Characterization of a newly identified lipase from a lipase-producing bacterium

Abstract

BACKGROUND: Lipases differ from one another with respect to certain properties, and such differences can be very important for various industrial applications. Considering the rapidly developing nature of the relevant industries, there is a need for new lipases with characteristics differing from those of existing enzymes.

METHODS: In this study, a bacterium was isolated from both the surface mucus layer and gills of rainbow trout (Oncorhynchus mykiss) from Giresun, Turkey. The bacterial species was identified based on its morphological and physiochemical properties, and on its 16S rDNA sequence. The qualitative activity of the bacterial lipase was determined on Rhodamine B and Tween-20 agar plates. The lipase was partially purified from the supernatant of bacterial cultures, and then characterized.

RESULTS: The bacterial strain was identified as Acinetobacter sp. strain SU15. The enzyme from Asp-SU15 exhibits maximum activity toward p-nitrophenyl dodecanoate (C₁₂) at 40°C and pH 8.0. The specific activity of the lipase was calculated to be 10.059 U·L^–1. The molecular mass of the enzyme was determined to be ~62 kDa via SDS-PAGE. However, native-PAGE indicated that the enzyme forms very large active aggregates, with molecular masses exceeding 250 kDa. The catalytic activity of the enzyme is enhanced in the presence of Co²⁺, Ca²⁺, and methanol, but is partially inhibited by Ni²⁺, ethyl acetate, and butanol.

CONCLUSIONS: Further research could examine possible industrial applications for the lipase from Asp-SU15.

Keywords

Acinetobacter sp. / lipase / Oncorhynchus mykiss / SDS-PAGE

Introduction

Lipases (EC 3.1.1.3) are a highly useful group of enzymes because of their versatility. They find use in many sectors, including the textile, food, biomedical, petrochemical, pharmaceutical, dairy, agrochemical, and cosmetics industries. They are also used in detergent formulations and the synthesis of novel compounds ( Haliru and Bukola, 2012; Adan and Arslanoglu, 2013). Lipases are produced by animals, plants, and microorganisms, including Archaea ( Wang et al., 2008; Adan and Arslanoglu, 2013). Of these, the lipases produced by microorganisms are the most industrially important and are used the most often ( Haliru and Bukola, 2012). Microbial lipases have superior properties; for example, they can be readily obtained in a short time because the nutritional requirements of microorganisms are relatively simple. Moreover, microorganisms generally grow much faster than plants or animals, and they are amenable to convenient lipase-screening procedures. In addition, lipases from microorganisms are generally more stable at an alkaline or neutral pH ( Schmidt-Dannert, 1999; Adan and Arslanoglu, 2013).

There are many lipase-producing bacterial strains, yet only a few such strains are commercially important. In particular, these include the genera Achromobacter, Alcaligenes, Arthrobacter, Bacillus, Burkholderia, Chromobacterium, and Pseudomonas ( Jaeger et al., 1994; Gupta et al., 2004). Furthermore, many studies have been conducted on the lipases obtained from the Acinetobacter genus, and several lipolytic strains of Acinetobacter spp. have been isolated. These include A. calcoaceticus ( Pandey et al.,1999; Pratuangdejkul and Dharmsthiti, 2000; Wang et al., 2012), A. radioresistens ( Liu and Tsai, 2003), A. junii ( Yoon et al., 2004; Anbu et al., 2011), A. johnsonii G23 ( Wang et al., 2011; Dai et al., 2013), A. baumannii ( Huda, 2013), and other strains in the genus Acinetobacter ( Wakelin and Forster, 1997; Kim et al., 2008).

In this study, a psychrotrophic bacterium was isolated from both the surface mucus layer and gills of rainbow trout. The bacterium was identified as Acinetobacter sp. strain SU15. It was found to produce a lipase, which was subsequently characterized. Lipase activity was unaffected by the addition of several ions or organic solvents, although its activity was partially inhibited by Ni²⁺, ethyl acetate, and butanol. The results of this study suggest that future research may investigate possible applications of the lipase secreted from Acinetobacter sp. SU15 in various industrial areas.

Further research will be needed to determine the complete biochemical properties of the lipases from the Acinetobacter sp. described in this study, and the genes that encode them. This newly described lipase may become commercially important since other lipases from the Acinetobacter genus remain very stable in the presence of ions and organic solvents, and at a variety of temperatures and pH ranges ( Chen et al., 1998; Wang et al., 2012).

