Production and partial characterization of a novel thermostable esterase from a thermophilic Bacillus sp.

Enzyme and Microbial Technology 38 (2006) 628–635

Production and partial characterization of a novel thermostable esterase from a thermophilic Bacillus sp. Z. Burcu Bakır Ates¸lier, Kubilay Metin ∗ Adnan Menderes University, Department of Biology, 09010 Aydın, Turkey Received 30 September 2004; received in revised form 27 June 2005; accepted 19 July 2005

Abstract A thermophilic bacterium, Bacillus sp. 4, newly isolated from Alang¨ull¨u thermal spring (Aydın, Turkey), showed a cell-associated esterase activity. Culture conditions in the growth and esterase production by the Bacillus sp. 4 were investigated using partially modified Thermus medium at different pHs (pH 5.00–9.00) and temperatures (50–70 ◦ C). The optimal growth and esterase production was obtained at pH 6.00 and 65 ◦ C. The maximal esterase production was obtained in the mid-stationary phase, and its activity was either intracellular or membrane associated. Optimum pH and temperature for esterase activity were 6.00 and 65 ◦ C, respectively. After 1 and 10 h incubation at 65 ◦ C, the enzyme exhibited approximately 70 and 50% of its original activity, respectively. After 100 h incubation at 40 ◦ C, the original activity of the enzyme was almost protected (83%). The esterase activities were about 99, 100, 100 and 81% of their original values after 1 h incubation at pH 4.00, 6.00, 8.00 and 10.00, respectively. When the pNPB (C4 ) was used as substrate, the Michaelis–Menten constant (Km ) and maximum velocity for the reaction (Vmax ) of esterase were 62.89 ␮M and 833.33 U/mg protein, respectively. Phenylmethanesulphonyl fluoride (PMSF), a serine-specific inhibitor, strongly inhibited the esterase activity, whereas ␤-mercaptoethanol, a thiol group inhibitor, did not show any effect on the activity. The molecular mass (Mr ) of the esterase was estimated to be 81.9 kDa using SDS-PAGE. These results strongly suggest the presence of a single enzyme responsible for pNPB activity in the crude enzyme extract. Of all substrates (C2 –C16 ) tested, the highest activity was towards pNPB, whereas no activity was observed on pNPP (C16 ). © 2005 Elsevier Inc. All rights reserved. Keywords: Esterase; Lipase; Bacillus; Thermophile; Enzyme characterization

1. Introduction Esterases (EC 3.1.1.1) and lipases (EC 3.1.1.3) are the enzymes catalysing the hydrolysis of ester bonds and are widely distributed in animals, plants and microorganisms. In organic media, they catalyse reactions such as esterification, interesterification and transesterification [1]. Lipases can be distinguished from esterases by the interfacial activation phenomenon and/or having a hydrophobic domain (lid) covering the active site of the enzyme [2]. Besides, esterases are defined as enzymes that hydrolyze the triglycerides with short chains and prefer water-soluble substrates, while lipases are enzymes that hydrolyze triglicerides with long chains and prefer water-insoluble substrates [3,4]. Most of these lipases ∗

Corresponding author. Tel.: +90 256 212 84 98; fax: +90 256 213 53 79. E-mail address: [email protected] (K. Metin).

0141-0229/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2005.07.015

and esterases can be used in organic solvents; therefore, they have become two of the most widely used enzymes in organic synthesis and various industrial applications (detergent industry, food industry, oleochemical industry, pulp and paper industry and resolution of chiral drugs) [3,5–7]. Biocatalysts are usually less stable under operational conditions compared to chemical catalysts. Both esterases and lipases have rarely been utilized in industrial processes due to their low stability under operational process conditions. The process stability is generally higher for the enzymes with high thermostability. Hence, the search for new enzymes with high stability under process conditions is required for industrial applications. Besides, according to a previous report, the enzyme resistance to denaturation in organic solvents is correlated with their thermostability in water [8]. For this reason, thermostable enzymes are of great importance in terms of their use in both aqueous and organic media.

