Purification and properties of enantioselective lipase from a newly isolated Bacillus cereus C71

Process Biochemistry 42 (2007) 988–994 www.elsevier.com/locate/procbio

Purification and properties of enantioselective lipase from a newly isolated Bacillus cereus C71 Shaoxin Chen*, Lili Qian, Bingzhao Shi Department of Biochemistry, Shanghai Institute of Pharmaceutical Industry, Beijing Road West 1320, Shanghai 200040, PR China Received 13 November 2006; received in revised form 14 March 2007; accepted 16 March 2007

Abstract A lipase from Bacillus cereus C71 was purified to homogeneity by ammonium sulfate precipitation, followed by Phenyl-Sepharose chromatography, DEAE ion exchange chromatography and CIM1 QA chromatography. This purification procedure resulted in a 1092-fold purification of lipase with 18% yield. The molecular mass of the purified enzyme was determined to be approximately 42 kDa by SDS-PAGE and mass spectrometer. The lipase was stable in the pH range of 8.5–10.0, with the optimum pH 9.0. The enzyme exhibited maximum activity at 33 8C and retained 92% of original activity after incubation at 35 8C for 3 h. The protein hydrolyzed p-nitrophenyl esters with acyl chain lengths between C4 and C12. Enzyme activity was strongly inhibited in the presence of Cu2+ and Zn2+ but promoted by non-ionic surfactants. The lipase demonstrated higher enantioselectivity toward R-isomer of ethyl 2-arylpropanoate than the commercial lipases, and can be used potentially as a catalyst to prepare optically pure pharmaceuticals. # 2007 Elsevier Ltd. All rights reserved. Keywords: Lipase; Bacillus cereus; Purification; Characterization; Enantioselectivity

1. Introduction In the recent years, the growing need for optically pure pharmaceuticals has stimulated a fast development of stereoselective synthetic methods with various enzymes [1–3]. Among the enzymes used for asymmetry organic synthesis reactions, lipases (triacylglycerol acylhydrolases, E.C. 3.1.1.3) are the most widely used enzymes which catalyze the chemo-, region- and stereoselective hydrolysis of carboxylic acid esters or the reverse reactions in organic solvents [4–6]. Lipases can be produced from animals, plants and microorganisms. Especially, microbial lipases have received a great deal of attention as biocatalysts due to their stability, selectivity and broad substrate specificity [7,8]. Although a number of lipases are commercially available, only a few can meet the optimal requirements considered from a technical scale viewpoint [9]. Since organic reactions performed by lipases are enormous, the supply of suitable enzyme candidate is of particular importance [10]. Therefore, there is an increasing demand to identify and

characterize new lipases with emphasis on their applications in enantioselective biotransformations [7,11,12]. In our previous work, a new strain producing an intracellular lipolytic enzyme was screened from soil samples and identified as Bacillus cereus C71 [13]. The strain displayed a high hydrolysis activity toward R-enantiomer of 2-arylpropanoate esters, and has been applied to prepare enantiopure Rflurbiprofen by resolution of racemic flurbiprofen ethyl ester with whole cells. Although the optically pure S-flurbiprofen had been prepared by hydrolyzing its corresponding racemic esters with S-stereospecific lipases as catalysts in most cases [14–16], very few reports are available on R-specific lipases for hydrolysis of the racemic compounds [13]. In this study, the purification and partial characterization of a R-stereospecific lipase from B. cereus C71 were investigated. In addition, an assessment of the enantioselectivity properties of the enzyme is presented. 2. Materials and methods 2.1. Materials

* Corresponding author. Tel.: +86 21 62479808 455; fax: +86 21 62890729. E-mail address: [email protected] (C. Shaoxin). 1359-5113/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2007.03.010

Yeast extract and tryptone were obtained from Oxoid (England). Candida rugosa lipase (CRL), p-nitrophenyl ( pNP) acetate, pNP butyrate were purchased from Sigma. pNP caprylate, pNP laurate, pNP palmitate and pNP stearate were purchased from Fluka. Novozyme 435 and Lipozyme TL IM

C. Shaoxin et al. / Process Biochemistry 42 (2007) 988–994 were obtained from Novozymes. Lipase PS was from Amano Enzyme Inc. All other chemicals and solvents were of analytical grade.

