Purification and characterization of a new inducible thermostable extracellular lipolytic enzyme from the thermoacidophilic archaeon Sulfolobus solfataricus P1

Accepted Manuscript Title: Purification and characterization of a new inducible thermostable extracellular lipolytic enzyme from the thermoacidophilic archaeon Sulfolobus solfataricus P1 Author: Yun-Ho Choi Young- Jun Park Sung-Jin Yoon Hee-Bong Lee PII: DOI: Reference:

S1381-1177(15)30116-8 http://dx.doi.org/doi:10.1016/j.molcatb.2015.11.023 MOLCAB 3287

To appear in:

Journal of Molecular Catalysis B: Enzymatic

Received date: Revised date: Accepted date:

22-9-2015 25-11-2015 26-11-2015

Please cite this article as: Yun-Ho Choi, Young- Jun Park, Sung-Jin Yoon, Hee-Bong Lee, Purification and characterization of a new inducible thermostable extracellular lipolytic enzyme from the thermoacidophilic archaeon Sulfolobus solfataricus P1, Journal of Molecular Catalysis B: Enzymatic http://dx.doi.org/10.1016/j.molcatb.2015.11.023 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Purification and characterization of a new inducible thermostable extracellular lipolytic enzyme from the thermoacidophilic archaeon Sulfolobus solfataricus P1 Yun-Ho Choia1, Young- Jun Parkb1, Sung-Jin Yoonb, Hee-Bong Leea* [email protected] a

Department of Biochemistry, College of Natural Sciences, Kangwon National University,

Chuncheon, Republic of Korea b

Immunotherapy Research Center, Korea Research Institute of Bioscience and Biotechnology,

Daejeon, Republic of Korea *

Corresponding author: Tel.: +82 33 250 8512; fax: +82 33 259 5664.

1

Both of these authors contributed equally to this work.

1

Graphical Abstract

①

TLC

② kDa

+ S. solfataricus Cultivation for 24 h, 80 ºC

①

SDS-PAGE M

1

2

70 50 40

②

30

20

0.5% Corn oil in 0.5% Glucose midium

15

(A)

2

(B)

Highlights

► A new inducible extracellular lipolytic enzyme from S. solfataricus was purified. ► The enzyme is a serine esterase containing a typical Ser-His-Asp catalytic triad. ► The enzyme showed remarkable stability against heat and protein denaturing chemicals. ► This stability of the enzyme indicates a high potential for industrial applications. ► The enzyme was the first purified inducible extracellular archaeal enzyme.

3

ABSTRACT A new thermostable extracellular lipolytic enzyme, induced from the thermoacidophilic archaeon Sulfolobus solfataricus P1 using corn oil as an inducer, was purified to apparent homogeneity by butanol extraction and two column chromatographies using DEAESepharose followed by Butyl-Sepharose. The purified enzyme assessed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and gel filtration was approximately 45 kDa and monomeric. The maximal activity examined using p-nitrophenyl palmitate as a substrate was observed at 98C and pH 6.0. The enzyme showed remarkable thermostability: It retained 51% of its activity after 120 h at 80C. In addition, the enzyme displayed extremely high stability against water-miscible alcohols, SDS, and urea, even at high concentrations. This high stability of the enzyme against protein-denaturing agents indicates a high potential in industrial applications. The enzyme has broad substrate specificity, exhibiting not only carboxylesterase activity toward short-chain acyl esters but also lipase activity toward long-chain acyl esters including triacylglycerols regardless of saturated and unsaturated fatty acids. The kcat/Km ratios of the enzyme for p-nitrophenyl palmitate (C16), the most preferable substrate among all tested, was 93.4 s−1∙μM−1. Together, it was identified by thin-layer chromatography (TLC) that the enzyme can hydrolyze all positions of the three ester bonds in triolein. The enzyme is a serine esterase belonging to the α/β hydrolase family containing a typical catalytic triad composed of serine, histidine, and aspartic acid in the active site of the enzyme. The enzyme is the first purified inducible extracellular lipolytic enzyme from archaea. Keywords:

Sulfolobus

solfataricus

P1;

Inducible

Thermostability; A serine esterase; Catalytic triad.

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extracellular

lipolytic

enzyme;

1. Introduction

Lipolytic enzymes are ubiquitous enzymes existing widely in animals, plants, and microorganisms [1]. They include two major groups, carboxylesterases (EC 3.1.1.1) and lipases (EC 3.1.1.3), which show a preference toward short-chain acyl esters (≤C10) and long-chain acyl esters (≥C10) containing triacylglycerols, respectively. Most of them belong to the α/β hydrolase family containing a characteristic Ser-Asp-His catalytic triad and the most conserved pentapeptide sequence, GXSXG (where X is any residue) around the activesite serine [2]. Lipolytic enzymes play an important role as biocatalysts for biotechnological applications. Bacterial lipolytic enzymes, particularly thermophilic and extracellular lipolytic enzymes, are attracting enormous attention because of their applications in industry, such as medical biotechnology, detergent production, synthetic chemistry, flavor and aroma synthesis, and other food-related processes [3,4]. Moreover, the enzymes from hyperthermophilic archaea, which inhabit extremely high-temperature environments, have recently attracted increasing attention due to their several possible applications. In fact, their exceptional stability to denaturing agents is specifically useful for industrial biotransformation applications. In addition, they could provide new study models for both evolutionary clarification of the function and basic elucidation of the structure–stability relationship of this class of protein [5,6]. Sulfolobus solfataricus (S. solfataricus) P1 used in this study belongs to the thermoacidophilic archaea, which is isolated from sulfur-rich volcanic hot springs and grows at around 80°C and pH 4 [7]. So far, only few lipolytic enzymes have been characterized from archaea in contrast to the large numbers from bacteria. Most of the archaeal lipolytic enzymes are intracellular 5

carboxylesterases, such as S. acidocaldarius [8–10], S. solfataricus P2 [11,12], S. solfataricus P1 (DSM 5354) [13], S. solfataricus P1 (DSM 1616) [14,15], S. solfataricus MT4 [16], Pyrobaculum calidifontis [17], Archaeoglobus fulgidus [18], and Pyrococcus furiosus [19]. On the other hand, a few extracellular enzymes showing lipolytic activity have been reported from halophiles such as Haloarcula marismortui [20] without any addition of inducer and from thermoacidophilic Sulfolobus shibatae [21] with the addition of Tween compounds as an inducer in culture medium. However, there have been no reports on the purification of their enzymes. In this study, we describe the purification and characterization of a new inducible extracellular thermostable lipolytic enzyme from S. solfataricus P1 (DSM 1616). The enzyme was induced using 0.5% corn oil as an inducer and purified from culture medium, and then its various biochemical properties were examined. This is the first report on the purification of the inducible extracellular lipolytic enzyme from archaea.

