5.6 12.0  1.0 46.0  6.8 88.0  8.8 16.2  0.6 60.0  2.4 95.0  5.9 37.0  7.7 40.0  4.0 90.0  4.5 89.0  4.7 16.0  1.6

S. marcescens ECU1010 lipase was incubated with various metal ions (1 mM and 10 mM) at 30 8C for 1 h before the activity was measured with pnitrophenyl butyrate. All assays were performed in duplicates. a The activity toward p-nitrophenyl butyrate without any metal ions was taken as 100%.

substrates for the enzyme, and p-nitrophenyl myristate (C14) was the best among all the substrates tested, as shown in Fig. 2. Therefore, the enzyme is a typical lipase which prefers a longchain lipid, though it also displayed a strong carboxylic esterase activity as well. Whereas in the literature [26,28], a lipase from S. marcescens Sr41 8000 showed the highest activity toward triacylglycerides of relatively shorter chain length (C4–C8), while another lipase from S. marcescens ES-2 preferably hydrolyzed the triacylglycerides of medium-chain length (C8– C12).

well as the reaction rates of kinetic resolution [30]. Therefore, the information concerning effects of various surfactants on the lipase is of importance. As listed in Table 3, the lipase activity was enhanced by some surfactants, such as polyvinyl alcohol (PVA), Pg350me and especially Gum Arabic (142% of relative activity), at merely a concentration of 1% (w/v). Most of the lipase activity was retained after the addition of Pg400de, Pg250de and Tween-80. However, Triton X series, such as Triton X-45 and Triton X-100, inhibited the activity seriously (data not shown).

3.3. Effect of metal ions on the lipase activity

3.5. The lipase stability against various organic solvents

Effects of various inorganic salts and metal chelators on the lipase activity are presented in Table 2. The S. marcescens ECU1010 lipase was resistant against most metal ions, except Fe2+, Fe3+ and Zn2+ at a concentration of 1.0 mM. This is somewhat similar to the lipase of S. marcescens ES-2 but different from the lipase of S. marcescens Sr41 8000 where at a concentration of 1.0 mM, Co2+ and Ni2+ did not show remarkable inhibitory effect on the lipase. Most of metal ions had a negative effect on the lipase activity, except Ca2+, Mg2+ and monovalent ions at a concentration of 10 mM. The chelating agent EDTA significantly reduced the lipase activity, possibly suggesting that the purified lipase may be a metaloenzyme. However, activity of the lipase from S. marcescens ECU1010 was not significantly enhanced by Ca2+ which was reported to activate many lipases [29].

It is well known that effect of organic solvents on lipase activity differs from lipase to lipase. In this study, some typically water-miscible solvents with very low or even negative log P values, such as dimethyl sulfoxide, acetone, methanol, ethanol and isopropanol, were tested. Although good

3.4. Effect of surfactants on the lipase activity and stability Surfactants have been widely applied to lipase-catalyzed reactions of insoluble substrates to increase the lipid–water interfacial area, which in turn enhances the enantioselectivity as

Table 3 Effect of various surfactants on activity of the lipase from S. marcescens ECU1010 Surfactants

Relative activity (%) a

None Gum Arabic Polyvinyl alcohol (PVA) Pg350me Pg400de Pg250de Tween 80

100 142  0 118  2 117  12 96  4 78  28 72  0

S. marcescens ECU1010 lipase was incubated with various surfactants at 30 8C for 1 h before the activity was measured with p-nitrophenyl butyrate. All assays were performed in duplicates. a The activity toward p-nitrophenyl butyrate without any surfactants was taken as 100%.

