Effect of Temperature, pH, Ions and Solvents on Glycerol Ester Hydrolase produced from Rhizopus sp. Momsia Teena1,*, Momsia Prerna2, Malik CP3 1Research Scholar, School of Life Sciences, Jaipur National University, Rajasthan, India 2Research Scholar, School of Life Sciences, Jaipur National University, Rajasthan, India 3Academic Advisor, School of Life Sciences, Jaipur National University, Rajasthan, India *Email id: teena.momsia9@gmail.com
Abstract The present study is concerned with the production of lipase by Rhizopus sp. in enzyme assay medium supplemented with 1% olive oil. Different parameters namely incubation time, temperature, pH, solvents, ions and inhibitors have been extensively studied. The maximum enzyme production was recorded on the 5th day at 45°C, whereas optimum pH was 6.5. Addition of Ca+2 and Ag+ ions enhanced the enzyme activity whereas Hg2+ and Co2+ showed the strongly inhibitory effect. On the study of solvents for the activity using 50 µl/ml concentration of assay mixture in shaking condition, acetaldehyde, chloroform and formic acid slightly enhanced the activity where as acetic acid and formic acid reduced the activity to as little as 10% of the original activity. Top Keywords Basal salt solution, Enzyme activity, Lipase, p-NPA, Rhizopus sp. Top |
Introduction Lipases are defined as glycerol ester hydrolases (EC 3.1.1.3), have the ability to hydrolyse ester linkages of glycerides present at oil–water interfaces, involving interfacial adsorption useful application in catalysis industries (Martinelle et al., 1995; synthesis of esters formed from glycerol and long chain fatty acids (Kohno et al.,1994) and also able to catalyze esterification, transesterification and enantioselective hydrolysis reactions (Nini et al., 2001; Shintre et al., 2002; Nagayama et al., 2002; Piao et al., 2003; Raku et al., 2003). These enzymes extensively occur in nature in animals, plants and microorganisms. Microorganisms include bacteria (Kulkarni and Gadre, 2002), fungi (Fodiloglu and Erkmen 1999; Shimada et al., 1992), Yeast (Corzo and Rewah, 1999) and actinomycetes (Sommer et al., 1997), among microorganisms fungi are preferred for lipase production because of their easy availability in large quantities, their specificity and their low cost of production (Yadav et al., 1998). The exploitation of fungal lipase in various industries and the increased demand in last decades because of diverse applications in medicines (digestive enzymes), food additives (flavour modifying enzymes), clinical reagents (glyceride hydrolysing enzymes), cleaners (detergent additives) and for synthesis of biodiesel and biopolymers are more common (Sugihara, 1984; Pandey, 1999). |
Due to high demand of lipases in various industries, fungi are the extensively exploited as lipase producers. Among fungi Aspergillus spp. and Rhizopus spp. are exploited as best sold commercial lipase preparations (Frost et al., 1987). Therefore, the present study is optimised for the production of lipases by an indigenous arid soil fungal isolate. |
Top Materials and Methods Isolation of Fungi A total of 53 fungal isolates were procured using soil dilution method (Booth, 1971), from oil rich soil samples, collected from different niches of Rajasthan (India) during an extensive survey conducted in the year 2009. All the isolated pure cultures were screened for lipase production, among these Rhizopus sp. showed high lipolytic zone. Therefore this strain was selected for further experiment. Growth Medium and Cultural Conditions 1mL of olive oil along with 100 ml basal salt solution containing (Peptone: 0.5 g; MgSO4.7H2O: 0.05 g; KCl: 0.05 g; KH2PO4: 0.2 g; NaNO3: 0.05 g) in 250 ml of Erlenmeyer flask were autoclaved at 15 psi for 15 min. These flasks were inoculated with 106 spores/ml suspension of culture and incubated at 28°C in a shaking incubator (80 rpm). Enzyme Extraction The fungal biomass was harvested from the culture grown as in the above basal salt solution. Crude enzyme extract was prepared from the culture supernatant which was centrifuged at 10,000 rpm for 15 min at 4°C and stored at 20°C. Enzyme Assay Lipase activity was measured spectrophotometrically using p-nitrophenyl acetate (pNPA) method reported by Licia et al. (2006). The substrate for this reaction was composed of two reagents, solution A and B. Solution A contained 40 mg of p-nitro phenyl actetate dissolved in 12 ml isopropanol, solution B contained 0.1 g of gum arabic and 0.4 ml of Triton X-100 dissolved in 90 ml distilled water. The substrate solution was prepared by adding 1 ml of solution A and 19 ml of solution B. The assay mixture contained 1ml of the substrate, 0.5 ml of phosphate buffer (10 mM, pH 7.0), 0.1 ml of enzyme and the final volume was made up to 3 ml with distilled water. The enzyme activity was stopped by adding 0.2 ml isopropanol and liberation of p-nitrophenol was detected in the UV spectrophotometer at 400 nm. One enzyme unit was defined as 1µmol of p-nitrophenol enzymatically released from the substrate per min. Top Effect of Various Parameters on Lipase Activity Effect of Incubation Periods Enzyme activity was observed from the first to the tenth day as per similar protocols described above. 0.1 ml of crude enzyme extract added in substrate solution, incubated at 37°C for 30 min and lipase activity was measured spectrophotometrically against control. Effect of pH on Lipase Activity and Stability The optimum pH was determined by measuring the hydrolysis of p-nitrophenyl acetate in the pH range 2.510. The buffers used were glycine-HCl buffer (pH 2.5), citrate buffer (pH 3.05.0), phosphate buffer (pH 5.57.5), tris-HCl buffer (8.08.5) and glycine-NaOH buffer (pH 9.0, 9.5, 10.0). 