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Purification and characterization of an organic solvent-stable lipase from Pseudomonas stutzeri LC2-8 and its application for efficient resolution of (R, S)-1-phenylethanol
Biochemical Engineering Journal 64 (2012) 55–60
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Regular article
Purification and characterization of an organic solvent-stable lipase from Pseudomonas stutzeri LC2-8 and its application for efficient resolution of (R, S)-1-phenylethanol Yan Cao, Yu Zhuang, Changjin Yao, Bin Wu, Bingfang He ∗ College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, 30 Puzhunan Road, Nanjing 211816, Jiangsu, China
a r t i c l e
i n f o
Article history: Received 1 December 2011 Received in revised form 24 February 2012 Accepted 11 March 2012 Available online 19 March 2012 Keywords: Organic solvent-stability Lipase Purification Pseudomonas stutzeri Enantioseparation Biocatalysis
a b s t r a c t An organic solvent-stable lipase from newly isolated solvent-tolerance bacterium Pseudomonas stutzeri LC2-8 was purified by acetone precipitation and anion exchange chromatography. The apparent molecular mass of the purified lipase was estimated by SDS-PAGE to be 32 kDa. The open reading frame (ORF) of lipase LC2-8 encodes 311 amino acids with 287 amino acid residues in the mature lipase which shared 96% homology at the amino acid level with the putative lipase LipC from Pseudomonas stutzeri A1501. The optimum pH and temperature for lipase activity were 8.0 and 30 ◦ C, respectively. Its hydrolytic activity was found to be highest towards p-nitrophenyl caproate (C8). Lipase LC2-8 showed high tolerance in the presence of various organic solvents. Most of the hydrophilic solvents tested strikingly enhanced the activity and stability of lipase LC2-8. The half-life of lipase LC2-8 was extended to 10-fold in the presence of isopropanol, acetone, ethanol and methanol. The transesterification resolution of (R, S)-1-phenylethanol by lipase LC2-8 was carried out with the yield of 47.6%, the enantiomeric excess of residual substrate (ees ) was 99.9%, giving an E-value over 200. The solvent-stable lipase LC2-8 showed an attractive potency for application in biocatalysis in non-aqueous systems. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) catalyzes triglyceride hydrolysis, ester synthesis, transesterification and other reactions. In recent years, its application in detergent additives and in the preparation of chemical and pharmaceutical intermediates, especially the preparation of chiral intermediates by enzymatic hydrolysis, transesterification or aminolysis reactions in organic solvents has been increasingly emphasized [1]. Lipases are diverse in their sensitivity to solvents, and are more destabilizing in polar water miscible solvents than in water immiscible solvents [2]. Therefore, enzymes that are naturally stable, especially with high activity in different types of organic solvents, have great potential in extending the practical catalysis system. Due to the particularity of existing environments, organic solvent-tolerant extremophiles have recently become a research focus for the isolation of organic solvent-stable proteases and lipases. Considerable effort has been made to exploit new species of solvent-tolerant microorganisms which secrete solventstable lipases such as Pseudomonas aeruginosa LST-03 [3], Bacillus
∗ Corresponding author. Tel.: +86 25 58139902; fax: +86 25 58139902. E-mail address: [email protected] (B. He). 1369-703X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2012.03.004
sphaericus 205y [4] and Serratia marcescens ECU1010 [5]. So far, the number of solvent-tolerant bacteria is limited and the properties of the lipases isolated are insufficient to satisfy the needs of diversity substrates. Therefore, it is necessary to explore novel lipases with high activity in organic solvents to expand their application in practical catalysis. In this paper, we report the purification and characteristics of a solvent-stable lipase from the newly isolated organic solventtolerant bacterium Pseudomonas stutzeri LC2-8. The efficient transesterification of (R, S)-1-phenylethanol with high enantioselectivity catalyzed by lipase LC2-8 in a non-aqueous system was also discussed. 2. Materials and methods 2.1. Materials All p-nitrophenyl fatty acid esters and high-performance liquid chromatography (HPLC)-grade solvents were purchased from Sigma (St. Louis, MO, USA). (R, S)-1-phenylethanol (98%) was purchased from Fluka (Germany). LA-Taq DNA polymerase and the PMD-18 Tcector were purchased from Takara (Dalian, China). DH5␣ (Clontech, Saint-Germain-en-Laye, France) was used as the E. coli host. All other chemicals were of analytical grade.
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2.2. Screening and isolation of organic solvent-tolerant lipase producing strains Soil samples from areas contaminated by crude oil and chemicals were collected. The microorganisms with organic solvent tolerance were screened using enriched medium (0.1% tryptone, 0.18% corn steep liquor, 0.35% (NH4 )2 SO4 , 0.3% KH2 PO4 , 0.25% NaCl, 0.1% MgSO4 ·7H2 O and 5% sunflower oil), both toluene and cyclohexane were added to the medium at a concentration of 20% (v/v), respectively. The cultures were then acclimated by being repeatedly transferred to sterile enrichment medium incubated at 30 ◦ C. Samples of repeated batch cultures were diluted and spread on tributyrin agar plates (0.3% yeast extract, 1.0% tryptone, 0.25% NaCl, 0.5% tributyrin and 1.8% agar). Colonies developing a clear zone were picked up and screened for their lipase production on rhodamine B agar plates (0.1% yeast extract, 0.1% K2 HPO4 , 0.05% MgSO4 ·7H2 O, 1.5% agar, 0.0024% rhodamine B, 0.5% (v/v) corn steep liquor, 6% (v/v) olive oil and 1.8% agar). The microbes showing high ratios of an orange fluorescent halo clear zone diameter to colony diameter under UV light at 350 nm were selected as potential highyield lipase producers for subsequent experiments. The extracellular lipase with remarkable organic solvent (25%, v/v) tolerance was selected for further research. The producing strain identified based on the analysis of the Microlog Microbial Identification System (Biolog Automated Micro-Station System, Biolog, USA) and 16S rDNA sequence BLAST in the Genbank Data Library. 2.3. Culture conditions for lipase LC2-8 production Strain LC2-8 was cultured in lipase-producing medium consisting of (w/v) 1.0% yeast extract, 0.8% glucose, 0.2% K2 HPO4 , 0.05% MgSO4 ·7H2 O, 0.05% (v/v) Triton X-100 and 0.5% (v/v) sunflower oil, with a pH of 8.0. The incubations were carried out at 30 ◦ C with shaking at 180 rpm. After 48 h of incubation, the culture supernatant was collected by centrifugation and used as the crude lipase. 2.4. Assay of lipase activity and protein concentration Lipase activity was measured with a modified spectrophotometric method with p-nitrophenyl palmitate (p-NPP) as substrate [6]. The substrate, p-NPP (3 mg), with a final concentration of 0.3 mg/mL was dissolved in 1 mL of isopropanol and mixed with 9 mL of 50 mM sodium phosphate buffer (pH 8.0) containing gum arabic (0.1%) and Triton X-100 (0.6%). The reaction mixture was composed of 240 L of substrate solution and 10 L of appropriately diluted enzyme solution, and incubated at 30 ◦ C for 10 min. The p-nitrophenol (p-NP) produced in the reaction mixture was quantified spectrophotometrically at 410 nm. One unit of enzyme activity was defined as the amount of enzyme that liberated 1 mol p-NP per min under standard assay conditions. The protein concentrations in this study were determined by the method of Bradford [7] using bovine serum albumin as a standard. 2.5. Purification and cloning of the lipase The lipase LC2-8 was purified in two steps. Chilled acetone was added slowly to the culture supernatant of strain LC2-8 up to 60% (v/v) concentration with continuous stirring and kept at 0 ◦ C for 4 h to allow protein precipitation. The precipitates were harvested by centrifuging at 10,000 × g for 30 min at 4 ◦ C, and resuspended in 50 mM Tris–HCl buffer (pH 8.0), and then applied to a DEAE-Sepharose FF column (1.6 cm × 20 cm, Amersham Biosciences, Sweden) that had been pre-equilibrated with 50 mM Tris–HCl buffer (pH 8.0), eluted with a NaCl gradient from 0.2 to 0.8 M. The fractions with lipase activity were concentrated by
centrifugation using a Centricon 10 kDa molecular weight cut-off device (Millipore, USA). The purified lipase was subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) according to the method of Laemmli [8], and the Coomassie-stained band of the purified lipase was excised from the gel and submitted to the National Center of Biomedical Analysis (NCBA, Beijing) for MS analysis. The trypsin-digested protein fragments of the lipase were subjected to LC/MS/MS analysis, and the sequences of the fragments were submitted to the Mascot program for possible identity matching [9]. The genomic DNA of P. stutzeri strain LC2-8 was obtained by the standard phenol/chloroform precipitation method. The sense primer 5 -TGCCCCATAGCCCGATTCACAT-3 and the antisense primer 5 -ATGCTCTGTTTGAGCGGCTCCT-3 were designed based on the sequence of the putative lipase from P. stutzeri, since MS analysis of the digest fragments of the purified lipase showed that some fragments were identical to that of putative lipase from genome of P. stutzeri A1501 [10]. PCR amplification was carried out with the primers described above. The amplified PCR fragment was cloned into the PMD-18T vector following User Manual from Takara and then sequenced.