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The aim of this study was to examine the thermostable esterase activity of thermophilic Bacillus sp. 4 and to determine the characteristics of the enzyme.

2. Materials and methods 2.1. Chemicals Analytical reagent grade chemicals were obtained from commercial sources. Unless mentioned otherwise, all chemicals were purchased from Merck (Darmstadt, Germany). p-Nitrophenyl acetate (pNPA), p-nitrophenyl laurate (pNPL) and phenylmethanesulphonyl fluoride (PMSF) were purchased from Fluka (Buchs, Switzerland). p-Nitrophenyl butyrate (pNPB), p-nitrophenyl palmitate (pNPP), N,N methylene bisacrilamide, bovine serum albumin (BSA) and molecular weight markers for SDS-PAGE were purchased from Sigma (Taufkirchen, Germany). Nitrilotriacetic acid was purchased from AVOCADO (Karlsruhe, Germany). (NH4 )2 SO4 and acetone were purchased from Pancreac (Barcelona, Spain). Yeast extract was purchased from idg (Lancashire, UK) and acetonitryl was purchased from sds (Peypin, France). Glycerol was purchased from RiedeldeHa¨en (Seelze, Germany). 2.2. Selection of lipolytically active isolates Five Bacillus strains have been previously isolated from Alang¨ul¨u thermal spring (Aydın, Turkey) [9]. These strains are registered in Culture Collection of Adnan Menderes University, Department of Biology. These isolates were investigated for their lipolytic activity according to Tween 80 agar method [10]. Nutrient agar medium was supplemented with 0.01% CaCl2 ·H2 O and Tween 80 sterilized (separately) for 20 min at 120 ◦ C. Tween 80 was added to the molten agar medium at 45 ◦ C to give a final concentration of 1%. The medium was shaken until the Tween 80 had dissolved completely and then was streaked onto Petri dishes. For positive test, an opaque halo occurred around the colonies. 2.3. Production media The composition of production medium (partially modified Thermus medium of Ramaley and Hixson) [11] used in this study was (per liter): triptone, 3 g; yeast extract, 1 g; Castenholz basal salts solution, 100 mL. The composition of Castenholz basal salts solution was (per liter): nitrilotriacetic acid, 1 g; CaSO4 ·2H2 O, 0.6 g; Na2 HPO4 , 1.11 g; KNO3 , 1.03 g; ferric chloride solution (0.28 g/L), 10 mL; Nitsch’s trace element solution, 10 mL (H3 BO3 , 0.5 g/L; CoCl2 ·6H2 O, 0.046 g/L; CuSO4 ·5H2 O, 0.016 g/L; MnSO4 ·H2 O, 2.2 g/L; NaMoO4 ·2H2 O, 0.025 g/L; H2 SO4 , 0.5 mL/L; ZnSO4 ·5H2 O, 0.5 g/L). The pH was adjusted to 6.00. The media were sterilized for 20 min at 120 ◦ C at 1.5 psi.

