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Int. J.Biotech Biosci, ISSN 2231-0304 Vol 1 (4): 410-415 (2011)

Original paper Madhikar et al

ISOLATION, PARTIAL PURIFICATION AND CHARACTERIZATION OF LIPASE FROM SUNFLOWER GERMINATING OIL SEEDS Madhikar SD1, Gadge PP1, Yewle JN1, Jadhav UU4, Chougale AD5, Zambare VP2* & Padul MV1,3 1 Department of Biochemistry, New Arts, commerce and Science College, Ahmednagar, Maharashtra, 414001, India 2 Center for Bioprocessing Research and Development, South Dakota School of Mines and Technology, Rapid City, SD, 57701 USA, Email : [email protected] 3 Department of Biochemistry, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad, Maharashtra, 431004, India 4 Department of Power Mechanical Engineering, National Tsing Hua University, No. 101, Sec. 2, Kuang Fu Rd, 30013, Hsinchu, Taiwan, Republic of China 5 Mass Spectrometry and Proteomics Group, Organic Chemistry Division, National Chemical Laboratory, Pune, India

Abstract The lipase was extracted and partially purified from germinating sunflower seeds using chilled acetone and ammonium sulfate precipitation. Partially purified and dialyzed enzyme profile was observed on native-polyacrylamide gel electrophoresis (PAGE). The lipase was optimally active at pH 7 and temperature of 40oC. In the presence of Ca2+ and Mg2+ enhance the activity at low concentration, while the Hg2+ and ethylenediaminotetracetic acid (EDTA) showed inhibitory effect. The enzyme was found to be metalloenzyme. Enzyme kinetics with olive oil emulsion substrate showed km and vmax of 6.71 mg and 0.011μm/mL/min, respectively. The mettaloenzyme enzyme was able to attack specifically on oil in seeds to generate free fatty acids as the major end product. This understanding may help in devising efficient method to overcome the problem of sunflower seed oil in stability. Keywords: Lipase, sunflower, germinating seeds, metal ions, characterization

INTRODUCTION In recent years the growing demand of lipolytic enzymes has been increased due to its potential use in the various manufacturing processes of industrial goods such as detergent industry, food industry, cosmetics, flavour enhancers and in pharmaceutical industry (Gandhi 1997; Corzo & Revah 1999; Cancino et al 2008). Lipase may be used to produce fatty acids

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(Linder et al 2002), polyesters (Kumar & Gross 2000), biomodified fats (Neklyudov et al 2002), aroma and flavor compounds (Athawale et al 2003), amides, thiol esters (Gandhi 1997), and lubricant and solvent esters (Hills 2003). Lipases are widespread in nature and have been found in animals, higher plants and microorganisms (Aravindan et al 2007). In plants lipase activity has been International Journal of Biotechnology and Biosciences

ISOLATION, PARTIAL PURIFICATION AND CHARACTERIZATION OF LIPASE FROM SUNFLOWER GERMINATING OIL SEEDS

identified in various tissues but relatively high concentration is found in seeds. Seeds are generally rich in triacylglycerols, which serve as compact source of energy for the newly emerging plant. During germination of the seed, the reserved triacylglycerols are disappeared, since the fatty acids can not be oxidized to provide energy until they are released from the triacylglycerol. Lipases are probably rate controlling during germination and the activity of the lipase is high during germination (Brockerhoff & Jensen 1974; Ejedegba et al 2007). Lipases are the enzymes, which can essentially contribute to deterioration of lipid fraction of oil seeds. They are considered to begin unfavourable changes of lipid fraction due to releasing fatty acids from glycerides thus increasing the acidity of the product and providing the substrate for oxygenation reaction catalysed by lipoxygenase (Mierzejewska et al 2003). In the present paper we report the isolation and characterization of a lipase from germinating sunflower seeds.

Lipase assay- The titrimetric method of Maliks et al (2000) was used for determination of lipase activity. Olive oil emulsion was prepared in 180 mL distilled water containing 20 mL olive oil, 0.4g of sodium benzoate and 1g gum-arabic. Assay mixture contained 5 mL olive oil emulsion, 5 mL 0.1M Tris buffer (pH 8), and 1 mL crude enzyme and incubated at 35oC for 10 min. The reaction was stopped by 10 mL of acetone and methanol mixture (1:1). Each sample was titrated against 0.025N NaOH using 1% phenolphthalein as indicator. The volume of NaOH used in the titration was noted and used for enzyme activity calculations. One unit of lipase is defined as the amount of enzyme required to liberate 1ìmol of free fatty acid from olive oil per min under the standard assay conditions.

