Production, Partial Purification and Characterization of an Extracellular Psychrotrophic Lipase from Pseudomonas Sp. ADT3

Psychrotrophic Pseudomonas ADT3 (NCBI GenBank Acc.no.JX914667) is capable of growth on lipid as the sole carbon source. In this paper, we report the purification and characterization of an extracellular lipase from psychrotroph, isolated from soil sample of NyAlesund, Svalbard, Arctic region. The Pseudomonas ADT3 isolate produces lipase enzyme in the extracellular minimal media with only 1% olive oil. The lipase was purified from the concentrated culture supernatant. The crude enzyme was partially purified by saturated ammonium sulphate precipitation followed by extensive dialysis. Enzyme activity was found to be induced 6-folds in presence of 1.2 mM lead ion but strongly inhibited by heavy metals Hg2+ as well as EDTA and β-mercaptoethanol. The purified lipase has activity at two pH optima of pH i.e. pH 3.5 and 8.5. Optimum temperature for lipase activity was recorded at 22cC. The purified active fraction of lipase exhibits specific activity of 527.8 U/mg. The Vmax and Km was 144.93 U/mg/min and 0.260 mM respectively determined using Lineweaver-Burk plot. Zymogram analysis revealed prominent lipase band at 13.9 kDa range in the 80% saturated ammonium sulphate purified enzyme fraction. Citation: Dey A, Chattopadhyay A, Mukhopadhyay SK, Saha P, Chatterjee S, et al. (2014) Production, Partial Purification and Characterization of an Extracellular Psychrotrophic Lipase from Pseudomonas Sp. ADT3. J Bioremed Biodeg 5: 242. doi:10.4172/2155-6199.1000242 Volume 5 • Issue 6 • 1000243 J Bioremed Biodeg ISSN: 2155-6199 JBRBD, an open access journal Page 2 of 2 useful with potential applications especially in low temperature bioremediation and food processing. Materials and Methods Chemicals All chemicals used were of analytical grade, obtained from Himedia (India) and Sigma-Aldrich Company. Sample collection The soil samples were collected from NyAlesund (78°55’N, 11°54’E) Svalbard, Arctic region during the Indian Arctic Expedition 2009, organized by National Centre for Antarctic and Ocean Research, Goa under the Ministry of Earth Science, Govt of India. Isolation of lipolytic bacteria Pseudomonas sp. ADT3 was isolated in our laboratory from the soil samples collected from Arctic. Soil samples have been serially diluted and plated on M9 minimal media containing 0.3%(w/v) KH2PO4, 0.6%(w/v) Na2HPO4, 0.05% (w/v) NaCl, 0.1% (w/v) NH4Cl, 2 mM MgSO4, 0.1 mM CaCl2, with 1.0% olive oil as the sole carbon source and 2% (w/v) agar [18] at pH 8.0 by spread plate method. Plates were incubated for 48 h at 22°C. Pure cultures of the isolate were maintained on M9 minimal medium agar slants containing olive oil and were routinely maintained by sub culturing. Screening of isolates for lipase activity Pseudomonas strain ADT3 was screened by qualitative plate assay. The strains were grown on olive oil agar base plate at 22°C for 48 h. Zone of clearance was observed maximal with ADT3 strain which was selected for further analysis. The zone of clearance was observed due to hydrolysis of olive oil to fatty acids [26]. Lipase production was monitored by irradiating plates containing fluorescent dye Rhodamine B with UV light at 350 nm. After 48 h of incubation bacterial colonies began to show an orange fluorescence, with continuing incubation time orange fluorescent halos were formed around the colonies. The orange fluorescence under UV illumination indicates the presence of lipolytic activity of ADT3 [27]. Preparation of crude enzyme extract ADT3 cells were grown in Erlenmeyer flasks (250 mL) with 50 mL of M9 minimal medium containing 1% olive oil as substrate and flasks were incubated in shaker incubator with 120 rpm agitation at 22oC for 48 h. Cells were harvested by centrifugation at 10,000 rpm for 10 minutes at 4°C. The supernatant obtained was filtered using 0.45 μm cellulose acetate filter membrane (Sigma-Aldrich). The cell free filtrate was used as the extracellular crude enzyme. The cell pellet obtained was suspended in 1.0 mL solution I (10 mM EDTA pH 8, 50 mM glucose, and 25 mM Tris-HCl, pH 8) with 100 μL 10 mg/mL lysozyme. The suspension was vortexed and kept at 4°C for 15 minutes followed by temperature