6 Purification and characterization of L- Asparaginase

Transcription

1 Purification and characterization of L- Asparaginase 93 6 Purification and characterization of L- Asparaginase 6.1 Introduction Purification of a protein is an important step for characterization of its physical and biological properties. Moreover, for effective therapeutic use of a protein, it must be free of any contaminants and impurities. Success of a downstream process mainly depends on the cost effectiveness, less sequential operations and overall yield of the product. Cost and yield of the product is mainly influenced by the number of sequential steps involved in purification. Downstream processing accounts for around 8% of overall production cost, which clearly indicates a need for process optimization. Above all, the purification process must be simple, easy and adaptable, particularly in large scale (125). Cytotoxicity studies are the preliminary step in anticancer drug screening. Many methods are available for screening the anticancer potential of agents viz., tryphan blue dye exclusion assay, microculture tetrazolium assay (MTT) and sulpho rhodamine assay ( SRB). The antileukemic effect of L-asparaginase is postulated to result from the rapid and complete depletion of the circulating pool of asparagine. Cytotoxicity is a result of depletion of non- essential amino acid, asparagine. The leukemic cell has repressed asparagine synthetase activity, whereby they depend on the circulating asparagine. The current study describes the methods adopted and results of purification, characterization and in vitro anti-leukemic activity of L-asparaginase produced by isolate SI99 (Aspergillus terreus).

2 94 Exploration of soil and marine sources for microbes producing asparaginase 6.2 Materials and Methods Production of L-asparaginase by submerged fermentation Production of L-asparaginase was carried out in Erlenmeyer flask containing optimized medium (Table 6-1) for 5 days at 165 rpm and 25ºC with 7.5 % inoculum. The cell free supernatant was collected by centrifuging at 15 rpm for 15 min and was used for estimating the extracellular enzyme activity. Enzyme assay was carried out as described in section The protein content was estimated by Lowry method(126). Specific activity of the enzyme was expressed as IUmg -1 protein. (Materials used are listed in appendix) Table 6-1: Optimized medium used for production of L-asparaginase S.No Ingredient gl -1 1 Sucrose Yeast extract Ammonium chloride 1 4 Magnesium sulphate.5 5 L-Asparagine 31.9 ph adjusted to Purification of L-asparaginase The sequential steps followed in purification included ammonium sulfate precipitation, ion exchange and gel filtration chromatography. The molecular weight of the L-asparaginase was determined by Sodium Dodecyl Sulphate Poly Acrylamide Gel Electrophoresis (SDS-PAGE).

3 Purification and characterization of L- Asparaginase Ammonium sulfate precipitation Ammonium sulfate removes water from the surrounding of the protein revealing hydrophobic patches, which come together and causes the protein to precipitate. The more a protein is hydrophilic, the more will be the ammonium sulfate needed. The fractionation range of ammonium sulfate needed to precipitate out the target protein was determined by performing analytical ammonium sulfate cut (127, 128). Ammonium sulfate was added to the supernatant in different concentrations ranging from 1 to 9%w/v saturation, with constant stirring in ice bath. The precipitate was removed by centrifuging at 1 rpm for 1 min in cooling centrifuge maintained at 4 C. The supernatant was used for estimation of enzyme activity and protein content. The fractionation range of ammonium sulfate needed was determined. The fractionation range of ammonium sulfate was found to be 4-7% w/v. The precipitation of the target protein was done using the fractionation range. Initially the cell free supernatant was brought to 4% saturation with ammonium sulfate and kept at 4-8 C overnight. After overnight equilibration, the precipitate was removed by centrifuging at 1 rpm for1 min in cooling centrifuge at 4 C. The supernatant was further brought to 7% saturation with ammonium sulfate and left overnight at 4-8 C. The precipitate was collected by centrifuging at 1 rpm and 4 C for 15 min. The precipitate was re-suspended in 1 ml cold 5mM Tris-HCl, ph 8.6 and desalted using sephadex G-25 column with the same buffer. An aliquot from this was used to determine the enzyme activity and protein content. The desalted protein solution was collected, stored at 4-8 C and used in further steps of purification.

