Purification and Characterization of an Alkali-Thermostable Lipase from Thermophilic Anoxybacillus flavithermus HBB 134

Abstract

An intracellular lipase from Anoxybacillus flavithermus HBB 134 was purified to 7.4-fold. The molecular mass of the enzyme was found to be about 64 kDa. The maximum activity of the enzyme was at pH 9.0 and 50℃. The enzyme was stable between pH 6.0 and 11.0 at 25℃, 40℃, and 50℃ for 24 h. The K_m and V_max of the enzyme for pNPL substrate were determined as 0.084 mM and 500 U/mg, respectively. Glycerol, sorbitol, and mannitol enhanced the enzyme thermostability. The enzyme was found to be highly stable against acetone, ethyl acetate, and diethyl ether. The presence of PMSF, NBS, DTT and β-mercaptoethanol inhibited the enzyme activity. Hg²⁺, Fe³⁺, Pb²⁺, Al³⁺, and Zn²⁺ strongly inhibited the enzyme whereas Li⁺, Na⁺, K⁺, and NH₄⁺ slightly activated it. At least 60% of the enzyme activity and stability were retained against sodium deoxycholate, sodium taurocholate, n-octyl-β-D-glucopyranoside, and CHAPS. The presence of 1% Triton X-100 caused about 34% increase in the enzyme activity. The enzyme is thought to be a true lipase since it has preferred the long-chain triacylglycerols. The lipase of HBB 134 cleaved triolein at the 1- or 3-position.

Introduction

Lipase enzymes (triacylglycerol acylhydrolases; E.C. 3.1.1.3) are produced by all living things, from microorganisms to animals [15]. Microbial enzymes are more demanding owing to their high enzyme yields, great variety of catalytic activities, continuous availability due to absence of seasonal fluctuations, ease of use for genetic manipulations, and fast growth of microorganisms on low-cost media. Moreover, microbial enzymes are superior with respect to their higher stability and convenience as well as safety during the production [15].

Bacterial lipases may be intracellular, extracellular, or membrane-bound, and they function for the storage and usage as carbon or energy source of the triacylglycerols, which are essential for the development of microorganisms. Since the extraction, purification, and immobilization of extracellular lipases are high-cost procedures, microorganisms bearing intracellular lipases are preferred as direct enzyme sources for industrial applications such as biodiesel and polyester production [1]. The primary function of lipases is to hydrolyze acylglycerides at the lipid-water interface. However, a reverse reaction catalyzed by lipase produces glycerides from fatty acids and glycerol under certain conditions [36]. Lipases are considered to be significant enzymes owing to their usefulness in organic solvents.

Consequently, lipases have been applied in various industrial applications and organic synthesis (i.e., detergent, food, oleochemical, pulp, and paper industries along with resolution of chiral drugs, wastewater treatment, synthesis of peptide, and production of biodiesel) [4,15,39]. Bacteria are generally preferred as enzyme sources since their enzymes possess higher catalytic activities. Bacterial enzymes tend to have optimum pH at neutral or alkaline pHs and they are often more thermostable compared with yeast and fungi [15].

Only a few studies have been done on the lipase production by Anoxybacillus strains. Esterases from Anoxybacillus gonensis A4 and G2 [9,12], a lipase from Anoxybacillus kamchatkensis [33], lipases from different Anoxybacillus species [34], a carboxylesterase from Anoxybacillus sp. PDF1 [2], a lipase from Anoxybacillus flavithermus [45], and an esterase/lipase from Anoxybacillus flavithermus [8] have been reported. In this study, in the effort of finding new enzyme sources, we describe a new lipase produced by the thermophilic strain Anoxybacillus flavithermus HBB 134. Purification, characterization, and determination of kinetic parameters are also performed.