shock at 37°C for 1 hour and further incubated at 4°C for 30 minutes. The suspension was centrifuged at 5000 rpm for 5 minutes at 4°C and the supernatant was stored as crude enzyme extract at −20°C for further studies as intracellular enzyme extract [28]. Partial purification of extracellular lipase Ammonium Sulphate Fractionation and Dialysis against Buffer (Desalting): The online program ammonium sulphate calculator from EnCor Biotechnology, Inc, Gainesville, Florida [29], was used for calculating the amount of solid ammonium sulphate to be added for getting desired fractions. Precipitation was done at 10%, 10-20% and 20-30% saturation for 2-3 h. and at 40-50%, 50-60%, 60-80%, and 8090% saturation of ammonium sulphate for overnight incubation, with magnetic stirrer at 4°C. After each precipitation step, the fraction was centrifuged at 12,000 rpm for 15 minutes at 4°C. All the precipitates obtained were resuspended in a minimal amount of buffer (10 mM Tris-HCl, pH 8.0). Then it is dialyzed against large volume of the same buffer by successive change in buffer after 2 hours at 4°C. The process was continued until the last trace of ammonium sulphate was removed. All the concentrated fractions were subjected to protein and enzyme activity assay to choose the fraction containing maximum activity. Activity staining was also done for confirmation with alphanapthyl acetate as substrate for lipase. Acetone precipitation: The crude enzyme was subjected to different fractions of acetone precipitation with continuous stirring with magnetic stirrer overnight at 4°C. The resulting precipitate was centrifuged (15,000xg) for 20 min at 4°C. The acetone precipitates were dissolved in minimal amount of 10 mM Tris HCl buffer of pH 8.0, and then dialyzed extensively against the same buffer overnight at 4oC. The enzyme activities in the dialysate were again assayed. Lipase assay Extracellular lipase activity in bacterial culture supernatants after centrifugation (12,000 g for 20 min) was determined by titrimetric assay using olive oil as substrate with pHstat autotitrator system. Lipase activity was examined by titrating free fatty acids liberated from triglycerides with alkali [30,31]. The reaction mixture contained 1 ml of 0.1 M TrisHCl buffer pH 8.0, 50 mM KCL, 200 μl Tween 20, 1 ml olive oil or other oil as substrate, 1 ml of culture supernatant at 22°C for 3 h. The reaction mixture was mixed well on a reciprocal shaker at 60 rpm. After incubation, the mixture was shaken vigorously with 3 mL of ethanol to stop the enzyme reaction and break down the emulsion. The amount of fatty acids liberated during the reaction was estimated by titrating with 10 mM NaOH on an autotitrator system using phenolphthalein indicator. Blank contained the same components except enzyme solution. One unit of lipase activity was defined as the amount of enzyme required to liberate 1 μM equivalent of free fatty acid per minute. Specific activity was defined as units of lipase activity per milligram protein. Specific activity was measure by using following formula. The results obtained were means of triplicates for each experiment. ml titrant/min x molarity of titrant x 1000 Specific activity Amount of protein (mg) Optimization of different parameters for enzymatic activity The different parameters like substrate, pH, temperature, incubation time and cofactors were optimized for enzymatic activity. During optimization of each parameter, all other factors were kept unaltered. Enzyme was extracted as described above. Substrate preference in reaction mixture for performing lipase activity was measured by the amount of fatty acids liberated by titrating with 10 mM NaOH using phenolphthalein indicator. Substrates used were mustard oil, coconut oil, sunflower oil, olive oil and tributyrin at 1% (v/v) concentration. For the determination of pH stability the pH of the reaction mixture was varied with different buffer systems, citratephosphate buffer for pH 2.0 6.0, phosphate buffer for pH 6.57.5 and Tris-HCl buffer for pH 8.0-11.0 respectively for lipase activity assay. For each pH, a blank Citation: Dey A, Chattopadhyay A, Mukhopadhyay SK, Saha P, Chatterjee S, et al. (2014) Production, Partial Purification and Characterization of an Extracellular