Research Article [Araştırma Makalesi]

Türk Biyokimya Dergisi [Turkish Journal of Biochemistry–Turk J Biochem] 2015; 40(2):116–124doi: 10.5505/tjb.2015.13008

Purification and characterization of a novel thermostable esterase from Thermus sp. NCCB 100425T

[Thermus sp. NCCB 100425T’den yeni bir ısıya dayanıklı esterazın saflaştırılması ve karakterizasyonu]

ABSTRACTObjective: The purpose of the present study was to purify and characterize an esterase from a thermophilic bacterium Thermus sp. NCCB 100425T and to check its suitability for industrial applications.Methods: Thermus sp. NCCB 100425T esterase was purified by using ammonium sulphate precipitation and hydrophobic interaction chromatography and then characterized biochemically.Results: The purity of the enzyme was observed as a single band on native- and SDS- PAGE. In the presence of p-nitrophenyl acetate (pNPA) as a substrate, the optimum pH and temperature of the enzyme were found to be 7.5 and 60°C, respectively. Km and Vmax values are calculated as 18.32 mM and 96.15 U/mg protein, respectively, with pNPA. pH stability was investigated in the range of pH 4.0-9.0 at 4°C and 60°C. After 7 days incubation, activity of pure enzyme was retained 85-90% for all pH at 4°C and 59±5.2% for pH 8.0 at 60°C. It was determined that approximately 80% of enzyme activity was retained between 30-60°C after 7 days incubation. In the presence of 10% ethanol and DMSO, the enzyme activity was retained 96±2.7% and 78±2.5%, respectively. Additionally, it was detected that some metal ions affect the enzyme activity at different ratios.Conclusion: It is clear that Thermus sp. NCCB 100425T esterase might have advantages for industrial and/or clinical applications in terms of especially its high thermal- and pH-stability.Key Words: Esterase, Thermus sp. NCCB 100425T, Thermophilic, Hydrophobic Interaction Chromatography, PurificationConflict of Interest: The authors have no conflict of interest.

ÖZETAmaç: Bu çalışmanın amacı termofilik bir bakteri olan Thermus sp. NCCB 100425T’den bir esteraz enziminin saflaştırılıp karakterize edilmesi ve endüstriyel uygulamalar için uygunluğu-nun incelenmesidir.Metod: Thermus sp. NCCB 100425T esterazı amonyum sülfat çöktürmesi ve hidrofobik etkile-şim kromatografisi kullanılarak saflaştırıldı ve biyokimyasal özellikleri incelendi.Bulgular: Doğal- ve SDS-PAGE’de tek bant şeklinde görülen saf enzimin, p-nitrofenil asetat (pNPA) varlığında optimum pH ve sıcaklık değerleri sırasıyla 7,5 ve 60°C olarak belirlendi. Yine pNPA bir substrat olarak kullanıldığında Km ve Vmax değerlerinin 18,32 mM ve 96,15 U/mg protein olduğu tespit edildi. pH kararlılığı pH 4,0-9,0 aralığında 4°C ve 60°C’de incelen-di. 7 günlük bir inkübasyondan sonra saf enzimin aktivitesinin 4°C’de bütün pH’larda yakla-şık %85-90 oranında korunduğu, bu oranın 60°C’de pH 8,0’de ise %59±5,2 olduğu görüldü. 30-60°C’ler arasında 7 gün inkübe edilen enzim, orijinal aktivitesini %80 oranında korudu. %10 nihai konsantrasyondaki etanol ve DMSO varlığında enzim aktivitesi sırasıyla %96±2,7 ve %78±2,5 oranlarında korundu. Ayrıca, bazı metal iyonlarının enzim aktivitesini farklı oran-larda etkilediği tespit edildi. Sonuç: Bütün bu sonuçlar değerlendirildiğinde Thermus sp. NCCB 100425T esterazının özel-likle yüksek sıcaklık- ve pH-kararlılığı nedeniyle endüstriyel ve/veya klinik uygulamalar için avantajlara sahip olabileceği açıkça görülmektedir.Anahtar Kelimeler: Esteraz, Thermus sp. NCCB 100425T, Termofilik, Hidrofobik Etkileşim Kromatografisi, SaflaştırmaÇıkar Çatışması: Yazarların çıkar çatışması yoktur.

http://www.TurkJBiochem.com 116 ISSN 1303-829X (electronic) 0250-4685 (printed)

Mehmet Ali Tekincanli,Melike Yildirim Akatin,Ahmet Colak

Karadeniz Technical University, Faculty of Science, Department of Chemistry, Trabzon

Correspondence Address[Yazışma Adresi]

Dr. Melike Yıldırım Akatın

Karadeniz Technical University, Faculty of Science, Department of Chemistry, 61080 Trabzon, TürkiyePhone: +90 462 3777653E-mail: melikey80@yahoo.com

Registered: 31 January 2014; Accepted: 17 September 2014[Kayıt Tarihi: 31 Ocak 2014; Kabul Tarihi: 17 Eylül 2014]

Yayın tarihi 5 Mart 2015 © TurkJBiochem.com

[Published online March 5, 2015]

Thus, this study can be considered as a step to obtain an industrially suitable esterase having excellent features.