 

Materials and Methods

Materials

Reagents used in this study were of analytical grade. Unless stated otherwise, all chemicals were purchased from Fluka (Switzerland). Olive oil, linseed oil, Tween 40 (polyoxyethylene sorbitan monopalmitate), tricaprylin, triolein, Tween 80 (polyoxyethylene sorbitan monooleate), p-nitrophenyl palmitate, p-nitrophenyl butyrate, Brij-35, Luria-Bertani (LB) broth, glyceryl trioleate, 1,3-diolein, 1,2-dioleoylglycerol, 1-oleoylglycerol, and cyclohexane were purchased from Sigma-Aldrich (Germany). Tween 60 (polyoxyethylene sorbitan monostearate), sodium dodecyl sulfate (SDS), Triton X-100, NH4Cl, AlCl3, CuCl2, BaCl2, FeCl3, ZnCl2, MgCl2, MnCl2, NiCl2, NaCl, KCl, HgCl2, PbCl2, CoCl2, EDTA, ammonium sulfate, ammonium persulfate, sodium hydroxide, p-nitrophenol, sodium hypochlorite, β-mercaptoethanol, methanol, ethanol, isopropanol, hexane, acetone, chloroform, glycerol, glycine, titrisol, and peptone were purchased from Merck (Germany). Tween 20 (polyoxyethylene sorbitan monolaurate), 1,4-dioxane, ethylene glycol, and dimethyl sulfoxide (DMSO) were purchased from Pancreac (Spain). CaCl2, diethyl ether, ethyl acetate, hydrogen peroxide, benzene, toluene, xylol, oleic acid, and sodium acetate were purchased from Riedel-de Haen (Germany). Na2O3Se, LiCl, sodium taurocholate, cetrimide, and 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate hydrate (CHAPS) were purchased from ABCR (Germany). Sodium deoxycholate, octyl-β-D-glucopyranoside, and sorbitol were purchased from Acros Organics (USA), and acetonitrile was purchased from S.D.S. (France).

Microorganism

From a screening study performed before, it was determined that Anoxybacillus flavithermus HBB 134 was a lipase-producing strain and the culture conditions were optimized and the HBB 134 strain was registered in the GenBank database system under the accession number GQ342689 [3]. LB medium was used for growth of the HBB 134 strain and it was stored at -80℃ in 20% skim milk solution. The strain was cultivated for 24 h in 50 ml of the optimized medium (0.5% olive oil, 0.5% peptone, 1% NaCl, and 0.5% gum arabic) at 150 rpm on an orbital shaker at 45℃ and pH 6.5. Culture samples were harvested at the 12th hour. The culture medium was centrifuged (15 min, 20,000 ×g, 4℃) (Sigma-3K30, Germany) and the cells were washed twice with ultra-pure water and were resuspended in 20 mM, pH 8.0 Tris-HCl buffer at 4℃. The cell suspension was placed in an ice bath and sonicated for 10 min, at 40% of the maximum power (Bandelin Sonopuls-HD2200, Germany). Following this, a centrifugation procedure was applied to the mixture for 15 min at 4℃ and 20,000 ×g, and the supernatant was removed and used for the intracellular lipolytic activity measurements.

Lipase Activity Assay

Lipase activities were measured either spectrophotometrically using p-nitrophenyl-laurate (pNPL) as substrate [40] or titrimetrically using a pH-stat (Radiometer, France) according to the method of Lesuisse et. al. [24] with some modifications. The mixture containing 0.1 ml of enzyme solution, 0.8 ml of 50 mM pH 8.0 Tris-HCl buffer, and 0.1 ml of 10 mM pNPL resolved in ethanol was used for spectrophotometric assay. This reaction was carried out for 30 min at 65℃. Following the incubation, the reaction was stopped by adding 0.25 ml of 0.1 M Na2CO3 solution. After centrifugation (10,000 ×g) for 15 min (Heraeus-Biofuge Pico, Germany), the absorbance was measured at 410 nm. The amount of enzyme that releases 1 μmol of p-nitrophenol (ε = 17.34 mM-1 cm-1) from pNPL in one minute under the stated experimental conditions was regarded as one unit of lipase activity. For the pH-stat method, 1 mM triacylglycerol or 1% oil (v/v) was emulsified by homogenization for 5 min in ultra-pure water solution of 0.1 M NaCl and 1% gum arabic. The pH of the substrate solution was adjusted to the desired pH. The reaction was initiated by adding 10 μl of enzyme solution into the test medium. The end point of titrator was set to the desired pH, and 0.01 N NaOH solution was employed as the titrant for the released fatty acids, continuously. Under the stated experimental conditions, the amount of enzyme responsible for the release of 1 μmol fatty acid per minute was defined as one unit of lipase.