2.6. Effect of pH and temperature on lipase activity and stability The activity of the lipase was determined at various pH values (6.0–10.0) at 30 ◦ C. The pH stability of the lipase was analyzed by incubating solutions of the enzyme (0.05 mg/mL) at different pH values for 3 h at 30 ◦ C. To keep the sample pH constant, the following 50 mM buffer systems were used: citric acid/sodium citrate, pH 3.0–6.0; Na2 HPO4 /KH2 PO4 , pH 6.0–8.5; Tris–HCl, pH 7.1–9.0; Gly/NaOH, pH 8.6–10.6; and Na2 HPO4 /NaOH, pH 10.0–12.0. The residual activity was measured according to the pNPP method described above. The effect of temperature on the lipase was measured under various temperature of range 15–50 ◦ C at pH 8.0. The thermal stability of the lipase was assayed by incubating the purified lipase (0.05 mg/mL) at different temperatures (20–50 ◦ C) for 3 h and its residual activity was then measured according to the pNPP method. Each measurement was performed three times and standard error was included.
2.7. Effects of metal ions, inhibitors and surfactants on the lipase activity The effects of different metal ions (Fe2+ , Ca2+ , Mg2+ , Cu2+ , Zn2+ , Li+ , Mn2+ , Co2+ and Ba2+ ) and inhibitors (phenylmethyl sulfonyl fluoride (PMSF), Woodwards reagent K (WRK), dithiothreitol (DDT), -mercaptoethanol and EDTA) were investigated by preincubating the purified lipase (0.05 mg/mL) with 1 and 10 mM solutions of these ions or inhibitors for 1 h at 30 ◦ C, and the residual activity was then tested using the pNPP method described above. Similarly, the effect of surfactants (Triton X-100, Tween 80, Tween 20 and SDS) at the concentration of 0.05% and 0.5% on the lipase was investigated. Each measurement was performed three times and standard error was included. K+ ,
2.8. Substrate specificity to various p-nitrophenyl esters The substrates, p-nitrophenyl fatty acid esters, of varying chain length (C2, C4, C8, C10, C14, C16 and C18) were used at the final concentration of 0.3 mg/mL and the lipase activity was measured according to the pNPP method.
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2.9. Effects of organic solvents on lipase LC2-8 The effects of organic solvents with different log P values at 25% (v/v) concentration on the activity and the stability of the purified lipase were investigated following Ogino et al. [11]. The purified lipase dissolve in 50 mM sodium phosphate buffer (pH 8.0) was filter sterilized (using cellulose acetate membrane, 0.22 m). Aliquots (1 mL) of the organic solvents were added to the purified lipase solution (3 mL) with protein concentration of about 0.05 mg/mL in a sealed glass vial. The mixture was incubated at 30 ◦ C with shaking at 150 rpm. The remaining activity was measured at appropriate time intervals over a period of 10 days. The buffer was added instead of organic solvent as a reference when hydrophilic solvents were studied. 2.10. Bioresolution of (R, S)-1-phenylethanol by lipase LC2-8 Lipase LC2-8 powder preparation: Chilled acetone was added to the culture supernatant of strain LC2-8 until the ratio was 0.8:1 (v/v) under magnetic stirring at 0 ◦ C for 4 h. The precipitated protein was obtained by centrifugation at 10,000 × g and 4 ◦ C for 30 min. Finally, the precipitate was air-dried and lipase LC2-8 powder was obtained. The kinetic resolution of (R, S)-1-phenylethanol was carried out in 1 mL n-hexane containing vinyl acetate 800 mM and (R, S)-1phenylethanol 200 mM catalyzed by 10 mg lipase LC2-8 powder at 30 ◦ C with shaking at 200 rpm. 2.11. Determination of the concentrations of isomers 1-phenylethanol and phenylethanol ester The amounts of optical isomers were determined by HPLC (Dionex P680) using Chiracel OJ column (250 mm × 4.6 mm) at 35 ◦ C. The mobile phase was n-hexane/isopropanol (95:5, v/v) and the flow rate was 0.8 mL/min. The wavelength of the UV detector was set at 254 nm. 2.12. Nucleotide sequence The nucleotide sequence of lipase LC2-8 has been assigned GenBank accession number JN681265. 3. Results 3.1. Isolation of organic solvent-tolerant lipase-producing bacteria
Fig. 1. SDS-PAGE of lipase LC2-8 at different stages of purification. Lane 1, protein molecular weight marker; lane 2, supernatant; lane 3, lipase concentrated by acetone precipitation; lane 4, lipase purified by DEAE-Sepharose FF.
recovery was achieved. The purified lipase was homogeneous with the apparent molecular mass of 32 kDa in SDS-PAGE (Fig. 1). The trypsin-digested peptides of lipase LC2-8 were identified by mass spectrometry and sequencing. The matching sequences included DGAQVYVTEVSQLNTSELR (fragment 1), GEELLAQVEEIVAISGKPK (fragment 2), YVAGVRPDLIASVTSVGAPHKGSD VADLIR (fragment 3), CSSHLGMVIR (fragment 4), and MNHLDEVNQFMGLTSLFETDPVSVYR (fragment 5). These sequences showed high homology to the sequences of the putative lipase LipC from the genome of P. stutzeri A1501. Based on the sequence of the putative lipase from P. stutzeri A1501, the gene encoding the organic solvent stable lipase of P. stutzeri LC2-8 was cloned. The full-length open reading frame (ORF) of lipase LC2-8 consisted of 936 bp encoding 311 amino acid residues, and the mature lipase contained 287 amino acid residues which shared 87% homology at the DNA level and 96% homology at the amino acid level with the putative lipase of P. stutzeri A1501 (NCBI accession no. YP001172529.1). Nine amino acids residues (Ile106 , Ile115 , Val148 , Ala167 , Asn182 , Lys217 , His222 , Val238 , Ser247 ) from lipase LipC from P. stutzeri A1501 were substituted with Val, Val, Ile, Ser, Asp, Phe, Arg, Ile and Ala in lipase LC2-8, respectively. 3.3. Effect of pH and temperature on lipase activity and stability
Cyclohexane and toluene were added to the enriched medium so that only solvent-tolerant microbes were able to grow. As a result, 123 strains of organic solvent-tolerant lipase producers were isolated. Of these strains, strain LC2-8 secreted extracellular lipase which showed highest activity and stability in the presence of hydrophilic organic solvents, and was isolated for further research. Organic solvent-tolerant strain LC2-8 was identified as P. stutzeri based on the analysis of the Biolog (SIM = 0.543, 16–24 h) and 16S rDNA sequences (NCBI GeneBank accession no. FJ345693), possessing the highest homology (99%) with that of P. stutzeri. Strain LC2-8 was deposited in the China Center for Type Culture Collection (Wuhan, China) with the accession number CCTCC M 2010279. 3.2. Purification and cloning of the lipase The extracellular lipase from P. stutezri LC2-8 was purified by acetone precipitation and DEAE-Sepharose FF anion-exchange chromatography (Table 1). About 21.2-fold purification with 32.8%
The optimal pH for lipase activity was found to be 8.0 (Fig. 2). Lipase LC2-8 showed good stability in the pH range 5.0–9.5. The temperature optimum for lipase activity was observed to be 30 ◦ C (Fig. 3). The enzyme retained 60% of its maximal activity at 20 ◦ C, showing the characteristics of a cold-active lipase. The lipase showed considerable thermo-sensitivity, and the activity decreased sharply when the temperature was above 40 ◦ C. 3.4. Effects of metal ions, inhibitors and surfactants on lipase activity The effects of various metal ions, inhibitors and surfactants on lipase activity are summarized in Table 2. Among the tested metal ions, K+ , Ba2+ and Ca2+ significantly activated lipase LC28; while Zn2+ and Cu2+ strongly inhibited lipase LC2-8. The metal chelator, EDTA, had a little influence on the activity of lipase at the low concentration, but inhibited lipase activity by 56.2% at a
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Table 1 Purification of the lipase from P. stutzeri LC2-8.