Materials and methods

Bacterial strain and qualitative lipase activity

Fresh rainbow trout (Oncorhynchus mykiss) were obtained from a fish farm in Giresun, Turkey. The fish were packed in ice and transferred to the laboratory within 2 h. Samples were obtained from both the surface mucus layer and gills of the fish, and these were then spread on nutrient agar using a sterile swab. Plates were incubated at 30°C and 10°C for 2–3 days, and then bacterial colonies were chosen according to various morphological characteristics such as colony color, shape, and structure. The qualitative lipase activity of the bacteria was determined via two methods ( Haba et al., 2000; Kukreja and Bera, 2005). The lipase from the bacterium was obtained as described by Rajan et al. (2011).

Identification of the bacterial strain

Gram staining was carried out and the bacterial strains were identified based on their morphological, physiological, and biochemical characteristics, as described in Bergey’s Manual of Systematic Bacteriology ( Krieg and Holt, 1984). The colors and shapes of the colonies were determined. The tolerance of the bacteria to NaCl (2%–12%), pH conditions (3.0–11.0), and temperatures (4–37°C) was then established.

For molecular identification of the strain, genomic DNA was extracted according to Sambrook et al. (1989). The 16S rDNA genes were amplified by PCR using genomic DNA and the following oligonucleotide primers: (UNI16SL: 5ʹATT CTA GAG TTT GAT CAT GGC TTC A3ʹ, and UNI16S-R: 5ʹATG GTA CCG TGT GAC GGG CGG TGT TGT A3ʹ).The amplified DNA fragments were sequenced by Macrogen, Europe. The 16S rDNA gene sequences were compared with entries in the updated GenBank database using the BLAST server. The sequences were then further characterized by phylogenetic analysis.

Phylogenetic analysis

The nucleotide sequences of the 16S rRNA genes were edited using EditSeq. The 16S rRNA gene sequences of one isolate from Oncorhynchus mykiss—together with those of 21 closely related species—were used in the phylogenetic analysis. The phylogenetic analysis was performed via the neighbor-joining method implemented using MEGA 5.0 software ( Tamura et al., 2004). The reliability of the phylogram was tested by bootstrap analyses of 1000 replicates using MEGA 5.0.

Quantitative lipase activity

Lipase activity was quantitatively analyzed using a spectrophotometric assay, with p-nitrophenyl dodecanoate (Sigma) as a substrate. The enzyme solution (100 µL) was added to the substrate solution (900 µL), which consisted of 1 part 30 mg p-nitrophenyl dodecanoate in 10 mL isopropanol, and 9 parts 0.1 g gum arabic and 2 mL Triton X-100 in 90 mL Tris-HCl buffer (pH 8.0). The reaction mixture was incubated for 15 min at 30°C and then immediately cooled for 10 min to 4°C. Lipase activity was measured at 410 nm ( Kumar et al., 2012), and the enzyme activity was calculated in U×L^–1 ( Kumar et al., 2012).

Partial purification of the lipase

The culture of the lipase-producing strain was centrifuged (10000 ×g for 10 min at 4°C) and the supernatant was used for precipitation. Solid ammonium sulfate was added to achieve 30% saturation of the supernatant, which was then stirred for 24 h at 4°C. The suspension was then centrifuged again (10000 ×g for 10 min at 4°C). The precipitate was re-suspended in 50 mM Tris-HCl buffer (pH 8.0) and then stored at 4°C. Following this, ammonium sulfate was added to 50%, then and 80%, saturation of the remaining supernatant. The precipitates were suspended in 50 mM Tris-HCl buffer and stored at 4°C ( Ugras et al., 2013). Protein concentration was determined colorimetrically using the Bradford assay (Bradford, 1976).

Effect of temperature and pH on lipase activity

The optimal temperature range for lipase activity was determined to be 10–90°C, at pH 8.0, after which the residual activity was determined. The effect of temperature on lipase stability was established by analyzing the residual activity after incubation for 5–60 min at the optimal temperature ( Lee et al., 1999). The optimal pH of the lipase was determined using various buffer solutions (50 mM), including sodium acetate (pH 4.0 and 5.0), potassium phosphate (pH 6.0 and 7.0), Tris-HCl (pH 8.0), and glycine-NaOH (pH 9.0 and 10.0) ( Lee et al., 1999).

