Characterization Of Thermostable Lipase From Thermophilic Geo: Purification And Kinetic Characterization

Materials and methods

Discuss about the Characterization Of Thermostable Lipase From Thermophilic Geo.

Belonging to the class 3 of hydrolytic enzymes, lipases are esterase enzymes which are further classified into the subclass and sub-subclass 1 and 1.3 respectively (EC 3.1.1.3). Animals, plants as well as microorganisms are involved in producing lipases. The lipases are involved in reversible hydrolysis and lipid synthesis. Recently there has been an elevated rate of commercial interest in lipase because of its characteristics which includes active organic interfaces, absence of cofactor requirements and the capacity to accommodate a wide range of substrates. However there is only a limited amount of lipases acquired from bacterial sources which is of commercial value. Lipases isolated from microbial resources has been found to be significant in biotechnological applications like in dairies, detergents making, and textile industries along with surfactant and oil-processing industries. Additionally it is useful in the pharmaceutical industries.

Thermostable enzymes are mostly isolated from the family of thermophilic organisms. These enzymes are also of high commercial value since it shows a property of inherent stability. A large number of thermophilic microorganisms have been isolated from various ecological zones. Lipase is one of the thermostable enzymes that finds in the industries. The study related to this area is recently finding importance in research. The Geobacillus species form a phenotypically and phylogenetically coherent group of thermophilic bacilli with high levels of 16S rRNA sequence similarity (98.5–99.2%). Established species of thermophilic bacilli such as Bacillus stearothermophilus, Bacillusthermocatenulatus, Bacillusthermoleovorans, Bacillusthermoglucosidasius and Bacillus thermodenitrificans are part of this group.

This paper highlights the purification and kinetic characterization of the thermophilic lipase that was isolated from the microbial source, Geobacillus thermodinitrificans IBRL-nra which was initially isolated from a hot spring in Labok region of Kelantan in Malaysia.

Geobacillus thermodenitrificans IBRL-nra that was used in the study had been isolated from a hot spring located in Malaysia in Labok, Kelantan. This bacteria strain was analysed by 16S rRNA molecular assay. The microorganism was cultured on the nutrient agar media and maintained at 65?C. In order to maintain the viability of the culture, it was subcultured at an interval of two weeks.

For determination of lipase activity, modified colorimetric methods were used as given by Kumar et al., 2005 [18]. 1.0 ml of culture filtrate was shake along with 2.5 ml of olive oil emulsion along with 1.48 ml of 100 mM phosphate buffer (pH 7.0), and 20 μL of 20 mM CaCl_2. All of these were taken in an orbital shaker and agitated at a speed of 200 to mix vigorously. Reagents were prepared by adjusting the solution of 5% (w/v) copper (II) acetate-1-hydrate to pH 6.1 with pyridine. The upper layer absorbance of the lipase was read at an absorbance of 715 nm.  In order to measure the lipase activity, the amount of free fatty acids released were measures in accordance to the standard curve of the fatty acid (oleic acid) [41]. The amount of enzyme releasing 1 μmole of fatty acid per minute refers to one unit of lipase activity.

Microorganism and Culture Maintenance

The Lowry method was employed in order to measure the protein content of the cell-free supernatant [42]. Bovine serum albumin was used as standard for conduction of the assay.

For proper purification of the extracellular crude lipase, three consecutive procedures were employed namely heat treatment followed by affinity chromatography and gel-filtration chromatography. Using the ultra-centrifugal filter (Milipore-Amicon), the crude lipase was concentrated. The filter has a membrane pore size of 3000 Da.

The heat treatment method was carried out by raising the temperature of the extracts to a specific temperature. This was maintained at a given time. This was then cooled down on ice, followed by centrifugation in order to produce the supernatants that will be further assayed [40].

The concentrated enzyme collected from the previous step was loaded on a HiTrap Heparin column (5.0 mL, 1.6 cm × 2.5 cm) equilibrated with 10 mM phosphate buffer (pH 7.0). The unbound protein was washed out with low ionic strength buffer (10 mM phosphate buffer, pH 7.0) until the protein was undetectable at absorbance 280 nm. Then, the enzyme was eluted with high strength buffer (10 mM phosphate buffer, 1-2 M NaCl, pH 7.0) using a step elution method. The flow rate was adjusted to 16 mL/hour and the fraction volume of 3.0 mL was collected.