Supernatant of culture Acetone precipitation DEAE-Sepharose FF
Enzyme activity (U/mL)
Protein (mg/mL)
Specific activity (U/mg)
Total activity (U)
Yield (%)
Purification (fold)
145 332 47.6
0.65 0.52 0.01
223.1 638.5 4760
2900 1992 951
100 68.7 32.8
1 2.9 21.2
Table 2 Effects of metal ions, inhibitors and surfactants on activity of the lipase.a Effectors
Relative activity (%)
Inhibitors EDTA PMSF WRK -Mercaptoethanol DTT Surfactants TritonX-100 Tween 20 Tween 80 SDS Controlb
1 mM 76.5 ± 1.24 91.7 ± 4.56 90.5 ± 2.43 98.9 ± 2.14 102.3 ± 1.09 0.05% 145.3 ± 3.55 103.3 ± 2.28 107.6 ± 3.98 38.8 ± 1.78 100
10 mM 43.8 ± 1.65 79.8 ± 2.12 83.1 ± 1.11 99.8 ± 0.76 96.0 ± 1.21 0.5% 107.8 ± 5.87 94.5 ± 1.45 98.2 ± 1.76 3.6 ± 0.34 100
Effectors
Relative activity (%)
Metal ions Li+ K+ Ba2+ Ca2+ Mg2+ Mn2+ Co2+ Fe2+ Cu2+ Zn2+
1 mM 100.9 ± 0.98 116.6 ± 0.23 115.2 ± 1.90 121.3 ± 1.43 101.5 ± 0.62 108.2 ± 0.75 102.7 ± 1.93 102.3 ± 2.28 78.1 ± 2.17 41.5 ± 1.61
10 mM 107.4 ± 1.27 110.2 ± 1.53 127.0 ± 1.12 117.8 ± 2.71 104.0 ± 0.66 19.5 ± 0.98 97.4 ± 1.65 94.2 ± 2.28 23.7 ± 0.55 5.9 ± 0.23
Values are means ± SD (n = 3). a The purified lipase LC2-8 was incubated with various inhibitors, metal ions (1 mM and 10 mM) and surfactants (0.05% and 0.5%) in 50 mM Tris–HCl buffer (pH 8.0) at 30 ◦ C for 1 h. b Lipase activity is shown as values relative to that measured without addition of effectors (control).
concentration of 10 mM. PMSF and WRK slightly inhibited activity of the lipase. The thiol reducing agents, DTT and -mercaptoethanol did not affect lipase activity. The nonionic surfactant, Triton X-100, enhanced the activity of the lipase, while Tween 20 and Tween 80 showed slight activation at low concentrations. In contrast, the anionic surfactant, SDS, strongly inhibited lipase LC2-8. 3.5. Substrate specificity The enzyme substrate specificity was examined using various fatty acid esters of p-nitrophenyl. The lipase preferentially hydrolyzed p-nitrophenyl esters with medium chain fatty acids and exhibited the highest hydrolytic activity toward p-nitrophenyl caproate (C8). Short chain substrates, especially p-nitrophenyl acetate (C2) were hydrolyzed very slowly (Fig. 4).
Fig. 2. Effect of pH on activity (solid) and stability (hollow) of the lipase. Lipase activities are shown as values relative to that measured in 50 mM sodium phosphate buffer pH 8.0 (taken as 100%). Stability of the lipase (0.05 mg/mL) was determined by incubating at 30 ◦ C for 3 h in the following buffer systems: 50 mM citric acid/sodium citrate (pH 3.0–6.0) (), 50 mM Na2 HPO4 /KH2 PO4 (pH 6.0–8.5) (䊉 ), 50 mM Tris/HCl (pH 7.1–9.0) (), 50 mM Gly/NaOH (pH 8.6–10.6) ( ♦), and 50 mM Na2 HPO4 /NaOH (pH 10.0–12.0) (). The remaining activities of the lipase are shown as values relative to the initial activity (taken as 100%) at pH 8.0.
3.6. Effects of organic solvents on the lipase activity The effects of various organic solvents with different log P values on the lipase are shown in Table 3. Most of the tested hydrophilic solvents at 25% concentration enhanced the activity of the lipase. Among these solvents, isopropanol, acetone, ethanol and DMF significantly increased the activity of lipase to 132.6%, 215.3%, 168.9% and 159.3%, respectively, compared to the initial activity without the addition of organic solvents. The activity of the purified lipase significantly decreased during incubation in the absence of organic solvents at 30 ◦ C, with a half-life of only 24 h; while in the presence of the tested organic solvents, the half-life of lipase LC2-8 was extended significantly by more than 10-fold in the presence of n-heptane, isopropanol, acetone, ethanol and methanol, respectively.
Fig. 3. Effect of temperature on activity and thermostability of the lipase. The activities () are shown as values relative to that measured at 30 ◦ C (taken as 100%). The thermostability of the lipase (0.05 mg/mL) in 50 mM sodium phosphate buffer (pH 8.0) was measured at various temperatures for 3 h. The remaining activities () of the lipase are shown as values relative to that before incubation (taken as 100%).
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Fig. 4. Substrate specificity of the lipase. The activities of lipase towards different pnitrophenyl esters were determined and showed as values relative to that measured towards p-nitrophenyl caproate (taken as 100%). Table 3 Effects of organic solvents on activity and stability of the lipase. Organic solvents
log P
Residual activity (%)a
Control Nonane Isooctane n-Octane n-Heptane n-Hexane Isopropanol Acetone Ethanol Methanol DMF DMSO
– 5.6 4.7 4.5 4 3.5 0.28 −0.23 −0.24 −0.76 −1 −1.35
49.90 76.5 70.5 73.4 94.2 89.8 132.6 215.3 168.9 121.0 159.3 106.0
± ± ± ± ± ± ± ± ± ± ± ±
5.65 3.85 2.32 1.23 4.98 0.76 1.42 2.15 1.51 0.76 0.52 1.41
Half-livesb 1 day 2 days 2 days 2 days >10 days 7 days >10 days >10 days >10 days >10 days 7 days 7 days
Values are means ± SD (n = 3). a The purified lipase LC2-8 was incubated with various solvents (25%, v/v) at 30 ◦ C for 24 h with shaking at 150 rpm. The initial activity of the lipase without the addition of organic solvents was taken as 100%. b During the first three days of incubation, the remaining activity was determined every 6 h, then, the activity was determined every day. The result of data regression showed that inactivation of the lipase in the presence and absence of organic solvent obeyed first-order kinetics. The half-lives were calculated from the exponential regression curves.
3.7. Bioresolution of (R, S)-1-phenylethanol by lipase LC2-8 in a non-aqueous system (R, S)-1-phenylethanol is one of the most important chiral blocks used in cosmetics and the pharmaceutical industry. After primary optimization, the 1-phenylethanol was esterified by lipase LC2-8 powder in hexane using vinyl-acetate as an acyl donor (Fig. 5). After 24 h of reaction, the 52.4% of substrate was conversed. The
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Fig. 6. Time course of bioresolution of (R, S)-1-phenylethanol catalyzed by lipase LC2-8. The kinetic resolution of (R, S)-1-phenylethanol was carried out in 1 mL n-hexane containing vinyl acetate 800 mM and (R, S)-1-phenylethanol 200 mM catalyzed by 10 mg (30U) lipase LC2-8 powder at 30 ◦ C and 200 rpm. Symbols: (䊉) conversion; () ees . The amounts of optical isomers of the esterified product and residual substrate were determined by HPLC.
lipase LC2-8 was found to be high activity of esterification towards (R)-1-phenylethanol. The residual substrate, (S)-1-phenylethanol, remained with the yield of 47.6%, and the enantiomeric excess of (S)-1-phenylethanol reached 99.9%, giving an E-value over 200 (Fig. 6). 4. Discussion Stability in the presence of organic solvents is a requisite property of enzymes used in non-aqueous system. However, many enzymes are easily inactivated or denature in organic solvents. Recently, solvent-tolerant bacteria as a relatively novel group of extremophilic microorganisms with unique ability to live in presence of organic solvents have attracted the great attention of many researchers [12]. It is found that these microbes are attuned to work under solvent rich environment, thus some of the enzymes from this kind of microbes are naturally stable towards organic solvents, especially the extracellular enzymes such as proteases and lipases [13]. In a similar effort, solvent tolerant bacteria Pseudomonas stutzeri LC2-8 was isolated using toluene and cyclohexane enrichment medium. Many solvent-tolerant lipases had been obtained from Pseudomonas and Bacillus genus. However, lipases from different genus, species and even strains have large variations in substrate specificity, enantioselectivity, as well as solvent stability. The amino acid sequence of lipase from P. stutzeri LC2-8 showed 95.5% and 96% homology to the putative lipase lip A from P. mendocina PK-12CS and the putative lipase lip C from P. stutzeri A1501, respectively. There is no report about the characteristics of these two lipases from P. stutzeri A1501 and P. mendocina PK-12CS. In addition, the lipase LC2-8 showed less than 80% homology with the lipase from
Fig. 5. Kinetic resolution of (R, S)-1-phenylethanol catalyzed by lipase LC2-8.
