Organic-Solvent and Surfactant Tolerant. Thermostable Lipase, Isolated from. a Thermophilic Bacterium, Geobacillus. thermodenitrificans IBRL-nra

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1 Advanced Studies in Biology, Vol. 5, 213, no. 9, HIKARI Ltd, Organic-Solvent and Surfactant Tolerant Thermostable Lipase, Isolated from a Thermophilic Bacterium, Geobacillus thermodenitrificans IBRL-nra Anuradha Balan 1, *, Darah Ibrahim 1 and Rashidah Abdul Rahim 2 1 Industrial Biotechnology Research Lab, School of Biological Sciences Universiti Sains Malaysia, 118, Pulau Pinang, Malaysia 2 School of Biological Sciences, Universiti Sains Malaysia 118, Pulau Pinang, Malaysia *Corresponding author tulasi79@yahoo.com darah@usm.my Copyright 213 Anuradha Balan, Darah Ibrahim and Rashidah Abdul Rahim. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

2 39 Anuradha Balan, Darah Ibrahim and Rashidah Abdul Rahim Abstract Lipases (triacylglycerol acylhydrolase, EC ), catalyze the hydrolysis of long-chain triglycerides with the formation of diacylglyceride, monoglyceride, glycerol and fatty acids. Lipases are actively used in various industries which include food and diary, pharmaceuticals, organic synthesis and detergent and cosmetics. Thermostable lipases are commercially significant for their potential use in industries as it is stable and active in organic solvents and resistance to high temperature and chemical denaturation. The production of thermostable lipase from Geobacillus thermodenitrificans IBRL-nra was carried out in a shake-flasks system at 65 ºC in cultivation medium containing (%; w/v or v/v): glucose 1.; yeast extract 1.25; NaCl.45 and olive oil.1 with agitation of 2 rpm for 24 hours. The extracted extracellular crude thermostable lipase was purified to homogeneity by using ultrafiltration, Heparin-affinity chromatography and Sephadex G-1 gel-filtration chromatography by 34 folds with a final yield of 9%. The molecular weight of the purified enzyme was estimated to be 3 kda after SDS-PAGE analysis. Purified thermostable lipase exhibited highest stability in the presence of acetone, ethanol and acetonitrile after 1h and 24h of incubation periods. Thermostable lipase showed elevated activity (22%) when pre-treated with Triton X-1 and could withstand 1% of its activity in the presence of protease up to 4 hours and could retain 7% of its initial activity after 24 hours of incubation. Keywords: Geobacillus thermodenitrificans IBRL-nra, thermostable lipase, affinity chromatography, gel filtration chromatography, surfactants, organic solvents Introduction Lipases (triacylglycerol acylhydrolases, EC ), are ubiquitous enzymes that catalyze the hydrolysis of triglycerides into fatty acids and glycerol at oil-water interface (Sharma et al., 21). Lipases had received great attention in industrial application due to its unique characteristics as they are stable and active in organic solvents, exhibit higher degree of enantioselectivity and regioselectivity and also possesses a broad range of substrate specificity (Sheikh et al., 23). Lipases play major role as biocatalyst in industrial application as lipasecatalysed processes are similar to natural metabolism pathway of living things and it is more safe and environment-friendly. However most of the industrial processes are carried out at elevated temperature, therefore enzymes with thermalstability had been the interest of researchers and industrialist. Thermostable lipases from thermophiles are highly stable and resistance to chemical and high temperature denaturation compared to their mesophilic counterparts.