Materials and Methods Chemicals Substrates (p-nitrophenyl acetate, p-nitrophenyl butyr-ate, p-nitrophenyl laurate and p-nitrophenyl palmitate), Bovine Serum Albumin (BSA), sodium dodecyl sulphate (SDS), N,N,N’,N’ tetramethyl ethylenediamine (TEMED) and Phenyl-Sepharose Fast Flow were purchased from Sigma Chem. Co. (St. Louis, MO, USA). Chloride salts of metal ions (Na+, Li+, Mg2+, Ni2+, Mn2+, Zn2+, Cu2+, Co2+ and Ca2+) and solvents were purchased from Merck A.G. (Darmstadt, Germany). All chemicals were analytical and microbiological grade.Bacterial strain and crude enzyme extraction Thermus sp. NCCB 100425T bacterial strain isolated from a geothermal source in Buharkent/Aydın in Turkey was provided from Department of Biology, Karadeniz Tech-nical University (Trabzon). Strain was grown aerobically at 65ºC in Nutrient-Broth (NB) medium for overnight. Grown cells were removed from NB medium by cen-trifugation at +4ºC, at 17136xg for 10 min (Rotina 35 R, Hettich Zentrifugen). Then, cells were suspended in 20 mM Tris-HCl (pH 8.0) buffer and 50 μL lysozyme (10 mg/mL) was added. After incubation at 37ºC for 30 min., cells were disrupted by sonication (Bandelin-Sonopuls, Germany) in an ice bath (80% amplitude, 1.0 cycle for 5 min). Finally, cell proteins were removed by centrifuga-tion at +4ºC, at 17136xg for 10 min and supernatant was used as crude enzyme extract.Purification procedureEsterase enzyme was purified from crude enzyme extract by two steps; ammonium sulphate precipitation and hy-drophobic interaction chromatography. Precipitation pro-cedure was applied as reported previously [13]. The crude enzyme extract was slowly precipitated with ammonium sulphate (20% (w/v) concentration) in an ice bath. Af-ter 1 hour incubation, precipitated protein was removed from suspension by centrifugation at +4ºC, 29068xg for 30 min (Hermle Z366 Centrifuge). Then, proteins were resuspended in 20 mM Tris-HCl (pH 8.0) buffer. Enzyme solution was ultrafiltrated by using 10,000 kDa cut-off centrifugal filter device (Millipore Amicon, USA). Hydro-phobic interaction chromatography (HIC) was applied as described previously [14,15]. Solid ammonium sulphate was slowly added to the enzyme solution to a final con-centration of 1.0 M. Phenyl-Sepharose Fast Flow (Sigma Chem. Co., USA) was loaded onto column (diameter 1.5 cm and height 30 cm) and column was equilibrated with 20 mM Tris-HCl (pH 8.0) buffer including 1.0 M ammo-nium sulphate. After enzyme solution was loaded column, column was washed with the same buffer. Proteins were eluted reverse gradient of ammonium sulphate (from 1.0 M to 0 M) in 20 mM Tris-HCl (pH 8.0) buffer at a flow

Turk J Biochem 2015; 40(2):116-124 117 Tekincanlı et al.

Introduction Esterases are the enzymes catalyzing the hydrolysis and formation of ester bonds and they are widely distributed in animals, plants and microorganisms. Among esterases, carboxyl ester hydrolases (EC 3.1.1.1) hydrolyze esters of short chain carboxylic acids (≤12) and triacylglycerol hydrolases or lipases (EC 3.1.1.3) display maximum ac-tivity towards insoluble long chain (≥12) acylglycerides [1]. In organic media, they catalyze reactions such as esterification, inter-esterification and trans-esterification [2]. Therefore, they have become two of the most widely used enzymes in organic synthesis and various industrial applications such as detergent industry, oleochemical in-dustry, pulp and paper industry [3,4,5]. Also, the modifi-cation of triglycerides for fat and oil industry, synthesis of flavor esters for food industry, resolution of racemic mixtures used for the synthesis of fine chemicals for the pharmaceutical industry can be performed with esterases [6]. It was reported that the carboxylesterase produced by Bacillus subtilis has been used in the synthesis of naproxen as a nonsteroidal anti-inflammatory drug [3] and 2- arylpropionic acids with high enantioselectivity [7]. An esterase cloned from Thermomyces lanuginosus exhibited high enantioselectivity (E=95) in the kinetic resolution of 2-carboxyethyl-3-cyano-5-methylhexa-noic acid ethyl ester into (3S)-2-carboxyethyl-3-cyano-5-methylhexanoic acid, a valuable chiral intermediate for Pregabalin [8], which is a lipophilic derivative of 4-aminobutyric acid and has been developed to a new blockbuster drug for the treatment of several central ner-vous system disorders [9].In spite of esterases have been isolated from various spe-cies, microbial esterases are attractive because the cost to grow and maintain them is less and they are easy to manipulate. But, the major reasons of limiting industrial usage of known esterases are their limited thermostabil-ity, mainly at high temperatures; pH stability; and insta-bility in the organic solvent in operating industrial condi-tions. For example, organic solvents are often needed to solubilize substrates and products. If the reaction can be performed at higher temperature, there is no need to add (and remove at the end) organic solvents, therefore the reaction can be performed at lower cost in an environ-mental friendly process [10]. From this point of view, the exploration for new microbial enzyme sources is vital for the advancement of new thermostable and organic solvent resistant enzymes and their applications [11]. The global market for enzymes (carbohydrases, proteases, lipases, and others) has showed a great increase in the last years. It was USD 4,411.6 million in 2013 and is expected to increase to USD 7,652.0 million by 2020, growing at a CAGR of 8.3% from 2014 to 2020 [12]. Because provid-ing novel enzymes to the market is very important, we purified and biochemically characterized a novel esterase from a thermophilic strain Thermus sp. NCCB 100425T.