2. Materials and methods

2.1. Strains, growth conditions, and chemicals

S. solfataricus P1 (DSM 1616) was purchased from American Type Culture Collection (ATCC). S. solfataricus P1 was aerobically grown at 75ºC, pH 4.0 in a 5-liter fermentor with moderate stirring (120 rpm). The growth medium containing 0.2% (w/v) yeast extract and mineral bases was used according to the description of ATCC media formulations. Cells were harvested by centrifugation (4,000  g) for 45 min at 4ºC. For the induction of the extracellular lipolytic enzyme from the S. solfataricus, the harvested cells from 15-liter 6

cultivation in the yeast extract medium were suspended into the new medium (1 liter, pH 4.0) containing 0.5% (w/v) glucose, using the mineral bases and 0.5% (v/v) corn oil as an inducer. The suspended cells were further cultured aerobically at 75ºC for 24 h. The induction by corn oil was designed by modifying the method of Huddleston et al. who used Tween compounds (e.g., Tween 20, 40, 60, and 80) as inducers for the induction of the extracellular esterase from S. shibatae [21]. In this study, the inductions by 0.5% (v/v) olive oil and 0.2% Tween compounds (Tween 20, 60, and 80) were also examined to compare them with that by 0.5% corn oil using the same method as described above. In addition, the inductions of the extracellular lipolytic enzyme from S. solfataricus by three kinds of Tween compounds (Tween 20, 60, and 80) as inducers to compare with those of the extracellular esterase from S. shibatae were examined after cultivation for 24, 48, 72, and 96 h. The general yeast medium containing S. solfataricus without an inducer and 0.5% glucose medium containing an inducer without the S. solfataricus P1 strain were used as controls after cultivation under the same conditions. All chemicals used in this study were purchased from Sigma Chemical Co. Ltd. unless otherwise stated.

2.2. Purification of extracellular lipolytic enzyme

All purification steps were performed at room temperature unless otherwise stated. The existence of the induced extracellular lipolytic enzyme in each fraction during purification procedures was traced preferentially by the formation of a clear zone on a tributyrinemulsified agar plate and further identified by the methods of activity staining and enzyme assay as described below. After cultivation in an inducible growth medium, cells were precipitated by centrifugation (6,000  g) for 30 min at 4ºC. When olive oil or corn oil was 7

used as an inducer in a culture medium, the supernatant contained a lipid layer upon a medium layer that was transferred to the separating funnel and left to stand for a while until the upper lipid layer was clearly separated. The lipid composition of the upper lipid layer separated from the lower medium layer using the funnel was later analyzed on a thin-layer chromatography (TLC) plate after the lipid extraction from the layer. The lower medium layer containing the induced extracellular lipolytic enzyme was collected from the separating funnel. The purification of the induced extracellular lipolytic enzyme from the collected medium layer was performed after butanol extraction to remove any possible hydrophilic (short-chain fatty acids) and/or micellar products produced by the enzyme reaction that could disturb the purification by the following column chromatography. The butanol extraction was done in another funnel by the addition of 10 mM sodium phosphate buffer (buffer A, pH 6.0)-saturated 1-butanol to the medium layer at the ratio of 1 : 1. The funnel containing two solutions was shaken vigorously to ensure it was mixed well and left to stand until the upper butanol layer was separated spontaneously. The 1-butanol of the upper butanol layer was evaporated using a rotary vacuum evaporator (EYELA, USA) for the TLC analysis of the lipid products in it. The lower medium layer containing the enzyme was collected from the funnel for the purification of the enzyme. The collected lower solution containing the enzyme was dialyzed twice to remove the remaining butanol against the 5 liters of buffer A for 16 h. The dialyzed enzyme solution was applied to a column of DEAE-Sepharose (5.5 by 30 cm) equilibrated with buffer A and eluted with 600 ml of buffer A to remove unbound proteins to an anion exchanger (DEAE). Subsequent elution was performed with continuous linear gradients using 720 ml of buffer A containing 0–0.15 M and 0.15–0.3 M NaCl, and finally with 240 ml of buffer A containing 0.3 M NaCl. 8

The fractions containing the lipolytic enzyme activity were collected and combined. The final salt concentration of the combined protein solution was adjusted to 2 M by powdered NaCl, loaded onto a Butyl-Sepharose column (3.0 × 15 cm) equilibrated with buffer A containing 2 M NaCl, and then washed with 500 ml of buffer A containing 2 M NaCl to remove unbound proteins. After washing, the bound proteins were eluted with 500 ml of buffer A containing 0 M NaCl. Subsequent elutions were performed with a series of step gradients using 150 ml of buffer A containing 30%, 40%, 50%, 60%, and 70% (v/v) ethylene glycol. The fractions showing lipolytic activity on a tributyrin-emulsified agar plate, and having a single band on a sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gel, were collected and dialyzed twice to remove ethylene glycol against 5 liters of buffer A for 18 h. The dialyzed protein solution was used as the purified enzyme solution. The protein concentration was determined by the method of Bradford [22], using bovine serum albumin as the standard.

2.3. Identification and determination of molecular mass

The molecular mass of the purified extracellular lipolytic enzyme was determined on 12.5% SDS-PAGE gel with and without mercaptoethanol in the sample buffer using the low molecular mass standards [23]. Gels were stained by silver [24]. The molecular mass of the native protein was also determined by high performance liquid chromatography (HPLC) [GPC column (Protein-PakTM 300SW, 7.8 × 300 mm, Waters)] using the molecular mass standards (Sigma MW-GF-200 kit).