2.2. Production of lipase The screening and culture conditions of B. cereus C71 were described previously [13], and the strain was deposited in China General Microbiological Culture Collection Center as B. cereus CGMCC 1909. For large-scale production of lipase, B. cereus C71 was cultivated in a 5 l bioreactor (New Brunswick Scientific). Bioreactor was inoculated with the seed culture generated in the seed flask. The initial volume of the culture was 3 l containing tryptone 10 g/l, yeast extract 5 g/l, NaCl 10 g/l and Tween-80 0.5 g/l. Temperature and pH were controlled at 37 8C and 7.0 with 3 M NaOH solution, respectively. After incubation for 18 h, the culture broth was centrifuged at 8000 rpm for 20 min. Cells were harvested and stored at 20 8C until use.

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2.7. Substrate specificity Substrate specificities toward different pNP esters were determined at 33 8C and 50 mM Tris–HCl buffer (pH 9.0) using the spectrophotometric assay. The synthetic pNP esters between C2 and C18 were pNP acetate (C2), pNP butyrate (C4), pNP caprylate (C8), pNP laurate (C12), pNP palmitate (C16), and pNP stearate (C18), respectively.

2.8. Enantioselective hydrolysis of ethyl 2-arylpropanoate A typical hydrolysis reaction was performed in a total volume of 5 ml containing 50 mM ethyl 2-ayrlpropanoate, 50 mM sodium phosphate buffer (pH 9.0), 20 U lipase and 0.5% Tween-40. The hydrolysis reaction was carried out at 30 8C with agitation (250 rpm). Aliquots (0.1 ml) were withdrawn to assay conversion and enantioselectivity by HPLC.

2.9. Polyacrylamide gel electrophoresis 2.3. Purification procedure The cell pellet collected was washed twice with distilled water, and then resuspended in 100 mM potassium phosphate buffer (pH 9.0). The suspension was treated by sonication to break cells wall. Unbroken cells and cell debris were removed by centrifugation for 30 min at 12,000 rpm. The resulting supernatant was precipitated with 60% saturation of ammonium sulfate at 4 8C overnight. The precipitant was dissolved in 250 ml of 10 mM potassium phosphate buffer (pH 7.5) containing 0.1 M (NH4)2SO4, and load onto a PhenylSepharose CL-6B column equilibrated with the same buffer. The column was then washed with 150 ml of 15 mM potassium phosphate buffer (pH7.5) and eluted with distilled water. Fractions containing lipase activity were applied to a DEAE-Sepharose Fast Flow column previously equilibrated with 150 mM Tris– HCl buffer (pH 8.0). The unbound protein was collected, and the active fractions were then put on the same DEAE column re-equilibrated with 50 mM Tris–HCl buffer (pH 8.0, buffer A). After the column was washed with 10 column volumes of the buffer A, the lipase was eluted with 50 ml 0.3 M NaCl in the buffer A. The active fractions were dialyzed and lyophilized. Then, the sample was dissolved in a minimal volume of 30 mM Tris–HCl buffer (pH 8.0, buffer B), and was applied to a CIM1 QA column (BIA Separations, Slovenia) equilibrated with buffer B. After washing with 10 column volumes of the buffer B, the bound proteins were eluted with a linear gradient of 0–0.5 M NaCl (20 ml) in the buffer B. The lipase-containing fractions were pooled, dialyzed and lyophilized.

The molecular mass and purity of lipase were analyzed by a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). These standards (phosphorylase b: 97.2 kDa; serum bovine albumin: 66.4 kDa; ovalbumin: 44.3 kDa, carbonic anhydrase: 29 kDa; trypsin inhibitor: 20.1 kDa) were used as protein markers in the method.