Determination of Protein Concentration

Bradford’s method was used to measure protein concentration [5] using bovine serum albumin as the standard.

Purification of Anoxybacillus flavithermus HBB 134 Lipase

Unless otherwise stated, all purification steps were accomplished at 4℃. Ammonium sulfate was added slowly into the intracellular enzyme fraction to 0-20%, 20-40%, 40-60%, and 60-70% saturation for fractional precipitation. The precipitate obtained by centrifugation (10,000 ×g for 30 min at 4℃) of the mixture was dissolved in 20 mM pH 8.0 Tris-HCl buffer at 4℃, containing 0.5 mM 1,4-dithiothreitol and 0.1 mM EDTA and dialyzed against the same buffer overnight. Each fraction was analyzed for protein content and enzyme activity. Fractions with high specific activity were chosen for the next purification step. The solution was loaded onto a Phenyl-Sepharose CL-4B column (1 × 20 cm) equilibrated with 20 mM pH 8.0 Tris-HCl buffer, containing 0.2 M (NH4)2SO4. Before loading onto the column, the (NH4)2SO4 concentration of the solution was adjusted to 0.2 M and unbound proteins were removed from the column at a flow rate of 60 ml/h with the same buffer. Then the column was eluted with a reverse gradient from 0.2 M to 0 M (NH4)2SO4 in 20 mM pH 8.0 Tris-HCl buffer. The lipase bound to the column was then eluted using 30% 2-propanol (v/v) in the same buffer. Fractions that exhibited lipase activity were mixed and dialyzed against 20 mM pH 8.0 Tris-HCl buffer at 4℃ for 24 h. The dialysate was concentrated with a 10,000 MWCO ultrafiltration membrane (Sartorius, Germany) and loaded onto a column (1 × 100 cm) of Sephadex G-100 (medium) pre-equilibrated with 20 mM pH 8.0 Tris-HCl buffer containing 0.15 M NaCl. In order to elute bound proteins, the column was washed with the same buffer at a flow rate of 13 ml/h and the fractions that exhibited lipase activity were collected for characterization of the enzyme.

Electrophoresis and Zymography

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (10%), under the reducing and non-reducing conditions described in Laemmli [21], was used to check the molecular weight and purity of the enzyme. Protein staining was carried out using Coomassie brilliant blue G-250. Zymograms were developed using tributyrin as the substrate [32]. After the gel was transferred to the tributyrin agar plate (pH 9.0), it was incubated at 50℃ until a clear zone occurred.

pH Effect on Lipase Activity and Stability

The effect of pH on A. flavithermus HBB 134 lipase activity was assayed by using a range of pH values (6–11). The potentiometric assay (pH-stat; Radiometer, France) was conducted by automatically titrating the free fatty acids liberated from triolein emulsion, using 0.01 N NaOH solution. Triolein emulsion was prepared at the concentration of 1 mM in ultra-pure water containing 0.1 M NaCl and 1% gum arabic and emulsified by homogenizing for 5 min. Then 15 ml of this substrate solution was transferred to the temperature-controlled vessel of the pH-stat. Temperature and end-point pH values were adjusted to 65℃ and the assay pH, respectively. The reaction was initiated by adding 50 μl of purified enzyme and its activity was measured.

To determine the effect of pH on lipase stability, the enzyme solution was diluted 5-fold in buffers at pH 6.0-11.0 following preincubation for 24 h at 25℃. The standard assay conditions were used to measure the residual lipase activities.