2.4. Enzyme assays and activity staining 9

A standard enzyme assay was carried out using an artificial chromogenic substrate, pnitrophenyl (PNP) palmitate (C16) [14,25]. The enzyme reaction was started by the addition of 0.1 ml of the purified extracellular lipolytic enzyme solution (0.6 μg/ml) to 0.1 ml of freshly prepared and prewarmed PNP-palmitate solution (5 mM) as a substrate and 0.8 ml of prewarmed 100 mM sodium phosphate buffer (pH 6.0) containing sodium taurocholate (2 mg/ml) and gum arabic (1 mg/ml) as emulsifiers at 70C. The medium as an enzyme source in the experiments using Tween compounds as inducers was used after the concentration with ammonium sulfate. The initial velocity of the reaction was measured by monitoring the changes in absorbance at 405 nm. One unit of lipolytic enzyme activity was defined as the amount releasing 1 μmol of p-nitrophenol per min from PNP esters at 70ºC. The extinction coefficients of pnitrophenol were determined prior to the measurements under every condition. Activities for all substrates were determined in the same way. The examination of the substrate specificity was performed by using PNP-butyrate (0.01–1 mM), PNP-caprylate (0.01–1 mM), and PNP-palmitate (0.01–1 mM) as substrates. In every measurement, the effect of the non-enzymatic hydrolysis of

substrates was considered and subtracted from the value measured when the enzyme was added. Measurements were carried out at least three times. Standard deviations never exceeded 10% of the mean values. Activity staining was carried out on tributyrin-emulsified agar plates containing 1.5% (w/v) agar powder, 0.01% (w/v) rhodamine B, and 1% (v/v) tributyrin in buffer A, as described previously [14,26]. The presence of active extracellular lipolytic enzymes in the sample solutions was primarily judged from the formation of the clear zone on a turbid tributyrinemulsified agar plate at 75C after simple spotting. Another activity staining method [14,16], that is useful for identifying the extracellular 10

lipolytic enzyme, was employed after SDS-PAGE, because enzymes from hyperthermophilic archaea are remarkably stable against high temperatures and high concentrations of general denaturants such as alcohols and SDS. A renaturation procedure was carried out to recover the enzyme activity in a gel. A gel was washed twice in a 2-propanol / 50 mM Tris-HCl (pH 7.0) (1 : 4, v/v) solution for 15 min, subsequently rinsed three times in 50 mM Tris-HCl (pH 7.0) buffer for 15 min, and then finally rinsed again with water. The gel was stained in the dark at 37ºC by immersing it in a solution consisting of 100 ml of 50 mM sodium phosphate buffer (pH 7.5) and 50 mg of Fast Blue BB plus 1 ml of acetone solution containing 10 mg αnaphthyl acetate until the dark gray band appeared on the gel. The stained gel was rinsed with water and fixed in 3% (v/v) acetic acid.

2.5. Catalytic properties of extracellular lipolytic enzyme

The optimum pH and temperature for the purified extracellular lipolytic enzyme activity were determined by the standard enzyme assay using PNP-palmitate. The effect of pH on the enzyme activity was examined at 70ºC in the pH range 3.0–9.0. The following buffers (100 mM) were used: sodium citrate (pH 3.0–6.0), sodium phosphate (pH 6.0–8.0), and Tris/HCl (pH 8.0–9.0). The effect of temperature on the enzyme activity was investigated at optimum pH (pH 6.0) at temperatures ranging from 20ºC to 98ºC, the maximal temperature enabling examination in a water bath. The thermal stability of the enzyme was investigated using the standard enzyme assay after incubating the enzyme for the designated time periods (0‒120 h) at two different temperatures (50ºC and 80ºC). The stability of the enzyme against several compounds [water-miscible organic solvents 11

(methanol, ethanol, and 2-propanol), detergent (SDS), and urea] was examined by the measurement of the residual activity using the standard enzyme assay immediately after each compound was mixed with the enzyme solution and incubated for 60 min at 70ºC. Blank samples were prepared with the buffer solution instead of the enzyme, and incubated in the same way as described above. Control experiments were performed in the absence of the compound. Each measurement was carried out with two different concentrations of the compounds: 50% and 90% (v/v) organic solvents, 1% and 5% (w/v) SDS, 4 M and 8 M urea. The inhibitory effect of chemical modifiers that are specific to particular amino acids [such as pyridoxal 5´-phosphate (PLP) to Lys, p-chloromercuribenzoate (PCMB) to Cys, phenylmethylsulfonylfluoride (PMSF) to Ser, and diethyl pyrocarbonate (DEP) to His] on the enzyme was examined. Enzyme activity was measured using the standard enzyme assay after each inhibitor (0.5 mM and/or 5 mM) was added to the enzyme and then incubated for 60 min at 70ºC. Moreover, 5 mM paraoxon and eserine known as inhibitors toward the lipolytic enzymes, were examined under the same conditions described above. To investigate the effect of divalent cations on the enzyme activity, 5 mM of divalent cations (CaCl2, CuSO4, FeSO4, MnCl2, MgCl2, and ZnSO4) were separately added to the enzyme and incubated for 60 min at 70°C. Enzyme activity was measured as described above. In order to clarify whether divalent cations are required for the reaction, 10 mM ethylenediaminetetraacetic acid (EDTA) was added to the enzyme and incubated for 60 min at 70ºC. The residual activities were measured using the standard enzyme assay after incubation.

