Aqueous conditions

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Lipases belong to the class hydrolase (EC and in aqueous condition hydrolyze acyl glycerides into free fatty acids and glycerol (Jaeger & Eggert 2002). Under reduced aqueous conditions, lipases catalyze variety of organic transformations which are chemo-, regio- enantio selective beside esterification (Rubin and Denis 1997; Kazlauskas and Bornscheuer 1998). Due to such multifaceted properties, it is one of the important industrial enzymes and has find usage in a wide array of industrial applications, such as food technology, detergent, chemical industry and biomedical sciences (Pandey et al. 1999).

Lipases are serine hydrolases as catalytic triad is composed of Ser-Asp/Glu-His, and usually contain consensus sequence (Gly-x- Ser-x-Gly) that are present around the active site serine while three-dimensional (3-D) structures reveal the characteristic α/β-hydrolase fold (Nardini and Dijkstra 1999).

Lipases are widely distributed in the earth's flora and fauna but found more abundantly in microbial flora comprising bacteria, fungi and yeast (Pandey et al. 1999) but for commercial bulk production, bacterial source is used as enzyme is extra cellular and easy to handle. (Gupta et al 2004). Lipases producing bacterial sources are enormous but only few wild or recombinant strains have been used for commercial production of lipases where as lipases of Pseudomonas are widely used for a variety of biotechnological applications (Beisson et al. 2000).

It has bee reported that lipase from Pseudomonas aeruginosa has shown thermo, pH stability, besides retaining activity in presence of organic solvent Karadzic et al (2006).

In present study an extracellular lipase from Pseudomonas aeruginosa BN-1 has been purified, characterized and compared with other bacterial lipases.

Materials & Methods


Pseudomonas aeruginosa BN-1 was isolated during sample analysis of well water and municipal supply and produced extracellular lipase (Noman et al 2004).

Production of Lipase

Pseudomonas aeruginosa BN-1 was grown in mustard oil medium (1% tryptone, 0.05% CaCl2, 0.05% MgSO4, 0.001% K2HPO4, 0.5% mustard oil) at 37 °C for 48 hours. The fermented broth was centrifuged (Sigma 3K30 centrifuge using 12150 rotor) at 30000 x g for 15 minutes at 4 °C to remove cells. A clear cell free supernatant was obtained containing crude lipase.

Purification of Lipase

Concentration of crude enzyme through ultrafiltration

Clear cell free supernatant (CFS) was concentrated by ultrafiltration (Amicon 8400 stirred ultrafiltration cell under nitrogen) using cut off membrane of 30 kD. Retentate was lyhpolized and stored at -80°C till further analysis. Retentate was reconstituted in 55 mM Tris-HCl buffer of pH 8.0 before use.

Size exclusion chromatography by Sephadex G-100

2.0 ml of retentate was applied 2.5 x 30 cm sephadex G-100 column pre equilibrated 55 mM Tris-HCl buffer of pH 8.0. Elution was carried out with same buffer with a flow rate of 60 ml/hour and 2 ml fractions were collected. Fractions having absorbance at 280 nm were assayed for lipase activity. Fractions showing enzymatic activity were pooled and lyhpolized.

Ion exchange chromatography by DEAE A-50

Active fractions were re-suspended in 20 mM Tris-HCl buffer of pH 8.0 and applied to pre 1.5 x 20 cm column of DEAE pre equilibrated with same buffer.

Elution of proteins was done using a linear gradient of NaCl from 0.0-1.0 M in 20 mM Tris HCl buffer (pH 8.0) while fractions (1.0 ml) were collected and monitored at 280 nm. Protein fractions were assayed for lipase activity and fractions with enzyme activity were pooled, subjected to centrifugal filtration (centricon using 10 kD cut off) to remove salt and lyhpolized.

Protein Estimation

Protein was estimated by the method described by Lowry et al (1951) using Bovine Serum Albumin (BSA) as standard.

Electrophoresis and molecular mass determination

Molecular weight of lipase was determined by SDS PAGE carried out according to method described by Laemmli (1970).

Native Electrophoresis and activity stain

Native PAGE gels for separation of non denatured proteins were carried out according to Hames & Rickwood (1981). Native gels were subjected to lipase or esterase activity according to Takahashi et al., (1998).

Lipase assays

Colorimetric assay

Colorimetric assay was based on hydrolysis of 4-nitrophenyl palmitate as described by Ruiz et al., (2004) In the reaction mixture 55 mM Tris-HCl (pH 8.0) buffer was used instead of Phosphate buffer. One lipase unit U is defined as mmol of 4 -nitrophenol released per minute under assay condition.