Effect of different substrates, metal ions, and organic solvents on lipase activity

The lipase activity was analyzed using various substrates, including p-nitrophenyl acetate, p-nitrophenyl dodecanoate, and p-nitrophenyl butyrate, via spectrophotometry ( Kumar et al., 2012). The enzyme solution obtained from the bacterial supernatant was incubated at 30°C for 1 h with 5 mM and 10 mM of the metal ions present in NiCl₂, ZnCl₂, CuCl₂, CoCl₂, MgCl₂, and CaCl₂ ( Lee et al., 1999), and then the residual activity was determined spectrophotometrically ( Kumar et al., 2012). The effect on lipase activity of organic solvents, such as hexane, butanol, isopropanol, methanol, ethyl acetate, and ethanol, was analyzed according to Lee et al. (1999); lipase activity was measured spectrophotometrically ( Kumar et al., 2012).

Detection of molecular weight via SDS-PAGE

The SDS-PAGE (12%) procedure was performed according to Laemmli (1970). Samples of the lipase were run on an SDS-PAGE gel and then compared with a marker, which contained 10–225 kDa peptides (Promega, USA). Following electrophoresis, the gel was stained with CBB R250.

Direct detection of lipase activity via native-PAGE

The native-PAGE (12%) procedure, which requires non-denaturing conditions, was performed as described by Laemmli (1970). Samples of partially purified lipase were run on a native-PAGE gel. Bovine serum albumin (BSA 1 mg mL^–1) was used as a marker. Following electrophoresis, the gel was sliced vertically. The first section, which included the samples of partially purified lipase and BSA, was stained with CBB R250. The other sections of the gel were assayed for direct detection. The direct lipase activity was performed as described in Park et al. (2007).

Results

The bacterial isolate was identified based on morphological, physiological, and biochemical characteristics, in addition to a phylogenetic analysis of 16S rDNA sequences. The 16S rRNA gene sequence consistently revealed a 99% similarity that from Acinetobacter sp. P162 (GenBank: KC904087); the phylogenetic analysis of the 16S rRNA genes also supports this identification (Fig. 1). Since the result agreed well with the findings of the biochemical and physiological analysis, the isolated bacterium was identified as Acinetobacter sp. P162, strain SU15. The isolate, Asp-SU15, is Gram-negative, psychrotolerant, catalase-positive, lipase-positive, and cellulase-positive. It forms smooth, opaque, irregularly shaped, cream-colored colonies on nutrient agar. The strain grows within a temperature range of 4–40°C. Its salt tolerance during growth was determined to be between 2% and 5% (w/v) NaCl, between pH 7.0–9.0, although growth was relatively weak at 45°C and pH 5.0 (Table 1).

In the current study, enzymatic activity was qualitatively indicated by the appearance of a visible precipitate (opaque halos), resulting from the deposition of calcium salt crystals, which was formed by the fatty acids released when either Tween 20 or Tween 80 was enzymatically hydrolyzed ( Gopinath et al., 2005). Thus, Acinetobacter sp. SU15 secretes a lipase (Fig. 2).

The lipase was partially purified from the supernatant of bacterial cultures via ammonium sulfate precipitation (30%, 50%, and 80%). The specific activity of each fraction was calculated to be 5.242 U×g^–1, 7.293 U×g^–1, 10.059 U×g^–1, respectively.

These results demonstrate that the enzyme exhibits optimum activity toward p-nitrophenyl dodecanoate (C₁₂) at 40°C, in Tris-HCl buffer at pH 8.0 (Table 2, and Figs. 3 and 4). The activity was calculated to be 9.793 U×L^-1.

The activity of lipase obtained from Asp. SU15 became enhanced in the presence of Ca²⁺ and Co²⁺, whereas enzymatic activity decreased in the presence of Ni²⁺, ethyl acetate, or butanol. Furthermore, there were no significant differences in activity following the addition of ions or organic solvents, with only partial inhibition being observed (Figs. 5 and 6).

As shown in Fig. 7, the molecular mass of the enzyme (~62 kDa) was established by SDS-PAGE (Fig. 7C). However, native-PAGE indicated that the enzyme forms very large, active aggregates with high molecular masses (>250 kDa). Direct enzymatic activity was observed on native-PAGE for the aggregates with masses>250 kDa; however, no such activity was seen in the 62 kDa region of the gel (Fig. 7A and B).