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2.4. Culture conditions Bacillus sp. 4 was grown overnight in 10 mL production medium in glass tubes at 65 ◦ C. The culture was inoculated (1%, v/v) at a starting cell density of D600 = 0.1 to 100 mL of production medium in 500 mL Erlenmayer flasks. The culture was incubated at 65 ◦ C on a shaker at 150 rpm for 24 h. Optimal conditions for growth and enzyme production were determined at different pHs and temperatures (three trials were conducted at each temperature and pH). Growth curves of Bacillus sp. 4 were determined by measuring the optical density during cell growth at 600 nm against the fresh medium. 2.5. Preparation of crude enzyme Cells from 100 mL culture in a 1 L Erlenmayer flask were harvested in the middle of stationary phase by centrifugation at 5500 × g, 4 ◦ C for 15 min (Hettich, Universal 32 R). The pellet was resuspended in 50 mM sodium phosphate buffer, pH 6.00, with a final volume of 0.5 g/mL wet cells and disrupted by ultrasonic sonicator (Bandelin HD 2200, MS 73) for 5 min. The disrupted cell debris was removed by centrifugation (5500 × g, 4 ◦ C, 15 min). The supernatant (supernatant 1) was recentrifuged at 23,900 × g, 4 ◦ C for 1 h and obtained supernatant (supernatant 2) was assayed for intracellular enzyme activity. 2.6. Enzyme and protein assays Enzyme activity was measured spectrophotometrically (Shimadzu UV-1601) using p-nitrophenyl butyrate as substrate. This assay was performed as described by Winkler and Stuckman [12] with some modifications. Fifty microliters of enzyme solution was mixed with 930 ␮L 50 mM sodium phosphate buffer, pH 6.00, and pre-warmed at 65 ◦ C for 5 min in a 1 mL spectrophotometer cuvette. The reaction was started by addition of 20 ␮L of substrate solution (50 mM pNPB dissolved in acetone and stored at −20 ◦ C). The increase in absorbance at 346 nm was read against a blank without enzyme for 5 min. Under these conditions, the molar extinction coefficient was determined to be 5.48 L mmol−1 cm−1 . One enzyme unit (IU) was the amount of enzyme liberating 1 ␮mol of p-nitrophenol (pNP) per minute under the assay conditions. The protein concentrations of the enzyme samples were measured using Lowry method [13] with bovine serum albumin as standard. All measurements were performed three times and the mean values were calculated. 2.7. Effect of temperature on activity and stability In order to assess the temperature effect on activity, the enzyme was pre-incubated at different temperatures (30–85 ◦ C) for 15 min at pH 6.00. The reaction was started by adding the substrate, pNPB, at a final concentration of

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1 mM and the residual activity was measured spectrophotometrically. The effect of temperature on the enzyme stability was determined by incubating the enzyme samples at 25, 40, 65 and 75 ◦ C in 50 mM sodium phosphate buffer, pH 6.00. The residual activity was measured at the time intervals for 100 h under the standard assay conditions. Activities were expressed as a percentage of activity at time 0 h. In both assays, the pH of sodium phosphate buffer was adjusted to 6.00 for every temperature applied. 2.8. Effect of pH on activity and stability The following buffers (50 mM) were used for the pH assays: sodium acetate buffer (pH 4.00–5.50), sodium phosphate buffer (pH 6.00–8.00) and sodium carbonate buffer (pH 10.00). The pH of each buffer was adjusted at the enzyme reaction temperature. To determine the optimum pH of enzyme activity, the enzyme was pre-incubated at different pHs (pH 4.00–7.50) for 15 min at 65 ◦ C. Enzymatic reaction was started by adding the substrate, pNPB, at a final concentration of 1 mM and the residual activity was measured spectrophotometrically. The effect of pH on enzyme stability was determined by incubating the enzyme samples in buffers of different pH values (pH 4.00, 6.00, 8.00 and 10.00) at room temperature (28 ◦ C). The residual activity was measured at the time intervals for 80 h under the standard assay conditions. Activities were expressed as a percentage of the activity at time 0 h. The molar extinction coefficient of pNP was determined at each pH tested: 6.32, 5.85, 5.72, 5.54, 5.24, 4.57 and 4.82 L mmol−1 cm−1 at pH 4.00, 4.50, 5.00, 5.50, 6.00, 6.50, 7.00 and 7.50, respectively. 2.9. Determination of kinetic parameters The effect of the substrate (pNPB) concentration (range from 10 to 200 ␮M) on the reaction rate was determined under the standard assay conditions. The Michaelis–Menten constant (Km ) and maximum velocity for the reaction (Vmax ) with pNPB were calculated by the Lineweaver–Burk plot. 2.10. Effect of PMSF and β-mercaptoethanol on esterase activity The effect of phenylmethanesulphonyl fluoride, a serinespecific inhibitor, and ␤-mercaptoethanol, a –SH group inhibitor, on esterase activity was tested by incubating the enzyme at room temperature (28 ◦ C) for 1 h in the presence of 1 and 5 mM of each compound. The residual enzyme activity was measured under the standard assay conditions.