at 650 nm were utilized to prepare calibration curve and protein concentration was calculated via calibration curve.Activity assay and substrate specificity Esterase activity was separately determined by using a spectrophotometric assay with p-nitrophenyl acetate (pNPA), p-nitrophenyl butyrate (pNPB), p-nitrophenyl laurate (pNPL) and p-nitrophenyl palmitate (pNPP) as substrates [19,20,21]. For the enzyme assay, a substrate solution was prepared by mixing stock substrate solution (10 mM), ethanol and 50 mM phosphate buffer (pH 8) in ratio of 1:4:95 (v/v/v), respectively. To determine the en-zymatic activity, 100 µL of the enzyme was added to 1400 µL of the substrate solution. After the incubation of the reaction mixture at 55°C for 15 min, the change in absor-bance at 410 nm was monitored spectrophotometrically. The background hydrolysis of the substrate was deducted by using a reference sample of identical composition to the incubation mixture except for the enzyme. One unit of enzymatic activity was defined as the amount of protein releasing 1 µmol of p-nitrophenol per minute. Effects of pH and temperature The activity of Thermus sp. NCCB 100425T esterase as a function of pH was assayed at 50°C by using p-nitro-phenyl acetate (pNPA) as a substrate and 50 mM buffer systems with overlapping values: Mcilvaine (pH 4.0-7.5)

rate of 1.0 mL min-1. Finally, the fractions with the highest esterase activity and the relatively lower protein contents were pooled and concentrated by using 10,000 kDa cut-off centrifugal filter device (Millipore Amicon, USA). For then specific activity were calculated.Native-PAGE, activity staining and SDS- PAGEGel electrophoresis was performed in a Mini-protean Tetra System (Bio-Rad, CA, USA). Purification of ester-ase enzyme was monitored by Native- and SDS-PAGE. Native-PAGE was carried out by using 5% stacking gel and 10% separating gel under non-denaturing conditions [16]. Gels were run at 25 mA for 90 min at 4°C and then stained with Coomassie Brillant Blue R-250. For activity staining, a gel prepared as describe above was stained by incubating in 100 mL of 20 mM Tris-HCl buffer (pH 8.0) including 2% (v/v) β-naphthyl acetate (30 mM stock solution) at 55ºC for 25 min. At last, 0.04% (w/v) Fast Blue B salt was added and esterase activity bands were observed [17]. SDS-PAGE was performed by using 12% separating gel and then stained with Coomassie Brillant Blue R-250 [16].Protein determination Protein concentration was determined according to Low-ry method by using bovine serum albumin (BSA, Sigma Chem. Co., USA) as a standard [18]. Absorbance values

Turk J Biochem 2015; 40(2):116-124 118 Tekincanlı et al.

Figure 1. Purification of Thermus sp. NCCB 100425T esterase by hydrophobic interaction chroma-tography. Phenyl-Sepharose Fast Flow (Sigma Chem. Co., USA) was loaded onto column (diameter 1.5 cm and height 30 cm). Flow rate and fraction volume was 1 mL/min and 1.5 mL, respectively.

Abs

orba

nce

(280

nm

)

Fraction number

Act

ivity

(U)

0.81.2

1.0

0.8

0.6

0.4

0.2

0.0

0.6

0.4

0.2

0.01 12 23 34 45 56

Absorbance (280 nm)

Activity (U)

Salt concentration (mM)

Table 1. Purification steps of Thermus sp. NCCB 100425T esterase

Fraction Fraction volume Protein Total protein Total activity Activity Specific activity Yield Fold

(mL) (mg/mL) (mg) (U) (U/mL) (U/mg protein) (%)

Crude extract 65 10.8 702 286.8 4.4 0.41 100 1

(NH4)2SO4 5 1.5 7.5 13.2 2.6 1.76 4.6 4.3

precipitation (20%)

Phenyl-Sepharose FF 0.5 0.15 0.075 2.15 4.2 28.67 0.75 69.9

To investigate the effect of metal ions on the enzyme activity, Na+, Li+, Mg2+, Mn2+, Zn2+, Ca2+, Co2+ and Cu2+ were directly added to the standard reaction mixture in a final concentration of 1 mM and 10 mM, separately. En-zyme activity determined in the absence of metal ion was defined as 100% [26].The effect of some organic solvents on the enzyme activ-ity was studied by adding methanol, ethanol, isopropanol, acetonitrile, acetone and dimethylsulphoxide (DMSO) to the standard reaction mixture in the final concentration of 10% and 30%. Enzyme activity determined in the absence of organic solvent was defined as 100% [26].Statistical analysisAll experiments were carried out in triplicates. Data are represented by the mean±standard deviation. Statistical significance was calculated using oneway analysis of variance and Duncan’s test. A value of p<0.05 was taken as statistically significant.

Results Enzyme purificationIn this study, Thermus sp. NCCB 100425T esterase was purified in successive steps including ammonium sul-phate precipitation and hydrophobic interaction chroma-tography (HIC) (Figure 1). The enzyme activity and total protein concentration were determined for all fractions collected. The fractions with the highest esterase activ-ity and the relatively lower protein contents (27-30) were pooled and concentrated.After the purification procedure, the total protein, total ac-tivity, specific activity, yield and purification fold of each fraction were determined (Table 1). The enzyme purified with ammonium sulphate precipitation and HIC showed the recovery of 4.6% and 0.75% respectively. The corre-sponding purification folds were seen to be 4.3 and 69.9. In addition, specific activity was seen to increase from 1.76 to 28.67 U/mg protein.Native-PAGE, activity staining and SDS- PAGEA single protein band observed in both native and SDS-PAGE indicated that the esterase enzyme was success-fully purified from Thermus sp. NCCB 100425T. Also, activity staining indicated that the enzyme was purified successfully as an active esterase (Figure 2).