Temperature Effect on Lipase Activity and Stability

The enzyme assay was carried out spectrophotometrically at different temperatures (5-95℃) at pH 8.0 to determine the effect of temperature on lipase activity. The enzyme was pre-incubated at temperatures from 25℃ to 95℃ for 24 h. The standard assay conditions were used to measure the residual lipase activities.

Kinetic Parameters

The lipase activity was measured using various concentrations (5-120 μM) of p-nitrophenyl laurate as substrate. Km and Vmax values of the enzyme were calculated using a Lineweaver–Burk plot.

Effect of Polyhydric Alcohols on Thermal Stability of Lipase

Effects of various polyhydric alcohols (ethylene glycol, glycerol, sorbitol, and mannitol) on the thermal stability of the enzyme were examined. Incubation of the enzyme was performed at 65℃ for 1 h in pH 9.0 glycine-NaOH buffer containing polyhydric alcohol under examination at a final concentration of 1, 2, and 3 M. Standard assay conditions were used for relative activity measurement (percentage of the activity at time 0 h). The activity measurements carried out in the absence of polyhydric alcohols were considered as a control.

Effects of Inhibitors and Metal Ions on Lipase Activity

The effects of phenylmethylsulfonyl fluoride (PMSF), β-mercaptoethanol, 1,4-dithiothreitol (DTT), N-bromosuccinimide (NBS), N-cyclohexyl-N-(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate (CMC), eserine, metal ions, and EDTA on lipase activity were determined using 1 and 5 mM of each at 50℃ and 30 min.

Effects of Detergents and Bleaching Agents on Lipase Activity

The effects of detergents and bleaching agents on lipase activity was studied by incubating enzyme in the presence of 0.1% of these reagents at 50℃ and 30 min. Standard assay conditions were used to measure enzyme activities.

Effects of Organic Solvents on Lipase Stability

The enzyme was incubated with organic solvents at final concentrations of 10% and 50% (v/v). The mixture was incubated for 1 h at 50℃ and 200 rpm in an orbital shaker. Standard assay conditions were used to measure enzyme activities.

Substrate Specificity

Lipase activity using various oils (1%) and triglycerides (1 mM) was studied using the pH-stat method as described above. Substrate specificities against different pNP esters were determined spectrophotometrically at 50℃ and 50 mM pH 7.0 Tris-HCl buffer at 0.2 mM substrate concentration. In the case of pNP palmitate assay, 0.1% gum arabic and 0.4% Triton X-100 were added to the buffer and sonicated for 5 min.

Positional Specificity

Thin layer chromatography (TLC) of the reaction products obtained with triolein as the substrate was used to examine the positional specificity of the lipase [24]. Triolein (2%) was sonicated in 0.1 M pH 9.0 glycine-NaOH buffer at 50℃ containing 1 mM CaCl2 for 3 min. The enzyme solution (100 μl) was added to the mixture. The reaction mixture was incubated at 30℃ at 200 rpm on an orbital shaker for 6 h. Then the reaction products were extracted twice with 1 ml of ethyl ether. Ether extracts were applied to a silica gel-60 TLC plate (Merck, Germany), and development was carried out with a mixture of chloroform/acetone/acetic acid (96:4:l (v:v:v)). The spots were visualized with saturated iodine vapor.

Statistical Analysis

The experiments were conducted in triplicates. The statistical significance of differences between the groups and comparisons of the control group and the assay group were determined using one-way ANOVA and the paired-samples t test, respectively. STATISTICA 7.0. computer software was used for these analyses.

 