4 96 Exploration of soil and marine sources for microbes producing asparaginase Ion exchange chromatography (IEC) The sample obtained after desalting was diluted to 5mL of 5mM Tris-HCl buffer and used in IEC. DEAE-sepharose anion exchange column was equilibrated with 5mM Tris-HCl buffer (ph 8.6). To the equilibrated column, the sample was applied and the column was washed with two bed volumes of the same buffer to remove any unbound protein. The sample was eluted as 6mL/fraction using NaCl gradient (.1-.5M) at flow rate of 3mLh -1. The protein content and enzyme activity were determined. The fractions showing peak L- asparaginase activity were pooled together and concentrated by dialysis against poly (ethylene glycol) 2, followed by dialysis against 5mM Tris HCl buffer at 4 C (129, 13) Gel filtration chromatography (GFC) The sample obtained after dialysis was chromatographed on a column of Sephacryl S 2, which was pre-equilibrated with.5m Tris-HCL buffer of ph 8.6. The sample was eluted with the same buffer at 24mLh -1 flow rate as 4mL fractions. The enzyme activity and protein content of the fractions were determined. Fractions with peak enzyme activity were pooled together and concentrated by dialysis against poly (ethylene glycol) 2, followed by dialysis against 5mM Tris HCl buffer and stored at 4-8 C (131) Determination of molecular weight of enzyme by SDS-PAGE The sample after purification steps was electrophoresed by SDS- PAGE (132), with 5% stacking gel and 1% resolving gel. Standard markers included phosphorylase (97.4kDa), bovine serum albumin (66.2kDa), ovalbumin (43kDa), carbonic anhydrase (31kDa), trypsin inhibitor (2.1kDa) and lysozyme (14.3kDa).

5 Purification and characterization of L- Asparaginase Partial characterization of the enzyme Effect of metal ions for the enzyme activity The effect of different metal ions on the enzyme was studied by pre-incubating enzyme with 1mM zinc sulphate, calcium chloride, sodium chloride, potassium chloride, ferrous sulphate and magnesium sulphate for 1h at 37 C. The relative enzyme activity was estimated Effect of inhibitors and chelators on enzyme activity The effect of different inhibitors viz., phenyl methyl sulphonyl fluoride (PMSF, 5mM), dithiothritol (DTT, 5mM), β- mercaptoethanol (MCE, 5mM) and chelator viz., 5mM ethylene diamine tetra acetic acid (EDTA) was determined. The enzyme was pre-incubated with respective inhibitor or chelator at 37 C for 1h and then the enzyme assay was performed under standard conditions Effect of ph on enzyme activity and stability The effect of ph on enzyme activity was studied by estimating the activity at ph ranging from 4. to 12. with different buffers viz., phosphate buffer mixed (ph4.), phosphate buffer ((ph 5.-7.), Tris-HCl (ph 8.) and glycine-naoh (ph ). The enzyme was incubated in contact with different relevant buffers (ph ) at 37 C for 48h to determine its stability in different ph. The relative activities were measured Effect of temperature on enzyme activity and stability The effect of temperature on enzyme activity was determined by incubating the reaction mixture at different temperatures ranging from 2 to 9 C.

6 98 Exploration of soil and marine sources for microbes producing asparaginase The stability of enzyme in different temperature was studied by incubating the enzyme at different temperatures for 1 h. The residual enzyme activities were determined Determination of substrate specificity and kinetic parameters The enzyme activity was determined with L-asparagine and L- glutamine as substrate at a final concentration of 1 mm. High performance thin layer chromatography of the digest was performed. The Michaelis constant (K m ) and maximal velocity (V max ) of the purified enzyme were determined using L-asparagine as substrate in the range of 5 16 µm with the help of Lineweaver-Burk plot. ( ) In vitro anti-leukemic activity of the purified L-asparaginase by MTT assay (136) The human ALL cell line MOLT-4 was cultivated as suspension culture in RPMI 164 medium supplemented with 1%(v/v) fetal bovine serum (FBS) at 37 C in a 5% CO2 incubator. The effect of L-asparaginase on the growth rate of MOLT-4 cells was determined by MTT. Briefly, exponentially growing cells were collected by centrifugation for 1 min at 2 rpm, washed twice in PBS, and re-suspended at a density of approximately 4x1 5 cells/ml in fresh medium. The cells were seeded in 96-well plates at a density of 4 cells/well in RPMI 164 medium supplemented with 1%(v/v) fetal bovine serum (FBS) and incubated at 37 C for 24 h in a 5% CO2 incubator. After 24 h, the plates were centrifuged at 2 rpm for 1 mins and the supernatant was carefully replaced with 1 µl of medium containing purified L-asparaginase at different concentrations (.1,.1, 1, 1 IU).