The fraction containing lipase with highest activity from affinity chromatography was loaded on Sephadex G-100 column (40.0 cm × 1.2 cm) equilibrated with 100 mM phosphate buffer, pH 7.0. The enzyme was then eluted with the same buffer with a flow rate of 1 mL/min. Fractions of 2 mL were collected [18].

For the optimum temperature determination, lipase activity was measured by colorimetric assay at different temperature in the range of 40–90°C at pH 7.0 in 100 mM phosphate buffer. For the thermostability, the purified lipase was incubated at 60, 65, 70, and 75°C for up to 24 h in 100 mM phosphate buffer, pH 7.0 and residual activity was measured at intervals of 1 h.

The lipase activity was determined at 65°C in a pH range of 4.5–10.5 using 100 mM different buffers: acetate buffer for pH 4.5, 5.0; phosphate buffer for pH 5.5, 6.0, 6.5, 7.0, and 7.5; Tris-HCl buffer for pH 8.0 and 8.5; glycine-NaOH buffer pH 9.0, 9.5, 10.0, and 10.5. To measure the pH stability, the purified lipase was incubated at pH 6.5, 7.0 and 7.5 in different buffers for 24 h at 65°C. At every 1 hour interval, the residual activity was measured [21].

The purified lipase was pre-incubated with each of the selected organic solvents. Three volume of the purified lipase was incubated with one volume of 25% (v/v) of organic solvents at 65ºC for one hour prior to lipase assay with agitation at 150 rpm [19] .The lipase activity was then determined by using colorimetric assay and the lipase activity without the addition of organic solvent was set as control.

Lipase Assay

For substrate specificity, triglycerides (C2–C18) with concentration of 10 mM and natural oils (corn oil, palm oil, soy bean oil, canola oil and sunflower oil were used as the substrates. The olive oil emulsion was substituted with the various substrates and lipase assay was carried out at 65°C with shaking at 200 rpm for 30 min using the calorimetric method as described earlier.

All the triglycerides tested, lipase from Geobacillus thermodinitrificans showed significant preference toward tripalmitin C16:0. The Km value and maximum reaction velocity (Vmax) for the enzyme were determined by the method of Line weaver–Burk plots, the Eddie-Hofstee plot and the Hanes plot. For determination of the Km and Vmax values, 1ml of purified lipase enzyme was added to the test tubes containing various concentration of tripalmitin ranging from0.1 to 1 mM [43]. The reaction mixture was incubated at 35 ?C for 10 min and the enzyme activity was measured at 540 nm.

The bacterial strain after 16S rRNA analysis was identified as Geobacillus thermodenitrificans IBRL-nra. This was recognised as a heat-loving that is a thermophilic bacteria that is capable of reducing nitrate to nitrogen. The strain is a gram-positive rod, forms flat, lobate and off-white colonies (Figure 1). It can grow at 45–70°C at pH 6–8 in 0.30% NaCl. The strain used in this study was isolated from a hot spring in Kelantan, Malaysia [17].