Results and Discussion

Purification of Anoxybacillus flavithermus HBB 134 Lipase

The intracellular lipase from HBB 134 was purified 7.4-fold with 3% recovery (Table 1). The 20-60% ammonium sulfate fractions were chosen for the next purification step owing to their higher specific lipase activity. Then a phenyl Sepharose CL-4B column was employed for the purification of this lipase. Since lipase enzymes are hydrophobic, they easily interact with hydrophobic supports [37] and this property makes hydrophobic interaction chromatography a good choice for the purification of most lipases. Following hydrophobic interaction chromatography, final purification was conducted using Sephadex G-100 gel filtration chromatography (Fig. 1A). The patterns of bands on the SDS–PAGE and PAGE of partially purified enzyme are given in Figs. 1B and 1C, respectively. The molecular mass of the enzyme was approximately 64 kDa. These results put forward that the lipase is monomeric. The molecular mass of the enzyme was different from most of the other microbial lipases such as those from Bacillus thermoleovorans CCR11 (11 kDa) [6], Bacillus subtilis 168 (19 kDa) [24], Bacillus coagulans BTS-3 (31 kDa) [20], Acinetobacter sp. ES-1 (32 kDa) [22], and Aeromonas sobria LP004 (97 kDa) [26]. In addition, the molecular masses of lipases from Bacillus sp. (60 kDa) [29], Pseudomonas aeruginosa PseA (60 kDa) [14], and Bacillus sp. THL027 (69 kDa) [10] are close to that of HBB 134 lipase.

Table 1.Lipase activity was determined using p-NPL as substrate.

Fig. 1.Purification of lipase from A. flavithermus HBB 134. (A) Purification on Sephadex G-100 column. SDS-PAGE (B) and zymogram (C) of A. flavithermus HBB134 lipase. Lane 1, molecular weight markers (Carbonic anhydrase 29 kDa, chicken egg albumin 45 kDa, bovine serum albumin 66 kDa, phosphorylase B 97.4 kDa, β-galactosidase 116 kDa, and myosin 205 kDa). Lane 2, crude enzyme. Lane 3, dialysate. Lane 4, phenyl Sepharose CL4B. Lane 5, Sephadex G-100.

pH and Temperature Effects on Enzyme Activity and Stability

The effects of pH on HBB 134 lipase activity were tested using emulsion of triolein as substrate with the pH-stat method. The lipase showed activity at pH 6.5–10.5 (Fig. 2A) with highest activity (100%) at pH 9.0. The enzyme had no activity below pH 6.5 and above pH 10.5. Moreover, the enzyme maintained 83% and 79% of its maximum activity at pH 8.5 and 9.5, respectively. It is known that lipases show their activity at alkaline pHs [13]. Several studies report that lipases from Bacillus sp. [29], Pseudomonas aeruginosa PseA [14], Pseudomonas sp. AG-8 [38], and Bacillus subtilis 168 [24] have optimum activity at pHs 8.0 or 10.0. The lipases of Bacillus thermoleovorans CCR11 [6] and Acinetobacter sp. RAG-1 [41] are reported to have catalytic activity at pH 9.0, similar to HBB 134 lipase. However, lipases that show activity at neutral and acidic pHs are also available [10,22,25]. The results obtained from the pH stability assay showed that lipase was stable (>94% of lipase activity) between pH 6.0 and 11.0 after 24 h incubation. Lipase exhibited maximum stability at pH range 8.0-11.0, retaining 100% activity after 24 h (Fig. 2B). The present results prove that the enzyme under investigation is an alkaline lipase. Lipases that are stable under alkaline conditions are considered as promising candidates for removable of fat stains in detergent formulation [11] and treating wastes from diary industries [26]. HBB 134 lipase protected its stability over a wider pH range compared with some other lipases [18,19,41,44].

Fig. 2.Effects of pH and temperature on the activity and stability of lipase from A. flavithermus HBB 134. (A) The effect of pH on the activity of the lipase. The lipase was incubated with substrate (triolein) at various pHs at 65℃. (B) pH stability of the lipase. The lipase was incubated at 25℃ for up to 24h . Buffers (50 mM) used were McIlvaine (pH 6.0-7.0), Tris-HCl (pH 8.0), glycine-NaOH (pH 9.0-10.0), and phosphate (pH 11.0). (C) The effect of temperature on the activity of lipase. The lipase was incubated with substrate (pNPL) in 50 mM Tris-HCl buffer (pH 8.0) at various temperatures. (D) Thermostability of the lipase. The lipase was incubated at 25-95℃ for up to 24h in 50 mM Tris-HCl buffer (pH 8.0). All measurements are the mean of three experimental data.