Turk J Biochem 2015; 40(2):116-124 119 Tekincanlı et al.

and Tris-HCl (pH 7.5-9.0). The activity was expressed as percent relative activity with respect to maximum activ-ity, which was considered as 100% [22]. The optimum temperature of the enzyme was determined at optimum pH value by measuring the activity at differ-ent temperatures in the range of 20-90°C with 10°C in-crements by using pNPA as a substrate. The activity was expressed as percent relative activity in relation to the tem-perature optimum, which was considered as 100% [22].Enzyme kineticsEnzyme kinetic parameters of Thermus sp. NCCB 100425T esterase were obtained by measuring the rate of p-nitrophenyl acetate (pNPA) hydrolysis at various sub-strate concentrations (5-400 μM) in the standard reaction mixture (at 60ºC in 50 mM Tris-HCl buffer, pH 7.5). The Michaelis-Menten constant (Km) and maximum velocity (Vmax) were determined from the Lineweaver-Burk plot using the Microsoft Excel software.pH- and thermal- stability To observe pH-stability of purified Thermus sp. NCCB 100425T esterase, two trails were made separately at 4ºC and 60ºC. The purified enzyme was incubated at various pH values Mcilvaine (pH 4.0-7.0) and Tris-HCl (pH 8.0-9.0) for 7 days. At the end of the storage period, the activ-ity was assayed under standard reaction conditions. The percentage residual enzyme activity was calculated by a comparison with non-incubated enzyme [20]. To determine thermal-stability of purified Thermus sp. NCCB 100425T esterase, the enzyme was incubated in 50 mM Tris-HCl buffer (pH 7.5) over the range of 30-90ºC for up to 7 days. The esterase activity was determined at stan-dard assay conditions. Control with non-incubated enzyme was used to determine the 100% activity value [23,24,25].Effect of some metal ions and organic solvents on the enzyme activity

Figure 2. Native-PAGE, activity staining and SDS-PAGE of puri-fied enzyme. (a) Native-PAGE Coomassie Brillant Blue staining: line 1, crude enzyme extract; line 2, 20% (NH4)2SO4 precipitation; line 3, purified esterase, (b) Native-PAGE activity staining: line 1, crude enzyme extract; line 2, 20% (NH4)2SO4 precipitation; line 3, purified esterase, (c) SDS-PAGE with Coomassie Brillant Blue staining: line 1, crude enzyme extract; line 2, 20% (NH4)2SO4 pre-cipitation; line 3, purified esterase; M: molecular weight marker.

1 1 12 2 23 3

-116.0

-66.2

-45.0

-35.0

-25.0

-18.4-14.4

3M kDa(a) (b) (c) Table 2. Activity of Thermus sp. NCCB 100425T esterase on

different substrates. 100% activity corresponds to 30.14 U/mg protein

Substrates Relative activity (%)

p-nitrophenyl acetate (pNPA) 100

p-nitrophenyl butyrate (pNPB) 79.7

p-nitrophenyl laurate (pNPL) 16.5

p-nitrophenyl palmitate (pNPP) 11.4

with 10°C increment. Data showed that highest activity was at 60°C (Figure 3b). It was observed that the enzyme retained 70% and 90% of its maximum activity at 50°C and 70°C, respectively. Enzyme kinetics Kinetic parameters of esterase purified from Thermus sp. NCCB 100425T were determined by using p-nitrophenyl acetate (pNPA) as substrate. Km and Vmax values were cal-culated from the Lineweaver–Burk plot as 18.32 mM and 96.15 U/mg protein, respectively (Figure 4). pH- and thermal- stability pH stability of esterase from Thermus sp. NCCB 100425T was analyzed by incubating the purified enzyme at 4°C and 60°C in the buffers having different pH values for up to 7 days. After incubation, the esterase activity assay was performed under standard reaction conditions. The pure enzyme was quite stable all pH value at 4°C. After 7 days

Substrate specificity Substrate specificity of the purified esterase was tested on various p-nitrophenyl esters: p-nitrophenyl acetate (pNPA), p-nitrophenyl butyrate (pNPB), p-nitrophenyl laurate (pNPL) and p-nitrophenyl palmitate (pNPP). Re-sults showed that the enzyme was more active to hydro-lyze short-chain carboxylic acids esters (Table 2). Effect of pH and temperature Effect of pH on the purified Thermus sp. NCCB 100425T esterase was analyzed at 50ºC between pH values of 4.0 and 9.0 using 50 mM buffer systems with overlapping values; Mcilvaine (pH 4.0-7.5) and Tris-HCl (pH 7.5-9.0) and p-nitrophenyl acetate (pNPA) was used as a substrate. As shown in Figure 3a, maximal relative esterase activity was measured at pH 7.5. Effect of temperature on the enzyme activity was inves-tigated at different temperatures between 20°C to 90°C

Turk J Biochem 2015; 40(2):116-124 120 Tekincanlı et al.