2.6. Analysis of substrate specificity and lipid products in lipid layer by TLC The substrate specificity and the positional specificity of the induced extracellular lipolytic enzyme were examined by the analysis of lipid products on a TLC plate after the enzymatic 12

reaction using tributyrin, tricaprylin, or triolein as a substrate [27]. The enzymatic reaction was started by the addition of 200 μl of the purified enzyme solution to 2.2 ml of 10 mM substrates (tributyrin, tricaprylin and triolein) emulsified in 10 mM sodium phosphate buffer (pH 6.0) and incubated at 70°C for 24 h with moderate shaking (135 rpm, 5-cm shaking distance) in a water bath. After incubation, reaction products were obtained from the chloroform layer by using the Bligh and Dyer method [28] as follows. First, 2.4 ml of total enzyme reaction mixture was transferred to the separating funnel, 3 ml of chloroform and 6 ml of methanol were added to the reaction mixture to make one phase (final ratio, CHCl3 : MeOH : H2O = 1 : 2 : 0.8, v/v/v) and then mixed well by shaking vigorously by hand. After it was left to stand for a while, another 3 ml of chloroform and 3 ml of distilled water were added to the one-phase solution of the funnel to make a two-phase solution (final ratio, CHCl3 : MeOH : H2O = 1 : 1 : 0.9, v/v/v). After it was shaken vigorously again, it was left to stand until the mixed solution spontaneously separated into two phases: a chloroform layer including non-polar solutes (e.g., lipids) in the lower part and a methanol/water layer including polar solutes in the upper part. Aliquots of the chloroform layer containing the enzyme reaction products were applied to a Silica Gel 60 plate (Merck KGaA, Darmstadt, Germany) and developed with a developing solvent mixture of chloroform : acetone : acetic acid = 95 : 4 : 1 (v/v/v) [27] or petroleum ether : diethyl ether : acetic acid = 80 : 20 : 1 (v/v/v) [29]. The spots on a TLC plate were visualized by spraying 50% (v/v) H2SO4 in ethanol followed by heating in an oven at 150°C until charred spots were revealed or by standing in an iodine chamber for the identification of lipids containing unsaturated fatty acids. In addition, the analyses of the lipid products in the chloroform layer from the upper lipid layer and in the butanol layer from the lower medium layer with 0.5% corn oil and olive oil as described previously were also performed on a TLC plate. The developing solvent system 13

was petroleum ether : diethyl ether : acetic acid = 65 : 25 : 4 (v/v/v) (11) and the spots were visualized by standing in an iodine chamber. Tributyrin, tricaprylin, triolein, 1(3),2-diolein, monoolein, oleic acid, olive oil, and corn oil were used as standards on the TLC plates.

3. Results and discussion

3.1. Induction of extracellular S. solfataricus lipolytic enzyme

The existence of the extracellular lipolytic enzymes after the cultivation of S. solfataricus P1 in the general yeast extract culture medium without an inducer and in the 0.5% glucose medium with an inducer (Tween 20, corn oil, or olive oil) was preferentially examined on a tributyrin-emulsified agar plate and a SDS-PAGE gel by two activity staining methods. The result showed that lipolytic activity was not observed in the general yeast extract medium (data not shown) but was detected in the 0.5% glucose media containing inducers, indicating that the extracellular lipolytic enzyme was produced by these inducers. However, we did not attempt the induction using the general yeast extract medium with an inducer, because it would have been difficult to purify the enzyme in the crude yeast, as it includes many proteins. The induction of the extracellular esterase from S. shibatae was done by using the growth medium containing 0.2% peptone, 0.13% ammonium sulfate, basal salts, and 1% Tween compounds (Tween 20, 40, 60, and 80) as inducers and sole carbon sources [21]. However, the induction of the extracellular lipolytic enzyme from S. solfataricus in this study was performed by further cultivation of S. solfataricus cells, pre-cultured in the original yeast extract medium to help adaptation and growth, in the medium containing glucose (0.5%) and 14

inducers [corn oil (0.5%), olive oil (0.5%), or Tween compounds (0.2%)] as carbon sources. Therefore, to compare the effect of these Tween compounds on the induction of the extracellular esterase from S. shibatae [21], the inductions of the extracellular lipolytic enzyme from S. solfataricus by three kinds of Tween compounds (Tween 20, 60, and 80) as inducers were examined. These inductions were performed by the standard enzyme assay using PNP-palmitate as a substrate and their concentrated media as enzyme sources after cultivation for 24, 48, 72, and 96 h. The induction effect of the extracellular lipolytic enzyme by Tween 20, 60, and 80 was expressed and compared with μmoles of p-nitrophenol released per min. As shown in Fig. 1, until 48 h of cultivation, the induction of the S. solfataricus lipolytic enzyme by Tween 20 was the highest and that by Tween 80 was slightly lower than that by Tween 20, but that by Tween 60 was very low. In addition, the best cultivation time for enzyme induction was 48 h for all three kinds of Tweens, but the induction after cultivation for 24 h also reached nearly 90% of that for 48 h. The reported inductions of the extracellular esterase from S. shibatae were the best by 24 h of culture by Tween 20, the next by Tween 60, and then the lowest by Tween 80. However, the inductions by Tween 20 and Tween 80 (but not Tween 60) after 48 h of culture decreased to almost zero [21]. The difference in the results from S. solfataricus and S. shibatae might be due to the culture conditions as described previously and/or the structure and function of each enzyme from S. solfataricus and S. shibatae. For both results, it was assumed that the induction of the extracellular lipolytic enzyme from S. solfataricus by inducers after cultivation for 24 h might be acceptable. Another point deduced from Fig. 1 was that the induced lipolytic enzyme from S. solfataricus prefers ester compounds containing monolaurate (C12:0) or monooleate (C18:1) much more than monostearate (C18:0). This is because Tween compounds are polyoxyethylene sorbitan esters containing acyl chains of various lengths: monolaurate (C12:0) in Tween 20, 15