The enzyme was active in a wide range of temperatures of 20-60℃, with maximum activity at 50℃ (Fig. 2C). Moreover, it showed 95% of the maximum activity at 55℃. The results were similar to those of Bacillus megaterium (55℃) [25], Bacillus sphaericus 205y (55℃) [42], and Bacillus coagulans BTS-3 (55℃) [20].

The thermostability of lipase was studied by incubating it at 25℃, 40℃, 50℃, 65℃, 80℃, and 95℃ and pH 9.0 for durations ranging from 0 to 24 h. Residual activities were determined with the standard assay procedure. The enzyme was stable for 24 h at 25℃ and retained 100% of its activity. The enzyme maintained 92% and 85% of its activity after incubation at 40℃ and 50℃ for 24 h, respectively (Fig. 2D). These results indicate that the enzyme may be useful for various processes such as detergent, leather, medical, cosmetic, textile, and food industries [15]. The stability of HBB 134 lipase was better than lipases of Bacillus thermoleovorans CCR11 [6], Acinetobacter sp. ES-1 [22], Bacillus sphaericus 205y [42], and Pseudomonas aeriginosa PseA [14]. However, Pseudomonas sp. KWI-56 lipase retained 96% of its activity after 24 h incubation at 60℃ [17] and Bacillus sp. lipase was stable at 60℃ for 1 h [29].

Kinetic Parameters

The Km and Vmax values for pNPL substrate were calculated to be 0.084 mM and 500 U/mg protein, respectively, using Lineweaver–Burk plots. These results showed that the Km value of HBB 134 lipase was remarkably lower than Km values of lipase from Bacillus sp. (0.5 mM) [30] and Bacillus sp. (0.19 mM) [29]. Since a lower Km value of the enzyme indicates higher affinity for its substrate and higher initial rate of the reaction, these results indicate that HBB 134 lipase may find use in industrial processes.

Polyhydric Alcohol Effect on Temperature Stability

The effect of various polyhydric alcohols was investigated to enhance the thermostability of the lipase. After 1 h incubation with no alcohols added to the reaction medium at 65℃, 50% of activity was retained (control sample). Ethylene glycol (with two –OH groups) did not enhance the thermostability of lipase. Glycerol (with three -OH groups) at 3 M concentration enhanced the lipase thermostability and 66% of activity was retained. Lipase thermostability was enhanced linearly with increasing concentration of sorbitol (with six -OH groups) and mannitol (with six -OH groups) and at 3 M concentration lipase retained 88% and 92% of its activity, respectively (Fig. 3). The results were similar to the lipase purified from Bacillus licheniformis MTCC 6824 [7]. It has been reported that polyhydric alcohols with a few numbers of free hydroxyl groups have detrimental effect on the lipase activity of Bacillus licheniformis MTCC 6824 [7]. In this study, it is demonstrated that HBB 134 lipase carries similar features of aforementioned lipases in the presence of polyhydric alcohols.

Fig. 3.Effect of polyhydric alcohols on the thermostability of the lipase from A. flavithermus HBB 134. The lipase was incubated at 65℃ for 1 h in the absence (control) and presence of polyhydric alcohols at a final concentration of 1, 2, and 3 M. All measurements are the mean of three experimental data.

Organic Solvent Effect on Enzyme Stability

The biotechnological approach addresses usage of lipases for bioconversions in organic solvents. Hence, protection of enzyme activity and stability in organic solvents has been regarded as a novel aspect in lipase studies [14]. The enzyme stability was investigated to determine the effects of various organic solvents with changing concentrations (10% and 50%) after 1 h incubation at 50℃ (Fig. 4). Lipases possess diverse sensitivity to solvents; however, it is generally accepted that water-immiscible solvents are less destabilizing than polar water-miscible solvents [28]. Although there are studies that support this view [16,29], some others do not comply with it. It has been suggested that lipase of Bacillus thermoleovorans CCR11 showed high stability toward water-miscible organic solvents but lost its activity completely with butanol [6]. Whereas the lipase of Pseudomonas fluorescens JCM5963 was inactivated with water-immiscible organic solvents like chloroform, benzene, and cyclohexane, it was activated with those water-miscible ones [44]. No significant correlation has been found between the stability of HBB 134 lipase and the solubility of the organic solvents in water. Similar results were found with Bacillus megaterium lipase [25].