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other Pseudomonas species. To our knowledge, this is the first report on the characteristics, especially solvent stability of the lipase from the species of P. stutzeri. Of particular most availability and novelty is that the lipase from P. stutzri LC2-8 showed significantly solvent-stable characteristics in both hydrophobic and hydrophilic solvent. Most of the hydrophilic solvents tested enhanced the activity of lipase LC2-8. Generally, enzymes are not stable in the presence of hydrophilic solvents, the higher its affinity to water, the more likely it is to strip the essential water from the enzyme molecules [2]. Only a few studies have reported that lipases from organic solvent-tolerant bacteria were stabilized and activated by hydrophilic solvents. In the presence of 30% (v/v) isoamyl alcohol for 1 h and 24 h, the remaining activity of the lipase from P. fluorescens JCM5963 [14] was reported to be 142% and 120%, respectively. The lipase from B. cepacia ATCC 25416 was shown to increase 1.7-, 1.8-, 1.7- and 2.0fold with 30% (v/v) acetone, chloroform, n-hexane, cyclohexane and n-heptane in the reaction mixture, respectively [15]. The activation of lipase in the presence of some hydrophilic organic solvents could be explained by the disruption of aggregates between the lipase molecules themselves [12]. The tendency of the lipase to aggregate has been observed during the experiment. On the other hand, some researchers conjectured that different interactions of some amino acid residues with various polar organic solvents may convert the lipase conformation to some degrees from closed form (active site is covered by a lid) to open form (active site is uncovered) causing the increase of the lipase activity and even enantioselectivity [16]. The prominent stability of lipase LC2-8 in organic solvents suggests that it may have potential for use in organic catalysis in non-aqueous systems. In addition to organic solvents, 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 well as the reaction rate of the kinetic resolution [17]. The lipase LC2-8 was also activated by some nonionic surfactants such as Triton X-100, Tween 20 and Tween 80. This characteristic further strengthens the application of lipase LC2-8 in biocatalysis and the resolution of drug intermediates in non-aqueous media. As mentioned before, 1-phenylethanol is regarded as a chiral building block in synthesis. Lipase LC2-8 was found to catalyze enantioselective transesterification of (R, S)-1-phenylethanol with high chiral selectivity in n-hexane. The enantiomeric excess of (S)-1-phenylethanol reached 99.9%, giving an E-value over 200. Habulin reported that commercial lipase Novozyme 435 was used for kinetic resolution (R, S)-1-phenylethanol with e.e. >99% at 50% conversion in ionic liquids [18]. Xue reported that commercial lipase Pseudomonas cepacia lipase (PSL) immobilized on HOOC-MCF was used for the transesterification resolution of (R, S)-1-phenylethanol with e.e. >99% at 50% conversion, while only 7% conversion was obtained with the free form [19]. Fortunately, the lipase LC2-8 in free form efficiently performed chiral resolution of (R, S)-1-phenylethanol. As well as its solvent stability, the lipase LC2-8 is considered as an alternative efficient biocatalyst for resolution of chiral secondary alcohols. Further researches are being conducted to investigate the overexpression of lipase LC28 in Pichia pastoria, the immobilization of lipase LC2-8 and the application in bioresolution. 5. Conclusion In this study, an extracellular lipase from the newly isolated organic solvent-tolerant bacterium P. stutzeri LC2-8 was purified
and identified. The lipase exhibited high activity and stability in a variety of organic solvents. Furthermore, the lipase showed good enantioselectivity towards (R, S)-1-phenylethanol. These indicated that lipase LC2-8 would be a very attractive enzyme for potential application in biocatalysis in non-aqueous media. Acknowledgements Financial supports for this research from the National Program on Key Basic Research Project (2011CB710800), the Key Program of the National Natural Science Foundation of China (20936002). We also acknowledge the support of the projects funded by PCSIRT and PAPD. References [1] S.N. Baharum, A.B. Salleh, C.N.A. Razak, M. Basri, M.B.A. Rahman, R. Rahman, Organic solvent tolerant lipase by Pseudomonas sp strain S5: stability of enzyme in organic solvent and physical factors affecting its production, Ann. Microbiol. 53 (2003) 75–83. [2] A.M. Klibanov, Enzymes that work in organic solvents, Chem. Tech. 16 (1986) 354–359. [3] H. Ogino, K. Miyamoto, H. Ishikawa, Organic-solvent-tolerant bacterium which secretes organic-solvent-stable lipolytic enzyme, Appl. Environ. Microbiol. 60 (1994) 3884–3886. [4] C.J. Hun, R. Rahman, A.B. Salleh, M. Basri, A newly isolated organic solvent tolerant Bacillus sphaericus 205y producing organic solvent-stable lipase, Biochem. Eng. J. 15 (2003) 147–151. [5] L.L. Zhao, J.H. Xu, J. Zhao, J. Pan, Z.L. Wang, Biochemical properties and potential applications of an organic solvent-tolerant lipase isolated from Serratia marcescens ECU1010, Process Biochem. 43 (2008) 626–633. [6] U.K. Winkler, M. Stuckmann, Glycogen, hyaluronate, and some other polysaccharides greatly enhance the formation of exolipase by serratia-marcescens, J. Bacteriol. 138 (1979) 663–670. [7] M.M. Bradford, Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254. [8] U.K. Laemmli, Cleavage of structural proteins during assembly of head of bacteriophage-T4, Nature 227 (1970) 680–685. [9] D.N. Perkins, D.J.C. Pappin, D.M. Creasy, J.S. Cottrell, Probability-based protein identification by searching sequence databases using mass spectrometry data, Electrophoresis 20 (1999) 3551–3567. [10] Y.L. Yan, J. Yang, Y.T. Dou, M. Chen, S.Z. Ping, J.P. Peng, W. Lu, W. Zhang, Z.Y. Yao, H.Q. Li, W. Liu, S. He, L.Z. Geng, X.B. Zhang, F. Yang, H.Y. Yu, Y.H. Zhan, D.H. Li, Z.L. Lin, Y.P. Wang, C. Elmerich, M. Lin, Q. Jin, Nitrogen fixation island and rhizosphere competence traits in the genome of rootassociated Pseudomonas stutzeri A1501, Proc. Natl. Acad. U. S. A. 105 (2008) 7564–7569. [11] H. Ogino, S. Nakagawa, K. Shinya, T. Muto, N. Fujimura, M. Yasuda, H. Ishikawa, Purification and characterization of organic solvent-stable lipase from organic solvent-tolerant Pseudomonas aeruginosa LST-03, J. Biosci. Bioeng. 89 (2000) 451–457. [12] N. Doukyu, H. Ogino, Organic solvent-tolerant enzymes, Biochem. Eng. J. 48 (2010) 270–282. [13] H. Ogino, H. Ishikawa, Enzymes which are stable in the presence of organic solvents, J. Biosci. Bioeng. 91 (2001) 109–116. [14] A.J. Zhang, R.J. Gao, N.B. Diao, G.Q. Xie, G. Gao, S.G. Cao, Cloning, expression and characterization of an organic solvent tolerant lipase from Pseudomonas fluorescens JCM5963, J. Mol. Catal. B: Enzym. 56 (2009) 78–84. [15] X.Q. Wang, X.W. Yu, Y. Xu, Homologous expression, purification and characterization of a novel high-alkaline and thermal stable lipase from Burkholderia cepacia ATCC 25416, Enzyme Microb. Technol. 45 (2009) 94–102. [16] I.J. Colton, S.N. Ahmed, R.J. Kazlauskas, A 2-propanol treatment increases the enantioselectivity of Candida rugosa lipase toward esters of chiral carboxylic acids, J. Org. Chem. 60 (1995) 212–217. [17] Z.D. Long, J.H. Xu, L.L. Zhao, J. Pan, S. Yang, L. Hua, Overexpression of Serratia marcescens lipase in Escherichia coli for efficient bioresolution of racemic ketoprofen, J. Mol. Catal. B: Enzym. 47 (2007) 105–110. [18] Habulin, Maja, Knez, Zeljko, Optimization of (R, S)-l-phenylethanol kinetic resolution over Candida antarctica lipase B in ionic liquids, J. Mol. Catal. B: Enzym. 58 (2009) 24–28. [19] P. Xue, X.H. Yan, Z. Wang, Lipase immobilized on HOOC-MCF: a highly enantioselective catalyst for transesterification resolution of (R, S)-1-phenylethanol, Chin. Chem. Lett. 18 (2007) 929–932.
