Expression and characterization of thermotolerant lipase with broad pH profiles isolated from an Antarctic Pseudomonas sp strain AMS3

Sources of bacteria

The bacterium was isolated from Antarctic soil. The isolated denoted as AMS3. Identification was performed using 16SrDNA and homology to Pseudomonas spp. The 16SrDNA sequence was sent to NCBI under accession no KR821141. The isolate was screened to produce lipase via qualitative approach using selective media tributyrin, Rhodamine B and Victoria Blue agar plates containing tributyrin, triolen and olive oil respectively as substrate (Samad et al., 1989). Lipase producer will hydrolyze the lipid to free fatty acid. Lipolysis is observed directly by changes in the appearance of the substrate such as forming a clearing zone and change in colour of indicator dye used (Scholze et al., 1999).

Quantitative assay for lipase activity

The lipase assay was performed with a colorimetric method using olive oil as the substrate as previously described by Kwon & Rhee (1986). A reaction mixture of 1.0 ml of enzyme, 2.5 ml olive oil emulsion (50% olive oil +50% phosphate buffer, the emulsion was mix using homogenizer at 2,500 rpm) and 0.02 ml CaCl₂.2H₂O was used. The reaction mixture was incubated for 30 min with shaking (250 rpm) at 37 °C. The reaction was stopped by the addition of 5.0 ml isooctane. The upper layer (4.0 ml) was moved to a test tube, and 1.0 ml of cupric acetate pyridine pH 6.1 was added. The concentration of free fatty acid dissolved in isooctane was determined by measuring the absorbence at 715 nm. One unit of lipase activity is defined as the rate of release one micromole of free fatty acid in one min.

Cloning of the lipase gene

Recombinant AMS3 lipase purification

The His-tagged recombinant AMS3 lipase was purified using Nickel Sepharose affinity chromatography. The cell pellet was resuspended using 100 mM Tris–HCl (pH 7.4) binding buffer containing 20 mM imidazole and 0.5 M NaCl. The sonicated cells were centrifuged at 12,000 × g for 30 min to separate the crude extract. The crude enzyme was filtered and subjected to affinity columns that were pre-equilibrated with binding buffer. The recombinant enzyme was eluted using elution buffer containing 100 mM Tris–HCl with 0.5 M imidazole and 0.5 M NaCl (pH 7.4). The active fractions were pooled together and the protein homogeneity was determined using SDS-PAGE.

Protein determination

The protein content was measured by the Bradford method (Bradford, 1976); BSA was used as a standard. During chromatography, the protein concentrations in the fractions were monitored by measuring the absorbance at 280 nm.

SDS-PAGE analysis

The purified fractions that showed lipase activity were pooled and subjected to SDS-PAGE according to the method described by Laemmli (1970). The sample was electrophoresed on a 12% resolving gel (2.5 ml 1.5 M Tris–HCl (pH 8.8), 100 µl 10% SDS, 4.4 ml distilled water, 3 ml Bis-acrylamide (12%), 50 µl APS and 10 µl TEMED) and a 6% stacking gel (2.5 ml 0.5 M Tris–HCl (pH 6.8), 100 µl 10% SDS, 5.9 ml distilled water, 1.5 ml 12% Bis-acrylamide and 50 µl APS). Electrophoresis was performed at a constant voltage (210 V) at room temperature. Coomassie Brilliant Blue R 250 was used to stain the gel for 10 min, and then the gel was destained with destaining solution. The molecular mass of the protein was estimated using a protein standard marker (Unstained protein marker from Thermo scientific USA).

Characterization of AMS3 lipase

Effect of pH

The optimum pH of the recombinant lipase was studied by emulsifying the substrate (olive oil) in buffers of different pH values (4.0–12.0). The following buffers (50 mM) were used: (1) sodium acetate (pH 4.0–6.0), (2) potassium phosphate (pH 6.0–8.0), (3) Tris–HCl (pH 8.0–9.0) and (4) glycine-NaOH (pH 9.0–12.0). To assess the pH stability of the lipase, the enzyme solution was pre-incubated in different pH buffers (pH 4.0–12.0) at 50 °C for 30 min, followed by the lipase assay.

Effect of metal ions

The effect of metal ions on lipase activity was studied using the method described by Kwon & Rhee (1986) after the enzyme was pre-incubated at 50 °C for 30 min in 50 mM Phosphate buffer (pH 8.0) and either 1 mM or 5 mM of various metal ions in different reaction vessels (Li⁺, Rb⁺, Na⁺, Mg²⁺, Ca²⁺, Fe²⁺, Mn²⁺, K⁺, Zn²⁺, Ni²⁺ or Co²⁺).