2.10. Protein concentration determination The protein concentration was measured using Coomassie-binding assay described by Bradford with bovine serum albumin as a standard [17].

2.11. Enzyme activity assay Enzyme activity was measured using a spectrophotometric method with pNP butyrate ( pNPC4) as substrate [18]. Hundred microlitres of lipase solution were added to 880 ml of reaction buffer (50 mM Tris–HCl, pH 9.0) and the reaction mixture was pre-warmed to 33 8C and then mixed with 20 ml of freshly prepared 25 mM pNPC4 in isopropanol. The reaction mixture was incubated at 33 8C for 10 min, and subjected to colorimetric assay at 405 nm. One enzymatic unit was defined as the amount of enzyme liberating 1 mmol p-nitrophenol per min under the above conditions. Enzyme activity was also determined using flurbiprofen ethyl ester as substrate according to the method described previously [13].

2.4. Effect of pH on enzyme activity and stability 2.12. Analytical methods To investigate the optimal pH, lipase activity was assayed at 33 8C at various pH values (7.0–10.0). The buffers used for the pH ranges of 7.0–8.5 and 9.0– 10.0 were 50 mM sodium phosphate buffer and 50 mM Tris–HCl buffer, respectively. pH stability in the range of 4.0–12.0 was examined by incubating the enzyme solution for 3 h at 33 8C with different buffers, and then determined the residual activity.

2.5. Effect of temperature on enzyme activity and stability The enzyme activity was measured in the range of 20–45 8C using the standard activity assay procedure at related temperature. Stability of the lipase was investigated by measuring the residual activity after incubating the enzyme solution at 20–45 8C for 3 h in 50 mM Tris–HCl buffer (pH 9.0).

2.6. Effect of metal ions and surfactants on enzyme activity Various metal ions at final concentration of 1 or 10 mM were added to the lipase in 50 mM Tris–HCl buffer (pH 9.0). The solution was incubated at 33 8C for 15 min and assayed the lipase activity. The enzyme solutions containing 0.05% (w/v) or 0.5% (w/v) of the surfactants Tween-20, Tween-40, Tween-80, Triton X-100, SDS in 50 mM Tris–HCl buffer (pH 9.0) were incubated at 33 8C for 15 min for lipase assay.

The concentrations and enantiomers of 2-arylpropanoic acids were determined by HPLC [13]. The eep was defined as the ration of ([R] [S])/ ([R] + [S])  100%, where [R] and [S] are concentrations of R- and S-enantiomer, respectively. The E-value defined as the ratio of the specificity constants of the enzyme for the fast-reacting and slow-reacting substrate enantiomers was calculated according to the equation E = ln[1 c(1 + eep)]/ln[1 c(1 eep)], where c is the extent of conversion [19].

3. Results and discussion 3.1. Purification The intracellular lipase from B. cereus C71 was purified by 60% ammonium sulfate precipitation, Phenyl-Sepharose CL6B, DEAE-Sepharose Fast Flow and CIM1 QA column chromatography. In order to facilitate the isolation of lipase from the partial purified fractions, the CIM1 QA disk was applied at the final purification step. The column is a novel monolithic column optimized for better purification than that achieved with conventional supports [20]. The result indicated

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C. Shaoxin et al. / Process Biochemistry 42 (2007) 988–994

Table 1 Purification of lipase from Bacillus cereus C71 Purification step

Total protein (mg)

Total activitya (U)

Specific activity (U/mg)

Purification fold

Yield (%)

Crude extract Ammonium sulfate Phenyl-Sepharose CL-6B DEAE-Sepharose F.F. 1 DEAE-Sepharose F.F. 2 CIM1 QA

1576.9 1022.7 85.03 23.1 3.8 0.26

315.4 286.4 221.1 204.1 113.5 56.8

0.2 0.28 2.6 8.84 29.9 218.5

1 1.4 13 44.2 149.5 1092.5

100 91 70 65 36 18

a

Activity was measured with flurbiprofen ethyl ester as a substrate.