Rel

ativ

e Act

ivity

(%)

Rel

ativ

e Act

ivity

(%)

100 100

80 80

60 60

40 40

20 20

0 03.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 1009080706050403020100

pH Temperature (°C)

McilvaineTris-HCl

(a) (b)

Figure 3. (a) pH-activity profile of Thermus sp. NCCB 100425T esterase. The activity of the purified esterase as a function of pH was as-sayed at 50°C by using pNPA as a substrate and over a pH range from 4.0 to 9.0 using 50 mM Mcilvaine (pH 4.0-7.5) and Tris-HCl (pH 7.5-9.0). 100% activity corresponds to 50.25 U/mg protein. (b) Temperature-activity profile of Thermus sp. NCCB 100425T esterase. The optimum temperature was determined at pH 7.5 by measuring the activity at different temperatures in the range of 20-90°C with 10°C increments by using pNPA as a substrate. 100% activity corresponds to 70.84 U/mg protein.

Figure 4. Lineweaver-Burk plot of purified Thermus sp. NCCB 100425T esterase.

1/V

(mg

prot

ein/

U)

y=0.1906x+0.0104R2=0.9934

0.06

0.05

0.04

0.03

0.02

0.01

-0.1 -0.05 0.05 0.1 0.15 0.2 0.2501/[S] (1/μM)

Mg2+, Mn2+, Zn2+, Ca2+ and Co2+ ions did not have signifi-cant effect on esterase activity at 1 mM final concentra-tion. Ni2+ and Cu2+ ions inhibited the enzyme 25±2.8% and 83±0.9% at 10 mM concentration. Effect of some organic solvents on the enzyme activityTo investigate the effect of some organic solvents on the es-terase activity, methanol, ethanol, isopropanol, acetonitrile, acetone and dimethylsulphoxide (DMSO) were added into the reaction mixture in the final concentration of 10% and 30%. The esterase activity were inhibited approximately 50% at the 10% final concentration of methanol, isopropa-nol and acetone (Table 4). There was 4±2.7% and 22±2.5% inhibition of esterase activity in presence of 10% ethanol and DMSO, respectively. There was no esterase activity in presence of methanol, isopropanol, acetonitrile and ac-etone at the 30% final concentration. Ethanol and DMSO also inhibited the original esterase activity by 25±3.1% and 57±3.5%, respectively at the 30% final concentration.

Turk J Biochem 2015; 40(2):116-124 121 Tekincanlı et al.

incubation, the enzyme was retained approximately 90% of its original activity for all pH values (Figure 5a). Af-ter 7 days incubation in different pH at 60°C, the enzyme was retained nearly 19±4.5%, 49±5.0% and 59±5.2% of its activity at pH 4.0, 6.0 and 8.0, respectively (Figure 5b). Thermal stability of Thermus sp. NCCB 100425T ester-ase was determined by pre-incubating for 1, 2 and 7 day at different temperature between 30-90°C (Figure 6). It observed that enzyme was highly stable at 30-60°C and conserved nearly 90% and 95% of its activity at 30 and 40°C after 7 days incubation, respectively. On the other hand, the enzyme pre-incubated at 80 and 90°C for 7 days lost its activity completely. Effect of some metal ions on the enzyme activityTo study of the effect of metal ion on activity of Thermus sp. NCCB 100425T esterase, Na+, Li+, Mg2+, Mn2+, Zn2+, Ca2+, Co2+, Cu2+ and Ni2+ ions were added separately at 1 mM and 10 mM final concentration (Table 3). Na+, Li+,

Figure 5. (a) pH stability profile of Thermus sp. NCCB 100425T esterase at 4°C. (b) pH stability profile of Thermus sp. NCCB 100425T at 60°C. The effect of pH on enzyme stability was determined by incubating the purified enzyme at 4°C and 60°C in the buffer solutions of different pH values: Mcilvaine (pH 4.0-7.0 and Tris-HCl (pH 8.0-9.0). At the end of the storage period, the activity was assayed under standard reaction conditions (60°C and pH 7.5). 100% activity corresponds to 85.98 U/mg protein.

Res

idua

l Act

ivity

(%) 100

80

60

40

20

00 1 2 3 4 5 6 7

Incubation Time (Day)

(a)

pH 4.0pH 5.0pH 6.0pH 7.0pH 8.0pH 9.0 R

esid

ual A

ctiv

ity (%

) 100

80

60

40

20

00 1 2 3 4 5 6 7

Incubation Time (Day)

(b)

pH 4.0pH 5.0pH 6.0pH 7.0pH 8.0pH 9.0

Figure 6. Thermal stability profile of Thermus sp. NCCB 100425T esterase. Thermal stability of the enzyme was studied by incubating the purified enzyme in 50 mM Tris-HCl buffer (pH 7.5) over the range of 30-90. The esterase activ-ity was determined at standard assay conditions. 100% activity corresponds to 85.98 U/mg protein.

Rel

ativ

e Act

ivity

(%)

100

80

60

40

20

00 1 2 3 4 5 6 7 8

Incubation Time (Day)