Secondary structure and thermal denaturation measurement of AMS3 lipase using Circular dichroism (CD) spectropolarimeter

Circular dichroism (CD) spectra were recorded using JASCO J-810 spectropolarimeter at 25 °C. The purified AMS3 lipase was dialysed over night with 10 mM phosphate buffer pH 7 prior to CD spectral analysis. The secondary content measurement was conducted from wavelength of 190 to 260 nm on a 1 mm path length. A few temperatures have been set to measure the changes of secondary structure from 10 °C to 90 °C. The protein concentration was 0.1 mg/ml and the cell pathlength 0.1 cm. The data been collected every 1 nm (band with) and the data pitch every 0.5 nm. Protein secondary structures content were estimated from the far-UV CD spectra based on the following link: http://perry.freeshell.org/raussens.html (Raussens, Ruysschaert & Goormaghtigh, 2003).

The thermal denaturation of AMS3 lipase was measured at 222 nm from 10 °C to 90 °C at a 1 °C/min heating rate. Wavelengths 222 nm measures α-helical content of the protein. Data collected from higher wavelengths usually have lower absorption. T_m is defined as a midpoint of sigmoidal melting curves using 0.5 mg/ml protein. The data was collected every 1 degree per min. Data pitch, bandwidth, response, scanning speed, and accumulation were set to be 0.1 degree, 1 nm, 1 s, 1 degree per min and 3 times, respectively.

Lipase gene isolation and expression in E. coli

To isolate the lipase gene, a genomic library approach was completed. To achieve this, a library was constructed via fragmentation or partial digestion of genomic DNA using Sau3A1. The plasmid (pET51b) was linearized using BamHI. Among thousands of transformants from the shotgun cloning, three of them produced a clearing zone. The positive recombinants harboring the putative lipase gene were assumed to have cold active lipolytic activity when the recombinant clone was initially grown at 37 °C for 24 h and transferred at 4 °C. The clearing zone only appeared after 3 days of incubation at 4 °C. The colonies were transferred to two selective media plates, triolein and Rhodamine B plates, to confirm lipase gene expression. Colonies 1 and 2 gave a positive result by the intense blue and orange coloration around the colonies. Colony 3, however, did not exhibit such coloration. These results show that these two colonies produced a putative true lipase compared to colony 3 that was a putative esterase producer (data shown in Data S1). To identify a true lipase producer the transformant must able to hydrolyse triglyceride that contain long chain fatty acid (Scholze et al., 1999). Colony 1 and 2 were sent for sequencing and the partial sequence from both colonies shown to contain putative gene from α∕β hydrolase family. Colony 1 (denoted as AMS3 lipase) was selected due to the high catalytic activity at 20 °C and was used for the remainder of the study.

Generally, protein functional and structural studies often require a large amount of pure, correctly folded protein, which is commonly produced in Escherichia coli expression systems. However, these overexpressed proteins are sometimes not efficiently processed by the E. coli post-translational machinery, resulting in protein misfolding. A dense insoluble aggregate of misfolded proteins is generally known as inclusion bodies (IBs). Most of the lipases from Pseudomonas are expressed in the form of IBs. Even under the control of pET-25b(+), a vector containing a signal sequence responsible for the translocation of expressed protein to the periplasm, the lipases from Pseudomonas sp. MIS38 and Pseudomonas sp. strain KB700A were still overexpressed in non-active, insoluble forms (Claudia et al., 2009; Rashid, Shimada & Ezaki, 2001).

Lipase sequence analysis

Putative plasmid contained recombinant lipase AMS3 was sent for sequencing. A few sets of primer were used to complete the sequence. The presence for open reading frame (ORFs) was analyzed using the ORF finder software from the NCBI. One open reading frame (ORF) was found. With a predicted size of 1353 bp encoding a 450 amino acid was determined. The protein sequence is made up by two different domains namely the GST C (KT215165) domain and lipase (KP744536) domain at the N and C terminal sections respectively. The lipase (AMS3 lipase) molecular mass without the GST C sequence was predicted to be 43.6 kDa with pI value of 6.3. The BLASTp result revealed that AMS3 lipase showed homology to α∕β hydrolase family. Multiple sequence alignment was done using Biology Workbench software to predict the conserve region of the lipase. Aligment between known lipase from various microorganisms exhibited the conserved region of the lipase (Fig. 1). A typical lipase conserved region contains the pentapeptide (GXSXG) sequence. The lipase catalytic triad which made up by Ser, Asp and His were also conserved. No signal peptide sequence was observed as predicted using SignalP software. Similarly, Group III lipases including lipases from Pseudomonas fluorescens and Serratia marcescens has no signal peptide. The protein origin can be reveal from G + C content. The G + C content was calculated using http://watson.nih.go.jp (FramePlot 2.3.2). AMS3 lipase content 55% of G + C almost similar to lipase from Pseudomonas fragi (59.3%) (Claudia et al., 2009). The N terminal section of AMS3 lipase contains a GST C (glutathione-S-transferase C family) similar to Pseudomonas Ag1. Glutathione have been reported as essential factor for chaperon to activate the lipase from Pseudomonas (Rosenau & Jaeger, 2000; Tanaka, Nihira & Yamada, 2000). According to Tanaka and co-workers, glutathione was found to be important for ‘lipase activator factor’ or Lif protein from Pseudomonas sp strain 109 to fold an active form (Tanaka, Nihira & Yamada, 2000).