Blot® Cell from Biorad). Samples of 10 ␮g (for protein staining) and 100 ␮g (for activity staining) bacterial proteins were used. Samples were not heated at 100 ◦ C to allow activity staining after migration. Calibration for molecular mass was made using the following standard proteins: myosin from rabbit muscle (205 kDa); ␤-galactosidase from E. coli (116 kDa); phosphorylase b from rabbit muscle (97 kDa); albumin from bovine (66 kDa); albumin from egg (45 kDa); carbonic anhydrase from bovine erythrocytes (29 kDa). Gels were run in duplicate, one being stained for protein and other for activity. Protein staining was done by using Coomasie Blue under standard conditions [14]. After electrophoresis, SDS was eliminated according to Blank et al. [15] by washing first with 20 mM Tris–HCl (pH 6.00) containing 0.08% (w/v) NaOH, 0.025% (w/v) maleic acid and 25% (v/v) isopropanol (30 min) and second by the same solution without isopropanol (15 min). Then, the renatured gel was laid on a Petri dish containing 2% (w/v) agar in 20 mM Tris–HCl (pH 6.00) and 10% (v/v) pNPB solution (16.5 mM) in isopropanol. Activity was detected by appearance of clear halos after 1 or 2 h of incubation at 40 ◦ C. Comparison of the two gels (protein and activity) assigned the molecular mass of the esterase. 2.12. Substrate specificity The substrate specifities of the esterase were determined using p-nitrophenyl acetate (pNPC2), p-nitrophenyl butyrate (pNPC4), p-nitrophenyl laurate (pNPC12) and p-nitrophenyl palmitate (pNPC16) as substrates at the standard assay conditions. The substrate stock solutions were prepared in acetone at a concentration of 50 mM. When using pNPP, Triton X-100 was added to the sodium phosphate buffer at a concentration of 1% and stock pNPP solution was added to this solution at a final concentration of 1 mM and sonicated for 2 min at room temperature before the kinetic measurements were started. 2.13. Statistical analysis The SPSS computer statistics program was used for statistics [16]. The protein concentrations, molar extinction coefficient of pNP and kinetic parameters were analyzed by linear regression analysis. One-way ANOVA was used for testing the differences between the groups. Paired-samples t-test was used to compare the means of the control group and the assay group.

3. Results and discussion 3.1. Effects of temperature and pH on esterase production

2.11. Electrophoresis and activity detection The SDS-PAGE system used was that described by Laemmli [14] with 10.5% (w/v) acrylamide gel (Mini Trans-

In order to determine the optimum temperature for growth and esterase production by Bacillus sp. 4, the isolate was grown on the production medium at various temperatures

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Fig. 3. Effect of pH on esterase production of Bacillus sp. 4.

Fig. 1. Effect of temperature on: (a) growth of Bacillus sp. 4 and (b) the esterase activity of Bacillus sp. 4.

(50, 55, 60, 65 and 70 ◦ C). Bacillus sp. 4 reached the logarithmic phase much rapidly at 60 and 65 ◦ C than the other temperatures assayed. The logarithmic phase was shorter at 65 ◦ C than 60 ◦ C (Fig. 1a). Maximum esterase production was observed at 65 ◦ C (Fig. 1b). These results showed that 65 ◦ C was the optimum temperature for growth and esterase production. The activity of esterase reached to a maximum in the middle of the stationary phase and then declined rapidly (Fig. 2). This result is somewhat different from that of Lactobacillus casei CL96 [17], showing maximum activity during the late logarithmic phase, while it demonstrated some sim-