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Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Regular article
Purification and characterization of an organic solvent-stable lipase from Pseudomonas stutzeri LC2-8 and its application for efficient resolution of (R, S)-1-phenylethanol Yan Cao, Yu Zhuang, Changjin Yao, Bin Wu, Bingfang He ∗ College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, 30 Puzhunan Road, Nanjing 211816, Jiangsu, China
a r t i c l e
i n f o
Article history: Received 1 December 2011 Received in revised form 24 February 2012 Accepted 11 March 2012 Available online 19 March 2012 Keywords: Organic solvent-stability Lipase Purification Pseudomonas stutzeri Enantioseparation Biocatalysis
a b s t r a c t An organic solvent-stable lipase from newly isolated solvent-tolerance bacterium Pseudomonas stutzeri LC2-8 was purified by acetone precipitation and anion exchange chromatography. The apparent molecular mass of the purified lipase was estimated by SDS-PAGE to be 32 kDa. The open reading frame (ORF) of lipase LC2-8 encodes 311 amino acids with 287 amino acid residues in the mature lipase which shared 96% homology at the amino acid level with the putative lipase LipC from Pseudomonas stutzeri A1501. The optimum pH and temperature for lipase activity were 8.0 and 30 ◦ C, respectively. Its hydrolytic activity was found to be highest towards p-nitrophenyl caproate (C8). Lipase LC2-8 showed high tolerance in the presence of various organic solvents. Most of the hydrophilic solvents tested strikingly enhanced the activity and stability of lipase LC2-8. The half-life of lipase LC2-8 was extended to 10-fold in the presence of isopropanol, acetone, ethanol and methanol. The transesterification resolution of (R, S)-1-phenylethanol by lipase LC2-8 was carried out with the yield of 47.6%, the enantiomeric excess of residual substrate (ees ) was 99.9%, giving an E-value over 200. The solvent-stable lipase LC2-8 showed an attractive potency for application in biocatalysis in non-aqueous systems. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) catalyzes triglyceride hydrolysis, ester synthesis, transesterification and other reactions. In recent years, its application in detergent additives and in the preparation of chemical and pharmaceutical intermediates, especially the preparation of chiral intermediates by enzymatic hydrolysis, transesterification or aminolysis reactions in organic solvents has been increasingly emphasized [1]. Lipases are diverse in their sensitivity to solvents, and are more destabilizing in polar water miscible solvents than in water immiscible solvents [2]. Therefore, enzymes that are naturally stable, especially with high activity in different types of organic solvents, have great potential in extending the practical catalysis system. Due to the particularity of existing environments, organic solvent-tolerant extremophiles have recently become a research focus for the isolation of organic solvent-stable proteases and lipases. Considerable effort has been made to exploit new species of solvent-tolerant microorganisms which secrete solventstable lipases such as Pseudomonas aeruginosa LST-03 [3], Bacillus
∗ Corresponding author. Tel.: +86 25 58139902; fax: +86 25 58139902. E-mail address: [email protected] (B. He). 1369-703X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2012.03.004
sphaericus 205y [4] and Serratia marcescens ECU1010 [5]. So far, the number of solvent-tolerant bacteria is limited and the properties of the lipases isolated are insufficient to satisfy the needs of diversity substrates. Therefore, it is necessary to explore novel lipases with high activity in organic solvents to expand their application in practical catalysis. In this paper, we report the purification and characteristics of a solvent-stable lipase from the newly isolated organic solventtolerant bacterium Pseudomonas stutzeri LC2-8. The efficient transesterification of (R, S)-1-phenylethanol with high enantioselectivity catalyzed by lipase LC2-8 in a non-aqueous system was also discussed. 2. Materials and methods 2.1. Materials All p-nitrophenyl fatty acid esters and high-performance liquid chromatography (HPLC)-grade solvents were purchased from Sigma (St. Louis, MO, USA). (R, S)-1-phenylethanol (98%) was purchased from Fluka (Germany). LA-Taq DNA polymerase and the PMD-18 Tcector were purchased from Takara (Dalian, China). DH5␣ (Clontech, Saint-Germain-en-Laye, France) was used as the E. coli host. All other chemicals were of analytical grade.
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2.2. Screening and isolation of organic solvent-tolerant lipase producing strains Soil samples from areas contaminated by crude oil and chemicals were collected. The microorganisms with organic solvent tolerance were screened using enriched medium (0.1% tryptone, 0.18% corn steep liquor, 0.35% (NH4 )2 SO4 , 0.3% KH2 PO4 , 0.25% NaCl, 0.1% MgSO4 ·7H2 O and 5% sunflower oil), both toluene and cyclohexane were added to the medium at a concentration of 20% (v/v), respectively. The cultures were then acclimated by being repeatedly transferred to sterile enrichment medium incubated at 30 ◦ C. Samples of repeated batch cultures were diluted and spread on tributyrin agar plates (0.3% yeast extract, 1.0% tryptone, 0.25% NaCl, 0.5% tributyrin and 1.8% agar). Colonies developing a clear zone were picked up and screened for their lipase production on rhodamine B agar plates (0.1% yeast extract, 0.1% K2 HPO4 , 0.05% MgSO4 ·7H2 O, 1.5% agar, 0.0024% rhodamine B, 0.5% (v/v) corn steep liquor, 6% (v/v) olive oil and 1.8% agar). The microbes showing high ratios of an orange fluorescent halo clear zone diameter to colony diameter under UV light at 350 nm were selected as potential highyield lipase producers for subsequent experiments. The extracellular lipase with remarkable organic solvent (25%, v/v) tolerance was selected for further research. The producing strain identified based on the analysis of the Microlog Microbial Identification System (Biolog Automated Micro-Station System, Biolog, USA) and 16S rDNA sequence BLAST in the Genbank Data Library. 2.3. Culture conditions for lipase LC2-8 production Strain LC2-8 was cultured in lipase-producing medium consisting of (w/v) 1.0% yeast extract, 0.8% glucose, 0.2% K2 HPO4 , 0.05% MgSO4 ·7H2 O, 0.05% (v/v) Triton X-100 and 0.5% (v/v) sunflower oil, with a pH of 8.0. The incubations were carried out at 30 ◦ C with shaking at 180 rpm. After 48 h of incubation, the culture supernatant was collected by centrifugation and used as the crude lipase. 2.4. Assay of lipase activity and protein concentration Lipase activity was measured with a modified spectrophotometric method with p-nitrophenyl palmitate (p-NPP) as substrate [6]. The substrate, p-NPP (3 mg), with a final concentration of 0.3 mg/mL was dissolved in 1 mL of isopropanol and mixed with 9 mL of 50 mM sodium phosphate buffer (pH 8.0) containing gum arabic (0.1%) and Triton X-100 (0.6%). The reaction mixture was composed of 240 L of substrate solution and 10 L of appropriately diluted enzyme solution, and incubated at 30 ◦ C for 10 min. The p-nitrophenol (p-NP) produced in the reaction mixture was quantified spectrophotometrically at 410 nm. One unit of enzyme activity was defined as the amount of enzyme that liberated 1 mol p-NP per min under standard assay conditions. The protein concentrations in this study were determined by the method of Bradford [7] using bovine serum albumin as a standard. 2.5. Purification and cloning of the lipase The lipase LC2-8 was purified in two steps. Chilled acetone was added slowly to the culture supernatant of strain LC2-8 up to 60% (v/v) concentration with continuous stirring and kept at 0 ◦ C for 4 h to allow protein precipitation. The precipitates were harvested by centrifuging at 10,000 × g for 30 min at 4 ◦ C, and resuspended in 50 mM Tris–HCl buffer (pH 8.0), and then applied to a DEAE-Sepharose FF column (1.6 cm × 20 cm, Amersham Biosciences, Sweden) that had been pre-equilibrated with 50 mM Tris–HCl buffer (pH 8.0), eluted with a NaCl gradient from 0.2 to 0.8 M. The fractions with lipase activity were concentrated by
centrifugation using a Centricon 10 kDa molecular weight cut-off device (Millipore, USA). The purified lipase was subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) according to the method of Laemmli [8], and the Coomassie-stained band of the purified lipase was excised from the gel and submitted to the National Center of Biomedical Analysis (NCBA, Beijing) for MS analysis. The trypsin-digested protein fragments of the lipase were subjected to LC/MS/MS analysis, and the sequences of the fragments were submitted to the Mascot program for possible identity matching [9]. The genomic DNA of P. stutzeri strain LC2-8 was obtained by the standard phenol/chloroform precipitation method. The sense primer 5 -TGCCCCATAGCCCGATTCACAT-3 and the antisense primer 5 -ATGCTCTGTTTGAGCGGCTCCT-3 were designed based on the sequence of the putative lipase from P. stutzeri, since MS analysis of the digest fragments of the purified lipase showed that some fragments were identical to that of putative lipase from genome of P. stutzeri A1501 [10]. PCR amplification was carried out with the primers described above. The amplified PCR fragment was cloned into the PMD-18T vector following User Manual from Takara and then sequenced.