Titrimetric assay

Titrimetric assay was carried out as described by (Watnabe et al 1977). One Unit of lipase activity is defined as mmol of fatty acid released per minute under assay conditions.

Characterization of enzyme

Optimum temperature and thermal stability

Optimum temperature assays were performed by the titremetric assay at temperatures ranging from 4ºC - 70ºC.

Thermostability of the enzyme was determined by preparing enzyme solutions in Tris-HCl buffer 55 mM (pH 8.0) and incubated for 1 h at temperatures ranging from 4 to 70º. After incubation, enzyme activity was assayed by colorimetric method at optimum temperature.

Optimum pH and pH stability

Optimum pH assays were performed by titremetric assay replacing the Tris-HCl buffer 55 mM (pH 8) in the reaction mixture by the following buffers (at a final concentration of 55 mM): citrate phosphate buffer (pH 4.0-7.0), phosphate buffer (pH 6.0-8.0), Tris-HCl buffer (pH 7.5-9.0) and glycine- NaOH buffer (pH 9.0-10.0). Enzyme activity was assayed at optimum temperature.

For pH stability assay, enzyme solutions were prepared in the corresponding buffers (at 55 mM): citrate phosphate buffer (pH 4-7), phosphate buffer (pH 6-8.0), Tris-HCl buffer (pH 7.5-9) and glycine- NaOH buffer (pH 9-10), and incubated for 1 h.

Substrate Range

The substrate range of the enzymes was determined by using p-NP derivatives (200 mM) with carbon chain length of 2-18: p-NP acetate (C2), p-NP butyrate (C4), p-NP valerate (C5), p-NP caproate (C6), p-NP caprylate (C8), p-NP caprate (C10), p-NP laurate (C12), p-NP palmitate (C16) and p-NP stearate (C18).

Activity against natural triglycerides

Mustard oil, castor oil, linseed oil, corn oil, soybean oil sunflower oil, peanut oil palm oil coconut oil and palm oil (1% in emulsion) were used as substrate to determine the lipolytic behavior of enzyme while olive oil (1% in emulsion) was taken as control and activity was determined by titrimetric assay as described earlier.

Effect of Metal ions

Purified enzyme was mixed with soluble solutions of metal salts (5 mM) and incubated for 30 minutes and assayed for residual activity.

Effect of Surfactants, Detergents & Protein modifying agents

Solutions of various surfactants, detergents and protein modifying agents were mixed with enzyme, preincubated for 30 minutes and then assayed for residual activity.

Solvent Stability

Stability of purified lipase in organic solvent was carried out according to Ogino et al (2000). Enzyme activity at 0 hour is taken as control (100%).

N-Terminal Sequencing

For N-terminal sequencing, Band exhibiting lipolytic activity determined through zymography was cut from native PAGE gel and send to Alta Biosciences University of Birmingham UK for N terminal sequencing.

Results & Discussion

Lipase from Pseudomonas aeruginosa BN-1 was purified to 42.99 fold (Table 1). The strategy adopted for purification of lipase isolated was comparable to that of Guar et al., (2008) who obtained 8.6 fold purification when enzyme from Pseudomonas aeruginosa PseA was concentrated using ultrafiltration followed by size exclusion chromatography. Additional step of purification by ion exchange chromatography using DEAE A-50 increased the purification fold. Lee and Rhee (1993) have reported 5.3 fold purification of lipase from Pseudomonas putida 3SK when used sephadex G-100 and DEAE -A50 chromatography simultaneously.

The molecular weight of lipase on SDS-PAGE was found to be 60.0 kD approximately. A single non smearing band was observed on native PAGE confirmed by activity staining (Figure 1). It has been reported that molecular weights of lipases from Pseudomonas aeruginosa varies considerable. The molecular weight of lipase in this study was found to be 60 kDa, identical to lipases from Pseudomonas aeruginosa S5 60 kDa (Rehman et al., 2005) and Pseudomonas aeruginosa Pse A (Gaur et al.,2008). Zymography in SDS PAGE was not visible may be due to inhibitory effect of SDS on enzyme under experimental condition. A single band was observed in native electrophoresis, suggesting that enzyme did not form aggregation which is contrast to the findings of Lessuise et al.,(1993).