Psychrotrophic Lipase from Pseudomonas Sp. ADT3. J Bioremed Biodeg 5: 242. doi:10.4172/2155-6199.1000242 Volume 5 • Issue 6 • 1000243 J Bioremed Biodeg ISSN: 2155-6199 JBRBD, an open access journal Page 3 of 2 without the enzyme was carried out and the values were subtracted from the experimental ones. The optimal temperature for activity was measured from 10°C80°C at pH 8.5 for 3 h using the standard assay method. For the determination of thermal stability, the purified lipase was incubated for 30, 60, 90,120, 150, 180, 210, 240 min at 20°C, 30°C, 50°C and 60°C and 70°C respectively, then the residual lipase activity was measured as described in the standard assay method. For determining the effect of incubation time on enzymatic activity the reaction mixture was incubated for various time interval i.e.30 min, 60 min, 90 min, 120 min, 150 min, and 180 min respectively at 22°C. The effects of various compounds and inhibitors on lipase activity were examined. The different cofactors like calcium ion, magnesium ions, zinc, iron, lead, silver, EDTA etc. at 1.0 mM concentration were added. Determination of kinetic parameters The kinetic parameters Km and Vmax were determined from Lineweaver Burk double reciprocal plots of substrate concentration vs. initial reaction rates [32]. The Michaelis-Menten constant (Km) of purified extracellular lipase was determined by varying the concentration of olive oil. The initial velocity measured by quantitatively measuring the amount of the product at various time intervals [33]. Extracellular protein profile of the isolate Extraction of extracellular proteins: The cells were grown in two separate flasks, one in M9 minimal media containing 1% (v/v) olive oil and in other set M9 with 1% (w/v) peptone for 48 h with shaking at 22°C. The cells were harvested by centrifugation at 12,000 rpm for 10 min. The cell free supernatant was collected. The extracellular proteins were precipitated by 30% acetone overnight at 4°C. Finally the proteins were dissolved in 0.1 M Tris-HCl buffer pH 6.8. Protein determination: The protein content of the extract was determined following the method of Bradford [34]. Determination of molecular mass: Molecular mass of the Pseudomonas ADT3 strain producing lipase was determined by SDSPAGE [35] using a 12% resolving and 5% stacking gel. 50 μg of protein was loaded in each well. For SDS-PAGE, standard molecular weight markers were used. The gel was stained with Coomassie Blue R 250 and destained with 30% methanolic destaining solution. The gel was observed in the gel documentation system (Vilbur Lourmat, France) and molecular mass was analysed with Quantum Capt software. Immunisation of Rabbit to Raise Specific Antibody A single oil inducible band of 13.9 kDa was cut from the gel and homogenised by crushing with a sterile glass rod in Eppendrof tube containing 500 μl autoclaved water. A rabbit was injected subcutaneously at 5 to 6 different sites with this protein homogenate mixed 1:1 with Freund’s complete adjuvant twice (once per month) followed by two more injections of the same protein homogenate mixed 1:1 with Freund’s incomplete adjuvant. Dot Blot: To check serum antibody titre, dot blot assay was done. 2 μl of the different dilutions (1:10, 1:100 and 1:1000) of antigen i.e., total bacterial protein obtained from oil grown cells was spotted onto PVDF membrane. 1 mg/ml BSA in different volumes (1 μl, 2 μl, 3 μl) was also spotted on the membrane as control. Western Blot: The SDS PAGE gel, after electrophoresis (unstained) was electroblotted onto PVDF membrane (Sigma Aldrich USA) at 45 volts for 3 hours. The PVDF membrane was blocked with 3% milk powder for 1 hour at room temperature and washed thrice with buffer A(10 mM tris HCl pH8, 1 mM EDTA pH8, 0.05% tween 20 and 0.9% NaCl), followed by incubation in 1:100 dilution of antiserum in the same buffer, at 4°C overnight. Then the membrane was washed with buffer A thrice, five minutes each and incubated in 1:15,000 dilutions of Goat anti-rabbit IgG coupled to alkaline phosphatase (Sigma Aldrich USA) in buffer A for 2 hours at room temperature. The membrane was again washed in buffer A, 5 minutes each, for three times and equilibriated for 30 minutes in alkaline phosphatase buffer (100 mM tris HCl