monostearate (C18:0) in Tween 60, and monooleate (C18:1) in Tween 80. The result indicates that the lipolytic enzyme from S. solfataricus prefers esters with unsaturated fatty acid (C18:1) or saturated fatty acid (C12:0) much more than those with saturated fatty acid (C18:0). In conclusion, based on the above results examined by using Tweens as inducers, the culture conditions for the induction of the extracellular lipolytic enzyme from S. solfataricus in this study were chosen as follows: 0.5% glucose medium containing 0.5% corn oil or olive oil as inducers and 24 h of cultivation time. Corn oil and olive oil were chosen instead of Tween compounds as inducers due to their better induction of the lipolytic enzyme supported by (i) natural lipids, (ii) triacylglycerols containing three ester bonds with three fatty acids, and (iii) high unsaturated fatty acid content. Corn oil is constituted by 99% triacylglycerols with proportions of approximately 55% polyunsaturated fatty acids (98% omega-6 linoleic acid, C18:2), 30% monounsaturated fatty acids (over 99% oleic acid, C18:1), and 15% saturated fatty acids (80% palmitic acid, C16:0). Olive oil is also a triacylglycerol containing major fatty acids such as oleic acid (55–83%), linoleic acid (3.5–21%), palmitic acid (7.5–20%), stearic acid (0.5–5%), and linolenic acid (C18:3, 0–1.5%). The induction of the extracellular lipolytic enzyme from S. solfataricus by corn oil or olive oil could be easily compared by observing the oil states of the lipid layer on the culture medium after cultivation for 24 h in the presence and absence of S. solfataricus P1 in the culture medium. As shown in Fig. 2, corn oil on the culture medium in the absence of S. solfataricus P1 remained without any significant change in its original color or volume before and after cultivation for 24 h (Fig. 2a). This result also indicates that the spontaneous hydrolysis of corn oil, a triacylglycerol with three ester bonds, under the acidic condition (pH 4) of the general culture medium of S. solfataricus did not even come close to occurring during the 24 h of cultivation. On the contrary, corn oil on the culture medium in the presence 16

of S. solfataricus P1 turned into a lipid layer with thick white foam after 24 h of cultivation (Fig. 2b). Both the color and volume of the corn oil were changed by S. solfataricus P1 during the 24 h of cultivation. This result indicates that the extracellular lipolytic enzyme able to hydrolyze corn oil was induced from S. solfataricus. The same phenomenon as above (Fig. 2) was also observed when olive oil was used instead of corn oil as an inducer (data not shown). Therefore, to compare the enzyme inductions from S. solfataricus by corn oil and olive oil used as inducers, the enzymatic products in the upper lipid layer and in the lower medium layer were examined by TLC after 1-liter cultivation for 24 h using 0.5% glucose medium containing each inducer. The enzymatic products in the upper lipid layer and in the lower medium layer were obtained from the chloroform layer by the Bligh and Dyer method and from butanol evaporation after butanol extraction, respectively. As shown in Fig. 3, after 24 h of cultivation, both olive oil and corn oil appeared to be hydrolyzed by the induced extracellular lipolytic enzymes from S. solfataricus (lanes 5 and 6) as compared with their controls on a TLC plate (lanes 1 and 2). However, although the hydrolysis degree produced by the corn oil seemed to be a little better than that produced by the olive oil, they were too similar to compare quantitatively on a TLC plate. Nonetheless, we chose to use corn oil as an inducer in this study, because the unsaturation degree of corn oil is slightly higher and the content of long-chain saturated fatty acids such as stearic acid (C18:0) is lower than that of olive oil, as described previously. The result agreed with the theory that the induced lipolytic enzyme from S. solfataricus prefers Tween 20 (C12:0) or Tween 80 (C18:1) much more than Tween 60 (C18:0) (Fig. 1). In addition, no lipid products were observed in the 1-butanol extracts of the lower medium layer (Fig. 3, lanes 3 and 4), which was expected, because both oils are composed of long-chain fatty acids. 17

3.2. Purification of extracellular S. solfataricus lipolytic enzyme

The induced extracellular lipolytic enzyme from the lower culture medium layer after cultivation for 24 h was purified to apparent homogeneity in three steps as shown in Table 1. The enzyme was purified 26-fold with a yield of 26.3%. The purified enzyme showed a specific activity of 2108 units/mg for the hydrolysis of PNP-palmitate at 70ºC, pH 6.0. The homogeneity of the purified enzyme was confirmed by SDS-PAGE, with a single band of approximately 45 kDa (Fig. 4, lane 1). The molecular mass of the enzyme, determined using a HPLC sizing column, was almost identical to the molecular weight assessed by SDS-PAGE, indicating that the enzyme is monomeric. In addition, SDS-PAGE followed by activity staining using α-naphthyl acetate showed that the purified enzyme has an esterase activity (Fig. 4, lane 2). This result indicates that heating the sample buffer containing the purified enzyme, 1% β-mercaptoethanol, and 1% SDS for 1 min in boiling water before performing a usual SDS-PAGE does not appear to significantly affect the enzyme activity due to its stability against temperature and SDS based on the results in Fig. 7 and Fig. 8. This stability against temperature and chemical reagents known as denaturing agents (e.g., urea, SDS, and organic solvents) are common with esterases from thermophilic archaea [11,14,17]. This is the first report of a new inducible extracellular lipolytic enzyme from S. solfataricus and of the purification of this enzyme from archaea. Results reported from the first inducible extracellular esterase of S. shibatae and from the extracellular esterase and lipase of Haloarcula marismortui were not obtained by using the purified enzyme [20,21].

3.3. Catalytic properties of induced extracellular S. solfataricus lipolytic enzyme

18

The optimum pH and temperature of the induced lipolytic enzyme activity were determined by a standard lipolytic enzyme assay using PNP-palmitate as a substrate. The induced extracellular lipolytic enzyme displayed the maximum activity at pH 6.0 (at 70ºC) (Fig. 5A). The initial pH of the cell culture of S. solfataricus is always adjusted to pH 4 at 75ºC at the beginning; however, the pH at late exponential phases usually changes to pH 6. Hence, it seems to be quite reasonable that the maximal activity of the enzyme was displayed at pH 6.0. In addition to this optimum pH, the maximum activity of the enzyme was detected at 98ºC (at the optimum pH of 6.0) (Fig. 5B). These enzyme activities were increased continuously with the increase of temperature up to 98ºC, the maximal temperature measurable in a water bath. This optimum pH, pH 6.0, of the induced extracellular lipolytic enzyme of S. solfataricus was the same as that of the inducible extracellular esterase from S. shibatae [21]. The optimum temperature, 98ºC, of the S. solfataricus lipolytic enzyme was slightly higher than the 90ºC of the S. shibatae esterase [21] and similar to those of esterases from S. solfataricus P1 (DSM 5354) [13] and P. furiosus [19]. However, it was higher than those of most other esterases from thermophilic archaea reported [8,10–12,14–18,20]. The thermostability of the purified lipolytic enzyme was examined at 50ºC and 80ºC with increasing incubation time up to 120 h (Fig. 6). The enzyme activity was maintained with little change until 120 h at 50ºC. Even at 80ºC, approximately 50% of the activity was still preserved after 120 h, although it was gradually lowered with increasing time. The induced S. solfataricus lipolytic enzyme exhibited considerable stability against high temperatures like all hyperthermophilic archaeal enzymes including esterases [8,10–21]. The effect of organic solvents, detergents, and urea on the extracellular lipolytic enzyme was examined using the standard enzyme assay after incubation for 60 min at 70ºC. As shown in Fig. 7, all three alcohols tested (methanol, ethanol, and 2-propanol) significantly 19