MATERIALS AND METHODS

Lipase characterization

Seed collection and germination- Germinating seeds of sunflower (Helianthus annus), safflower (Carthamus tinetorus), mustard (Brassica Juncea), sesame (Sesamum indicum), and flax (Linum usitatissimum) were purchased from local market. All seeds were soaked in water for six hours and were allowed to germinate for 24 h at room temperature (28 ± 2oC) on moist germination papers.

Polyacrylamide gel electrophoresis- Enzyme purification or purity was checked on non denaturing native PAGE using 10% gel concentration with slight modification on method described by Holt & Hartman (1994). Gel was stained using the Coomassie Brilliant Blue R250 staining solution. Total enzyme protein used for PAGE was of 10 μg for crude as well as partially purified lipase.

Extraction and partial purification of lipase- After 24 h of seed germination, the seed coats were removed manually and 20g seed cotyledons were homogenized in chilled acetone at 4°C. The suspension was centrifuge at 3000 rpm and residue obtained was dissolved in 100mL distilled water followed by centrifugation at 7500 rpm. The supernatant was used as source of crude enzyme and was precipitated by ammonium sulfate (80% saturation) according to Michael et al (2001) The precipitate was obtained by centrifugation at 10,000 rpm for 20 min. Precipitate was dissolved in 20 mL Tris-Cl buffer (10mM, pH 8.5) and dialyzed overnight against the same buffer. The dialyzed enzyme was used as partially purified enzyme and used for enzyme characterization.

Effect of pH and temperature- Optimum pH for lipase activity was determined covering the range (3-9) using 0.1M buffers of different pH. The buffers were: pH 3-6 (acetate); pH 7 (phosphate); pH 8-9 (Tris-Cl). For optimum temperature, the enzyme assay was performed as discussed above except that incubation was done at temperatures from 20-70oC.

Vol. 1 (4) ™ October - December 2011

Protein estimation- Protein concentration of soluble enzyme preparation was quantified by method of Lowry et al (1951) using bovine serum albumin (BSA) as a standard.

Enzyme kinetics- Lipase was assayed in reaction buffer (pH 8) at 24oC with different concentrations (10-120 mg/ml) of olive oil emulsion as a substrate. The values of vmax (maximum velocity) and km (Michaelis constant) were calculated from Lineweaver-Burk (LB) plot. Statistical analysis- All experiments were conducted

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in triplicates and results were represented with standard deviation calculated by Microsoft Excel program.

mum caused a rapid decrease in the enzyme activity (Fig. 2). Using sunflower lipase, (Sagiroglu & Arabaci (2005) observed an optimum pH of 7.5.

RESULTS AND DISCUSSION

A

B

Lipase screening- Among the all five germinating seeds, only sunflower germinating seeds (11.12 ± 0.32 x10-3 U/mL) showed maximum lipase activity followed by mustard (9.37 ± 0.14 U/ml) and sesame (9.12 ± 0.32 U/ml) seeds (Table 1). Very less lipase activities were found in safflower and flax seeds. A review on seed lipases shoed in detailed applications, mechanism and properties by Barros et al (2010). Lipase activity from sunflower with various substrata was also studied under different conditions by Aramiuc, (2011). Table 1. Lipase activity profile of various Indian germinating oil seeds Plant source

Botanical names

Lipase activity x 10(U/mL/min) 11.12 ± 0.32 5.37 ± 0.42 9.37 ± 0.14 9.12 ± 0.32 3.87 ± 0.21 3

Sunflower seeds Safflower seeds Mustard seeds Sesame seeds Flax seeds

Helianthus annus Carthamus tinetorus Brassica Juncea Sesamum indicum Linum usitatissimum

Fig 1. Electrophoresis pattern of crude and partially purified lipase isolated from sunflower germinating seeds. A, crude enzyme; B, partially purified dialyzed enzyme

± indicate the values of activities and standard deviation of triplicate analysis.