In order to purify the extracellular lipase, a three step procedure was followed of heat treatment, affinity chromatography followed by gel-filtration chromatography. The highest lipase activity was detected at fraction 10 with 308 U/mL in affinity chromatography. Affinity chromatography helped to minimize the purification steps and the loss of enzyme [6].  Use of heparin enabled highly sulphated glucosaminoglycan with a broad affinity for lipase. The partially purified lipase was then chromatographed on Sephadex G-100 gel filtration. A single peak of lipase was detected at fraction 17 with 92.2 U/mL activity. SDS-PAGE analysis of lipase exhibited a single-band with molecular mass estimated to be 30 kDa (Figure 2). The purification summary is tabulated in Table 1. After a three step purification procedures, crude lipase was purified to homogeneity by 34 fold from the culture supernatant with specific activity of 36.7 U and a final recovery of 11%. Sifour et al. 2010 [24] reported that a thermostable lipase was purified from Geobacillus stearothermophilus using ultrafiltration, Q-Sepharose ion exchange chromatography, Sephadex G-100 gel filtration, and adsorption on hydroxyl apatite to 22.6 fold with 8.8% recovery and molecular weight of 61 kDa. Smaller lipases have been reported by Chartrain et al. 1993 [25] (29 kDa), Kohno et al. 1994 [26] (30 kDa), Ohnishi et al. 1994b [27] (24 kDa), Mase et al. 1995 [28] (24 kDa), Lee et al. 1999 (35 kDa) [29], and Sharma et al. 2002 [30] (37 kDa). Kumar et al., 2005 [18], reported that an alkaline thermostable lipase was purified from B. coagulans BTS-3 with molecular weight of 31 kDa. In contrast, thermotolerant metallolipase from B. coagulans MTCC-6375 was reported to be 103 kDa in size [31]. However, there are also relatively higher Mr lipase that have been reported from B. stearothermophilus [13], B. thermocatenulatus BTL2 [32], Bacillus sp. 398 [33], and Bacillus sp. J33 [34] possessing Mr of 62.5 kDa, 69 kDa, 50 kDa, and 60 kDa, respectively. The final yield of thermostable lipase was detected as quite low, however this study is mainly focuses on the kinetic characterization of the enzyme.

Purification       Step	Volume     (ml)	Activity (V/ml)	Total activity     (V)	Protein content (mg/ml)	Total protein   (mg)	Specific activity	Yield     %	Fold
Crud enzyme	100	2.715	271.5	6.701	670.1	0.405	100	1
Heat treatment	95	1.612	153.14	3.208	304.76	0.502	56.4	1.24
His tag  affinity   Chromatography	30	3.150	94.5	1.601	48.03	1.97	34.8	4.86
First step gel filtration	5	4.162	20.81	0.810	4.05	5.13	7.7	12.7
Second step gel filtration	1	4.712	4.712	0.240	0.24	19.6	1.7	48.3

Determination of Protein Content

Table 1. Summary of purification of thermostable lipase from G. thermodenitrificans IBRL

Lane M: protein marker

Lane 1: crude lipase.

Lane 2: uninduce pET-lipGt

Lane 3,4: heat treatment at 70°C  

Lane5,6,7: purified lipase using gel filtration

Lane 8, 9: concentrated purified lipase

Figure 3 shows the effect of temperature on lipase activity. The temperature range was detected at 40–90°C (Figure 3(a)) at a pH 7.0. 65°C was detected at the optimum temperature followed by 60 and 70°C. There was drop in activity above 75°C with only 20% of activity left at 80°C. Four temperatures (60, 65, 70, and 75°C) with highest activity were then chosen for thermostability study. The results showed no loss of activity for the first 60 minutes at 65 and 70°C (Figure 3(b)), after which the activity dropped slightly. However, for 3 h and 2 h at 65 and 70°C, original activity was retained at around 90%. The result obtained shows that the thermostable lipase of G. thermodenitrificans IBRL-nra is highly stable compared to lipase from Geobacillus stearothermophilus [24] with only 87.5% of its original activity retained after 15 minutes of exposure at 70°C. The half-life of the purified lipase was 8 h at 60°C, 16 h at 65, 70, and 75°C, respectively (Figure 3(b)). In studies by Sharma et al. 2002 [30] it was reported that the lipase from Bacillus sp. RSJ-1 had optimum activity at 50°C and it retained 96, 92, 78 and 34% of its maximum activity and half-life of 150, 90, 55, and 45 minutes at 55, 60, 65, and 70°C, respectively. Wang et al., 1995 [36] isolated another thermostable lipase from Bacillus strain for which the half-life of the enzyme was 8 hour at 75°C and it retained at least 90% of the original activity for 15 hour at 60°C. The characteristics of the lipase from G. thermodenitrificans IBRL-nra shows evidence that it is a thermostable enzyme as it showed an optimal activity at 65°C.

