CHAPTER 3 PURIFICATION OF L-ASPARAGINASE FROM STRAIN EPD INTRODUCTION

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1 43 CHAPTER 3 PURIFICATION OF L-ASPARAGINASE FROM STRAIN EPD INTRODUCTION L-asparaginase (L-asparagine amidohydrolase E.C ) is an effective antineoplastic enzyme, used in the acute lymphoblastic leukemia (ALL) chemotherapy (Narta et al 2007). The earlier efficacy studies of the enzyme was performed with guinea pig serum L-asparaginase but bulk preparation turned out not to be feasible. Yellin and Wriston (1966) succeeded in partial purification of two isoforms of L-asparaginase from the guinea pig serum and only one isoform exhibited antilymphoma activity in vivo. Owing to the difficulties in extraction of this enzyme from Guinea pig serum in sufficient amount, the quest for other sources is going on. Bacteria are proved to be very efficient and inexpensive source of L-asparaginase. The ease of their cultivation has facilitated the large scale production of the enzyme. Campbell and Mashburn (1969) purified the Escherichia coli L-asparaginase and demonstrated its antitumour activity which was similar to that of guinea pig sera. These findings provided a practical base for bulk preparation of enzyme for preclinical and clinical studies (Campbell et al 1967, Roberts et al 1966 Whelan and Wriston 1969, Ho et al 1978). Oettegen et al (1967) proved the value of L-asparaginase treatment of childhood leukemia. Subsequently, a wide variety of microbes such as bacteria, fungi, yeast and algae were screened for antitumour

2 44 L-asparaginase production. It was found that enzyme properties vary between organisms. L-asparaginases have only been obtained in large quantities from two bacterial sources, such as E. coli and Erwinia caratovora for clinical purpose (Aung et al 2000, Aghaipour et al 2001, Kozak et al 2002). L-asparaginase as an important chemotherapeutic agent in ALL and the use of L-asparaginase in the treatment of leukemia and other lymphoproliferative disorders has expanded immensely in recent years (Narta et al 2007). However, clinical employments of L-asparaginase are accompanied with fatal effects (Duval et al 2002, Kozak et al 2002). These effects are mainly due to L-asparaginase associated L-glutaminase activity and bacterial endotoxins in enzyme preparations (Manna et al 1995, Kotzia and Labrou 2005). As an antitumour agent, sensitivity of L-asparaginase application demands high degree of enzyme purity and there is a continuing need to screen newer bacteria with high yield of antitumour L-asparaginase (Kotzia and Labrou 2007). The bacterial source and purification procedures are determining the possibility of sufficient quantity and homogeneity of the antitumour L-asparaginase enzyme for therapeutic purposes. L-asparaginases have been purified from several different bacteria such as E.coli (Robert et al 1968), Serratia marcescens (Boyd and Phillips 1971), Vibrio succinogens (Distasio and Niederman 1976), Azotobacter vinelandii (Gaffer and Shethna 1977), Pseudomonas acidovorans (Davidson et al 1977), Pseudomonas geniculata (Kitto et al 1979), Corynebacterium glutamicum (Mesas et al 1990), Pseudomonas stutzeri MB-405 (Manna et al 1995), and Pseudomonas aeruginosa (Bessoumy et al 2004). These L-asparaginases were known to contain up to 10% L-glutaminase activity (Narta et al 2007). All purification procedures to get rid of this L-glutaminase activity proved to be unsuccessful so far. As discussed earlier, the study on L-asparaginase activity by marine Streptomyces has been reported in detail.

3 45 Yet, the study on purification of L-asparaginase from marine Streptomyces has not been reported so far. The successful isolation and identification of high L-asparaginase potential marine Streptomyces sp strain EPD 27 was described in the previous chapter. This prompted to study further on antitumour property of the enzyme with reference to therapeutic purposes. Hence, attempt has been made to purify the L-asparaginase from strain EPD 27 and to develop a modified purification procedure to obtain homogenous form of enzyme. The procedure and techniques used for the purification of L-asparaginase from the strain EPD 27 and the results obtained are discussed in this chapter. 3.2 MATERIALS AND METHODS The methodology in this chapter is sequentially organized as growth and harvesting of strain EPD 27, preparation of crude enzyme and purification of L-asparaginase Growth and Harvesting of Strain EPD 27 The strain EPD 27 was grown in 200 ml of starch casein broth in a 500 ml conical flask and incubated at 30 ºC in a shaking incubator (Orbit, NEOLAB) at 120 rpm for 7 days. The cells were collected after the incubation and centrifuged at rpm for 20 min at room temperature. The growth of the cells was measured and expressed as wet weight Preparation of Crude Enzyme The cells were washed twice with 0.05 M Tris buffer (ph 8.6), suspended in ice cold buffer for two h (twice the volume of the cell pellet) followed by sonication at 5 cycles for 3 min by using Sonicator (Digital sonifier, Brandson). A pale white cell lysate obtained was centrifuged at