Enzyme characterization- Lipase was partially purified by acetone and ammonium sulfate precipitation followed by dialysis. Partial purification showed cut off of unnecessary proteins and was evidenced on native PAGE (Fig. 1). Lipases were produced from a germinating seeds by various researchers (Ben et al 2000; Abigor et al 2002; Eze & Chilaka 2010) and purified with the help of acetone precipitation and different chromatographic steps as reported earlier (Opute 1975; Bahri 2000; Sammour 2005; Su et al, 2010). Sammour (2005) showed SDS electrophoresis pattern of germinating linseeds. As per literature lipases belongs to sunflower seedlings were of high molecular weight (78kDa) and were showed immunoreactivity (Mierzejewska et al 2003). Effect of pH on lipase activity- The enzyme activity increased with an initial increase in pH and optimum activity was noted at pH 7 suggesting neutral nature of the enzyme. Further increase in pH beyond opti-

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Fig 2. Effect of pH on lipase activity isolated from sunflower germinating seeds. Lipase assay was performed at 35oC and at various pH values. Error bars indicate standard deviation between replicates (n=3).

Effect of temperature on lipase activity- The temperature activity profile of sunflower germinating seed lipase is shown in Fig. 3. The lipase had an optimum temperature with a sharp peak at 40°C. Above and below this temperature the activity was dropdown. An

International Journal of Biotechnology and Biosciences

ISOLATION, PARTIAL PURIFICATION AND CHARACTERIZATION OF LIPASE FROM SUNFLOWER GERMINATING OIL SEEDS

optimum temperature of 50oC has previously been reported for purified lipases from same sunflower seeds (Sagoroglu & Arabaci 2005).

Arabaci 2005). Thus, the present study lipase showed more substrate specificity. Effect of metal ions and inhibitors- Metal ions as Ca+2, Mg2+ at lower concentration (0.001mM) showed enhanced effect on lipase activity where as at higher concentration the lipase activities were found to be inhibited. EDTA and Hg+2 inhibited the enzyme activity (Table 2). In literature, calcium and ATP were required for lypolytic activity in sunflower seedlings (Bahri, 2000). Our results were in agreement with the Enujiugha et al (2004) which showed Ca+2 as activator while Hg+2 and EDTA as inhibitors of African oil beans lipase. Table 2. Effect of metal ions on lipase activities from germinating sunflower seeds Metal ion

Fig 3. Effect of temperature on activity of the lipase isolated from sunflower germinating seeds. Lipase assay was performed at pH 8 and at various temperatures. Error bars indicate standard deviation between replicates (n=3).

Effect of substrate concentration- The km and vmax for the lipase were determined using olive oil emulsion as a substrate. The Km value for the free enzyme, estimated from Lineweaver-Burk plot (Fig 4) was 6.71 mg with olive oil emulsion as substrate. The vmax value obtained from the L.B. plot was 0.011 μm/mL/min. A km of 1.33 mM and vmax of 555U/mg was observed for purified lipase from sunflower oil seed (Sagoroglu &

Concentration (mM)

Control CaCl2 MgCl2 EDTA HgCl2

Residual lipase activity (%)

0.001 0.005 0.001

100.00 111.32 86.14 109.89

0.005

81.76

0.001

90.52

0.005

64.67

0.001 0.005

83.11 67.87

EDTA- Ethylenediaminotetraacetic acid

CONCLUSION

Fig 4. Lineweaver-Burk plot for lipase from sunflower germinating seeds. Lipase assay was conducted at various substrate concentrations at pH 7 and temperature 40oC. The data were plotted according to Lineweaver-Burk. Each value is average of three independent experiments.

Vol. 1 (4) ™ October - December 2011

Lipase was isolated and partially purified from germinating sunflower (Helianthus annus) seeds. A neutral lipase (optimum pH 7.0) with a moderate thermoactivity was isolated from the cotyledons of germinating sunflower seeds. The germinating sunflower seed lipase could prove useful in industrial biocatalytic hydrolysis. It could also be inferred from the present preliminary characterization that the germinating sunflower seed lipase could prove useful in processes that require lower cooling costs and minimal corrosion problems. Ca2+ and Mg2+ enhanced the enzyme activity at lower concentrations, while EDTA and Hg+2 caused various degrees of inhibition. The results show

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that the germinating sunflower seed lipase could be exploited in industrial processes. This understanding may broaden the use of lipases in industry and medicine and may help in devising efficient methods to overcome the problem of sunflower seed oil instability.

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ISOLATION, PARTIAL PURIFICATION AND CHARACTERIZATION OF LIPASE FROM SUNFLOWER GERMINATING OIL SEEDS

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