Purification and characterization of extracellular lipase from pseudomonas aeruginosa KF 853103.
MATERIALS AND METHODS
The present study was carried out at Biodiesel Laboratory, School of Energy Studies for Agriculture, Punjab Agricultural University, Ludhiana (Punjab).
Procurement of microbial cultures
Pseudomonas aeruginosa KF 853103 was procured from the Pulses Microbiology Laboratory, Department of Plant Breeding and Genetics, Punjab Agricultural University, Ludhiana. The bacterial strain was repeatedly sub-cultured and maintained on nutrient agar slants.
Determination of growth and lipase activity of bacterial strain
Pseudomonas aeruginosa KF 853103 was studied for its growth and extracellular lipase activity on minimal salt media using olive oil as carbon source. Lipase production in liquid culture was carried out by the method of Gokbulut and Arslanoglu (2013) (6) with slight modifications. The minimal medium used for lipase production consisted of 200 ml of 5X minimal salt solution ([Na.sub.2]HP[O.sub.4]O, 64 g/l; K[H.sub.2]P[O.sub.4], 15 g/l; NaCl, 2.5 g/ l and [NH.sub.4]Cl, 5 g/l), 800 ml of water and 0.02 per cent gum arabic. The medium was adjusted to pH 7.0, autoclaved and cooled. Then 2 ml 1M MgSO4 (sterile filtrated) and 0.1ml 1M Ca[Cl.sub.2] (sterile filtrated) was added to medium.
The inoculum was prepared by transferring a loopful of stock culture to minimal media broth (20 ml) in 150 ml flasks containing 1 per cent (w/v) olive oil as carbon source. The cultivation was performed at [+ or -] 28[degrees]C with constant shaking at 150 rpm in an orbital shaking incubator until absorbance at wavelength of 600 nm ([A.sub.600]) reached to 0.6. Lipase production was carried out by transferring 10 ml of prepared inoculum into 250 ml triplicate flasks containing 100 ml of minimal media broth supplemented with olive oil (1 % w/v) as carbon source. The inoculated flasks were incubated at [+ or -] 28[degrees]C with constant shaking at 150 rpm in an orbital shaking incubator. The aliquots of culture broth were withdrawn at an interval of 24, 36, 48, 60, 72, 96 and 120 h to determine the lipase activity. For this the bacterial cells were harvested from broth by centrifugation at 10,000 rpm for 15 min at 4[degrees]C. The cell free supernatant, thus, collected was used as crude extract to determine the activity of extracellular lipase. The relative growth of bacterial strain was measured in terms of absorbance ([A.sub.600 nm]) using visible spectrophotometer at different hours after incubation (24, 36, 48, 60, 72, 96 and 120 h).
Determination of lipase activity
Lipase activity was determined spectrophotometrically using p-nitrophenyl palmitate (p-NPP) as substrate by the method of Winkler and Stuckmann (1979) (7) with slight modifications. The substrate solution consisted of solution A and solution B. Solution A was prepared by adding 40mg of p-NPP in 12 ml of isopropanol. Solution B was prepared by adding 0.1 g of gum arabic and 0.4 ml Triton X-100 in 90 ml of distilled water. Finally, 1 ml of solution A was added to 19 ml of solution B drop wise with continuous stirring to obtain a substrate emulsion that remained stable for 2 h. The reaction mixture for lipase assay contained 1 ml of substrate emulsion, 0.5 ml of buffer (50 mM Tris HCl, pH8.0), 0.1 ml of crude enzyme and volume was made up to 3 ml with distilled water. The reaction mixture was incubated at 40[degrees]C for 45 min in a temperature-controlled water bath. The reaction was stopped by adding 0.5 ml of isopropanol and the absorbance was measured at 410 nm against blank. The standard curve was prepared by using p-nitrophenol (0.01 to 0.2 [micro]moles). The amount of enzyme activity was expressed as pkat of p-nitrophenol (p-NP) produced per ml under standard assay conditions. The protein content of enzyme extracts was estimated by method of Lowry et al (1951) (8). The specific activity was expressed as pkat of p-nitrophenol (p-NP) produced per mg protein.