2.6. Effect of pH and temperature on lipase activity and stability The activity of the lipase was determined at various pH values (6.0–10.0) at 30 ◦ C. The pH stability of the lipase was analyzed by incubating solutions of the enzyme (0.05 mg/mL) at different pH values for 3 h at 30 ◦ C. To keep the sample pH constant, the following 50 mM buffer systems were used: citric acid/sodium citrate, pH 3.0–6.0; Na2 HPO4 /KH2 PO4 , pH 6.0–8.5; Tris–HCl, pH 7.1–9.0; Gly/NaOH, pH 8.6–10.6; and Na2 HPO4 /NaOH, pH 10.0–12.0. The residual activity was measured according to the pNPP method described above. The effect of temperature on the lipase was measured under various temperature of range 15–50 ◦ C at pH 8.0. The thermal stability of the lipase was assayed by incubating the purified lipase (0.05 mg/mL) at different temperatures (20–50 ◦ C) for 3 h and its residual activity was then measured according to the pNPP method. Each measurement was performed three times and standard error was included.
2.7. Effects of metal ions, inhibitors and surfactants on the lipase activity The effects of different metal ions (Fe2+ , Ca2+ , Mg2+ , Cu2+ , Zn2+ , Li+ , Mn2+ , Co2+ and Ba2+ ) and inhibitors (phenylmethyl sulfonyl fluoride (PMSF), Woodwards reagent K (WRK), dithiothreitol (DDT), -mercaptoethanol and EDTA) were investigated by preincubating the purified lipase (0.05 mg/mL) with 1 and 10 mM solutions of these ions or inhibitors for 1 h at 30 ◦ C, and the residual activity was then tested using the pNPP method described above. Similarly, the effect of surfactants (Triton X-100, Tween 80, Tween 20 and SDS) at the concentration of 0.05% and 0.5% on the lipase was investigated. Each measurement was performed three times and standard error was included. K+ ,
2.8. Substrate specificity to various p-nitrophenyl esters The substrates, p-nitrophenyl fatty acid esters, of varying chain length (C2, C4, C8, C10, C14, C16 and C18) were used at the final concentration of 0.3 mg/mL and the lipase activity was measured according to the pNPP method.
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2.9. Effects of organic solvents on lipase LC2-8 The effects of organic solvents with different log P values at 25% (v/v) concentration on the activity and the stability of the purified lipase were investigated following Ogino et al. [11]. The purified lipase dissolve in 50 mM sodium phosphate buffer (pH 8.0) was filter sterilized (using cellulose acetate membrane, 0.22 m). Aliquots (1 mL) of the organic solvents were added to the purified lipase solution (3 mL) with protein concentration of about 0.05 mg/mL in a sealed glass vial. The mixture was incubated at 30 ◦ C with shaking at 150 rpm. The remaining activity was measured at appropriate time intervals over a period of 10 days. The buffer was added instead of organic solvent as a reference when hydrophilic solvents were studied. 2.10. Bioresolution of (R, S)-1-phenylethanol by lipase LC2-8 Lipase LC2-8 powder preparation: Chilled acetone was added to the culture supernatant of strain LC2-8 until the ratio was 0.8:1 (v/v) under magnetic stirring at 0 ◦ C for 4 h. The precipitated protein was obtained by centrifugation at 10,000 × g and 4 ◦ C for 30 min. Finally, the precipitate was air-dried and lipase LC2-8 powder was obtained. The kinetic resolution of (R, S)-1-phenylethanol was carried out in 1 mL n-hexane containing vinyl acetate 800 mM and (R, S)-1phenylethanol 200 mM catalyzed by 10 mg lipase LC2-8 powder at 30 ◦ C with shaking at 200 rpm. 2.11. Determination of the concentrations of isomers 1-phenylethanol and phenylethanol ester The amounts of optical isomers were determined by HPLC (Dionex P680) using Chiracel OJ column (250 mm × 4.6 mm) at 35 ◦ C. The mobile phase was n-hexane/isopropanol (95:5, v/v) and the flow rate was 0.8 mL/min. The wavelength of the UV detector was set at 254 nm. 2.12. Nucleotide sequence The nucleotide sequence of lipase LC2-8 has been assigned GenBank accession number JN681265. 3. Results 3.1. Isolation of organic solvent-tolerant lipase-producing bacteria
Fig. 1. SDS-PAGE of lipase LC2-8 at different stages of purification. Lane 1, protein molecular weight marker; lane 2, supernatant; lane 3, lipase concentrated by acetone precipitation; lane 4, lipase purified by DEAE-Sepharose FF.
recovery was achieved. The purified lipase was homogeneous with the apparent molecular mass of 32 kDa in SDS-PAGE (Fig. 1). The trypsin-digested peptides of lipase LC2-8 were identified by mass spectrometry and sequencing. The matching sequences included DGAQVYVTEVSQLNTSELR (fragment 1), GEELLAQVEEIVAISGKPK (fragment 2), YVAGVRPDLIASVTSVGAPHKGSD VADLIR (fragment 3), CSSHLGMVIR (fragment 4), and MNHLDEVNQFMGLTSLFETDPVSVYR (fragment 5). These sequences showed high homology to the sequences of the putative lipase LipC from the genome of P. stutzeri A1501. Based on the sequence of the putative lipase from P. stutzeri A1501, the gene encoding the organic solvent stable lipase of P. stutzeri LC2-8 was cloned. The full-length open reading frame (ORF) of lipase LC2-8 consisted of 936 bp encoding 311 amino acid residues, and the mature lipase contained 287 amino acid residues which shared 87% homology at the DNA level and 96% homology at the amino acid level with the putative lipase of P. stutzeri A1501 (NCBI accession no. YP001172529.1). Nine amino acids residues (Ile106 , Ile115 , Val148 , Ala167 , Asn182 , Lys217 , His222 , Val238 , Ser247 ) from lipase LipC from P. stutzeri A1501 were substituted with Val, Val, Ile, Ser, Asp, Phe, Arg, Ile and Ala in lipase LC2-8, respectively. 3.3. Effect of pH and temperature on lipase activity and stability
Cyclohexane and toluene were added to the enriched medium so that only solvent-tolerant microbes were able to grow. As a result, 123 strains of organic solvent-tolerant lipase producers were isolated. Of these strains, strain LC2-8 secreted extracellular lipase which showed highest activity and stability in the presence of hydrophilic organic solvents, and was isolated for further research. Organic solvent-tolerant strain LC2-8 was identified as P. stutzeri based on the analysis of the Biolog (SIM = 0.543, 16–24 h) and 16S rDNA sequences (NCBI GeneBank accession no. FJ345693), possessing the highest homology (99%) with that of P. stutzeri. Strain LC2-8 was deposited in the China Center for Type Culture Collection (Wuhan, China) with the accession number CCTCC M 2010279. 3.2. Purification and cloning of the lipase The extracellular lipase from P. stutezri LC2-8 was purified by acetone precipitation and DEAE-Sepharose FF anion-exchange chromatography (Table 1). About 21.2-fold purification with 32.8%
The optimal pH for lipase activity was found to be 8.0 (Fig. 2). Lipase LC2-8 showed good stability in the pH range 5.0–9.5. The temperature optimum for lipase activity was observed to be 30 ◦ C (Fig. 3). The enzyme retained 60% of its maximal activity at 20 ◦ C, showing the characteristics of a cold-active lipase. The lipase showed considerable thermo-sensitivity, and the activity decreased sharply when the temperature was above 40 ◦ C. 3.4. Effects of metal ions, inhibitors and surfactants on lipase activity The effects of various metal ions, inhibitors and surfactants on lipase activity are summarized in Table 2. Among the tested metal ions, K+ , Ba2+ and Ca2+ significantly activated lipase LC28; while Zn2+ and Cu2+ strongly inhibited lipase LC2-8. The metal chelator, EDTA, had a little influence on the activity of lipase at the low concentration, but inhibited lipase activity by 56.2% at a
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Table 1 Purification of the lipase from P. stutzeri LC2-8.