3 Organic-solvent and surfactant tolerant thermostable lipase 391 Thermophilic Bacillus species previously assigned to rrna group 5 have been transferred to a new genus Geobacillus (Ash et al., 1991 and Nazina et al., 21). The Geobacillus species form a phenotypically and phylogenetically coherent group of thermophilic bacilli with high levels of 16S rrna sequence similarity ( %). This group comprises established species of thermophilic bacilli such as Bacillus stearothermophilus (Kambourova et al., 23), Bacillus thermocatenulatus (Kauffmann and Schmidt-Dannert, 21), Bacillus thermoleovorans (Dong-Woo et al., 1999), Bacillus thermoglucosidasius and Bacillus thermodenitrificans (Nazina et al., 21). Thermostable lipases are employed in various industries which includes food and diary, fat and oil modification, detergent, cosmetic, pharmaceuticals, organic synthesis, oleo-chemical and biodiesel production. The high potential of lipase application in various industries has resulted in increasing demands for new lipase which is highly active with specific stability to temperature, ph, ionic strength, surfactants and organic solvents. The aim of this study was to purify the thermostable lipase from Geobacillus thermodenitrificans IBRL - nra and to determine its properties for industrial application. Materials and Methods Microorganism and culture maintenance The bacterial strain, IBRL-nra used in this study was isolated from a Malaysian hot spring in Labok, Kelantan and identified by 16SrRNA as Geobacillus thermodenitrificans (Raikhan, 23). It was cultured on nutrient agar and maintained at 65ºC. The strain was subcultured every two weeks to maintain its viability. Cultivation of microorganism G. thermodenitrificans IBRL-nra was grown in a culture medium consisted of 1.% (w/v) glucose, 1.25% (w/v) yeast extract and.45% (w/v) NaCl and the ph was adjusted to 6.8. After sterilization,.1% of olive oil was added together with the 5.% (v/v; 5 x 1 6 cells/ml) of inoculum which was prepared earlier (the inoculum was prepared by transferring 1-2 colonies of G. thermodenitrificans IBRL-nra into 3 ml of culture medium, incubated at temperature 65ºC with agitation 2 rpm for 24 hours). The inoculated culture medium was then incubated at temperature 65ºC with agitation 2 rpm for 24 hours. The fermentation culture was harvested, filtered and centrifuged at 6g for 15 minutes. The cell free supernatant was collected and used as the crude enzyme. Lipase assay Lipase activity was determined by using the modified colorimetric method of Kwon and Rhee, Culture filtrate (1. ml) was shaken with 2.5 ml of olive oil emulsion, 1.48 ml of 1 mm phosphate buffer (ph 7.) and 2 µl of 2 mm CaCl 2 in an orbital shaker at an agitation speed of 2 rpm for 3 minutes at

4 392 Anuradha Balan, Darah Ibrahim and Rashidah Abdul Rahim 65 C. The emulsion was prepared by mixing together 1% polyvinyl alcohol and olive oil (3:1; v/v) in a homogenizer. The enzyme reaction in the emulsion system was stopped by adding 6 M HCl (1. ml) and isooctane (5. ml), followed by mixing using a vortex mixer for 3 s. The upper isooctane layer (4. ml) containing the fatty acid was transferred to a test tube containing copper reagent (2 µl) and mixed vigorously. The reagent was prepared by adjusting the solution of 5% (w/v) copper (II) acetate-1-hydrate to ph 6.1 with pyridine. The absorbance of the upper layer was read at 715 nm. Lipase activity was measured by measuring the amount of free fatty acids released based on a standard curve of free fatty acid (oleic acid). One unit of lipase activity was defined as the amount of enzyme releasing 1µmole of fatty acid per minute. Lipase activity (U/ml) = / Protein determination Protein content of cell free supernatant was determined according to Lowry et al., 1951), using bovine serum albumin as standard. Purification The collected extracellular crude lipase was purified using a three step procedures: Ultrafiltration, followed by Affinity Chromatography and Gelfiltration Chromatography. The crude lipase was concentrated using the ultracentrifugal filter (Milipore - Amicon) with membrane pore size of 3 Da about 3 times. Affinity chromatography The concentrated enzyme collected from the previous step was loaded on a HiTrap Heparin column (5. ml, 1.6 cm x 2.5 cm) equilibrated with 1 mm phosphate buffer (ph 7.). The unbound protein was washed out with low ionic strength buffer (1 mm phosphate buffer, ph 7.) until the protein was undetectable at absorbance 28 nm. Then, the enzyme was eluted with high strength buffer (1 mm phosphate buffer, 1-2 M NaCl, ph 7.) using a step elution method. The flow rate was adjusted to 16 ml/hour and the fraction volume of 4. ml was collected. Gel-filtration chromatography The fraction containing lipase with highest activity from affinity chromatography was loaded on Sephadex G-1 column (4. cm x 1.2 cm) equilibrated with 1 mm phosphate buffer, ph 7.. The enzyme was then eluted with the same buffer with a flow rate of 1 ml/min. Fractions of 4 ml were collected. Determination of molecular weight The molecular mass of the purified lipase was determined by SDS-PAGE as described by Laemmli, 197 using 12.5% acyrlamide gel. Unstained standard protein range: ß- galactosidase(116.kda), bovine serum albumin (66.2kDa), ovalbumin (45.kDa), lactate dehydrogenase (35.), Rease Bsp981 (25.kDa) and