Production of lipase enzyme
The bacterial strain was grown in 1.0 litre minimal medium broth containing 1 per cent olive oil (v/v) at [+ or -] 28[degrees]C with constant shaking at 150 rpm in an orbital shaking incubator. The culture broth was collected after 72 h of incubation and centrifuged at 10,000 rpm at 4[degrees]C for 20 min. The cell-free culture supernatant was used as crude lipase and partially purified by ammonium sulfate precipitation followed by DEAE-cellulose column chromatography.
Partial purification and characterization of lipase enzyme
Ammonium sulphate precipitation
The ammonium sulfate precipitation was carried out by adding solid ammonium sulfate to crude enzyme mixture until 0-30 per cent saturation (w/v) with continuous stirring at 4[degrees]C. The mixture was then allowed to stand overnight at 4[degrees]C with gentle stirring and centrifuged at 10,000 rpm for 15 min. The precipitates were dissolved in minimum volume of 0.1M sodium phosphate buffer, pH 7.2 and was dialyzed against the same buffer (20 times diluted) overnight at 4[degrees]C. The supernatant thus obtained, was subsequently fractionated to 30-60 and 60-80 per cent ammonium sulphate saturation. The enzyme activity of these fractions (0-30%, 30-60% and 60-80%) was determined as described earlier.
DEAE-cellulose coloumn chromatography
The ammonium sulphate fraction exhibiting maximum lipase enzyme activity was applied to DEAE-cellulose column (50 x 1.8 cm) pre-equilibrated with 0.1M sodium phosphate buffer (pH 7.2). The enzyme was eluted with the same buffer using stepwise gradient (0.1 M each) of increasing molarity of NaCl (0.1-1.0 M) at a flow rate of 0.5 ml [min.sup.-1]. From this, 5 ml fractions were collected and assayed for lipase activity and protein content.
Effect of pH on enzyme activity
The effect of pH on lipase activity was determined in the pH range of 4.0-10.0. The various pH buffer systems, viz. sodium acetate buffer (50 mM, pH 4.0-5.0), phosphate buffer (50 mM, pH 6.0-7.0), Tris-HCl buffer (50 mM, pH 8.0-9.0) and glycine- NaOH buffer (pH 10.0) were used. The optimum pH was determined by plotting relative activity (%) versus pH.
Effect of temperature on activity and stability of the enzyme
The effect of temperature on lipase activity was determined by measuring the enzyme activity at different temperatures (30-80[degrees]C) at optimum pH. The optimum temperature was determined by plotting relative activity (%) versus temperature. Similarly, the thermostability of partially purified enzyme was determined by measuring the lipase activity after pre-incubating the enzyme at different temperatures (30-80[degrees]C) for 2 h. The lipase activity without any prior incubation at its optimum pH and temperature was taken as 100 per cent.
Effect of organic solvents on the enzyme activity
The effect of various organic solvents, viz. methanol, ethanol, propanol, hexane and acetone on the enzyme activity was investigated. The enzyme was incubated in the presence of organic solvents (30% v/v) at optimum pH and temperature for 30 min. The relative activity of the enzyme was expressed by taking activity without the addition of organic solvent as 100 per cent. Determination of [K.sub.m] and [V.sub.max]
The effect of substrate concentration on the reaction velocity of lipase was studied by using p-nitrophenyl palmitate (p-NPP) as substrate. The enzyme was incubated with different concentrations of p-NPP ranging from 0.12 to 0.37 mM. Lineweaver-Burk plots were plotted to determine [K.sub.m] and [V.sub.max] values. Similar studies were performed at different temperatures (30[degrees], 35[degrees] and 40[degrees]C) in order to determine the effect of temperature on [K.sub.m] and [V.sub.max] of enzyme. The free energy change [DELTA]G (kJ [mol.sup.-1]), enthalpy change [DELTA]H (kJ [mol.sup.-1]), entropy change [DELTA]S (J [K.sup.-1] [mol.sup.-1]) and energy of activation [E.sub.a] (kJ [mol.sup.-1]) were calculated for the enzyme catalyzed reaction at its optimum pH and temperature.