30°C

40°C

50°C

60°C

70°C

80°C

90°C

palmitate (pNPP, 16 carbons). This confirms the assump-tion that the enzyme is an esterase rather than a lipase. Similarly, Pseudomonas citronellolis ATCC 13674 ester-ase hydrolytic activity decreased as carbon chain length increased where it exhibited higher substrate specificity for pNPB [29]. A thermostable esterase from Bacillus li-cheniformis was active on short chain fatty acids esters containing 2 to 8 carbon atoms. The activity was very low with longer chain length substrates (C10 to C18) [30]. Effect of pH on the purified Thermus sp. NCCB 100425T esterase was analyzed at 50ºC between pH values of 4.0 and 9.0. Maximal relative esterase activity was measured at pH 7.5. Similar results were earlier reported that Ther-mus thermophilus HB27 esterase and Geobacillus sp. TF17 esterase had maximal activity at pH 8.0 and pH 7.5 in presence of p-nitrophenyl dodecanoate and pNPB, re-spectively [31,32].Effect of temperature on the enzyme activity was investi-gated at different temperatures between 20°C to 90°C and the highest activity was measured at 60°C. Similar results were also reported for esterases of Streptococcus thermo-philes and Bacillus licheniformis S-86 [28,33].Km and Vmax values of esterase purified from Thermus sp. NCCB 100425T were calculated from the Lineweaver–Burk plot as 18.32 mM and 96.15 U/mg protein, respec-tively, by using pNPA as substrate. It was previously re-ported that Bacillus subtilis DR8806 esterase acting on pNPA had Km of 4.2 mM and Vmax of 151 μmol min−1 mg−1 respectively [34]. The Km values of enzymes range wide-ly, but for most industrially used enzymes they lie in the range of 10-1 to 10-5 M when acting on biotechnologically important substrates, under normal reaction conditions. Km is a measure of the affinity of an enzyme for a particu-lar substrate, a low Km value representing a high affinity and a high Km a low affinity [35]. A high affinity for a substrate may be important especially some applications where the substrate is limiting. Because, low Km means that low concentration of the substrate is enough to run

DiscussionBecause industrial processes proceed often under extreme conditions, the broad application of enzymes in industry is hindered by the very limited repertoire of available en-zymes, their low activity and stability, and high costs. The special characteristics of enzymes are exploited for their commercial interest and industrial applications, which in-clude: thermophilic nature; tolerance to a varied range of pH, metal ions and organic solvents; stability of enzyme activity over a range of temperature and pH; and other harsh reaction conditions. Such enzymes have proven their utility in bio-industries such as food, leather, textiles, animal feed, and in bio-conversion. For these reasons, we aimed to obtain a novel esterase having features above mentioned.In this study, Thermus sp. NCCB 100425T esterase was successfully purified with ammonium sulphate precipita-tion and hydrophobic interaction chromatography (HIC). Then, detailed biochemical characterization of the en-zyme was carried out in terms of substrate specificities, thermal activation and stability, pH optimum and stability. In addition, the effects of some metal ions and organic solvents on esterase activity were studied. The esterase was purified 69.9 fold with a specific activ-ity of 28.67 U/mg protein. Various purification folds by using different chromatographic systems were previously reported about esterase purification such as 73.0 fold from Sparassis crispa, 52.0 fold from Melanocarpus albomy-ces, 36.2 fold esterase I and 37.0 fold esterase II from Streptococcus thermophiles [13,27,28]. The purified esterase had maximal relative activity to-wards p-nitrophenyl acetate (pNPA, 2 carbons) and p-nitrophenyl butyrate (pNPB, 4 carbons), respectively, whereas it could slightly cleavage ester bonds of p-ni-trophenyl laurate (pNPL, 12 carbons) and p-nitrophenyl

Turk J Biochem 2015; 40(2):116-124 122 Tekincanlı et al.

Table 3. Effect of metal ions on activity of Thermus sp. NCCB 100425T esterase. 100% activity corresponds to 82.63 U/mg protein

Residual Activity (%)

Metal Ion 1 mM Final 10 mM Final

Concentration Concentration

None 100 100

Mn2+ 90±2.3 89±2.5

Ca2+ 95±1.5 97±1.4

Cu2+ 102±3.2 17±0.9

Co2+ 96±1.2 94±1.2

Na+ 99±1.6 89±1.0

Li+ 95±0.9 94±2.0

Mg2+ 94±2.1 95±2.1

Zn2+ 97±1.8 95±1.6

Ni2+ 87±3.1 75±2.8

Table 4. Effect of some organic solvents on the activity of Thermus sp. NCCB 100425T esterase. 100% activity corresponds to 82.63 U/mg protein

Residual Activity (%)

Solvent 10% Final 30% Final

Concentration Concentration

None 100 100

Methanol 54±3.2 0

Ethanol 96±2.7 75±3.1

İsopropanol 49±2.2 0

Acetonitrile 25±3.4 0

Acetone 49±2.8 0

DMSO 78±2.5 43±3.5

Thermus sp. NCCB 100425T. The enzyme had high speci-ficity towards p-nitrophenyl acetate. The resistance of es-terase to some metal ions and organic solvents, along with the high pH- and thermal- stability, can make it a good candidate for applications in some industrial processes.AcknowledgementsThis work was financed by a research grant KTU-BAP (Proje Number: 8189). The authors would like to thank Prof. Dr. Ali Osman Beldüz, Assoc. Prof. Dr. Sabriye Canakci and their research group for providing bacterium strain.This study originates from the master thesis of Mehmet Ali Tekincanli.Conflict of InterestThere are no conflicts of interest among the authors.

References[1] Eggert T, Pouderoyen GV, Pencreach G, Douchet I. Biochemical

properties and three dimensional structures of two extracellular lipolitic enzymes from Bacillus subtilis. Colloid Surface B 2002; 26:37-46.

[2] Kawamoto T, Sonomoto K, Tanaka A. Esterification in organic sol-vents: Selection of hydrolases and effects of reaction conditions. Biocatalysis 1987; 1:137-45.

[3] Quax WJ, Broekhuizen CP. Development of a new Bacillus car-boxyl esterase for use in the resolution of chiral drugs. Appl Micro-biol Biotechnol 1994; 41(4):425-31.