ilarities with that of Propionibacterium freudenreichii ssp. freudenreichii ITG14 [18], showing maximum activity in the mid-logarithmic phase and followed by a rapid fall. It could be suggested that the esterase of Bacillus sp. 4 was not stable at the assay temperature or at the elevated pH of the culture medium or alternatively, some proteases may be responsible of this downfall. The effect of pH on the esterase production is determined by growing Bacillus sp. 4 on the production medium at different pHs (5.00, 5.50, 6.00, 7.00, 8.00 and 9.00) up to the middle of the stationary phase at 65 ◦ C (Fig. 3). The isolate, Bacillus sp. 4, did not show any growth on the medium at pH 5.00. Maximum esterase activity was observed at pH 6.00 and 84% of this maximum activity was observed at pH 5.50. The esterase activity was declined dramatically above pH 6.00. 3.2. Cellular localization of the esterase The esterase activity of Bacillus sp. 4 was assayed in different cellular fractions using pNPB as substrate (Table 1). Eighty-two percent of the activity was observed in the cell fraction. In addition, the specific activity of cellular fraction (pellet) was 13-fold higher than the culture supernatant. From these results, it could be suggested that the esterase was either cytoplasmic or cell membrane associated. After the cell breakage with sonication, the specific activity was increased about 30%. Therefore, the cell-associated esterase activity could be easily assayed using unbroken cells. This result suggested that the substrate was transferred through the cell wall and then hydrolyzed. Similar results have been reported in previous studies on the intracellular esterase activity [18,19]. 3.3. Effect of temperature on esterase activity and stability

Fig. 2. Growth curve and esterase activity of Bacillus sp. 4 at 65 ◦ C, pH 6.00, and change of the culture pH during the growth.

Esterase activity was tested at temperatures ranging from 30 to 85 ◦ C (Fig. 4). Activity increased linearly from 30 to 65 ◦ C and reached to a maximum at 65 ◦ C. Almost 70% of maximum activity was retained at 70 ◦ C suggesting a high thermal stability. The esterase activity decreased

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632 Table 1 Cellular localization of the esterase Fractions

Fraction volume (mL)

Protein (mg/mL)

Total protein (mg)

Esterase activity (U/mL)

Total activity (U)

Specific activity (U/(mg protein))

Yield (%)

Purification factor

Whole culture Supernatant 1 (culture supernatant)a Pelletb Supernatant 2c

200 186

0.87 0.76

174.00 140.74

137.77 33.15

27554.00 6165.90

158.51 43.87

100 22.37

1 0.28

0.19 0.20

0.29 1.20

113.14 143.25

169.71 859.50

587.40 767.07

0.62 3.13

3.70 4.82

a b c

1.5 6

Centrifugated at 5500 × g for 15 min. Resuspended in 50 mM sodium phosphate buffer. Centrifugated at 23,900 × g for 1 h.

rapidly above 70 ◦ C and 14% of maximum activity was observed at the highest temperature (85 ◦ C) tested. These results indicated that the esterase of Bacillus sp. 4 could be used with any temperature from 40 to 70 ◦ C in biotechnological applications. The temperature for maximal activity was similar to that of esterase from P. freudenreichii spp. freudenreichii ITG14 (65 ◦ C) [18], showing only 50% of maximum activity at 70 ◦ C and higher than those of Bacillus licheniformis (45 ◦ C) [20], Bacillus circulans (60 ◦ C) [21], Arthrobacter nicotianae 9458 (30 ◦ C) [22] and L. casei CL96 (37 ◦ C) [17]. However, it is noteworthy to point out that the esterase of Pyrobaculum calidifontis VA1, a hyperthermophilic archaeon, showed maximum activity at 90 ◦ C [23]. Furthermore, the optimum temperature of the esterase observed in this study was higher than that of the thermophilic lipases of some Bacillus species [24–27] and lower than those of Bacillus sp. THL027 [28] and Bacillus thermoleovorans ID-1 [29]. The thermostability of esterase was determined by measuring the residual activity at the time intervals during 100 h at 25, 40, 65 and 75 ◦ C (Fig. 5). The esterase retained about 79 and 83% of its maximum activity after incubation for 30 h (at 25 ◦ C) and for 100 h (at 40 ◦ C), respectively. When the enzyme was incubated at 65 ◦ C for 1 h, it retained 70% of its maximum activity, whereas the incubation at 75 ◦ C depicted 18% of the maximum activity. Half of the inactivation was observed after 10 h incubation at 65 ◦ C. In the light of the above-mentioned findings, it can be concluded that esterase employed in our study is quite thermostable, exhibiting more