Optimum activity of purified lipase was observed at 37°C while it retained more than 70% of its activity at 50°C for at least 1 hour (Figure 2). The lipase has better thermostability when compared with Pseudomonas aeruginosa MB 5001 lipase which retained only 10% of activity at 60°C (Chartrain et al., 1993).

Purified lipase from Pseudomonas aeruginosa BN-1 showed maximum activity at pH 8.0. (Figure 3) The stability of enzyme varied with variation in buffer composition. In citrate buffer (pH ranges from 4.0-7.0) residual activity was around 30%, increased to 70% in Phosphate buffer (pH ranges from 7.0-8.0). Maximum stability was observed with Tris-HCL buffer (pH ranges from 7.5-9.0) where 98% activity was retained at pH 8.0 and 70% at pH 9.0. In glycine-NaOH buffer, 50% of activity was retained at pH 9.0, while activity was completely lost at pH 10.0(Figure 4).

Lipase from Pseudomonas aeruginosa BN-1 has shown alkliophilic character as it retained more than 60% of its activity for 1 hour 37°C at pH 9.0, however lower to the findings for a lipase from pseudomonas sp. PK-12 CS reported by Jinwal et al., (2003).

Broad lipolytic activity was observed against natural triacylglycerols of plants sources (Table 2). Results revealed that as the chain length increased, the activity was also increased a character of true lipase. Mustard oil, palm oil, olive oil were the best substrates while lower activity was observed with tributyrin, and other short acyl chain glycerides. It had been proposed that lipolytic activity has dependence on appropriate surface tension and viscosity (Gargouriet al., 1989) which ultimately affect micelle formation. Kordel et al (1991) observed that low micelle formation by short chain glycerides due to low hydrophobicity and viscosity lower the hydrolytic activity. As the acyl chain length increases, there is increase in hydrophobicity and viscosity and ultimately increase in hydrolysis of glycerides (Kordel et al., 1991).

High lipolytic activity of Pseudomonas aeruginosa BN-1 lipase against mustard oil, imitate lipase from Pseduomonassp. (Rathi et al., 2000). Lipase from Pseudomonas pseudoalcaligenes F-111 had relative activity of 62% with sunflower as substrate (Lin et al., 1996), while Lipase from Pseudomonas aeruginosa LST-03 has highest activity against coconut oil (Ogino et al., 2000). Purified lipase from Pseudomonas aeruginosa has highest activity against palm oil (Rahman et al., 2005).

Lipase hydrolyzed para nitrophenyl esters of various acyl chains with preference for higher fatty acyl chains i.e. C6 and above. Lipase from Burkholderia sp. HY-10 has been reported to have broad substrate range from C4 to C18 4-nitrophenyl esters, with maximum hydrolytic activity against ρ-nitrophenyl caproate (C6) (Park et al.,2007).

Ca2+ and Ba2+ increased the lipolytic activity while Na+, K+ and Mg2+ have no effect.

Al 3+, Fe3+, Hg2+, Cu2+ Ni2+ and Mn2+ on the other hand markedly decreased the enzyme activity (Figure 6). Ca2+ ions have been known to stimulate the lipase activity in varying concentrations and a finding of this study is in accordance with it. It has been reported that lipase activity from B. licheniformis strain H1 increased up to 120% (Khyami-Horani et al., 1996) while a lipase from a Pseudomonas sp increased by 250% (Dong et al., 1999) in the presences of Ca2+ ions (10 mM).

Similarly, Ba2+ ions also enhanced lipase activity isolated from Burkholderia sp has been reported (Rathi et al., 2001).

Metal ions like Hg2+, Zn2+ and Cu2+ are reported to have inhibitory effect on Pseudomonas lipases by several workers (Iizumi et al.,1990; Kumura et al.,1993)

Lipase from Bacillus sp. lost 70% of its activity in presences of Hg2+ (Sughiara et al.,1991). Inhibition of Pseudomonas sp lipase by Al3+, Mn2+, Ni2+ and Fe3+ ions has also been reported (Dong et al., 1999).

1,4-Dithio-DL-threitol(DTT) and 2- Mercaptoethanol has no effect on the enzyme, while PMSF markedly inhibit the enzyme. Pseudomonas aeruginosa BN-1 lipase was not found to be EDTA sensitive. Na-deoxycholate and Tween 80 increased the lipolytic activity but SDS strongly inhibited the enzyme (Figure 7).