0.1 ml of enzyme was incubated with different buffers along with substrate solution at 37°C for 30 min and assayed for lipase activity. Effect of Temperature The optimum temperature of the enzyme was determined with p-nitrophenyl acetate by incubating the assay system in the temperature range of 10°C-70°C using citrate buffer pH 6.5 (0.5 M). To ascertain the stability, 0.5 mL of citrate buffer (0.5 M, pH 6.5) and 0.1 ml of enzyme along with the substrate was incubated at different temperatures (10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C) for 30 min and assayed for lipase activity. Effect of Inhibitors on Lipase Activity For determining the effect of different inhibitors on lipase activity they were incubated with (50 µg/ml and 100 µg/ml) two concentrations of EDTA, β-mercaptoethanol, SDS and Urea at 65°C for 30 min (pH 6.5) under standard assay conditions. Effect of Metal Ions on Lipase Activity For determining the effect of different metal ions on lipase activity they were incubated with two concentrations (50 µg/ml and 100 µg/ml) of Ca+2 (CaCl2), Ag+ (AgNO3), Fe+2 (FeCl3), Mg+2 (MgCl2), Mn+2 (MnCl2), Zn+2 (ZnSO4), K+ (KCl), Co+2 (CoCl2), Cu+2 (CuSO4), Hg+2 (HgCl2) and Na+(NaCl) for 30 min at 37°C under standard assay conditions. Effect of Solvents on Lipase Activity For determining the effect of different solvents on lipase activity they were incubated with (50 µl/ml and 100 µl/ml) two concentrations of (toluene, iso-amyl alcohol, acetic acid, benzene, formaldehyde, aniline, acetaldehyde, chloroform, formic acid, ethanol) for 30 min at 37°C under standard assay conditions. Top Results and Discussion The incubation period was optimised for lipase production by Rhizopus sp. in a basal salt medium, which was determined by batch culture. The extract was prepared from culture supernatant, which acts as crude extracellular enzyme extract. The production of enzyme increased during cell growth and reached a maximum value of 2868 U/ml on the 5th day of incubation thereafter decreasing the enzyme activity as it increases periodically (Figure 1). |
Effect of pH and Temperature The pH plays a crucial role for the detection of enzymatic activity, the effect of pH on enzymatic activity and stability provides valuable information about the enzyme. In this study the optimum pH was recorded at 6.5, and then rapidly dropped the activity (Figure 2). Enzymes are affected by changes in pH, and the optimum point where the enzyme showed more activity, at extremely high or low pH, generally resulted in complete loss of the activity in most of the enzymes. The optimum temperature for lipase production of Rhizopus sp. was tested at a temperature range from 10 to 70°C found that 45°C was the optimum incubate temperature (Figure 3). Effect of Various Metal Ions, Inhibitors and Solvents Certain enzymes besides the coenzyme require a metal ion for their full activity. Metal ions (when used 50 µg/ml) Cu+2 and Hg+2 completely inhibited lipase activity, while ions Fe+3 Mg+2, Mn+2, Zn+2, K+ reduced activity to as little as 20% of the original activity. In contrast to these, Ca+2 and Ag+, enhanced activity showed a stronger inhibitory effect (Hiol et al., 2000). When concentration was 50 µg/ml, all of the ions inhibited the enzyme activity except Ag+ ion (Figure 4). Similar observation was recorded by Hiol et al. (2000) while studying thermophilic lipases. A total of four inhibitors were studied with crude enzyme extract at the concentration of 50 µg/ml and 100 µg/ml, EDTA strongly inhibited the activity of enzyme and the other inhibitors namely urea, SDS, β-mercaptoethanol showed 50% relative The solvents, acetic acid and formic acid at 50 µl/ml concentration, showed a strongly inhibitory effect whereas toluene, iso-amylalcohol and ethanol partially reduced activity to as little as 20% of the original activity. In contrast to aniline, acetaldehyde and chloroform did not affect the activity at the same concentration (Figure 6). The enhancement of lipase activity in some solvents have many applications in oil-based fuel manufacture, biodegradation, pulp and paper industries, dairy and food industries (Kaieda et al., 1999). The present investigation on lipase produced by Rhizopus sp., showed that fungi are best producers of lipase in batch culture, which are active at pH 6.5, acidic lipase are used in medical applications and many esterification processes (Preetha Sasi et al., 2006). The optimum temperature was at 45°C and thereby proves to be advantageous in industrial applications such as industries like food additives, cosmetics and therapeutics used thermo tolerant lipases. The high region and spacio specificities of these enzymes have application in the kinetic resolution of optical isomers for synthesis of optically pure substances in pharmaceutical and chemical industries (Hasan et al., 2006; Bornscheuer 2002). Moreover, the physiochemical properties of lipase produced by Rhizopus sp. make it a suitable candidate for food additives and detergent formulation. Top Figures | Figure 1:: Effect of various incubation times on lipase enzyme production
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| | Figure 2:: Effect of various pH on lipase enzyme production
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| | Figure 3:: Effect of various temperatures on lipase enzyme production
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| | Figure 4:: Effect of various ions on lipase activity
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| | Figure 5:: Effect of various solvents on lipase production, whereas (A) control, (B) Toluene, (C) Iso-amyl alcohol, (D) Acetic acid, (E) Benzene, (F) Formaldehyde, (G) Aniline, (H) Acetaldehyde, (I) Chloroform, (J) Formic acid, and (K) Ethanol
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| | Figure 6:: Effect of various inhibitors on lipase enzyme production (A) Control, (B) SDS, (C) β-mercaptoethanol, (D) EDTA, (E) Urea
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