Fig. 4. Effect of temperature on Bacillus sp. 4 esterase activity.

thermostability compared to those of B. licheniformis [20], A. nicotianae 9458 [22] and L. casei CL96 [17], but less than those of Bacillus stearothermophilus [30] and B. circulans [21]. 3.4. Effect of pH on esterase activity and stability In general, esterases show maximum activity at pHs closed to neutrality (generally around pH 6.00) and in this respect they differ from lipases (typically above pH 8.00) [2]. The esterase from Bacillus sp. 4 showed an optimum activity at pH 6.00 suggesting this view (Fig. 6). The esterase demonstrated about 83, 98 and 69% of its maximum activity at pH 5.00, 5.50 and 6.50, respectively. At pH 7.50, only 16% of the maximum activity was observed. Similarly, the esterases of Arthrobacter viscosus [31], P. freudenreichii ssp. freudenreichii ITG 14 [18], Arthrobacter nicotianae 9458 [22], L. casei CL96 [17] and P. calidifontis VA1 [23] showed maximum activity near neutral pHs. However, some esterases have exhibited a maximum activity at alkaline pHs as reported elsewhere (pH 7.50–9.50) [19,20,32–34]. The effect of pH on enzyme stability was determined by measuring the residual activity at the time intervals at pH 4.00, 6.00, 8.00 and 10.00 (Fig. 7). The enzyme was stable at

Fig. 5. Effect of temperature on Bacillus sp. 4 esterase stability.

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Fig. 6. Effect of pH on Bacillus sp. 4 esterase activity.

Fig. 8. Lineweaver–Burk plot.

pHs ranging from 4.00 to 8.00 and a slight destabilization was observed at pH 10.00 after 1 h incubation at room temperature. After 10 h incubation at pH 4.00, 6.00, 8.00 and 10.00, it retained about 82, 98, 84 and 45% of maximum activity, respectively. When the enzyme is incubated, pH 6.00, for 24 and 30 h, a rapid decline in the activity is observed. This decline does not depict that the enzyme is not stable at pH 6.00. It may be speculated that the reason for this decline is that the enzyme is in crude extract form. The assay mixture does not include protease inhibitors. Therefore, the proteases that exist in the extract are active at this pH. And possible digestion of the enzyme by these proteases may be the main reason for this decline in enzyme activity. High pH stability detected in this study can be an indicative for a new esterase that can be useful for several industrial applications.

value when pNPB was used as substrate [34]. It was reported that the Km values of most industrial enzymes are varied in the range of 10−1 to 10−5 M when acting on biotechnologically important substrates [35].

3.5. Kinetic parameters The effect of p-nitrophenyl butyrate concentration on the hydrolysis rate was measured at substrate concentrations ranging from 10 to 200 ␮M. The values of Km and Vmax of the esterase, calculated from Lineweaver–Burk plot, were 62.89 ␮M and 833.33 U/mg protein, respectively (Fig. 8). This Km value was lower than the esterase from the thermophilic Bacillus strain G18A7 which had a 0.1 mM Km