that CIM1 QA could simplify purification of proteins but with high resolution. The purification steps and the yield are summarized in Table 1. The lipase was purified more than 1000-fold with a yield of 18% from the crude extract. The final pooled fractions gave a single band on SDS-PAGE electrophoresis with an apparent molecular mass of approximately 42 kDa (Fig. 1). The molecular mass of purified lipase was confirmed to be 41778.5 Da using AutoFlex MALDI-TOF mass spectrometer (Bruker). These results demonstrated that the native lipase form B. cereus C71 is a monomer. The NH2terminal amino acids analyzed by PE-ABI 491A protein sequencer (Applied Biosystems, USA) had the sequence I-AG-P-S-V-P-D-E-T-L-R. Compared with the known proteins database by the BLAST program, the sequence did not showed significant similarity to that of known lipases, suggesting this protein is a novel lipase. 3.2. Effects of pH on lipase activity and stability The effect of pH on lipase activity with pNPC4 as substrate was examined at various pH values at 33 8C (Fig. 2). The

Fig. 1. SDS-PAGE analysis of purified lipase from Bacillus cereus C71. M, proteins standards; lane 1, purified lipase.

enzyme was active in the range of 7.5–9.5, and the maximal activity was shown at pH 9.0. It is a characteristic of most microbial lipases that the optimum pH falls on the alkaline side [21]. The pH stability of the enzyme is also shown in Fig. 2. After incubation at 33 8C for 3 h, more than 70% of the original activity could be retained at pH 8.5–10.0, but it decreased slightly at pH lower than 8.5. This high activity and stability make the lipase applicable at alkaline pH conditions. 3.3. Effects of temperature on activity and stability The effect of temperature on lipase activity is shown in Fig. 3. The lipase was active in the temperature range of 30– 45 8C with a maximal activity at 33 8C (Fig. 3). The thermostability of the enzyme was examined by measuring the residual activity for a period of incubation at different temperatures at pH 9.0. After incubation for 3 h, the enzyme was stable at 20–35 8C with residual activity greater than 90% of the initial activity. However, the loss of activity increased with increasing temperature, indicating that the lipase from B. cereus C71 was not stable at high-temperature compared to other lipases from thermophilic Bacillus species [22,23]. Moreover, it was found that the thermostability of B. cereus

Fig. 2. Effect of pH on lipase activity and stability. The following 50 mM buffers were used: NaH2PO4–Na2HPO4 (pH 7.0–8.5) and Tris–HCl (pH 9.0– 10.0). Activity (&) was measured at different pH values and 33 8C with pnitrophenyl butyrate as a substrate. The maximum activity of the enzyme was taken as 100%. pH stability (*) was assayed after incubating lipase solution at different pH values for 3 h at 33 8C. The enzyme activity before incubation was defined as 100%.

C. Shaoxin et al. / Process Biochemistry 42 (2007) 988–994

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Table 3 Effect of surfactants on the purified lipase

Fig. 3. Effect of temperature on lipase activity and stability. Activity (&) was determined at pH 9.0, and the maximum activity of the enzyme was taken as 100%. Stability (*) was determined by incubating the purified lipase at various temperatures for 3 h. The enzyme activity before incubation was defined as 100%.

Surfactants

Concentration (%, w/v)

Relative activity (%) a

Control

0

100

Tween-20

0.05 0.5

112 128

Tween-40

0.05 0.5

134 153

Tween-80

0.05 0.5

122 141

Triton X-100

0.05 0.5

136 160

SDS

0.05 0.5

86 79

a Average value of triplicate assays using pNP butyrate as substrate. The activity of enzyme without added surfactant was taken as 100%.