activated the enzyme as compared to control, although the 50% alcohols increased the enzyme activity much more than the 90% alcohols. On the other hand, SDS inactivated the enzyme in a concentration-dependent manner. However, even after incubation for 60 min at 70ºC with 5% SDS, the enzyme was not significantly inactivated, and it retained 60% residual activity. Like alcohols, 4 M urea activated the enzyme as compared to control. Furthermore, the enzyme was not inactivated even by 8 M urea at 70ºC. Our data clearly showed that the enzyme possesses a remarkable stability against these chemicals well known as denaturing agents that can affect protein folding toward general enzymes from mesophiles. The enzyme shows much higher stability against detergents or organic solvents than the lipase from Penicillium expansum, a mesophile [30]. Moreover, the enzyme displays higher stability against organic solvents than other esterases from P. calidifontis [17], S. solfataricus P1 [14], and S. solfataricus P2 [11] and against SDS or urea than carboxylesterase from S. solfataricus P1 [14]. The high stability against SDS, organic solvents, and urea indicates that the strong hydrophobic interactions may make up the stable core in the enzyme. Moreover, even the activation of the enzyme in all concentrations of the alcohols and urea tested might be due to the partial unfolding of the enzyme in the assay buffer upon dilution. This extreme stability of the inducible extracellular lipolytic enzyme from S. solfataricus P1 against denaturing agents such as various alcohols, SDS, and urea and against high temperatures make it very attractive for future applications in industry as well as in biotechnology. The investigation of the substrate specificity of the purified extracellular lipolytic enzyme from S. solfataricus was investigated using two kinds of substrates: PNP-esters and triacylglycerols. The substrate specificity was determined by the standard enzyme assay using PNP-esters containing acyl chains of various lengths. As shown in Table 2, the highest Km value was observed with PNP-butyrate (C4), whereas the lowest was observed with PNP20

palmitate (C16). However, the reverse pattern was found for the kcat value; that is, the highest value was observed with PNP-palmitate and the lowest was observed with PNP-butyrate. Among the PNP-esters tested, PNP-palmitate showed the highest specificity constant (kcat/Km), 93.4 s−1μM−1, indicating that the enzyme has lipase activity preferring PNP-esters with long chain lengths (C16) to those with short or medium chain lengths. In addition, another substrate specificity of the enzyme toward simple triacylglycerols containing saturated and unsaturated acyl chains of various lengths [e.g., tributyrin (C4:0), tricaprylin (C8:0), and triolein (C18:1)] was examined on a TLC plate using the reaction products after the in vitro enzymatic reaction of these substrates and the purified enzyme. As shown in Fig. 8A, tributyrin and triolein were hydrolyzed to some extent by the enzyme, but tricaprylin was hardly hydrolyzed. Tributyrin, containing the shortest saturated fatty acid (C4:0), was the best substrate among the three triacylglycerols tested, and the next substrate was triolein containing an unsaturated fatty acid (C18:1). The result showed that the lipolytic enzyme induced from S. solfataricus prefers simple triacylglycerols containing short-chain fatty acids and long-chain unsaturated fatty acids. This result was similar to that of the purified thermostable lipase from Pseudomonas cepacia [31]. The P. cepacia lipase showed different activities toward simple triacylglycerols containing saturated and unsaturated acyl chains of various lengths. Caprate (C10:0) was the highest; the second-highest group included butyrate (C4:0) and laurate (C12:0); the next group included caprylate (C8:0), caproate (C6:0), and oleate (C18:1); and the lowest group included myristate (C14:0), palmitate (C16:0), and stearate (C18:0). This result from P. cepacia was also in line with that from S. solfataricus obtained using Tween compounds as described in Fig. 1. These results (Table 2 and Fig. 8A) show that the purified extracellular enzyme induced from S. solfataricus P1 prefers PNP-palmitate and tributyrin/triolein among the PNP esters 21

and simple triacylglycerols tested, respectively. Further, these results suggest that the choice of corn oil as an inducer in this study was quite reasonable, because the palmitate and oleate contents in corn oil are a little higher than those in olive oil. In connection with the result of substrate specificity (Fig. 8A), the positional specificity of the purified lipolytic enzyme from S. solfataricus was examined further using triolein as a substrate in the same way as above. The result is shown in Fig. 8B. The enzyme was found to be able to hydrolyze all positions of the three ester bonds in triolein as compared with their possible products used as controls, indicating that the induced extracellular enzyme may be divided into nonspecific lipase rather than regiospecific or fatty acid-specific lipase among the three categories of lipases. This result was similar with those of lipases from Pseudomonas sp. Strain KB700A and P. cepacia [27,31]. The results on a TLC plate (Figs. 8A and 8B) seemed to indicate that the in vitro enzymatic hydrolysis reactions were insufficient compared to their controls. However, this result was natural, because the reactions occurred in the interface between the enzyme solution and the water-immiscible substrates, lipids. These results showed the comparable differences between the reaction products. In conclusion, all results of the substrate specificities suggest that the extracellular enzyme induced from S. solfataricus P1 is a lipolytic enzyme showing broad substrate specificity with not only a carboxylesterase activity toward PNP-butyrate and tributyrin containing short-chain acyl esters (≤C10) but also a lipase activity toward PNP-palmitate and triolein containing long-chain acyl esters (≥C10). To examine the amino acids involved in the catalytic mechanism of the enzyme, the inhibitory effects of various chemical compounds that are specific to particular amino acids were measured using the standard enzyme assay after incubation for 60 min at 70ºC. As shown in Table 3, the induced S. solfataricus lipolytic enzyme was completely inhibited by 5 mM DEP. The same concentration (5 mM) of PMSF, PCMB, paraoxon, and eserine decreased 22