5 Organic-solvent and surfactant tolerant thermostable lipase 393 ß-lactoglobulin (18.4kDa) were used as a molecular weight marker. The gels were then silver stained (Bollag et al., 1996). Effect of organic solvent on lipase activity The purified lipase was pre-incubated with 25% of the selected organic solvents at 65ºC for 1h and 24h prior to lipase assay. The lipase activity was then determined by using colorimetric assay and the activity without the addition of organic solvent (control) was set as 1%. The tested organic solvents were acetone, acetonitrile, benzene, chloroform, diethyl-ether, DMSO, ethanol, isooctane, methanol,propanol, n-hexane and n-heptane. Effect of surfactants on lipase activity For the effect of surfactants, purified lipase was pre-incubated with selected surfactants (1:1) at a concentration of 1 mm for 3 minutes at 65ºC prior to lipase assay. The surfactants tested were Tween 2, Tween 4, Tween 8, Span 4, Triton X-1 and SDS. The control, purified lipase without the addition of surfactants was set as 1% and the lipase activity was assayed using colorimetric method. Effect of protease on lipase activity For the effect of protease on lipase activity, purified lipase was pre-treated with 1% of protease in the ratio of 1:4 at 65 C and the activity was determined at interval of 1, 2, 4, 8, 16 and 24h. Lipase activity without the addition of protease was set as 1%. Results and Discussion Purification The extracellular lipase from G. thermodentrificans IBRL-nra was purified using a three step procedures; ultrafiltration, affinity chromatography and gel filtration chromatography. The highest lipase activity was detected at fraction 18 with 38 U/ml (Figure 1) in affinity chromatography. Affinity chromatography was used in this study to minimize the purification steps and the loss of enzyme (Sharma et al., 21). Heparin used is a highly sulphated glucosaminoglycan with a broad affinity for lipase. The partially purified lipase was then chromatographed on Sephadex G-1 gel filtration. A single peak of lipase was detected at fraction 17 (Figure 2) with 92.2 U/ml activity. SDS-PAGE analysis of lipase exhibited a single-band with molecular mass estimated to be 27 kda (Figure 3). Lipases from Bacillus are reported to have low molecular weight of ~2 kda (Sugihara et al., 1991). Lipases with lower molecular weight have advantage as smaller enzymes are more stable due to smaller changes (unfolding) in tertiary structure (Sharma et al., 22). 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 36.7U and a final recovery of 9%. Effect of organic solvent on lipase activity In the organic solvent tolerant study, lipase was pre-treated with watermiscible organic solvents (acetone, acetonitrile, DMSO, ethanol, methanol and propanol) and water-immiscible organic solvents (benzene, chloroform, diethylether, isooctane, n-hexane and n-heptane) for 1h and 24h. Lipase showed