[4] Jaeger KE, Reetz MT. Microbial lipases form versatile tools for biotechnology. Trends Biotechnol 1998; 16(9):396-403.

[4] Sharma S, Gupta MN. Alginate as a macroaffinity ligand and an additive for enhanced activity and thermostability of lipases. Bio-technol Appl Biochem 2001; 33(Pt 3):161-5.

[6] Molinari F, Brenna O, Valenti M, Aragozzini F. Isolation of a novel carboxylesterase from Bacillus coagulans with highenantioselec-tivity toward racemic esters of 1,2-Oisopropylideneglycerol. En-zymeMicrob Tech 1996; 19:551-6.

[7] Azzolina O, Vercesi D, Collina S, Ghislandi V. Chiral resolution of methyl 2-aryloxypropionates by biocatalytic stereospecific hy-drolysis. II Farmaco 1995; 50:221-6.

[8] Li XJ, Zheng RC, Wu ZM, Ding X, Zheng YG. Thermophilic es-terase from Thermomyces lanuginosus: molecular cloning, func-tional expression and biochemical characterization. Protein Expr Purif 2014; 101:1-7.

[9] Silverman RB. From basic science to blockbuster drug: the discov-ery of Lyrica. Angew Chem Int Ed Engl 2008; 47(19):3500-4.

[10] Lagarde D, Nguyen HK, Ravot G, Wahler D, Reymond JL, et al. High-throughput screening of thermostable esterases for industrial bioconversions. Organic Process Res Develop 2002; 6:441-5.

[11] Ghati A, Sarkar K, Paul G. Isolation, characterization and molecu-lar identification of esterolytic thermophilic bacteria from an in-dian hot spring. Cur Res Microbiol Biotechnol 2013; 1:196-202.

[12] PREweb Online Visibility from Focus, Global Enzymes Market to Reach USD 7,652 Million by 2020-Industry Outlook, Size, Trends, Analysis and Forecast to 2020: GrandViewResearch,Inc, http://www.prweb.com/releases/Enzymes-Market/GrandViewResearch/prweb11670412.htm ((Last accessed: Thursday, August 7, 2014).

[13] Chandrasekaran G, Kim GJ, Shin HJ. Purification and characteri-sation of an alkaliphilic esterase from a culinary medicinal mush-room, Sparassis crispa. Food Chem 2010; 124:1376-81.

[14] Monti D, Ferrandi EE, Righi M, Romano D, Molinari F. Pu-

Turk J Biochem 2015; 40(2):116-124 123 Tekincanlı et al.

the reaction at half of its maximum speed. When the pH stability of the enzyme was examined, it was seen that the pure enzyme was quite stable both 4°C and 60°C. It was previously reported that esterase activ-ity of Geobacillus caldoxylosilyticus TK4 phosphotries-terase was quite stable at all pH values between 3.0 and 9.0 and retained over 90% of its original activity after 2 days incubation at 50°C [19]. Biocatalysts are usually less stable under operational conditions compared to chemi-cal catalysts. Although it sometimes is beneficial to adapt industrial processes to mild and environmentally benign conditions favored by the enzyme, the use of more ex-treme conditions is often desirable. Both esterases and li-pases have rarely been utilized in industrial processes due to their low stability under operational process conditions [36]. For this reason, pH-stability of Thermus sp. NCCB 100425T may be an advantage in industrial applications. Thermal stability of Thermus sp. NCCB 100425T esterase was determined by pre-incubating for 1, 2 and 7 day at dif-ferent temperature between 30-90°C. The result showed that Thermus sp. NCCB 100425T esterase is more stable than esterases from B. licheniformis (50% at 64°C for 1 h), Bacillus sp. (50% at 65°C for 10 h), and Thermotoga maritima (50% at 80°C for 30 min) [30,37,38]. Effect of some metal ions on the activity of Thermus sp. NCCB 100425T esterase was examined. Ni2+ and Cu2+ ions inhibited the enzyme 25±2.8% and 83±0.9% at 10 mM concentration. These results are consistent with the literature. It was reported that G. caldoxylosilyticus TK4 PHP, Picrophilus torridus and Geobacillus sp. HBB-4 es-terases were also inhibited by Cu2+ [19,26,39].Organic solvents can be advantageous in various indus-trial enzymatic processes; e.g. the reaction media used in biocatalytic esterification and trans-esterification contains less than 1% water. The use of organic solvents can in-crease the solubility of non-polar substrates, increase the thermal stability of enzymes, decrease water-dependent side reactions, or eliminate microbial contamination [40]. For these reasons, the effect of some organic solvents on the esterase activity was also investigated. The esterase activity was inhibited in different ratios in different sol-vents. It was previously reported that 18% and 24% in-hibitions was observed on the activity of Kluyveromyces marxianus CBS 15532 esterase in presence of methanol and ethanol at the 15% final concentration [14]. L. pyri-forme esterase activity was severely inhibited by acetoni-trile [20]. It was also reported that Geobacillus sp. HBB-4 esterase was inhibited by 50% with acetone, acetonitrile and ethanol [39]. Methanol in the 10% final concentra-tion caused 21% inhibition of G. caldoxylosilyticus TK4 esterase [19].

Conclusion In conclusion, the present study reports the purification and characterization of an esterase for the first time from

ces. Enzyme Microb Tech 2006; 39:265-73.[28] Liu SQ, Holland R, Crow VL. Purification and properties of in-

tracellular esterases from Streptococcus thermophilus. Int Dairy J 2001; 11:27-35.