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Table 4 Stability of S. marcescens ECU1010 lipase against various organic solvents Organic solvents

DMSO Methanol Isopropanol Ethanol Acetone

log Pa

1.3 0.76 0.28 0.24 0.23

Residual activity (%) b At 10% concentration

At 50% concentration

At 100% concentration

108.0  2.4 97.4  0.5 85.0  3.1 88.0  8.1 86.0  3.8

1.0  0.2 1.0  0.4 1.1  0.4 1.2  0.4 1.7  0.0

0 0 83.5  0.7 0 83.0  4.2

S. marcescens ECU1010 lipase was incubated in various solvents at 30 8C for 24 h under shaking condition (160 rpm) before the lipase activity was measured by the p-nitrophenyl butyrate ( pNPB) substrate. All assays were performed in duplicates. a log P value is the partition coefficient of an organic solvent between water and n-octanol phases. b Residual activities were measured by the standard assay with pNPB.

stability of bacterial and fungal lipases in hydrophilic organic solvents is rare, the S. marcescens ECU1010 lipase displayed extremely high stability (Table 4), with no drastic decreases in residual activity after incubation for 24 h at 10% concentration of water-miscible organic solvents. In addition, DMSO which is widely used to dissolve proteins to a certain extent, slightly activated the enzyme at 10% concentration. Simon [31] had also found similar effects of DMSO on some other hydrolases. Interestingly, the enzyme was quite stable, remaining active even in neat hydrophilic solvents such as isopropanol and acetone, respectively with 83.5 and 83.0% of initial activity after 24 h of preincubation. The lipases from B. megaterium CCOC-P2637 [8] and Pseudomonas mendocina PK12CS [18] are two of the reported examples of microbial lipases that were reasonably stable against hydrophilic solvents. When the lipase of B. megaterium CCOC-P2637 was incubated for 1 h in neat isopropanol, the residual activity was 97%, and when the lipase of P. mendocina PK12CS was incubated for 2.5 h in neat ethanol, the residual activity was 83%. In addition, solvents of high log P, such as cyclohexane, nheptane, isooctane and toluene, were also investigated. The S. marcescens ECU1010 lipase was apparently more stable in water-immiscible organic solvents than in water-miscible organic solvents (data not shown). This profile is similar to those of many lipases reported [32]. The organic solvent tolerance of any of the lipases from Serratia species has not yet been reported so far. It is the high stability of this lipase in either water-miscible or waterimmiscible organic solvents that makes it potentially useful for practical application in many synthetic reactions in nonconventional media. 3.6. Potential applications of the lipase for kinetic resolution of chiral acids Methyl mandelate ()-1a, naproxen methyl ester ()-2a, and ()-MPGM ()-3a were chosen as typical examples for kinetic resolution of chiral carboxylic acids. The results were shown in Table 5. The lipase was most active towards the ()-MPGM, 3a. The reaction proceeded with 60% conversion at a substrate concentration of 1.0 M, resulting in a highly pure product, trans-(2R, 3S)-MPGM, with 100% enantiomeric excess (ee),

which is known as a key intermediate for production of an important antihypertensive agent Diltiazem. (S)-Naproxen is a nonsteroidal anti-inflammatory drug which belongs to the family of 2-arylpropionic acids and is widely used for treatment of human connective tissue diseases. The S. marcescens ECU1010 lipase also showed very good enantioselectivity (E = 83) toward the substrate ()-2a. Whereas, the lipase from Candida antarctica [33] hydrolyzed the (R,S)-naproxen vinyl ester with merely a low selectivity (E = 22). 3.7. Potential application of the lipase for kinetic resolution of chiral alcohols Menthyl acetate ()-1b, glycidyl butyrate ()-2b, 4hydroxy-3-methyl-2-(2-propeny1)-2-cyclopentenon acetate (HMPCA) ()-3b, propylene carbonate ()-4b and 4phenyl-[1,3] -dioxolan-2-one ()-5b, were chosen as representatives of chiral alcohols. Especially, ()-4b and ()-5b were examples for carbonic acid esters. The results were shown in Table 5. Interestingly, the enzyme presented a significant enantiopreference towards (R)-2b, in contrast to the enantiopreference displayed by most of other lipases. 2b is a very attractive intermediate for preparation of optically active b-blockers and a wide range of other products. Most of lipases presented a stereochemical preference towards (S)-2b, producing (R)glycidyl butyrate and (R)-glycidol [34], including the lipase from Rhizopus sp. Bc0-09 [20] and the lipase from Rhizopus oryzae [35]. However, (S)-glycidyl derivatives are also very useful as starting materials for synthesis of many different drugs, for example, (R)-()-argentilactone [36] which exhibits both antileishmanial activity and cytotoxic activity against mouse leukemia cells. In this sense, the lipase from S. marcescens ECU1010 is more useful than the other lipases for the enantiomeric resolution of ()-2b. The lipase from C. antarctica (fraction B) (CALB) had the same phenomena [34]. However, the free CALB afforded only 3% of ees (ee of substrate) for the same substrate. Even though different techniques of immobilization as well as an adequate reaction medium were used, the E-value of the reaction was merely 9.0 (ees = 91% at 64% conversion). Without thinking about the enantiopreference, a lipase from Rhizopus sp. Bc0-09 [20] gave