The results show that changes in pH will affect the structure of protein as well as enzyme activity [27]. Figure 4 shows the effect of pH on lipase activity, showing activity in the pH range of 6.0-8.0 (Figure 4(a)). Maximal activity was seen at pH 7.0 followed by 6.5 and 7.5 (phosphate buffer) however the activity dropped from pH 8.0 onwards. Over 16 h, there was no activity loss, when the was preincubated at pH 7.0 and 7.5 (Figure 4(b)). 100% of activity was retained for 8 h at pH 6.5. However, lipase retained 85%, 90%, and 80% of its original activity for 24 h  at  pH 6.5, 7.0, and 7.5, respectively. Purified lipase from Geobacillus stearothermophilus, retained 95–100% of its original activity for 30 minutes at 60°C after incubation at pH 5–9. Studies by Kumar et al., 2005 [18] showed that the purified lipase from B. coagulans BTS-3 was stable within a pH range of 8.0–10.5 with optimum activity at pH 8.5. Lipase from B. stearothermophilus MC7 had pH optimum within the range of 7.5–9.0 and was stable at alkaline pH range 7.0–11.0 at 60°C [22]. In another study on lipase from Bacillus sp. RSJ-1, there was a maximum activity at pH 8.0 (100%), which followed by pH 9.0 (99%) and it retained 84% and 82% of its maximum activity at pH 8 and 9, respectively, for 2 hours at 50°C [30].

Purification of lipase

Figures (5) shows the effect of organic solvent on recombinant thermostable lipase LipGt. This was investigated as most of the reactions which involved lipase are carried out with the presence of organic solvents. When the enzyme was treated with ethanol, acetone and acetonitrile at 160%, 145% and 115% of relative activity, highest lipase LipGt activity was observed. There was no much enzyme activity when this was treated with the remaining organic solvents however the activity was inhibited while chloroform treatment.

The substrates specificity of purified lipase is shown in Figures 6 and 7. The activity of lipase increased from C8 to C16 where the highest lipase activity was observed at C16 (tripalmitin) showing 100% activity. This was` followed by C18 (tristearin) and C16 (triplamitin) with 60% activity. The enzyme hydrolysed triacylglycerols with acyl-group chain lengths between C8 and C18 with better activity compared to short chain acyl-group (C2–C6). The affinity of the enzyme towards long-chain triacylglycerols makes it is a true lipase. The natural oil study shows that lipase hydrolyzed olive oil has optimal activity 100%, followed by palm oil (96%), corn oil (90%), and sunflower oil (80%). The lowest lipase activity was obtained using soybean oil, castor oil and canola oil were used as the substrate. Reports showed that Lipase from G. stearothermophilus Strain-5 had increased affinity towards tributyrin [38]. Whereas thermoalkaliphilic lipase of Geobacillus sp. T1 showed rapid hydrolyzation of sunflower oil [39]. This ability of the thermostable enzyme for vegetable oil hydroloysis could be used in fat and oil industry. Applications like upgrading the vegetable oils into nutritionally important products like PUFA and cocoa butter replacer are present.

 For determination of the kinetic activity, Lineweaver-Burk plot for lipase from G.T .Lipase assay was conducted at various substrate concentrations at pH 7.0 and temperature 65°C. Woolf-Augustinsson-Hofstee plot for lipase from G.T. Lipase assay was conducted at various substrate concentrations at pH 7.0 and temperature 65°C. Hanes-Woolf plot for lipase from G.T. Lipase assay was conducted at various substrate concentrations at pH 7.0 and temperature 65°C. Out of the three methods of Lineweaver-Burk plot, Woolf-Augustinsson-Hofstee plot and Hanes-Woolf plot, the Lineweaver-Burk plot was the most suitable since its R² value is highest. Therefore it suggests that this graph is the most fitting graph out of all the three plots. The km value obtained was 0.60Mm and Vmax value was 101Mm min-1 when the substrate used was tripalmitin.

Conclusion

From the above study it can be concluded that thermostable lipase showed highest lipase LipGt activity was obtained when the enzyme was treated with ethanol, acetone and acetonitrile with 160%, 145% and 115% of relative activity, respectively.  Lipase hydrolysed tripalmitin (C16) and olive oil with optimal activity (100%) in comparison to other substrates. In the kinetic parameter determination, the km value obtained was 0.60Mm and Vmax value was 101Mm min-1 when the substrate used was tripalmitin. The properties of the enzyme are unique and therefore it holds a promising role in the biotechnological and industrial applications.

Heat treatment

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