Supernatant of culture Acetone precipitation DEAE-Sepharose FF
Enzyme activity (U/mL)
Protein (mg/mL)
Specific activity (U/mg)
Total activity (U)
Yield (%)
Purification (fold)
145 332 47.6
0.65 0.52 0.01
223.1 638.5 4760
2900 1992 951
100 68.7 32.8
1 2.9 21.2
Table 2 Effects of metal ions, inhibitors and surfactants on activity of the lipase.a Effectors
Relative activity (%)
Inhibitors EDTA PMSF WRK -Mercaptoethanol DTT Surfactants TritonX-100 Tween 20 Tween 80 SDS Controlb
1 mM 76.5 ± 1.24 91.7 ± 4.56 90.5 ± 2.43 98.9 ± 2.14 102.3 ± 1.09 0.05% 145.3 ± 3.55 103.3 ± 2.28 107.6 ± 3.98 38.8 ± 1.78 100
10 mM 43.8 ± 1.65 79.8 ± 2.12 83.1 ± 1.11 99.8 ± 0.76 96.0 ± 1.21 0.5% 107.8 ± 5.87 94.5 ± 1.45 98.2 ± 1.76 3.6 ± 0.34 100
Effectors
Relative activity (%)
Metal ions Li+ K+ Ba2+ Ca2+ Mg2+ Mn2+ Co2+ Fe2+ Cu2+ Zn2+
1 mM 100.9 ± 0.98 116.6 ± 0.23 115.2 ± 1.90 121.3 ± 1.43 101.5 ± 0.62 108.2 ± 0.75 102.7 ± 1.93 102.3 ± 2.28 78.1 ± 2.17 41.5 ± 1.61
10 mM 107.4 ± 1.27 110.2 ± 1.53 127.0 ± 1.12 117.8 ± 2.71 104.0 ± 0.66 19.5 ± 0.98 97.4 ± 1.65 94.2 ± 2.28 23.7 ± 0.55 5.9 ± 0.23
Values are means ± SD (n = 3). a The purified lipase LC2-8 was incubated with various inhibitors, metal ions (1 mM and 10 mM) and surfactants (0.05% and 0.5%) in 50 mM Tris–HCl buffer (pH 8.0) at 30 ◦ C for 1 h. b Lipase activity is shown as values relative to that measured without addition of effectors (control).
concentration of 10 mM. PMSF and WRK slightly inhibited activity of the lipase. The thiol reducing agents, DTT and -mercaptoethanol did not affect lipase activity. The nonionic surfactant, Triton X-100, enhanced the activity of the lipase, while Tween 20 and Tween 80 showed slight activation at low concentrations. In contrast, the anionic surfactant, SDS, strongly inhibited lipase LC2-8. 3.5. Substrate specificity The enzyme substrate specificity was examined using various fatty acid esters of p-nitrophenyl. The lipase preferentially hydrolyzed p-nitrophenyl esters with medium chain fatty acids and exhibited the highest hydrolytic activity toward p-nitrophenyl caproate (C8). Short chain substrates, especially p-nitrophenyl acetate (C2) were hydrolyzed very slowly (Fig. 4).
Fig. 2. Effect of pH on activity (solid) and stability (hollow) of the lipase. Lipase activities are shown as values relative to that measured in 50 mM sodium phosphate buffer pH 8.0 (taken as 100%). Stability of the lipase (0.05 mg/mL) was determined by incubating at 30 ◦ C for 3 h in the following buffer systems: 50 mM citric acid/sodium citrate (pH 3.0–6.0) (), 50 mM Na2 HPO4 /KH2 PO4 (pH 6.0–8.5) (䊉 ), 50 mM Tris/HCl (pH 7.1–9.0) (), 50 mM Gly/NaOH (pH 8.6–10.6) ( ♦), and 50 mM Na2 HPO4 /NaOH (pH 10.0–12.0) (). The remaining activities of the lipase are shown as values relative to the initial activity (taken as 100%) at pH 8.0.
3.6. Effects of organic solvents on the lipase activity The effects of various organic solvents with different log P values on the lipase are shown in Table 3. Most of the tested hydrophilic solvents at 25% concentration enhanced the activity of the lipase. Among these solvents, isopropanol, acetone, ethanol and DMF significantly increased the activity of lipase to 132.6%, 215.3%, 168.9% and 159.3%, respectively, compared to the initial activity without the addition of organic solvents. The activity of the purified lipase significantly decreased during incubation in the absence of organic solvents at 30 ◦ C, with a half-life of only 24 h; while in the presence of the tested organic solvents, the half-life of lipase LC2-8 was extended significantly by more than 10-fold in the presence of n-heptane, isopropanol, acetone, ethanol and methanol, respectively.
Fig. 3. Effect of temperature on activity and thermostability of the lipase. The activities () are shown as values relative to that measured at 30 ◦ C (taken as 100%). The thermostability of the lipase (0.05 mg/mL) in 50 mM sodium phosphate buffer (pH 8.0) was measured at various temperatures for 3 h. The remaining activities () of the lipase are shown as values relative to that before incubation (taken as 100%).
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Fig. 4. Substrate specificity of the lipase. The activities of lipase towards different pnitrophenyl esters were determined and showed as values relative to that measured towards p-nitrophenyl caproate (taken as 100%). Table 3 Effects of organic solvents on activity and stability of the lipase. Organic solvents
log P
Residual activity (%)a
Control Nonane Isooctane n-Octane n-Heptane n-Hexane Isopropanol Acetone Ethanol Methanol DMF DMSO
– 5.6 4.7 4.5 4 3.5 0.28 −0.23 −0.24 −0.76 −1 −1.35
49.90 76.5 70.5 73.4 94.2 89.8 132.6 215.3 168.9 121.0 159.3 106.0
± ± ± ± ± ± ± ± ± ± ± ±
5.65 3.85 2.32 1.23 4.98 0.76 1.42 2.15 1.51 0.76 0.52 1.41
Half-livesb 1 day 2 days 2 days 2 days >10 days 7 days >10 days >10 days >10 days >10 days 7 days 7 days
Values are means ± SD (n = 3). a The purified lipase LC2-8 was incubated with various solvents (25%, v/v) at 30 ◦ C for 24 h with shaking at 150 rpm. The initial activity of the lipase without the addition of organic solvents was taken as 100%. b During the first three days of incubation, the remaining activity was determined every 6 h, then, the activity was determined every day. The result of data regression showed that inactivation of the lipase in the presence and absence of organic solvent obeyed first-order kinetics. The half-lives were calculated from the exponential regression curves.
3.7. Bioresolution of (R, S)-1-phenylethanol by lipase LC2-8 in a non-aqueous system (R, S)-1-phenylethanol is one of the most important chiral blocks used in cosmetics and the pharmaceutical industry. After primary optimization, the 1-phenylethanol was esterified by lipase LC2-8 powder in hexane using vinyl-acetate as an acyl donor (Fig. 5). After 24 h of reaction, the 52.4% of substrate was conversed. The
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Fig. 6. Time course of bioresolution of (R, S)-1-phenylethanol catalyzed by lipase LC2-8. The kinetic resolution of (R, S)-1-phenylethanol was carried out in 1 mL n-hexane containing vinyl acetate 800 mM and (R, S)-1-phenylethanol 200 mM catalyzed by 10 mg (30U) lipase LC2-8 powder at 30 ◦ C and 200 rpm. Symbols: (䊉) conversion; () ees . The amounts of optical isomers of the esterified product and residual substrate were determined by HPLC.
lipase LC2-8 was found to be high activity of esterification towards (R)-1-phenylethanol. The residual substrate, (S)-1-phenylethanol, remained with the yield of 47.6%, and the enantiomeric excess of (S)-1-phenylethanol reached 99.9%, giving an E-value over 200 (Fig. 6). 4. Discussion Stability in the presence of organic solvents is a requisite property of enzymes used in non-aqueous system. However, many enzymes are easily inactivated or denature in organic solvents. Recently, solvent-tolerant bacteria as a relatively novel group of extremophilic microorganisms with unique ability to live in presence of organic solvents have attracted the great attention of many researchers [12]. It is found that these microbes are attuned to work under solvent rich environment, thus some of the enzymes from this kind of microbes are naturally stable towards organic solvents, especially the extracellular enzymes such as proteases and lipases [13]. In a similar effort, solvent tolerant bacteria Pseudomonas stutzeri LC2-8 was isolated using toluene and cyclohexane enrichment medium. Many solvent-tolerant lipases had been obtained from Pseudomonas and Bacillus genus. However, lipases from different genus, species and even strains have large variations in substrate specificity, enantioselectivity, as well as solvent stability. The amino acid sequence of lipase from P. stutzeri LC2-8 showed 95.5% and 96% homology to the putative lipase lip A from P. mendocina PK-12CS and the putative lipase lip C from P. stutzeri A1501, respectively. There is no report about the characteristics of these two lipases from P. stutzeri A1501 and P. mendocina PK-12CS. In addition, the lipase LC2-8 showed less than 80% homology with the lipase from
Fig. 5. Kinetic resolution of (R, S)-1-phenylethanol catalyzed by lipase LC2-8.