7 Purification and characterization of L- Asparaginase 99 After incubation for 48 at 37 C and 5% CO2, the plates were centrifuged at 2 rpm for 1 mins and inverted on tissue paper to remove the media. The cells were washed once with 1 µl PBS and the supernatant were removed after centrifugation at 2 rpm for 1 mins. To each well 1 µl MTT was added (2mg/mL) and the cells were incubated for an additional 2 h. Later, the formazan crystals formed were dissolved in 1 µl of isopropanol and incubated at 37 C for 3 mins. The optical density was measured at 54nm using Biotek ELx plate reader. The experiments were performed as duplicates and the percentage growth inhibition was calculated. 6.3 Results Purification of L-asparaginase Activity guided analytical ammonium sulphate cut method was used to determine the fractionation range for precipitation of the target protein. The fractionation range was found to be 4-7%w/v saturation of ammonium sulfate. With preparative ammonium sulphate precipitation, the yield was 58.3 and purification fold was The desalting was performed using sephadex G25 and the elute was diluted to 5mL with 5mM Tris-HCl buffer (ph 8.6) and further purified by IEC. The chromatogram of IEC was given in Chart 6-1. Five different protein peaks were observed and the target protein was eluted with.2m NaCl (Fraction no.12-16). The results of purification are given in Table 6-2. The purification fold and yield after IEC was found to be 23.8 and 34.95, respectively. Elute from IEC was subjected to dialysis and concentrated. It was further purified by GFC and the chromatogram was given in Chart 6-2. The target protein was eluted in three fractions (1-12). The

8 1 Exploration of soil and marine sources for microbes producing asparaginase yield and purification fold of the pooled fractions was found to be and 86.75, respectively. SDS-PAGE of the purified sample was performed and the results were given in Figure 6-1. The molecular weight of the denatured enzyme was calculated from the Rf values and was found to be kda (approx. 35kDa) Partial characterization of L-asparaginase The effect of ph, temperature, metal ions, inhibitors and chelators on the enzyme activity was studied. The results are given as Charts 6-3 to 6-6. Among the metal ions tested, enzyme activity was greatly affected by ferrous ions. Sodium, potassium and calcium ions enhanced the enzyme activity. The enzyme was found to be inhibited by PMSF (serine protease inhibitor) and DTT (cysteine protease inhibitor). The enzyme was found to be stable at wide ph of There was 2% decrease in activity when stored at ph 12. for 48h. The optimum ph for enzyme activity was found to be ph 8.. The enzyme was stable when stored at varied temperatures ranging from 2 to 5 C. Storage temperature above 5 C affected the enzyme activity. The optimum temperature for enzyme activity was found to be 4 C. The relative activity of the enzyme using L-glutamine was found to be 7%. The result of HPTLC was given in Figure 6-2 and the Rf values are given in Table 6-3. The K m and V max of the enzyme was found to be µm substrate and 4.39 µm/min (See Charts 6-7 & 6-8).

9 Purification and characterization of L- Asparaginase In vitro anti-leukemic studies The percentage inhibition of cell proliferation by L-asparaginase at different concentrations was calculated. The results were expressed as mean values in Table 6-4. The concentration of L-asparaginase causing inhibition of 5% of viable cells (IC5) was calculated. The results were compared with marketed formulation of L- asparaginase.