Fig. 4.Effects of organic solvents on the stability of the lipase from A. flavithermus HBB 134. The lipase was incubated at 50℃ for 1 h under shaking condition (200 rpm) in the absence (control) and presence of organic solvents at a final concentration of 10% and 50%. Lipase activities are expressed as the percentage of control, which was taken as 100%. All measurements are the mean of three experimental data.

HBB 134 lipase retained at least 90% of its activity after treatment with 10% concentration of methanol, DMSO, hexane, cyclohexane, and benzene. There was no loss in residual activities after treatment with acetone and ethyl acetate at 10% concentration and diethyl ether at 10% and 50% concentration. Since HBB 134 lipase is intracellular and the activity of the intracellular enzyme is close to the lipase activity in intact cells [3], it may be stated that the enzyme possesses an advantage in biodiesel production. As is known, immobilized extracellular microbial lipases are common in biodiesel production [1,15]. Extraction and immobilization of extracellular enzymes from microorganisms is a costly and time-consuming procedure. The results of this work suggest that HBB 134 lipase may be used for biodiesel production without employing immobilization procedures. Additionally, it can be suggested that the enzyme produced and purified in this study might be useful in other industries such as leather industry. In addition, the hydrolysis of water-insoluble esters for the separation of racemic mixtures via stereospecific hydrolysis may also be possible by means of such purified enzymes. Clearly, there is need to do further studies on this issue.

Metal Ion and Inhibitor Effects on Enzyme Activity

The effects of metal ions and inhibitors on the activity of the lipase are presented in Table 2. In most of the lipases and esterases, there is a serine residue located at the active site of the enzyme [13]. However, lipases have lid structures that cover the entrance of the active site. Therefore, some lipases were not inhibited or were slightly inhibited by PMSF [14,41]. HBB 134 lipase activity was decreased about 76%, 72%, 51%, 29%, and 30% in the presence of PMSF, NBS, DDT, β-mercaptoethanol, and CMC, respectively, which suggests the presence of serine, tryptophan, and cysteine residues and carboxyl groups at the active site of the enzyme. Similar results were found with Bacillus licheniformis [31], Bacillus thermoleovorans CCR11 [6], Bacillus sphaericus 205y [42], and Bacillus licheniformis MTCC 6824 [7] lipases. Li+, Na+, K+, and NH4+ increased the enzyme activity slightly whereas Hg+2, Zn+2, Fe+3, Pb+2, and Al+3 at the high concentrations strongly inhibited the enzyme activity. Similar to HBB 134 lipase, lipases of Bacillus sp. [29], Bacillus thermoleovorans CCR11 [6], and Acinetobacter sp. RAG-1 [41] were inhibited by Hg+2. The enzyme retained 96% of the activity after incubation with EDTA (5 mM), indicating that the lipase is not a metalloenzyme. It was reported that lipases of Pseudomonas aeruginosa PseA [14] and Aspergillus carneus [37] have also conserved their activities against EDTA.

Table 2.The lipase was incubated at 50℃ for 30 min in the absence (control) and presence of additives at a final concentration of 1 and 5 mM. Lipase activities are expressed as the percentage of control, which was taken as 100%. All measurements are the mean of three experimental data.

Detergent and Bleaching Agent Effects on Enzyme Activity

The effects of detergents and bleaching agents on the activity of the lipase are demonstrated in Table 3. After incubation with sodium taurocholate, enzyme activity was slightly increased; on the other hand, the presence of Brij 35, Tween 20, Tween 80, SDS, and Triton X-100 inhibited the enzyme. Lipases of Bacillus thermoleovorans CCR11 [6] and Bacillus thermocatenulatus [35] were also inhibited by Tween 20 and Tween 80. Sodium deoxycholate and CHAPS caused only 4% and 5% reduction in activity. The bleaching agent H2O2 strongly inhibited the enzyme. However, following the incubation with 0.1% NaOCl, the lipase retained 61% of its activity. Pseudomonas aeruginosa PseA lipase retained 62% and 85% of its activity after treatment with H2O2 and NaOCl, respectively [14].