6 394 Anuradha Balan, Darah Ibrahim and Rashidah Abdul Rahim highest activity in the presence of acetone, ethanol and acetonitrile after 1h and 24 of incubation periods (Figure 4). In the presence of acetone, lipase activity was enhanced 17% for the first hour and slightly decreased to 158% after 24 hours. While in the presence of ethanol and acetonitrile, lipase activity was enhanced 16% and 11% for the first hour and decreased to 11% and 1% respectively after 24 hours. However lipase activity was slightly inhibited by chloroform and diethylether after 24 hours of incubation with activity of 6% and 64% respectively. Kambourova et al., 23 also reported that thermostable lipase from B. stearothermophilus MC 7 was enhanced by sorbitol and methanol but completely inhibited by butanol, chloroform and diethylether. The results obtained in this study indicate that water-miscible organic solvents enhance thermostable lipase activity compared to water-immiscible organic solvents. The results obtained is in contrast with the statement that water-miscible solvents tend to strip off water from the enzyme, leading to the unfolding of the molecule and cause the enzyme to be less stable (Rahman et al., 25). However there is no significant correlation between stability of lipase in the presence of organic solvents (Ogino et al., 2) and its performance differs from lipase to lipase (Sugihara et al., 1991). Thermostable lipase of G. thermodenitrificans IBRL nra is highly stable and active in the presence of organic solvents, which holds a potential application in organic synthesis, fat and oil modification industries. Effect of surfactants on lipase activity All the tested surfactants significantly enhanced lipase activity. There was 22% increment in lipase activity when pre-incubated with Triton X-1, followed by 16% and 14% in Span 4 and Tween 8 respectively (Figure 5). SDS has no effect on lipase activity. The positive effect of surfactants could be due to the surfactant s role in decreasing the surface tension of the liquid and also preventing the aggregation of the enzyme and hence increase the enzyme activity. It is also reported that surfactants increase the accessibility of the substrates (Polizelli et al., 28). However thermostable lipase activity from G. stearothermophilus Strain-5 was not enhanced by surfactants and lipase activity could only retain 8% of its activity after 3 minutes incubation in Triton X-1 (Sifour et al., 21). Leow et al., 27 reported thermoalkaliphilic lipase of Geobacillus sp. T1 was significantly enhanced by Tween 8 (188%) followed by Tween 6 (126%) but was inhibited by Triton X-1 (72%) and SDS (5%). Thermostable alkaline lipase from Bacillus sp. DH4 was activated by Triton X- 11 and Triton X-114 with 164% and 148% activity respectively but was inhibited by SDS (Bora and Kalita, 28). The high tolerance of thermostable lipase from G. thermodenitrificans IBRL-nra in various surfactants suggests for its application as additive in detergent industry. Effect of protease on lipase activity Protease did not inhibit lipase activity whereby lipase could withstand 1% of its activity in the presence of protease up to 4 hours and still could retain 7% of its activity after 24 hours of incubation (Figure 6). Saxena et al., 23 reported that 8% of lipase activity of Aspergillus carneus could be retained after 24 hours of incubation. Proteases present in most of the food and dairy products

7 Organic-solvent and surfactant tolerant thermostable lipase 395 and detergents and it is reported that lipases enhance the removal of fatty food stains from fabrics and increase the washing capacity of detergents which contains protease (Polizelli et al., 28). The lipase in this study was found to be tolerant and stable in the presence of protease and therefore it can be employed in detergent, food and diary industries. Conclusion Thermostable lipase from Geobacillus thermodenitrificans IBRL-nra was successfully purified to homogeneity by using ultrafiltration, affinity chromatography and gel-filtration and characterized. The findings show that the enzyme is tolerant and stable in the presence of organic solvents, surfactants and protease. This indicates that the thermostable enzyme could be employed as potential catalyst for biotechnological processes in industries. Acknowledgement The authors would like to extend their appreciation to Universiti Sains Malaysia for awarding the Fellowship Scheme and Post Graduate Research Grant Scheme to support this research. References [1] A. Sugihara, T. Tani and Y. Tominaga, Purification and characterization of a novel thermostable lipase from Bacillus sp., Journal of Biochemistry, 19 (1991), [2] C. Ash, J. A. E. Farrow, S. Wallbanks and M. D. Collins, Phylogenetic heterogeneity of the genus Bacillus revealed by comparative analysis of smallsubunit-ribosomal RNA sequences, Letters in Applied Microbiology, 13 (1991), [3] D. Y. Kwon and J. S. Rhee, A simple and rapid colorimetric method for determination of free fatty acids for lipase assay, Journal of American Oil and Chemistry Society, 63 (1986), [4] H. Ogino, S. Nakagawa, K. Shinya, T. Muto, N. Fujimura, N., M. Yasudo and H. Ishikawa, Purification and characterization of organic solvent tolerant lipase from organic solvent tolerant Pseudomonas aeruginosa LST3, Journal of Bioscience and Bioengineering, 89 (2),