Discussion

In this study, a psychrotolerant bacterium was isolated and identified as Acinetobacter sp. strain SU15. It was determined that Acinetobacter sp. SU15 produces a lipase. Many studies have been conducted on the lipases obtained from the Acinetobacter genus, and several lipolytic strains have been isolated ( Liu and Tsai, 2003; Yoon et al., 2004). The present results demonstrated that the enzyme exhibits optimum activity toward p-nitrophenyl dodecanoate (C₁₂) at 40°C, in Tris-HCl buffer at pH 8.0. As reported, the optimal conditions for enzymatic activity differ among the lipases of the Acinetobacter genus. For example, the optimal activity of the lipase from Acinetobacter sp. O16 occurs at 35°C and pH 7.5 (Breuil and Kushner, 1975a, 1975b), that from A. calcoaceticus BD413 achieves its optimum at pH 7.8–8.8 ( Kok et al., 1995), that from A. radioresistens CMC-1 achieves its optimum at pH 10.5 ( Hong and Chang, 1988; Chen et al., 1998), that from Acinetobacter nov. sp. KM109 achieves its optimum at pH 8.0 ( Mitsuhashi et al., 1999), while that from Acinetobacter sp. SY-01 achieves its optimum at 50°C and pH 10.0 ( Han et al., 2003). The properties of the lipase described in the current study are similar to those of the others. Although other enzymes from bacteria in the Acinetobacter genus are generally stable at alkaline pH (such as 9.0–10.0), and thus are especially suited to applications in the detergent industry ( Hong and Chang, 1988; Wang et al., 2012), this enzyme lost 50% of its activity at pH 9.0. Nonetheless, the stability of this enzyme remained unaffected following 1 h at 40°C in Tris-HCl buffer (pH 8.0), permitting its application in a variety of areas.

The activity of the lipase was enhanced in the presence of Ca²⁺ and Co²⁺. No significant difference in activity occurred upon the application of organic solvents or other ions, with only partial inhibition from butanol, ethyl acetate, and Ni²⁺. Many studies have shown that the activity of lipases from Acinetobacter sp. O16, A. calcoaceticus BD413, and Acinetobacter sp. SY-01 can be increased Ca²⁺ ions ( Breuil and Kushner, 1975a; Kok et al., 1995; Han et al., 2003). Similarly, in the current findings, the activity of the lipase increased upon addition of Ca²⁺ ions, which may arise from a dependence on Ca²⁺ for fatty acids release, or from stabilization of the structure of the enzyme upon binding of Ca²⁺ ( Kouker and Jaeger, 1987; Wang et al., 2012).

The molecular mass of the enzyme was judged to be ~62 kDa by SDS-PAGE. The molecular masses of lipases from the Acinetobacter genus have been reported in previous studies, for example, 32 kDa from both Acinetobacter sp. ES-1 ( Kasana et al., 2008) and A. calcoaceticus BD413 ( Kok et al., 1995), 23 kDa from A. calcoaceticus LP009 ( Pratuangdejkul and Dharmsthiti, 2000), 38 kDa from A. radioresistens CMC2 (Ng et al. 1999), 43.8 kDa from Acinetobacter sp. SY-01 ( Han et al., 2003), 45 kDa from A. radioresistens CMC-1 ( Hong and Chang, 1988; Chen et al., 1998), 53 kDa from A. johnsonii LP28 ( Wang et al., 2011), and 62 kDa from Acinetobacter sp. KM109 ( Mitsuhashi et al., 1999). The molecular mass of the newly described enzyme matches that of the lipase from Acinetobacter sp. KM109; however, as was discovered during native-PAGE, the new lipase forms very large, active aggregates (>250 kDa). An earlier study reported that Acinetobacter sp. O16 produces a lipase that forms aggregates greater than 200 kDa ( Breuil and Kushner, 1975a, 1975b). Furthermore, Salameh and Wiegel found that the thermophilic alkaline lipases from Thermosyntropha lipolytica also form high molecular mass aggregates (≥280 kDa) on a gradient native-PAGE ( Salameh and Wiegel, 2010). In addition, that study found that SDS prevented aggregation, and a monomeric species was observed following SDS-PAGE. Moreover, several other lipases have been found to form aggregates, including those from Burkholderia cepacia ( Dunhaupt et al., 1992), Geobacillus thermocatenulatus ( Rúa et al., 1997), Bacillus subtilis ( Lesuisse et al., 1993) and a Moraxella sp. ( Feller et al., 1990).

As a result of this study, a psychrotolerant bacterium was identified, and shown to be Acinetobacter sp., strain Asp-SU15. The strain Asp-SU15 produces industrially important enzymes such as lipases, cellulases, and catalases. The lipase was characterized, and found to exhibit optimal activity toward p-nitrophenyl dodecanoate (C₁₂) at 40°C, in Tris-HCl buffer at pH 8.0. Its properties are both similar to, and different from, other lipases obtained from Acinetobacter strains. The results of this study suggest that future research should investigate possible applications of the lipase secreted from Acinetobacter sp. SU15 in different industrial processes.

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