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Table 5 Enantioselective hydrolysis of selected chiral esters by S. marcescens lipase

Substrate

()-1a ()-2a ()-3a ()-1b ()-2b ()-3b ()-4b ()-5b a b c d

Concentration (mM)

10 10 1000 10 50 100 50 20

Conv. (%)

a

33 41 a 60 a 25 b 49 c 50 b 0b 9.6 a

Residual ester

Product formed

Config. and ees (%)

Config. and eep (%)

a

(R), 15 (R), 66 a (2R,3S), 100a (R), 15 b (S), 93 b (S), 100b ND, ND ND, 0.25a

(S), 13 a (S), 95 a NDd, ND (S), 13 b ND, ND ND, ND ND, ND ND, 9.3 a

Enantio-selectivity (E-value)

1.4 83 >100 1.1 >100 >100 ND 1.2

Calculated from HPLC peaks, representing area percentage. Determined from GC peaks, representing area percentage. Determined by neutralizing titration. ND: not determined.

an E-value of 57 for the resolution reaction, affording (R)glycidyl butyrate and (R)-glycidol. Apart from the high enantioselectivity (E > 100), the concentration of substrate ()-2b was higher, as compared with other lipases. The substrate concentration was 50 mM in our case, while it was merely 10 mM for R. oryzae lipase [35] and for CALB [34], or even 3 mM for a lipase-like enzyme purified from the pancreatic porcine lipase (PPL) [37]. A higher substrate concentration is more preferable for industrial applications. This is perhaps because of the good tolerance of S. marcescens ECU1010 lipase against organic solvents or hydrophilic substrate. The substrate concentration in our work may be further increased after careful optimization. 4-Hydroxy-3-methyl-2-(2-propeny1)-2-cyclopentenone (HMPC) is the alcohol moiety of prallethrin, a widely employed synthetic pyrethroid insecticide for household use. The insecticidal activity of (S)-isomer prallethrin is several times higher than the (R)-antipode [19]. Therefore, much effort has been made to produce optically pure (S)-HMPC. Comparing with the resolution result by a lipase from Acinetobacter sp. CGMCC 0789 [19], the S. marcescens ECU1010 lipase showed better enantioselectivity to substrate ()-3b. As shown in Table 5, when the conversion reached 50%, the enantiomeric excess of residual substrate (ees) was 100%, giving an E-value of >100. This is a preferable property