Table 3.The lipase was incubated at 50℃ for 30 min in the absence (control) and presence of additives at a final concentration of 0.1%. Lipase activities are expressed as the percentage of control, which was taken as 100%. All measurements are the mean of three experimental data.

Substrate Specificity

HBB 134 lipase was active on a large number of substrates, with the highest affinity for Span 80 (Fig. 5A). The enzyme preferred long-chain triacylglycerol, indicating that it is a true lipase [13]. Since HBB 134 lipase showed higher activity with tristearin (C 18:0) than with triolein (C 18:1), it may be stated that the enzyme prefers saturated fatty acids as substrates. Additionally, the enzyme showed highest activity with the cottonseed oil, which has the highest saturated fatty acid content of the oils assayed. A different behavior was observed for Streptomyces rimosus lipase, which carried higher specificity toward unsaturated fatty acids [23]. Bearing high substrate specificity indicated that HBB 134 lipase carries a high potential for use in detergent, food, and pharmacology industries.

Fig. 5.Substrate specificity of the HBB 134 lipase. (A) Substrate specificity of the lipase toward various substrates. Lipase activities are expressed as the percentage of that of triolein, which was taken as 100%. (B) Substrate specificity of the lipase towards p-nitrophenyl fatty acid esters (pNP acetate, C2; pNP butyrate, C4; pNP caprylate, C8; pNP caprate, C10; pNP laurate, C12; and pNP palmitate, C16) with various acyl chain lengths. Lipase activities are expressed as the percentage of that of p-nitrophenyl laurate (C12), which was taken as 100%. All measurements are the mean of three experimental data.

p-Nitrophenylesters with a variety of alkyl chain lengths (C2-C16) were employed as substrates in order to measure HBB 134 lipase specificity (Fig. 5B). The enzyme demonstrated the highest hydrolytic activity towards p-nitrophenyl caprylate (pNPC8). It has been suggested that the lipase prefers medium acyl chain lengths. Similar results were found for Pseudomonas fluorescens JCM5963 [44] lipase.

Positional Specificity

Thin-layer chromatography was used for determination of the positional specificity of the lipase using glyceryl trioleate as a substrate (Fig. 6). If the enzyme is 1,3-specific, 2-monoglyceride and 1,2(2,3)-diglyceride will be the main products. On the other hand, for an enzyme with 2-specific regiospecificity, the main products are 1,3-diglyceride and 1(3)-monoglycerides [26]. The majority of the microbial lipases, such as those of Bacillus sp. THL027 [10], Bacillus subtilis 168 [24], Aspergillus carreus [37], Bacillus sphaericus 205y [42], and Penicillum camembertii Thom PG-3 [43], showed 1,3-positional specificity, and some others [14,23] showed random specificity. HBB 134 lipase hydrolyzed the glyceryl trioleate into 1,2(2,3)-diolein and oleic acid, which suggests that the enzyme has the 1- or 3-position specificity. The lipases that possess this position specificity are very rare in the literature. Mhetras et. al. [27] reported that Aspergillus niger NCIM 1207 lipase cleaved triolein at only the 3-position, releasing 1,2-diolein as major product. Owing to this position specificity property, HBB 134 lipase carries the potential to be used for inter- or transesterification reactions where useful oils are produced from cheaper oils. This application potential of this enzyme in this field should be further investigated.

Fig. 6.Positional specificity of the lipase from A. flavithermus HBB 134. Lane 1, glyceryl trioleate. Lane 2, 1,3-diolein. Lane 3, 1,2 (2,3)-diolein. Lane 4, 1(3)-monoolein. Lane 5, oleic acid. Lane 6, control (without enzyme). Lane 7, 1 h. Lane 8, 6 h.