3.6. Effect of PMSF and β-mercaptoethanol on esterase activity The effect of phenylmethanesulphonyl fluoride and ␤mercaptoethanol on the esterase activity is given in Table 2. PMSF, a typical serine inhibitor, strongly inhibited the reaction at the concentration of 1 mM. Most of the esterases and lipases have a serine residue at the active site of the enzyme [36]. But lipases can be distinguished from esterases by having a hydrophobic domain (lid) covering the active site [2–4]. Therefore, lipases are either slightly or not inhibited by PMSF because the “lid” structure makes the active site of the enzyme inaccessible to the reagent. It was reported that the lipases from Aeromonas sobria LP004 [35], Acinetobacter radioresistens CMC-1 [38], Ophistoma piceae [39], Streptomyces simosus [40], Rhizopus oryzae [41] and B. licheniformis [26] were not inhibited by PMSF. However, the esterases of Bacillus sp. strain G18A7 [34], Sulfolobus acidocaldarius [32], A. nicotianae 9458 [22] and P. calidifontis VA1 [23] were inhibited by PMSF similar to the esterase used in this study. On the contrary of these, the esterase from B. licheniformis [20] was slightly inhibited and A. viscosus NRRL B-1973 [31] was not inhibited by PMSF. As reported in the aforecited studies, the esterase of B. licheniformis shared some properties with lipases and the esterase of A. viscosus might contain tryptophan and cysteine within its active site. Table 2 Effect of various agents on the esterase activity Substance

Concentration (mM)a

PMSF

1

19

␤-Mercaptoetanol

1 5

98 100

a

Concentration in pre-incubation mixture. The activity is expressed as a percentage of the activity of untreated control. b

Fig. 7. Effect of pH on Bacillus sp. 4 esterase stability.

Residual activity (%)b

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Fig. 10. Substrate specifity of Bacillus sp. 4 esterase.

Fig. 9. SDS-PAGE profile of Bacillus sp. 4 esterase. Lane A: molecular mass standards. Lane B: crude enzyme extract. Lane C: esterase activity.

So as to determine the presence of a thiol group in the esterase molecule, the enzyme was incubated with ␤-mercaptoethanol at the concentration of 1 and 5 mM (Table 2). The enzyme activity was not influenced by ␤mercaptoethanol. From this result, it could be said that thiol group was not present or not critical for the catalytic function. Similar results were found in some lipases [26,29,37–39,42,43]. 3.7. Electrophoresis and activity detection The crude culture medium was used as enzyme source. It contained only one active band on pNPB as detected after SDS-PAGE (Fig. 9). The molecular mass (Mr ) of the esterase was estimated to be 81.9 kDa from SDS gels. These results strongly suggested the presence of a single enzyme responsible for pNPB activity in the enzyme extract, which validates the use of the crude medium as enzyme source. 3.8. Substrate specificity The substrate specificity of the enzyme was studied with p-nitrophenyl acetate (C2 ), p-nitrophenyl butyrate (C4 ), p-nitrophenyl laurate (C12 ) and p-nitrophenyl palmitate (C16 ) (Fig. 10). Maximum activity was observed on pNPB. Regarding the activity on pNPB as maximum, the activity on pNPA and pNPL was about 70 and 11%, respectively, and no activity was detected on pNPP. Since lipases hydrolyze esters in emulsion and usually water-insoluble substrates, typically triglicerides composed of long-chain fatty acids, whereas esterases preferentially hydrolyze “simple” esters and usually only triglicerides bearing fatty acids shorter than C6 [3], these results strongly suggest

that the enzyme used in this study showed an esterase activity. It must be pointed out that the finding about the significant activity demonstrated by the enzyme on pNPL (∼11%) is quite interesting. This value is higher from that of esterases from A. viscosus NRRL B-1973 [29] (0%), B. licheniformis [20] (less than 3% of maximum), A. nicotianae 9458 [22] (0%) and B. circulans [21]. Moreover, when pNPB is used as substrate, the esterase of P. freudenreichii ssp. freudenreichii ITG 14 [18] showed only 10% of maximum activity obtained by pNPA. Therefore, the new esterase is quite different from the known esterases which usually showed no activity on the substrates higher than C10 .

Acknowledgments This work was supported by the Adnan Menderes University, Research Foundation (FEF 02006). We are grateful to Dr. H. Halil Bıyık for technical support.

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