0

100

NaCl

1 10

98 96

concentration, respectively. The negative effect of ions on the lipase is generally the result from direct inhibition of the catalytic site like many other enzymes [24]. In addition, unlike most other lipases from other microorganisms [12,21,25], the catalytic activity of lipase from B. cereus C71 was not enhanced by Ca2+, indicating this enzyme is calcium-independent. It was well known that lipases activities from different sources are affected by surfactants [26]. Our previous results also showed that the enzyme activity and enantioselectivity of cells of B. cereus C71 were significantly affected by the presence of the surfactants [13]. To examine the effects of surfactant on activity of the purified lipase, a few surfactants were chosen based on our previous report. As shown in Table 3, in the presence of 0.05% (w/v) or 0.5% (w/v) surfactants, no inhibitive effect on activity was observed, except for SDS, which reduced 79% of the initial activity at 0.5% concentration. The other non-ionic surfactants, especially Triton X-100, could increase the activity by 60%. Surfactants are known to increase the lipid-water interfacial area, which, in turn, enhance the observed rate of lipase-catalyzed reactions [27]. So, it gives a simple method to improve the reaction efficiency by addition of surfactants.

KCl

1 10

83 75

3.5. Substrate specificity

CaCl2

1 10

99 92

MgCl2

1 10

93 88

NiCl2

1 10

80 71

CoCl2

1 10

78 65

MnCl2

1 10

73 61

ZnCl2 CuCl2

1 1

49 39

C71 lipase was related to the purity of the enzyme. The partially purified lipase displayed higher thermal stability than that of highly purified enzyme (data not shown). 3.4. Effect of metal ions and surfactants on lipase activity The effect of various metal ions on the activity of lipase is shown in Table 2. The results indicated that tested metal ions could not significantly increased the activity of lipase, but inhibited by most of the ions, especially Cu2+ and Zn2+, which reduced the lipase activity to 39 and 49% at 1 mM

Table 2 Effect of metal ions on the purified lipase Metal ions Control

Concentration (mM)

Relative activity (%)a

a Average value of triplicate assays using pNP butyrate as substrate. The activity of enzyme without added metal ion was taken as 100%.

Lipases were defined as the enzymes hydrolyzing long-chain acyglycerols (10 carbon atoms) [28]. However, it was known that most of lipases are also active on short-chain fatty acid esters [29]. Substrate specificity of B. cereus C71 lipase was evaluated by hydrolyzing p-nitrophenyl esters with different chain lengths of acid moiety of the substrates (Fig. 4). The results showed that the enzyme has a broad spectrum towards substrates from C2 to C18 with higher activity toward pNP butyrate (C4), pNP caprylate(C8) and pNP laurate(C12). Although the lipase activity decreased greatly on substrate of longer carbon chain (>12 carbon atoms), it still had 20% activity on pNP stearate (C18). The lipase from B. thermoleovorans ID-1 demonstrated a preference for p-nitrophenyl esters with acyl chain lengths of C4–C6 [30]. Similarly, lipase

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C. Shaoxin et al. / Process Biochemistry 42 (2007) 988–994 Table 5 Comparison of enantioselectivity of lipase from Bacillus cereus C71 with commercially available lipase for enzymatic hydrolysis of (R, S)-flurbiprofen ethyl estera Lipase

t (h)

Conversion (%)

eep% (configuration)

E

Novozyme 435 Lipozyme TL IM CRL Lipase PS B. cereus C71 (this study)

12 15 40 40 22

48  1.2 45  1.8 15  0.4 16  0.8 47  1.5

50.3 70.5 62.3 56.7 97.7

4.7 10.2 4.7 4 243.6

(R) (S) (S) (S) (R)

a

Reactions were conducted in 10 ml sodium phosphate buffer (50 mM, pH 9.0) containing 50 mM ester, 60 U lipase and 0.5% Tween-40 (w/v) at 30 8C.