pH 9.5, 100 mM NaCl and 5 mM MgCl2). The membrane was stained with BCIP/ NBT (5-Bromo, 4-Chloro, 3-Indolyl phosphate/Nitrobluetetrazolium) in alkaline phosphatase buffer and kept in the dark. Preadsorption of the Serum Antibody: 10 μl of the peptone grown cellular proteins were spotted on small strips of nitrocellulose membrane and air dried. These strips were immersed in 1:100 dilution of the antiserum in buffer A and incubated by gentle shaking for 1 hour. The strips were removed and the antiserum thus obtained contained antibodies specific only for oil induced proteins. This process was repeated until all the antibodies against the common antigens were removed. Native PAGE for Zymogram Analysis For zymogram studies, ADT3 cells were grown in M9 minimal medium with 1.0% olive oil for 48 hours. Extracellular proteins were extracted by ammonium sulfate precipitation and partially purified, and hence the protein concentration was measured by the Bradford assay. About 100 μg of proteins was resolved by 10% native gel [36] under nondenaturing condition. Electrophoresis was carried out at 4°C at 150 V of constant voltage. One part of gel was stained with coomasie blue and the unstained counterpart of the gel was assayed in situ for lipase activity by using a-naphthyl acetate (a-NA). Lipolytic active zone was identified by staining the gel with 0.03% Fast Blue RR salt, 0.05% α-naphthyl acetate, and 1% acetone in 25 mM Tris–HCl buffer at pH 7.4 [37]. Statistical analysis For statistical analysis, standard deviations for each of the experimental results were calculated using Microsoft Excel software. Results were expressed as the means of three independent determinations. Significance has been presented in the form of probability (p 0.05) values Results and Discussion Isolation and identification of psychrotrophic lipase producing bacteria Among numerous colonies detected, 10 colonies were randomly selected from the culture plate with lipid as sole carbon source. Most of the colonies were colourless with a few forming pigments. Among 10 isolates, one strain ADT3 showed maximal zone of clearance in olive oil agar base plate (Figure 1a) which is further confirmed by Rhodamine B plate assay where orange fluorescence under UV illumination indicates the presence of lipolytic activity of ADT3 (Figure 1b). Optimization of conditions for enzymatic activity Effect of pH on lipase activity: Two pH optima for enzymatic activity were noted at pH 3.5 (acidic) and pH 8.5 (alkaline), respectively. Citation: Dey A, Chattopadhyay A, Mukhopadhyay SK, Saha P, Chatterjee S, et al. (2014) Production, Partial Purification and Characterization of an Extracellular Psychrotrophic Lipase from Pseudomonas Sp. ADT3. J Bioremed Biodeg 5: 242. doi:10.4172/2155-6199.1000242 Volume 5 • Issue 6 • 1000243 J Bioremed Biodeg ISSN: 2155-6199 JBRBD, an open access journal Page 4 of 2 Figure 2 clearly shows that first, there is a sharp increase in enzyme activity at pH 3.5 which drops around neutral pH followed by a peak at pH 8.5 that gradually falls into a plateau. The most probable explanation may be the presence of two isoforms of the same lipase enzyme. The pH-stability profile made them useful for their application in industrial processes to be carried out at a pH range close to alkalinity. Effect of temperatures on lipase activity: The specific activities of the enzyme were determined at various temperatures from 10-80°C. Optimum lipase activity was observed at 22°C as shown in Figure 3. In recent studies, some cold-active lipase showing temperature optima of 20–25°C [38].As shown in Figure 4, the thermal stability of lipase was assessed for several time periods at various temperatures (20–70°C). The stability of the lipase was very high at 20°C, enzyme activity decreased slightly when temperature was at 30°C. It retained half of its activity after 150 mins and 90 min incubation at 50°C and 60°C respectively. Lipase activity was completely inactivated at 70°C at 120 mins. This enzyme is more stable than other lipase from Pseudomonas sp. The rapid inactivation at high temperature of lipase was a typical property of cold-adapted enzymes. Lipase enzyme from Pseudomonas ADT3 was a little more stable than lipases from other psychrotrophs at high