10 Protein (mg/ml) Enzyme activity (IU/mL) Protein (mg/ml) Enzyme activity (IU/mL) 12 Exploration of soil and marine sources for microbes producing asparaginase Protein Enzyme Fraction No (5mL each) Chart 6-1 Ion Exchange Chromatography: Elution Profile Protein Enzyme Fraction No (4mL each) Chart 6-2 Gel Filtration Chromatography: Elution Profile

11 Relative activity (%) Relative Enzyme Activity (%) Purification and characterization of L- Asparaginase Metal Ions (1mM) Calcium Magnesium Zinc Ferrous Sodium Potassium Chart 6-3 Effect of metal ions on the enzyme activity PMSF MCE DTT EDTA Chart 6-4 Effect of inhibitors and chelators on enzyme activity

12 Relative Activity (%) Relative activity (%) 14 Exploration of soil and marine sources for microbes producing asparaginase ph Activity Stability Chart 6-5 Effect of ph on stability and activity of enzyme Activity Stability Temperature ( C) Chart 6-6 Effect of temperature on stability and activity of enzyme

13 1/v v (µm/min) Purification and characterization of L- Asparaginase [S] (µm) Chart 6-7 Plot of reaction velocity (v) vs. Substrate concentration [S] 2 y = x R² = /S Chart 6-8 Lineweaver-Burk plot

14 16 Exploration of soil and marine sources for microbes producing asparaginase A-Elute from IEC; B- Elute from GFC, M-Marker Figure 6-1 Silver stained SDS-PAGE: Molecular weights of the bands Figure 6-2: HPTLC of the digests

15 Purification and characterization of L- Asparaginase 17 Table 6-2: Results of Purification of L-asparaginase Total Enzyme Total Protein Specific Activity Purification S.No Purification Step activity(iu) (mg) (IUmg -1 protein) Fold Yield 1 Crude (NH4)2SO DEAE-Sepharose Sephacryl 2HR Table 6-3: Rf values observed in HPTLC Track Spot Peak Rf 1 L-Asparagine L- Glutamine L-aspartic acid L-glutamic acid Digest- L-Asparagine Digest- L-Asparagine Digest- L-Glutamine Digest- L-Glutamine 2.43

16 18 Exploration of soil and marine sources for microbes producing asparaginase S.No Table 6-4: Percentage of Growth inhibition in MTT assay Enzyme concentration (IUmL -1 ) Growth Inhibition (%) Purified L- asparaginase from SI99 Marketed L- asparaginase preparation IC IUmL IUmL Discussion Purification of L-asparaginase was carried out using appropriate chromatographical techniques. The specific activity before purification was 3.17 IUmg -1 protein. Upon precipitation with ammonium sulphate the specific activity was found to be increased by 11 fold and the total protein content was found to be decreased by 19 fold. This indicates that the fractionation range of ammonium sulfate used for precipitation was effective enough in removing proteins which are contaminants, with subsequent loss of total activity (approximately 42%). With IEC, only 1.5% of the total protein from crude extract was eluted (66 fold decrease), but the specific activity was found to be increased by 23 fold. The specific activity after IEC was IUmg -1 protein. Five different protein peaks observed in the elution profile indicates an efficient removal of contaminants. GFC was performed as final step of purification. The specific activity after GFC was found to be increased by fold, with total protein content decreasing by 352 fold. Three bands were observed in SDS-PAGE after IEC. And after GFC, only one band was observed. From the bands observed, it can be claimed that the enzyme was purified to near homogeneity.

17 Purification and characterization of L- Asparaginase 19 The enzyme activity was severely affected by PMSF, a serine protease inhibitor and DTT, a cysteine protease inhibitor. The stability of enzyme at varied ph and temperature reduces the cost involved in storage and transportation. The low Km value indicates that the enzyme has high specificity for the substrate L- asparagine. The results of chromatography indicate no or little effect on L-glutamine. Overall, the enzyme has high L- asparaginase activity and no or little L-glutaminase activity. The IC5 value of L-asparaginase from isolate SI99 is comparable with the marketed L-asparaginase preparation. The IC5 values were quite high when compared to earlier results on HL6 cell lines with different L-asparaginase. 6.5 Conclusion The sequential purification of L-asparaginase from isolate SI99 (Aspergillus terreus) resulted in fold pure enzyme. The molecular weight of the purified denatured enzyme was found to be kda. The enzyme was considerably stable at ph range 7-1. and temperature range 2-5 C. The peak activity was at ph 8. and 4 C. The enzyme was found to be substrate specific to L- asparagine with 6% relative activity with L-glutamine as substrate. The purified L-asparaginase from isolate SI99 has an IC5 value of IUmL -1

18 11 Exploration of soil and marine sources for microbes producing asparaginase