the enzymatic activity to 11%, 51%, 40%, and 8%, respectively, of that of untreated enzyme. However, PLP had no significant effect on the enzyme activity. These results of enzyme inhibition by a serine-specific inhibitor (PMSF), histidine-specific inhibitor (DEP), and cysteine-specific inhibitor (PCMB) suggest that Ser, His, and Cys residues are located at or near the active site closely related to the catalytic activity of the enzyme. In particular, the result that Ser and His are crucial in the activity of the enzyme leads us to deduce that the induced S. solfataricus lipolytic enzyme may contain a catalytic triad consisting of Ser, His, and Asp, typically found in its active sites of bacterial and archaeal esterases. The result indicates that the induced S. solfataricus lipolytic enzyme is a serine esterase belonging to the α/β hydrolase family. Another possibility is that the inhibition by PCMB was caused by the conformational change of the active site in the enzyme due to the modification of cysteine residue by PCMB. In addition, the induced S. solfataricus lipolytic enzyme was inhibited significantly by organophosphate, paraoxon, and eserine. Bergmann et al. made a classification based on the interaction of esterases with organophosphates such as paraoxon [32].

A-esterases

(arylesterases)

hydrolyze

organophosphates,

B-esterases

(carboxylesterases/serine esterases) are inhibited by them, and C-esterases (cholinesterases) are not inhibited by organophosphates and do not hydrolyze them. Therefore, based on this inhibition pattern, the induced extracellular enzyme from S. solfataricus can be classified as a carboxylesterase and a serine esterase. The enzyme activity was not affected by incubation with 5 mM of various divalent cations for 60 min at 70ºC (data not shown). In addition, the incubation of the enzyme with 10 mM EDTA for 60 min at 70ºC did not result in a significant change of the enzyme activity (Table 3), suggesting that the enzyme does not require any divalent cation for activity.

23

4. Conclusion

The present study shows that the extracellular lipolytic enzyme from S. solfataricus P1 (DSM 1616) was induced using corn oil as an inducer in 0.5% glucose medium. The inducible extracellular lipolytic enzyme from S. solfataricus was purified from culture medium to apparent homogeneity and was estimated as a 45-kDa monomeric enzyme. The enzyme showed remarkable stability against high temperatures and high concentrations of chemical compounds generally known as denaturants to proteins, such as water-miscible alcohols (methanol, ethanol, and 1-propanol), SDS, and urea. In addition, the enzyme showed broad substrate specificity, exhibiting not only carboxylesterase activity toward short-chain acyl esters but also lipase activity toward long-chain acyl esters including triacylglycerols regardless of saturated and unsaturated fatty acids. The inducible extracellular lipolytic enzyme is a serine esterase belonging to the α/β hydrolase family containing a typical SerHis-Asp catalytic triad. Its extreme stability against heat and organic solvents indicates its high potential for use in industrial applications. Most importantly, the inducible extracellular thermostable lipolytic enzyme from S. solfataricus P1 (DSM 1616) reported in this study is the first purified archaeal enzyme.

Acknowledgments

This study is supported by a 2015 Research Grant from Kangwon National University (No. 520150407), and by the KRIBB Research Initiative Program, Republic of Korea.

24

References

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[18] G. Manco, E. Giosuè, S. D’Auria, P. Herman, G. Carrea, M. Rossi, Arch. Biochem. Biophys. 373 (2000) 182–192. [19] M. Ikeda, D.S. Clark, Biotechnol. Bioeng. 57 (1998) 624–629. [20] R.M. Camacho, J.C. Mateos, O. González-Reynoso, L.A. Prado, J. Córdova, J. Ind. Microbiol. Biotechnol. 36 (2009) 901–909. [21] S. Huddleston, C.A. Yallop, B.M. Charalambous, Biochem. Biophys. Res. Commun. 216 (1995) 495–500. [22] M.M. Bradford, Anal. Biochem. 72 (1976) 248–254.

[23] U.K. Laemmli, Nature. 227 (1970) 680–685.

[24] K. Ohsawa, N. Ebata, Anal. Biochem. 135 (1983) 409–415.

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Acta 1214 (1994) 43–53. [26] K.E. Jaeger, A. Steinbüchel, D. Jendrossek, Appl. Environ. Microbiol. 61 (1995) 3113– 3118. [27] N. Rashid, Y. Shimada, S. Ezaki, H. Atomi, T. Imanaka. Appl. Environ. Microbiol. 67 (2001) 4064–4069. [28] E.G. Bligh, W.J. Dyer, Can. J. Biochem. Physiol. 37 (1959) 911–917. [29] M. Kates, Techniques of lipidology: Isolation, analysis and identification of lipids, American Elsevier, NY, 1972. [30] W. Stöcklein, H. Sztajer, U. Menge, R.D. Schmid, Biochim. Biophys. Acta 1168 (1993) 181–189. 26

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27

Figure Captions Fig. 1. Effects of various Tween compounds used as inducers and cultivation times on the induction of extracellular lipolytic enzyme from S. solfataricus. The culture medium was assayed for enzyme activity with the substrate PNP-palmitate (see materials and methods). White, gray, and black bars indicate Tween 20, 60, and 80, respectively. Error bars indicate standard deviations.

Fig. 2. Aspects of upper lipid layers after cultivation for 24 h in absence (A) and presence (B) of S. solfataricus in 0.5% glucose medium containing 0.5% corn oil.

Fig. 3. Thin-layer chromatogram of lipid products in chloroform layer from upper lipid layer and in butanol extracts from lower medium layer. The upper lipid layer and the lower medium layer were obtained after cultivation for 24 h using corn oil and olive oil as inducers. The lipid products from the upper lipid layers and from the lower mediums were obtained from the chloroform layers by the Bligh and Dyer method and from butanol evaporation after butanol extraction, respectively. Lanes 1 and 2 were controls of corn oil and olive oil, respectively; lanes 3 and 4 were the butanol extracts from the lower medium layers after cultivation using corn oil and olive oil, respectively; lanes 5 and 6 were the chloroform layers from the upper lipid layers after cultivation using corn oil and olive oil, respectively. The developing solvent system was petroleum ether : diethyl ether : acetic acid = 65 : 25 : 4 (v/v/v). The spots were visualized by standing in an iodine chamber.