Purification of AMS3 lipase

E. coli BL21 cells (DE3) harboring the pET51b/lipAMS3 plasmid was used to overexpress the recombinant AMS3 lipase under the control of a T7 promoter. The crude AMS3 lipase containing 10x histidine and Strep tags was produced in a soluble form allowing for rapid purification of the recombinant protein. The addition of fusion affinity tags and GST C Pseudomonas A3 increased the size of AMS3 lipase to ∼60 kDa. Purification was accomplished using the XK16 affinity chromatography column (GE Healthcare, USA) packed with Nickel Sepharose resin. After the column was equilibrated with binding buffer, the crude sample was loaded onto the column. Elution was accomplished using increasing concentrations of imidazole. Pooled fractions resulted in a 50% yield. Some protein was retained in the low-yield fractions and not included in the pooled fraction, resulting in some loss. Pooled fractions had an overall purification factor of approximately 1.52 fold. In 2007, Yan et al. (2007) reported that their organic solvent-stable and thermostable lipase from Galactomyces geotrichum Y05 had up to a 32% recovery and a 3.2-fold purification factor using ammonium sulfate, an ion exchange column and gel filtration purification. SDS-PAGE analysis indicated that the molecular weight of the AMS3 lipase was approximately ∼60 kDa (Fig. 2). Generally, lipases have molecular weights in the range of 16–69 kDa. According to Schmidt-Dannert (1999), the Staphylococcal lipase family, which includes lipase BTL-2 from B. thermocatenulatus, possesses molecular weights of 40–45 kDa (Schmidt-Dannert, 1999).

Biochemical characteristics of AMS3 lipase

Effect of temperature

Lipase activity was determined from 5 to 70 °C. The optimum temperature for the lipase was activity was at 50 °C, and the activity dropped above 60 °C (Fig. 3A). Interestingly, the purified lipase exhibited a broad temperature profile, whereby the lipase was still active at temperatures ranging from 10–60 °C. This finding is unlike the typical characteristics of cold active lipase. Most cold active lipases have been found to be active at temperature ranges from 15–35 °C (Gerday et al., 2000). However, few have reported the broad temperature observation from psychrophilic/psychrotolerant bacteria. A Pseudomonas lipase from Greenland exhibited an almost similar activity at wide temperatures (Schmidt, Larsen & Stougaard, 2010). In contrast, AMS3 lipase exhibited higher optimum activity compared to other reported lipases isolated from psychrophilic bacteria. r-LipA from Sorangium cellulosum (Cheng et al., 2011), lipA1 from Psychrobacter sp. 7195 (Zhang et al., 2009), lipase from Pseudomonas fragi strain IFO 3458 (PFL) (Alquati et al., 2002) and lipX from Psychrobacter sp. C18 (Alquati et al. 2011) were all reported to show maximum activity at 30 °C. AMS3 lipase was mostly stable at 30 °C and below (Fig. 3B). The organic solvent-stable and thermostable lipase from Galactomyces geotrichum Y05 was stable below 60 °C at a pH of 8.0 for at least 12 h; however, above 60 °C, rapid inactivation occurred with only 36% residual activity. These characteristics differ from those of the AMS3 lipase and may be due to the effects of the nature of the enzyme itself, which originated from Antarctica.

Effect of metal ions

The effect of metal ions was studied to determine which metal ions could enhance or reduce the activity of AMS3 lipase. All of the metal ions studied enhanced lipase activity at 1 and 5 mM (Fig. 5). For Li⁺ and Rb⁺ lipase activity was heavily enhanced, with relative activities over then 160%. Li⁺ have been reported to improve the enantioselectivity of the lipase (Okamoto et al., 2006). Na⁺ and K⁺ were also found to enhance AMS3 lipase activity. Metal ion activation of enzymes is important in industrial applications for obtaining maximal catalytic efficiency. However, at 5 mM concentration, Li⁺, Ni²⁺ and Zn²⁺ reduced lipase activity to less than 50%. In contrast, Sharma et al. (2002) reported that Ca²⁺ ions increased the lipase activity of Bacillus sp. RSJ-1 by 16%, similar to the lipase activity of B. coagulans MTCC-6375 (Sharma et al., 2002).