8 396 Anuradha Balan, Darah Ibrahim and Rashidah Abdul Rahim [5] I. Kauffmann and C. Schmidt-Dannert, Conversion of Bacillus thermocatenulatus lipase into an efficient phospholipase with increased activity towards long-chain fatty acyl substrates by directed evolution and rational design, Life Sciences and Medicine, 14 (21), [6] L. Bora and M. C. Kalita, Production of thermostable alkaline lipase on vegetable oils from a thermophilic Bacillus sp. DH4, characterization and its potential applications as detergent additive, Journal of Chemical Technology and Biotechnology, 83 (28), [7] L. Dong-Woo, K. You-Seok, K. Ki Jun, K. Byung-Chan, C. Hak-Jong, K. Doo-Sik, T. Maggy and P. Yu-Ryang, Isolation and characterization of thermophilic lipase from Bacillus thermoleovorans ID-1, FEMS Microbiology Letters, 179 (1999), [8] M. Kambourova, N. Kirilova, R. Mandeva and A. Derekova, Purification and properties of thermostable lipase from a thermophilic Bacillus stearothermophilus MC 7, Journal of Molecular Catalysis B: Enzymatic, 22 (23), [9] M. Sifour, H. M. Saeed, T. I. Zaghloul, M. M. Berekaa and Y. R. Abdel- Fattah, Purification and properties of a lipase from thermophilic Geobacillus stearothermophilus Strain-5, International Journal of Biology and Chemistry, 4(4) (21), [1] N. A. H. Sheikh, H. B. Zen, B. T. Ong, M. Yasin, N. S. Halifah and A. B. Fatimah, Screening and identification of extracellular lipase producing thermophilic bacteria from a Malaysian Hot Spring, World Journal of Microbiology and Biotechnology, 19(9) (23), [11] N. N. H. Raikhan, Penghasilan Enzim Lipase Termotoleran daripada Aktinomiset Streptosporangium roseum, Master of Science Thesis, Universiti Sains Malaysia (23). [12] O. H. Lowry, A. Rosebrough, L. Farr and R. J. Randall, Protein measurement with the Folin Phenol Reagent, Journal of Biology and Chemistry, 193 (1951), [13] P. P. Polizelli, M. J. Tiera and G. O. Bonilla-Rodriguez, Effect of surfactants and polyethylene glycol on the activity and stability of a Lipase from Oilseeds of Pachira aquatica, Journal of American Oil and Chemistry Society, 85 (28),