Fig. 4. Substrate specificity of the lipase from B. cereus C71 again p-nitrophenyl esters of different acyl chain lengths. Enzyme activity was determined by changing the substrates from pNP acetate (C2) to pNP stearate (C18). The maximum activity of the enzyme was taken as 100%.

toward p-nitrophenyl esters with acyl chain lengths of between C8 and C14. Herein, the specificity of the lipolytic enzyme from B. cereus C71 toward p-nitrophenyl esters of longer fatty acids indicated that the enzyme is a true lipase. 3.6. Enantioselectivity of lipase from B. cereus C71 for hydrolyzing ethyl 2-arylpropanoates

Fig. 5. Chiral HPLC chromatogram of (R, S)-flurbiprofen ethyl ester, Sflurbiprofen and R-flurbiprofen in reaction mixture. The hydrolysis reaction was carried out for 18 h.

from Bacillus sp. BP-6 [31], B. thermocatenulatus [32] and Bacillus sp. HI [33] were the most active on esters with shorter fatty acid (C4), and lipase from Bacillus stearothermophilus P1 [22] and Bacillus sp. Tp10A.1 [34] showed higher activity

The enantioselectivity of the lipase was evaluated by hydrolysis of racemic ethyl 2-arylpropanoates. As shown in Table 4, although reaction rates were different, the enzyme was able to enantioselectively hydrolyze the racemic esters, yielding (R)-configuration acids with eep >97% (Fig. 5 shows the HPLC chromatogram of hydrolysis products of flurbiprofen ethyl ester). The compound 3 was also hydrolyzed by commercially available lipases (Table 5). It was found that only the Novozyme 435 produced R-flurbiprofen with 50.3%eep at 48% yield, corresponding to an E-value of 4.7. The same reaction catalyzed by the lipase from B. cereus C71 for 22 h resulted in R-flurbiprofen in 97.7% eep and 47% yield, corresponding to an E-value of 243.6. To our knowledge, among the lipases described in the literature for hydrolysis of 2-arylpropanoate esters, only few enzymes of microbial origin demonstrated R-enantiomeric acid selectively [26,35], therefore, it is of particular importance to explore novel lipases for certain bioconversion reactions. In general, enantiomeric excess values of the products >95% at high conversion and E > 100 are considered as synthetically useful enzymecatalyzed reactions [34]. The lipase from B. cereus C71 showed

Table 4 Hydrolysis of ethyl 2-arylpropanoate by purified lipase from B. cereus C71a

Compoundb

t (h)

Conversion (%)

ee

1 2 3

5 16 18

48  0.6 35  1.1 31  0.8

97.8 97.7 97.7

a b

p

% of R-acid

Reactions were conducted in 5 ml sodium phosphate buffer (50 mM, pH 9.0) containing 50 mM ester, 20 U lipase and 0.5% Tween-40 (w/v) at 30 8C. Compound 1, ibuprofen ethyl ester; 2, ketoprofen ethyl ester and 3, flurbiprofen ethyl ester.

C. Shaoxin et al. / Process Biochemistry 42 (2007) 988–994

higher enantioselectivity for the R-isomer of ethyl 2-arylpropanoate, thereby providing an enzymatic route to prepare the R-flurbiprofen which is a novel drug for anticancer and antiAlzheimer’s disease [36,37].

[13]

[14]

4. Conclusion In this study, the purification and characterizations of an enantioselective lipase from B. cereus C71 are reported. To our knowledge, the lipase from B. cereus has not been reported previously. The biochemical characterization and enantioselectivity of the lipase investigated in this paper will be provided to improve the performance of the lipase in future biotechnological applications. For its high enantioselectivity, this lipase is very interesting as a biocatalyst to obtain optically pure pharmaceuticals or intermediates that are in great demand in the pharmaceutical industry. Additionally, because of its wide substrate specificity, the enzyme can be used not only for short-chain fatty acids but also for mediumchain fatty acids, which will greatly broaden its industrial applications.

[15]

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[19]

[20] [21]

Acknowledgement This work was supported by Creative Foundation Grant of Shanghai Institute of Pharmaceutical Industry.

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