Substrate specificity

Assays addressing the specificity of the AMS3 lipase towards natural oils were conducted. The results showed that the AMS3 lipase hydrolyzed all of the natural oils tested, and the highest activity was seen with sunflower oil (Fig. 6). Olive oil is composed of mixed triglycerides (monounsaturated C18:1). Most lipases show a preference for long-chain fatty acids. The extracellular lipase Lip2 from Yarrowia lipolytica showed a much higher lipolytic activity toward triglycerides in olive oil and safflower oil than toward hydrophilic esters such as methyl fatty acid asters (Jaeger, Dijkstra & Reetz, 1999).

The AMS3 lipase displayed the second highest activity with coconut oil, with a relative activity of 110%, followed by canola oil, olive oil and sesame oil with relative activities of 106%, 100% and 90%, respectively. Similarly, AMS3 lipase exhibited the same substrate specificity with an extracellular lipase from Pseudomonas sp S5 (Rahman et al., 2005). In contrast, a Pseudomonas aeruginosa EF2 lipase reported olive oil as its preferred substrate (Gilbert, Cornish & Jones, 1991). AMS3 lipase has a broader substrate specificity ranging from C12 to C18. The ability of AMS3 lipase to hydrolyze long carbon chain triglycerides (C12 above) showed that the enzyme is a true lipase (Jaeger et al., 1994). According to Jaeger et al. (1994), surface pressure and lipid distribution are physical factors that can impact the determinants of lipase activity (Hiol et al., 1999). Therefore, as reported by Hiol et al. (1999), the differences in substrate specificity can arise not only from specific substrate binding but also from differential activation of the enzyme at the interface (Lanser, Manthey & Hou, 2001).

Effect of organic solvents

Persson et al. (2002) reported that the use of organic solvents in enzymatic catalysis is now well recognized with several advantages in water-poor mediums including improved solubility of hydrophobic substrates, a shift in thermodynamic equilibrium to synthesis from hydrolysis and increased thermostability of the enzyme (Persson et al., 2002). Assays measuring the effects of solvents were conducted to understand AMS3 lipase activity in organic solvents. The enzyme was incubated in a 25% v/v solvent at a temperature of 50 °C (Fig. 7). The results showed that AMS3 lipase exhibited some enhancements in stability expressed as relative activity in 25% v/v solvent compared to the control (without solvent). For instance, AMS3 lipase has a relative activity of 74%, 89% and 116% in DMSO, methanol and n-hexane, respectively, compared with its original activity at a temperature of 50 °C. The stability in the said organic solvents makes AMS3 lipase a good candidate for synthesis of biodiesel and transesterification reactions. Lipases have a sheath of water molecules tightly bound to them. This sheath protects the enzyme’s hydrophilic surface and possibly retains the native conformation even in non-polar solvents. Moreover, the changes in enzyme stability in organic solvents are also greatly affected by the structure of the enzymes (Gomez-puyou, 1992).

Secondary structure analysis of AMS3 lipase

As illustrated in Fig. 8A, the resulting denaturation (sigmoidal graph shape) curve indicated a structural transition of AMS3 lipase within the tested temperature range. The T_m value for AMS3 lipase for this transition was 62 °C (Fig. 8A). The Tm measurement using circular dichroism is close to the AMS3 lipase deactivation profile, which was at 55 °C. CD measurements have been widely used to follow the equilibrium between helical structures and unordered conformations. The CD spectra (molecular ellipticity) of AMS3 lipase was analyzed as a function of temperature between 190 and 260 nm. The wavelength 220 nm was set to monitor the transition of α-helical to unordered structures as they exhibited characteristic signals at this wavelength. The high-tension voltage (HT) value gradually increased until the T_m of AMS3 lipase and then decreased as the lipase unfolded above the melting temperature point. This outcome might be due to loss of protein secondary structure, followed by an increase in unordered conformations. This observation was supported by the secondary structure content determination at different temperatures. As depicted in Fig. 8B the increased formation of loop structures or disordered regions were observed notably at temperatures from 50–90 °C. In fact, AMS3 lipase exhibited a decreasing content of the alpha helices. This behavior could turn out to be the deactivation factor for AMS3 lipase. The enzyme gradually lost its activity beyond 50 °C. The random secondary structure conformation appears to be increasing as temperature drops together with β-pleat contents. The increased loop confirmation was found to be one of the factors for enzymes to work at low temperatures (Amico et al., 2002). These findings could explain the AMS3 lipase broad temperature profile.