Fig. 4. SDS-PAGE analysis of purified inducible extracellular lipolytic enzyme from S. solfataricus. Samples were loaded on a SDS-polyacrylamide gel in duplicate, and after being 28

run the gel was cut. One was stained with silver (A), and the other was stained by the activity staining method using the chromogenic substrate, α-naphthyl acetate, after renaturation (B). M, molecular weight standards; lane 1 in (A) and lane 2 in (B), the purified inducible extracellular lipolytic enzyme from S. solfataricus.

Fig. 5. Effects of pH and temperature on activity of purified inducible extracellular lipolytic enzyme from S. solfataricus. The effects of pH and temperature on the activity of the purified inducible

extracellular

lipolytic

enzyme

from

S.

solfataricus

were determined

spectrophotometrically using PNP-palmitate as a substrate. The enzyme activity was examined at 60°C at required pH (A) or at pH 6.0 at each indicated temperature (B). The required pH was obtained using 100 mM sodium citrate buffer () for the pH range of 3.0– 6.0; 100 mM sodium phosphate buffer () for the pH range of 6.0–8.0 and 100 mM Tris-HCl buffer (▲) for the pH range of 8.0–9.0. Error bars indicate standard deviations.

Fig. 6. Stability of inducible extracellular S. solfataricus lipolytic enzyme at different temperatures. The residual activity was determined after incubation of the enzyme for the indicated times at 50°C (◆) or 80°C (). The activity measurement was carried out using the standard enzyme assay. Error bars indicate standard deviations.

Fig. 7. Effects of several chemical compounds on stability of inducible extracellular S. solfataricus lipolytic enzyme at 70ºC. The enzyme was incubated at 70ºC in 100 mM sodium phosphate buffer (pH 6.0) with different concentrations of compounds. After 60 min, aliquots were removed from each incubation mixture and assayed for residual activity using the standard enzyme assay. White bars indicate controls without alcohols, SDS, and urea. Gray 29

bars indicate the concentrations of 50% alcohols, 1% SDS, and 4M urea. Black bars indicate the concentrations of 90% alcohols, 5% SDS, and 8M urea. Error bars indicate standard deviations.

Fig. 8. Thin-layer chromatographic analysis of substrate specificity using in vitro enzyme reaction. The substrate specificity of the purified S. solfataricus lipolytic enzyme using tributyrin (4:0), tricaprylin (8:0), and triolein (18:1) as substrates (A) and the positional specificity of the enzyme using triolein as a substrate (B) were examined by TLC analysis of products in the chloroform layers obtained using the Bligh and Dyer method after in vitro enzymatic reactions. (A) Lanes 1, 3, and 5 were controls of tributyrin, tricaprylin, and triolein, respectively; lanes 2, 4, and 6 were chloroform layers after in vitro enzymatic reactions using tributyrin, tricaprylin, and triolein as substrates, respectively. (B) Lane 1, monoolein; lane 2, 1(3),2-diolein; lane 3, oleic acid; lane 4, triolein; lane 5, chloroform layer after in vitro enzymatic reaction using triolein as a substrate. The developing solvent systems were chloroform : acetone : acetic acids = 95 : 4 : 1 (v/v/v) (A) and petroleum ether : diethyl ether : acetic acid = 80 : 20 : 1 (v/v/v) (B). The spots on a TLC plate were visualized by spraying 50% (v/v) H2SO4 in ethanol followed by charring (A) and by standing in an iodine chamber (B).

30

Fig. 1.

Activity (μmoles of ρnitrophenol/min)

18

16 14 12 10 8

6 4 2 0

24

48

72

Cultivation time (h)

31

96

Fig. 2.

32

Fig. 3.

33

Fig. 4.

kDa

M

1

2

70 50 40 30

20

15

(A)

(B)

34

Fig. 5.

Relative activity (%)

120

(A)

100 80 60 40 20 0 3

4

5

6

7

8

9

Relative activity (%)

pH 120

(B)

100 80 60 40 20 0 20

30

40

50

60

70

80

Temperature (℃)

35

90

95

10

Relative activity (%)

Fig. 6.

120 100 80 60 40 20 0 0

20

40

60

Time (h)

36

80

100

120

Fig. 7. 160

Relative activity (%)

140 120 100 80 60 40

20 0 Methanol

Ethanol 2-Propanol

SDS

Chemical compounds

37

Urea

Fig. 8.

38

Tables Table 1. Purification of induced extracellular lipolytic enzyme from S. solfataricus Purification step

Total protein (mg)

Total activity (U)

Specific activity (U/mg)

Culture medium

54.2

4024

74.24

Bu-OH extraction

48.3

3867

80.06

98.4

DEAE-Sepharose

5.1

3104

608.62

51.6

Butyl-Sepharose

0.6

1265

2108.33

38.1

39

Yield (%) Purification (fold) 100

1

1.07 8.19 26.3

Table 2. Kinetic parameters for hydrolysis of various PNP esters Mean ± SD

p-Nitrophenyl substrate

Km (μM)

kcat (s-1)

kcat/Km (s-1∙ μM-1)

Butyrate (C4)

129.4±2

1135±23

8.8

Caprate (C10)

46.6±5

1576±36

33.8

Palmitate (C16)

24.1±1

2252±35

93.4

40

Table 3. Effects of various inhibitors on activity of induced extracellular lipolytic enzyme from S. solfataricus

Inhibitor Control DEP

PMSF PLP PCMB EDTA Paraoxon Eserine

Concentration (mM) 0 0.5 5 0.5 5 5 5 10 5 5

% Residual activity (mean ± SD) after incubation for 60 mina 100 15±3 0 46±2 11±2 98±6 51±3 96±4 40±2 8±5

a

The enzyme was incubated at 70ºC in 100 mM sodium phosphate buffer (pH 6.0) with different concentrations of inhibitors. After 60 min, aliquots were removed from each incubation mixture and assayed for residual activity using the standard enzyme assay.

41