[29] Chao YP, Fu H, Wang YL, Huang WB, Wang JY. Molecular clon-ing of the carboxylesterase gene and biochemical characterization of the encoded protein from Pseudomonas citronellolis ATCC 13674. Res Microbiol 2003; 154(7):521-6.

[30] Alvarez-Macarie E, Augier-Magro V, Baratti J. Characterization of a thermostable esterase activity from the moderate thermo-phile Bacillus licheniformis. Biosci Biotechnol Biochem 1999; 63(11):1865-70.

[31] Fuciños P, Atanes E, López-López O, Esperanza Cerdán M, Isabel González-Siso M, et al. Production and characterization of two N-terminal truncated esterases from Thermus thermophilus HB27 in a mesophilic yeast: effect of N-terminus in thermal activity and stability. Protein Expr Purif 2011; 78(2):120-30.

[32] Ayna Ç, Kolcuoğlu Y, Öz F, Colak A, Ertunga NS. Purification and characterization of a pH and heat stable esterase from Geobacillus sp. TF17. Turk J Biochem 2013; 38:329-36.

[33] Torres S, Baigori MD, Swathy SL, Pandey A, Castro GR. Enzy-matic synthesis of banana flavour (isoamyl acetate) by Bacillus licheniformis S-86 esterase. Food Res Int 2009; 42:454-60.

[34] Asoodeh A, Ghanbar T. Characterization of an extracellular ther-mophilic alkaline esterase produced by Bacillus subtilis DR8806. J Mol Catal B: Enzym 2013; 85-6, 49-55.

[35] Fullbrook PD. Practical applied kinetics. In (Ed. Godfrey T, West S) Industrial enzymology 1996; pp. 483-50, Stockholm Pres, New York.

[36] Eijsink VG, Gåseidnes S, Borchert TV, van den Burg B. Directed evolution of enzyme stability. Biomol Eng 2005; 22(1-3):21-30.

[37] Ateslier ZBB, Metin K. Production and partial characterization of a novel thermostable esterase from a thermophilic Bacillus sp. En-zyme Microb Tech 2006; 38:628-35.

[38] Kakugawa S, Fushinobu S, Wakagi T, Shoun H. Characterization of a thermostable carboxylesterase from the hyperthermophilic bacterium Thermotoga maritima. Appl Microbiol Biotechnol 2007; 74(3):585-91.

[39] Metin K, Burcu Bakir Ateslier Z, Basbulbul G, Halil Biyik H. Characterization of esterase activity in Geobacillus sp. HBB-4. J Basic Microbiol 2006; 46(5):400-9.

[40] Heitmann P. Industrielle enzyme, Behr’s Verlag, Hamburg, Ger-many. 1994; pp. 913.

rification and characterization of the enantioselective esterase from Kluyveromyces marxianus CBS 1553. J Biotechnol 2008; 133(1):65-72.

[15] Torres S, Baigorí MD, Pandey A, Castro GR. Production and puri-fication of a solvent-resistant esterase from Bacillus licheniformis S-86. Appl Biochem Biotechnol 2008; 151(2-3):221-32.

[16] Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227(5259):680-5.

[17] Zhou X, Scharf ME, Sarath G, Meinke LJ, Chandler LD, et al. Partial purification and characterization of a methyl-parathion resistance-associated general esterase in Diabrotica virgifera vir-gifera (Coleoptera: Chrysomelidae). Pestic Biochem Phys 2004; 78:114-25.

[18] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951; 193(1):265-75.

[19] Yildirim M, Colak A, Col M, Canakci S. A new recombinant phos-photriesterase homology protein from Geobacillus caldoxylosilyti-cus TK4: An extremely thermo- and pH-stable esterase. Process Biochem. 2009; 44:1366-73.

[20] Yildirim Akatin M, Colak A, Saglam Ertunga N. Characterization of an esterase activity in Lycoperdon pyriforme, an edible mush-room. J Food Biochem 2011; 37:177-84.

[21] Lee D, Koh Y, Kim K, Kim B, Choi H, et al. Isolation and charac-terization of a thermophilic lipase from Bacillus thermoleovorans ID-1. FEMS Microbiol Lett 1999; 179(2):393-400.

[22] Yildirim Akatin M. Characterization of a β-glucosidase from an edible mushroom, Lycoperdon pyriforme. Int J Food Prop 2013; 16:1565-77.

[23] Nawani N, Khurana J, Kaur J. A thermostable lipolytic enzyme from a thermophilic Bacillus sp.: purification and characterization. Mol Cell Biochem 2006; 290(1-2):17-22.

[24] Yildirim M, Col M, Colak A, Güner S, Dülger S, et al. Dipheno-lases from Anoxybacillus kestanbolensis strains K1 and K4T. World J Microbiol Biotechnol 2005; 21:501-7.

[25] Colak A, Sahin E, Yildirim M, Sesli E. Polyphenol oxidase poten-tials of three wild mushroom species harvested from Lişer High Plateau, Trabzon. Food Chem 2007; 103:1426-33.

[26] Hess M, Katzer M, Antranikian G. Extremely thermostable ester-ases from the thermoacidophilic euryarchaeon Picrophilus torri-dus. Extremophiles 2008; 12(3):351-64.

[27] Kontkanen H, Tenkanen M, Reinikainen T. Purification and char-acterization of a novel steryl esterase from Melanocarpus albomy-

Turk J Biochem 2015; 40(2):116-124 124 Tekincanlı et al.