9 Organic-solvent and surfactant tolerant thermostable lipase 397 [14] R. K. Saxena, W. S. Davidson, A. Sheoran and B. Giri, Purification and characterization of an alkaline thermostable lipase from Aspergillus carneus, Process Biochemistry, 39 (23), [15] R. N. Z. R. A. Rahman, S. N. Baharum, M. Basri and A. B. Salleh, Highyield purification of an organic solvent-tolerant lipase from Pseudomonas sp. Strain S5, Analytical Biochemistry, 341(25), [16] R. Sharma, S. K. Soni, R. M. Vohra, L. K. Gupta and J. K. Gupta, Purification and characterization of a thermostable alkaline lipase from a new thermophilic Bacillus sp. RSJ-1, Process Biochemistry, 37 (22), [17] R. Sharma, Y. Chisti and U. C. Banarjee, Production, purification, characterization and applications of lipases, Biotechnology Advance, 19 (21), [17] T. C. Leow, R. N. Z. A. Rahman, M. Basri and A. B. Salleh, A thermoalkaliphilic lipase of Geobacillus sp. T1. Extremophiles, 11 (27), [18] T. N. Nazina, T. P. Tourova, A. B. Poltaraus, E. V. Novikova, A. A. Grigoryan, A. E. Ivanova, A. M. Lysenko, V. V. Petrunyaka, G. A. Osipov, S. S. Belyaev and M. V. Ivanov, Taxonomic study of aerobic thermophilic bacilli: descriptions of Geobacillus subterraneus gen. nov., sp. Nov. and Geobacillus uzunensis sp. Nov. from petroleum reservoirs and transfers of Bacillus stearothermophilus, Bacillus thermocatenulatus, Bacillus thermoleovorans, Bacillus kaustrophilus, Bacillus thermoglucosidasius and Bacillus thermodenitrificans to Geobacillus as the new combinations G. stearothermophilus, G. thermocatenulatus, G. thermoleovorans, G. kaustrophilus, G. thermoglucosidasius and G. thermodenitrificans, International Journal of Systemic and Evolutionary Microbiology, 51 (21), [19] U. K. Laemmli, Most commonly used discontinuous buffer system for SDS electrophoresis, Nature, 227 (197),

10 398 Anuradha Balan, Darah Ibrahim and Rashidah Abdul Rahim Table 1: Summary of purification of thermostable lipase from G. thermodentrificans IBRL nra Purification step Total activity Total Protein Specific activity Yield Fold (U) (mg) (U/mg) (%) Crude lipase Ultrafiltration Affinity Chromatography Gel-filtration Abs 28nm,8,7,6,5,4,3,2,1 Protein Lipase Fractions Lipase (U/ml) Figure 1: Purification profile of thermostable lipase from G. thermodenitrificans IBRL-nra on Hitrap Heparin Column affinity chromatography. Lipase was eluted with step elution using 1-2M NaCl.

11 Organic-solvent and surfactant tolerant thermostable lipase 399 Protein (Ab28nm) 3 2,5 2 1,5 1,5 Protein Lipase Lipase (U/ml) Fractions Figure 2: Purification profile of thermostable lipase from G. thermodenitrificans IBRL-nra on Sephadex G-1 gel filtration chromatography. MW(kDa) Lipase Figure 3: SDS-PAGE(12.5%) of thermostable lipase from G. thermodenitrificans IBRL-nra. Lane 1: unstained protein molecular weigth marker are ß-galactosidase(116.kDa), bovine serum albumin (66.2kDa), ovalbumin (45.kDa), lactate dehydrogenase (35.kDa), Rease Bsp981 (25.kDa) and ß- lactoglobulin (18.4kDa). Lane 2: crude lipase. Lane 3: concentrated lipase, Lane 4: partially purified lipase, Lane 5: purified lipase

12 4 Anuradha Balan, Darah Ibrahim and Rashidah Abdul Rahim Relative activity (%) h 24h Organic Solvent (25%) Figure 4: Stability of thermostable lipase in organic solvents. Lipase activity without the addition of organic solvent (control) was set as 1%. 25 Relative Activity (%) Control Tween 2 Tween 4 Tween 8 Span 4 Triton X-1 SDS Surfactants (1 mm) Figure 5: Effect of surfactants on thermostable lipase activity. The activity of purified lipase without the addition of surfactants was set as 1%.

13 Organic-solvent and surfactant tolerant thermostable lipase 41 Residual Activity (%) Hour (h) Figure 6: Effect of protease on thermostable lipase activity. Lipase activity without the addition of protease was set as 1%. Received: June 19, 213