RESULTS AND DISCUSSION
Growth and lipase activity of bacterial strain in olive oil liquid culture
The growth of bacterial strain measured in terms of absorbance of bacterial cells at 600 nm ([A.sub.600]) in olive oil liquid culture ranged from 0.21 to 0.96 at different hours after incubation and the growth was observed to be maximum at 72 h and thereafter, a decline in the growth was observed (Fig. 1). Lipase production was correlated with growth and the lipase activity of bacterial strain ranged from 88.46 to 710.05 pkat [ml.sup.-1]. Maximum extracellular lipase activity (726.35 pkat [ml.sup.-1]) was observed at 72 h of incubation, i.e the stationary phase of growth and thereafter, the lipase activity decreased. The decrease in lipase activity at later stages could be due to changes in pH as well as proteolytic degradation of enzyme by proteases released into the culture medium at the end of exponential phase of growth (7). These results are in accordance with the work done by Gokbulut and Arslanoglu (2013) (6) who reported maximum lipase production during stationary phase of growth (80 to 96 h after incubation). Saeed et al (2005) (9) studied that the extracellular lipase enzyme production of P. aeruginosa increased gradually at the end of log phase and reached its maximum level (74.66 U/ml) after 48 h of inoculation.
Partial purification and characterization of lipase enzyme
The crude lipase of P. aeruginosa was purified by ammonium sulphate precipitation, followed by dialysis and DEAE-cellulose coloumn chromatography. Among the three different levels of ammonium sulphate saturation, the maximum activity of enzyme was detected at 30-60 per cent ammonium sulphate saturation (Table 1). The enzyme was purified to 2.8-fold with a specific activity of 312.0 pkat [mg.sup.-1] protein relative to crude extract. The enzyme solution obtained after DEAE-cellulose column chromatography was purified to 15-fold with a specific activity of 1671.9 pkat [mg.sup.-1] protein. The elution profile of enzyme (Fig. 2) purified by DEAE-cellulose column chromatography revealed a single protein peak that overlapped with the lipase activity.
The present studies are in concordance with Zouaoui and Bouziane (2011) (10) who purified the extracellular lipase produced by P. aeruginosa using ammonium sulphate precipitation and DEAE cellulose column chromatography. The enzyme in the broth was purified 11-fold with an overall yield of 65.51 per cent after single step purification by ion-exchange column chromatography. However, Sharon et al (1998) (11) reported that the extracellular lipase produced by P. aeruginosa KKA-5 was purified by 518-fold with a specific activity of 2238.16 U [mg.sup.-1] protein using ammonium sulphate precipitation followed by hydroxylapatite column chromatography. Gaur et al (2008) (12) purified a lipase from solvent tolerant strain of P aeruginosa PseA by gel exclusion chromatography. The enzyme was purified to 8.6-fold with a specific activity of 143.3 IU [mg.sup.-1] protein.
Effect of different pH on the activity of purified lipase
Structure of proteins is greatly influenced by pH and is therefore, an important parameter affecting enzyme activity. Lipase activity from P. aeruginosa was examined in the pH range of 4.0-10.0 at 40[degrees]C and the optimal activity was observed at pH 8.0 (Fig. 3). The lipase retained 90.9 and 85.2 per cent of its maximum activity at pH 9.0 and 10.0, respectively. In the acidic range, significant reduction in the enzyme activity was observed. The enzyme retained only 50.0 and 50.8 per cent of maximum activity at pH 4.0 and 5.0, respectively.
The present findings are in accordance with Chakraborty and Paulraj (2009)5 who reported that the purified lipase produced by P. fluorescens MTCC 2421 was active in the pH range of 7.0 to 9.0 and the optimum activity was exhibited at pH 8.0. The enzyme retained only 27.44 and 18.89 per cent of maximum activity at pH 6.0 and 7.0, respectively. Gaur et al (2008) (12) reported that P. aeruginosa PseA lipase showed pH optima at 8.0. The lipase showed good pH stability retaining 100 per cent of the activity in pH range of 6.5-8.5.