Pseudomonas aeruginosa BN-1 lipase was not found to be EDTA sensitive, a character contrary to the lipase earlier reported (Rashid et al.,2001). No effect of 2-Mercaptoethanol can be explained as lipases contain very few sulfhydryl groups, important for lipase activity (Gupta et al., 2004). Similar findings were observed in case of lipases from C. viscosum (Sugiura et al.,1974), and S. aureus 226 (Muraoka et al., 1982).

No effect of DTT on Pseudomonas aeruginosa BN-1 lipase, even at a 10 mM concentration may be indicative of the fact that, lipase may not be a metalloprotein. No Burkholderia cepacia ATCC 25416 lipase remained unaffected in presences of DTT alone on but marked inhibition was observed in conjunction with SDS (Wang et al., 2009).

Lipase belongs to the class of serine hydrolases and thus inhibited in presences of PMSF. Extracellular lipase from B. subtilis 168 is strongly inhibited with 0.1 mM PMSF when preincubated only 10 min at 20°C (Lesuisse et al., 1993).

The presences of surfactants enhanced the micelle formation of hydrophobic substrate, facilitating more access to the enzyme thus enhancing the lipolytic activity. Complete activity lost was observed when SDS was added to lipase from Lipase from Burkholderia sp. HY-10 (Park et al., 2007). Extracellular lipase (Lip 2) from Yarrowia lipolytica also lost complete activity in the presence of SDS, but activity was enhanced in the presence of 0.1% Tween 80 while SDS completely inhibited it (Yu et al., 2007).

Lipase from pseudomonas aeruginosa BN 1 has shown tolerance towards DMSO, n-octane, n-decane, n-heptane, n-hexane, retaining 80% or greater activity, in cyclohexane, ethanol, methanol and butanol retaining more than 60% of activity, while 50% and 40% of activity was retained in presences of isopropanol and acetone respectively after 24 hours. Very low tolerance was observed with chloroform, benzene, toluene and xylene (Figure 8).

Organisms that produce lipases in medium containing oils, fats, and surfactants exhibit tolerance towards organic solvent (Ogino et al., 2001) and varied from lipases to lipases (Sughiara et al., 1991). Highest relative stability was observed with DMSO, n-decane and n­-octane, similar to lipase from Pseudomonas aeruginosa PseA (Gaur et al., 2008) and Pseudomonas aeruginosa LST-03(Ogino et al., 2000). Lowering of lipolytic activity with butanol and other long chain alcohols were due to absorption on the substrate blocking enzyme interfacial action with the substrate (Mattson et al.,1970). Klibanov (1986) explained the effect of organic solvents with variable hydrophobicity on enzyme. Polar solvents remove the essential water molecules from active sites of enzyme required for conformational flexibility, that causes denaturation.

N-terminal sequence for first 10 residues revealed the presences of hydrophobic amino acids, positively and negatively charged amino acid (Table 3).

There was no homology observed when the sequence obtained matched with sequences of lipases reported earlier (Table 3). Generally less than 10% homology has been observed when N-terminal amino acid sequences have been aligned (Jaeger et al., 1994). However, results were similar to the N-terminal amino acids of a chaperonin. There are reports that lipase and chaperone are coded together and is essential for proper lipase activity in some organisms (Pauwels et al., 2006; Sullivan et al., 1999). Pseudomonas sp lipases need a chaperone whose gene is located down stream of the lipase gene for efficient secretion and folding of active lipase (Hobson et al., 1993). This suggests that lipase from Pseudomonas aeruginosa BN-1 may contain a chaperone at its N-terminal for proper folding, secretion and lipolytic activity.


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Table 1: Purification of Lipase From Pseudomonas aeruginosa BN-1



Total protein


Lipase activity (U)

Specific Activity


Purification fold

Cell Free Supernatant













G 100






DEAE Sephadex A50






Table 2: Relative Enzymatic Activity Towards Various Substrate


Relative Activity (%)

Olive oil

(Taken as Reference)


Mustard oil


Castro oil


Linseed oil


Corn oil


Soybean oil


Sunflower oil


Peanut oil


Palm oil


Coconut oil


Groundnut oil


Table 3: N-terminal amino acid sequence of purified lipase

N-Terminal Sequence


P. aeruginosaBN-1











This Study

P. flourescens HU 380











Kojima and Shimizu, 2003

P. flourescens B 52












Tan and Miller, 1992

P. flourescens SIK WI












Chung et al , 1991

P. glumae











Batenberg et al ,1991

Burkholderia sp HY-10











Park et al, 2007

P. cepacia











Jorgensen et al, 1991