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other Pseudomonas species. To our knowledge, this is the first report on the characteristics, especially solvent stability of the lipase from the species of P. stutzeri. Of particular most availability and novelty is that the lipase from P. stutzri LC2-8 showed significantly solvent-stable characteristics in both hydrophobic and hydrophilic solvent. Most of the hydrophilic solvents tested enhanced the activity of lipase LC2-8. Generally, enzymes are not stable in the presence of hydrophilic solvents, the higher its affinity to water, the more likely it is to strip the essential water from the enzyme molecules [2]. Only a few studies have reported that lipases from organic solvent-tolerant bacteria were stabilized and activated by hydrophilic solvents. In the presence of 30% (v/v) isoamyl alcohol for 1 h and 24 h, the remaining activity of the lipase from P. fluorescens JCM5963 [14] was reported to be 142% and 120%, respectively. The lipase from B. cepacia ATCC 25416 was shown to increase 1.7-, 1.8-, 1.7- and 2.0fold with 30% (v/v) acetone, chloroform, n-hexane, cyclohexane and n-heptane in the reaction mixture, respectively [15]. The activation of lipase in the presence of some hydrophilic organic solvents could be explained by the disruption of aggregates between the lipase molecules themselves [12]. The tendency of the lipase to aggregate has been observed during the experiment. On the other hand, some researchers conjectured that different interactions of some amino acid residues with various polar organic solvents may convert the lipase conformation to some degrees from closed form (active site is covered by a lid) to open form (active site is uncovered) causing the increase of the lipase activity and even enantioselectivity [16]. The prominent stability of lipase LC2-8 in organic solvents suggests that it may have potential for use in organic catalysis in non-aqueous systems. In addition to organic solvents, 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 well as the reaction rate of the kinetic resolution [17]. The lipase LC2-8 was also activated by some nonionic surfactants such as Triton X-100, Tween 20 and Tween 80. This characteristic further strengthens the application of lipase LC2-8 in biocatalysis and the resolution of drug intermediates in non-aqueous media. As mentioned before, 1-phenylethanol is regarded as a chiral building block in synthesis. Lipase LC2-8 was found to catalyze enantioselective transesterification of (R, S)-1-phenylethanol with high chiral selectivity in n-hexane. The enantiomeric excess of (S)-1-phenylethanol reached 99.9%, giving an E-value over 200. Habulin reported that commercial lipase Novozyme 435 was used for kinetic resolution (R, S)-1-phenylethanol with e.e. >99% at 50% conversion in ionic liquids [18]. Xue reported that commercial lipase Pseudomonas cepacia lipase (PSL) immobilized on HOOC-MCF was used for the transesterification resolution of (R, S)-1-phenylethanol with e.e. >99% at 50% conversion, while only 7% conversion was obtained with the free form [19]. Fortunately, the lipase LC2-8 in free form efficiently performed chiral resolution of (R, S)-1-phenylethanol. As well as its solvent stability, the lipase LC2-8 is considered as an alternative efficient biocatalyst for resolution of chiral secondary alcohols. Further researches are being conducted to investigate the overexpression of lipase LC28 in Pichia pastoria, the immobilization of lipase LC2-8 and the application in bioresolution. 5. Conclusion In this study, an extracellular lipase from the newly isolated organic solvent-tolerant bacterium P. stutzeri LC2-8 was purified
and identified. The lipase exhibited high activity and stability in a variety of organic solvents. Furthermore, the lipase showed good enantioselectivity towards (R, S)-1-phenylethanol. These indicated that lipase LC2-8 would be a very attractive enzyme for potential application in biocatalysis in non-aqueous media. Acknowledgements Financial supports for this research from the National Program on Key Basic Research Project (2011CB710800), the Key Program of the National Natural Science Foundation of China (20936002). We also acknowledge the support of the projects funded by PCSIRT and PAPD. References [1] S.N. Baharum, A.B. Salleh, C.N.A. Razak, M. Basri, M.B.A. Rahman, R. Rahman, Organic solvent tolerant lipase by Pseudomonas sp strain S5: stability of enzyme in organic solvent and physical factors affecting its production, Ann. Microbiol. 53 (2003) 75–83. [2] A.M. Klibanov, Enzymes that work in organic solvents, Chem. Tech. 16 (1986) 354–359. [3] H. Ogino, K. Miyamoto, H. Ishikawa, Organic-solvent-tolerant bacterium which secretes organic-solvent-stable lipolytic enzyme, Appl. Environ. Microbiol. 60 (1994) 3884–3886. [4] C.J. Hun, R. Rahman, A.B. Salleh, M. Basri, A newly isolated organic solvent tolerant Bacillus sphaericus 205y producing organic solvent-stable lipase, Biochem. Eng. J. 15 (2003) 147–151. [5] L.L. Zhao, J.H. Xu, J. Zhao, J. Pan, Z.L. Wang, Biochemical properties and potential applications of an organic solvent-tolerant lipase isolated from Serratia marcescens ECU1010, Process Biochem. 43 (2008) 626–633. [6] U.K. Winkler, M. Stuckmann, Glycogen, hyaluronate, and some other polysaccharides greatly enhance the formation of exolipase by serratia-marcescens, J. Bacteriol. 138 (1979) 663–670. [7] M.M. Bradford, Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254. [8] U.K. Laemmli, Cleavage of structural proteins during assembly of head of bacteriophage-T4, Nature 227 (1970) 680–685. [9] D.N. Perkins, D.J.C. Pappin, D.M. Creasy, J.S. Cottrell, Probability-based protein identification by searching sequence databases using mass spectrometry data, Electrophoresis 20 (1999) 3551–3567. [10] Y.L. Yan, J. Yang, Y.T. Dou, M. Chen, S.Z. Ping, J.P. Peng, W. Lu, W. Zhang, Z.Y. Yao, H.Q. Li, W. Liu, S. He, L.Z. Geng, X.B. Zhang, F. Yang, H.Y. Yu, Y.H. Zhan, D.H. Li, Z.L. Lin, Y.P. Wang, C. Elmerich, M. Lin, Q. Jin, Nitrogen fixation island and rhizosphere competence traits in the genome of rootassociated Pseudomonas stutzeri A1501, Proc. Natl. Acad. U. S. A. 105 (2008) 7564–7569. [11] H. Ogino, S. Nakagawa, K. Shinya, T. Muto, N. Fujimura, M. Yasuda, H. Ishikawa, Purification and characterization of organic solvent-stable lipase from organic solvent-tolerant Pseudomonas aeruginosa LST-03, J. Biosci. Bioeng. 89 (2000) 451–457. [12] N. Doukyu, H. Ogino, Organic solvent-tolerant enzymes, Biochem. Eng. J. 48 (2010) 270–282. [13] H. Ogino, H. Ishikawa, Enzymes which are stable in the presence of organic solvents, J. Biosci. Bioeng. 91 (2001) 109–116. [14] A.J. Zhang, R.J. Gao, N.B. Diao, G.Q. Xie, G. Gao, S.G. Cao, Cloning, expression and characterization of an organic solvent tolerant lipase from Pseudomonas fluorescens JCM5963, J. Mol. Catal. B: Enzym. 56 (2009) 78–84. [15] X.Q. Wang, X.W. Yu, Y. Xu, Homologous expression, purification and characterization of a novel high-alkaline and thermal stable lipase from Burkholderia cepacia ATCC 25416, Enzyme Microb. Technol. 45 (2009) 94–102. [16] I.J. Colton, S.N. Ahmed, R.J. Kazlauskas, A 2-propanol treatment increases the enantioselectivity of Candida rugosa lipase toward esters of chiral carboxylic acids, J. Org. Chem. 60 (1995) 212–217. [17] Z.D. Long, J.H. Xu, L.L. Zhao, J. Pan, S. Yang, L. Hua, Overexpression of Serratia marcescens lipase in Escherichia coli for efficient bioresolution of racemic ketoprofen, J. Mol. Catal. B: Enzym. 47 (2007) 105–110. [18] Habulin, Maja, Knez, Zeljko, Optimization of (R, S)-l-phenylethanol kinetic resolution over Candida antarctica lipase B in ionic liquids, J. Mol. Catal. B: Enzym. 58 (2009) 24–28. [19] P. Xue, X.H. Yan, Z. Wang, Lipase immobilized on HOOC-MCF: a highly enantioselective catalyst for transesterification resolution of (R, S)-1-phenylethanol, Chin. Chem. Lett. 18 (2007) 929–932.