Effect of temperature on the activity and thermostability of lipase
The effect of temperature on the lipase activity was studied within the range of 30-80[degrees]C (Fig. 4). Lipase showed maximum activity at 40[degrees]C, whereas at higher temperatures, i.e. 70 and 80[degrees]C, the enzyme activity decreased up to 58.3 and 46.5 per cent of its maximum activity.
Similarly, Chakraborty and Paulraj (2009)5 reported that the purified extracellular lipase from P. fluorescens MTCC 2421 was active in the temperature range of 35-50[degrees]C with maximum activity at 40[degrees]C. The activity was found to be reduced to 50.7 and 93.0 per cent of its initial value at 30[degrees]C and 20[degrees]C, respectively. The lipase activity declined rapidly above 55[degrees]C with only 13 per cent of the activity remained at 60[degrees]C. However, the purified lipase from P. aeruginosa SRT 9 exhibited maximum activity at 55[degrees]C and the enzyme was found to be stable up to 65[degrees]C. As per Borkar et al (2009) (13), the activity of enzyme declined above 65[degrees]C, thus, indicating the thermal denaturation of enzyme.
The thermostability of lipase was examined by incubating the enzyme at different temperatures (30-80[degrees]C) for 2 h at optimum pH (Fig. 5). The enzyme was relatively stable within the temperature range of 30-40[degrees]C and retained 97.2 and 100.0 per cent of its activity at 30 and 40[degrees]C, respectively. The enzyme retained about 61.9 and 51.9 per cent of its maximum activity after exposure at 70 and 80[degrees]C, respectively for 2 h. Similarly, Gaur et al (2008) (12) reported the temperature optima of P. aeruginosa PseA lipase to be 40[degrees]C and the enzyme was found be stable at this temperature for at least 4 h.
Effect of different organic solvents on the activity of lipase
The effect of various organic solvents at 30 per cent (v/v) concentration on the stability of lipase is shown in the Fig. 6. The enzyme retained about 96.5, 80.3 and 93.4 of its maximum activity in the presence of methanol, ethanol and acetone, respectively. However, the relative activity of enzyme was comparatively less in the presence of hexane and propanol (64.8 and 52.8%). The stability of this enzyme in methanol indicated the potential use of methanol for transesterification reaction and biodiesel production.
The low relative activity of enzyme in the presence of propanol and hexane indicated that the long chain alcohols and non-polar organic solvents may have inhibitory effect on the enzyme activity14. Similarly, Sharon et al (1998)11 reported that P. aeruginosa KKA-5 was found to be stable in the presence of methanol, ethanol and acetone. The study conducted by Ugur et al (2014)14 on stability of lipase Lip SB 25-4 isolated from Streptomyces bambergiensis OS 25-4 in hydrophilic and hydrophobic solvents revealed that the enzyme was stable in methanol and ethanol retaining more than two-third of its activity for 60 h.
Determination of kinetic constants
The [K.sub.m] and [V.sub.max] values for lipase at its optimum pH 8.0 and temperature 40[degrees]C from Lineaweaver-Burk plot were found to be 0.136 mM and 71.4 pkat [ml.sup.-1], respectively (Fig. 7). The lower [K.sub.m] values indicated that the partially purified lipase had higher affinity for p-NPP. Borkar et al (2009) (13) determined the kinetic constants of purified lipase from P. aeruginosa SRT9. The [K.sub.m] and [V.sub.max] values for lipase catalyzed reaction using p-NPP as substrate were reported to be 0.037 mM and 188.6 mmol [l.sup.-1] [min.sup.-1], respectively. The [K.sub.m] values decreased with rise in temperature, which was a reflection of higher affinity of enzyme for substrate (Table 2). The values of log [V.sub.max] and [pK.sub.m] (-log [K.sub.m]) at different temperatures were plotted against 1/T (Fig. 8). The energy of activation ([E.sub.a]), enthalpy change ([DELTA]H) and entropy change ([DELTA]S) were calculated from these graphs using following formulae
[E.sup.a] = -2.303 xRxslope (Fig. 8b)
[DELTA]H = -2.303xRxslope (Fig. 8a)
[DELTA]G = -RTx2.303xlog [K.sub.eq]
[DELTA]G = [DELTA]H-T[DELTA]S
The free energy change ([DELTA]G), enthalpy change ([DELTA]H), entropy change ([DELTA]S) and energy of activation ([E.sub.a]) for the lipase catalyzed reaction were found to be -23.2 kJ [mol.sup.-1], 15.9 kJ [mol.sup.-1], 124.7 J [K.sup.-1] [mol.sup.-1] and 19.1 kJ [mol.sup.-1], respectively.