for chiral biocatalysts. In addition, S. marcescens ECU1010 lipase could also catalyze the stereoselective transesterification of racemic HMPC with vinyl acetate, with a similar E-value (>100) but a slower reaction rate than the hydrolysis. Although a chiral ester may be composed either of a chiral carboxylic acid or of a chiral alcohol or even the both, there were no apparently corresponding relationship between the lipase enantioselectivity and its substrate structure. The lipase showed high enantioselectivity towards ()-3a (E > 100), ()2b (E > 100), ()-3b (E > 100) and ()-2a (E = 83), while for ()-1a and ()-1b which also have carbon rings near the chiral centers, the E-values were as low as 1.4 and 1.1, respectively. As to carbonic acid esters (4b & 5b), the S. marcescens ECU1010 lipase showed very low enantioselectivity (E = 1.2) on ()-5b and no activity on ()-4b. The lipase from S. marcescens ECU1010 seems to be the first example reporting the kinetic resolution of ()-2b (E > 100), ()-3b (E > 100) and ()-2a (E = 83) among those from S. marcescens strains, although the lipase from S. marcescens SM6 [27] and S. marcescens ES-2 [26] had also been used for kinetic resolution of other racemic mixtures, including isopropylideneglycerol acetate, 2-phenyl-1-propanol and (S)-flurbiprofen. Further improvement of the reactivity and selectivity of S. marcescens ECU1010 lipase may be expected to achieve by optimizing the reaction environment (e.g.,

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immobilization support, solvent and water content) and operation conditions (pH & temperature, etc.) [38]. 4. Conclusions The lipase from S. marcescens ECU1010 was purified to homogeneity. The pH and temperature optima were pH 8.0 and 45 8C, respectively. The lipase showed the maximum activity on p-nitrophenyl myristate (C14) among various p-nitrophenyl esters. The lipase was not activated by Ca2+. However, it was activated by Gum Arabic. In addition, the most important property of the lipase was its high tolerance against organic solvents. Besides the high stability in hydrophobic organic solvents, the lipase also showed quite good stability in hydrophilic cosolvents such as acetone and 2-propanol. The high stability in hydrophilic solvents makes the lipase very promising for reactions involving the hydrolysis of esters for production of chrial alcohols or chiral acids. Furthermore, the lipase has been successfully applied to several typical resolution reactions of chiral esters with industrial relevance. The S. marcescens ECU1010 lipase is the first successful example reported for kinetic resolution of glycidyl butyrate, HMPCA and naproxen methyl ester. Especially for glycidyl butyrate, the lipase presents a significant enantioselectivity towards the (R)-glycidyl butyrate, which is different from most of other lipases. All the above results demonstrated that S. marcescens ECU1010 lipase accepts a wide variety of stereochemically different substrates, and gives moderate to high enantiomeric excesses on 4 of 8 chiral substrates tested. In conclusion, the high tolerance against organic solvent along with a broad substrate spectrum makes the S. marcescens ECU1010 lipase a very attractive enzyme for potential application in industry, particularly in the field of biocatalytic resolution to produce enantiopure building blocks for chiral pharmaceuticals or agrochemicals. Acknowledgements This research was financially supported by Ministry of Science and Technology, PR China (No. 2007AA02Z225) and Science and Technology Commission of Shanghai Municipal Government (No. 05DZ19350). References [1] Jaeger KE, Ransac S, Dijkstra BW, Colson C, Heuvel MV, Misset O. Bacterial lipases. FEMS Microbiol Rev 1994;15:29–63. [2] Jaeger KE, Dijkstra BW, Reetz MT. Bacterial biocatalysts: molecular biology, three-dimensional structures and biotechnological applications of lipases. Annu Rev Microbiol 1999;53:315–51. [3] Pandey A, Benjamin S, Soccol CR, Nigam P, Krieger N, Soccol UT. The realm of microbial lipases in biotechnology. Biotechnol Appl Biochem 1999;29:119–31. [4] Theil F. Lipase-supported synthesis of biologically active compounds. Chem Rev 1995;95:2203–27. [5] Chin JH, Rahman RNZA, Salleh AB, Basri M. A newly isolated organic solvent-tolerant Bacillus sphaericus 205y producing organic solventstable lipase. Biochem Eng J 2003;15:147–51.

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