In the present work extracellular lipase from P aeruginosa KF 853103 was produced, purified and characterized. The purified enzyme showed pH optima of 8.0 and temperature 40[degrees]C and was relatively stable within the temperature range of 30-40[degrees]C. The enzyme retained about 96.5, 80.3 and 93.4 of its maximum activity in the presence of methanol, ethanol and acetone, respectively. Due to its stability in a wide range of organic solvents, P aeruginosa KF 853103 lipase could be of significant biotechnological potential, particularly in enzyme-catalyzed transesterification reactions for biodiesel production from plant oils.
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Baljot Kaur , Monica Sachdeva Taggar  *, Poonam Sharma  and Manjeet Kaur Sangha 
 Department of Biochemistry,  School of Energy Studies for Agriculture, 'Department of Plant Breeding and Genetics, Punjab Agricultural University, Ludhiana--141004, India
(Received: 03 February 2016; accepted: 09 March 2016)
* To whom all correspondence should be addressed. E-mail: email@example.com
Caption: Fig. 1. Time course dependent lipase activity and bacterial growth curve of P. aeruginosa
Caption: Fig. 2. Elution profile of extracellular lipase from P. aeruginosa using DEAE cellulose column chromatography
Caption: Fig. 3. Effect of pH on the activity of purified lipase from P. aeruginosa
Caption: Fig. 4. Effect of temperature on the activity of purified lipase from P. aeruginosa
Caption: Fig. 5. Effect of temperature on the stability of purified lipase from P. aeruginosa
Caption: Fig. 6. Effect of different organic solvents on the activity of purified lipase from P. aeruginosa
Caption: Fig. 7. Lineaweaver-Burk plot of P. aeruginosa lipase at its optimum pH and temperature
Caption: Fig. 8. Effect of temperature on Km and [V.sub.max] of purified lipase from P. aeruginosa
Table 1. Purification of extracellular lipase from P. aeruginosa Purification steps Total Total activity (pkat protein (mg) p-nitrophenol) Crude 114420.0 1028.4 [(N[H.sub.4]).sub.2] S[O.sub.4] saturation 0-30% 4000.0 17.1 30-60% 6426.6 20.6 60-80% 2273.3 10.4 DEAE-Cellulose 535.0 0.32 Column Chromatography Purification steps Specific Purification activity (pkat fold p-nitrophenol [mg.sup.-1] protein) Crude 111.3 1.0 [(N[H.sub.4]).sub.2] S[O.sub.4] saturation 0-30% 233.9 2.1 30-60% 312.0 2.8 60-80% 218.6 2.0 DEAE-Cellulose 1671.9 15.0 Column Chromatography Table 2. Effect of temperature on [K.sub.m] and [V.sub.max] of purified lipase from P. aeruginosa Temperature [K.sub.m] p[K.sub.m] [V.sub.max] log ([degrees]C) (mM) (-log (pkat [V.sub.max] [K.sub.m]) p-nitrophenol [ml.sup.-1]) 30 0.166 0.78 55.5 1.74 35 0.153 0.82 66.6 1.82 40 0.136 0.87 71.4 1.85
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|Author:||Kaur, Baljot; Taggar, Monica Sachdeva; Sharma, Poonam; Sangha, Manjeet Kaur|
|Publication:||Journal of Pure and Applied Microbiology|
|Date:||Jun 1, 2016|
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