4 rpm at 4 ºC for 20 min and the supernatant fluid was collected. This was considered as crude enzyme and was stored at 4 ºC for the purification of L-asparaginase Protein Estimation (1951). Protein contents were estimated by the method of Lowry et al L-asparaginase Assay L-asparaginase was assayed by the method of Imada et al (1973) as described in previous section Purification of Enzyme The crude enzyme prepared from strain EPD 27 was used for further purification of L-asparaginase at 4 to 8 ºC. L-asparaginase was purified by a modified method of Davidson et al (1977) Organic solvent fractionation The stored crude enzyme was frozen, thawed and subjected to centrifugation at rpm at 4 ºC for 20 min repeatedly four times, separating the supernatant liquid each time and collecting it together. To the collected supernatant, equal volume of absolute ethanol was added slowly with stirring. The resultant mixture was kept stirring for 30 min and allowed to stand for 1 h. Then it was centrifuged at rpm at 4 ºC for 20 min and the precipitate was collected separately. The supernatant collected was centrifuged as before after adding three fourth volume of absolute ethanol to it. The precipitate formed was collected with the precipitate obtained previously and suspended in 0.05 M tris buffer (ph 8.6).

5 Diethylaminoethyl (DEAE) cellulose anion exchange chromatography The enzyme solution was applied on to a DEAE cellulose column (DEAE cellulose, 1.5 X 50 cm, Whatman microgranular DE 52) which was previously washed with 0.01 M Tris (ph 8.6) containing 0.05 M KCl and eluted with 0.1 M KCl in 0.01 M Tris buffer (150 ml each). A gradient elution was performed with 0.1 M KCl to 0.4 M KCl. A total of 50 fractions of 3 ml each were collected at a flow rate of 6 ml per h. and assayed for protein and enzyme activity. The high activity fractions were pooled and the protein was precipitated by adding equal volume of ethanol. It was centrifuged at rpm at 4 ºC for 20 min. The precipitate collected was suspended in 0.1 M Tris buffer (ph 8.6) Carboxymethyl (CM) Sepharose affinity chromatography The pooled high activity fractions were applied on to a second column CM-sepharose 6LB covalently linked to D-asparagine (1.5 X 12 cm) which had been previously equilibrated with 0.1M Tris buffer (ph 8.6) (Kotzia and Labrou 2005). It was eluted by linear gradient of M KCl (75 ml each) in the 0.1 M Tris buffer. A total of fifty fractions of 3 ml each were collected at a flow rate of 6 ml per h and assayed for protein and enzyme activity. Fractions of high activity were pooled and the protein was precipitated by adding equal volume of ethyl alcohol. It was centrifuged at rpm at 4 ºC for 20 min and the collected pellet was subjected to dialysis using the dialysis membrane (Himedia). The ethanol precipitated protein was dialyzed against 0.01 M Tris buffer (ph 8.6) overnight. The dialyzed protein was centrifuged at rpm at 4 ºC for 20 min and the pellet was suspended in 0.1 M Tris buffer (ph 8.6).

6 Sephadex G 200 gel filtration chromatography The pooled high active fractions were applied on to the third column, sephadex G 200 (1.5 X 50 cm) column which had been equilibrated with 0.05 M Tris containing 0.01 M KCl (ph 8.6). It was eluted by the same buffer and 50 fractions of 3 ml each were collected as before and assayed for protein and enzyme activity. The high activity fraction was lyophilized using LYODOL-Freeze dryer (Bio-Rad) and the powder formed was stored at 4 ºC High Performance Liquid Chromatography (HPLC) HPLC analysis of purified L-asparaginase was performed on an Agilent series 1100 HPLC system fitted with a reversed phase high performance liquid chromatography (RP-HPLC) column, 4.6 mm X 75 mm filled with Atlantis C18, 5 µm (Agilent). The solvent system was prepared as solvent A containing 0.1% (v/v) formic acid in H 2 O and solvent B containing 0.1% (v/v) acetonitrile (ACN). It was eluted by gradient elution of solvent B in 6 min as 70% at 0 to 1 min, 70 to 95% at 1 to 1.5 min, 95% at 1.5 to 2.5 min, 95 to 70% at 2.5 to 3 min and 70% at 3.0 to 6.0 min at flow rate 0.8 ml per min. 3.3 RESULTS AND DISCUSSION There is an immense demand for homogenous form of L-asparaginase from new bacterial source which lacks glutaminase and other toxins for the treatment of leukemia (Avramis and Panosyan 2005). The purification procedure adopted in this work is a modified method of Davidson et al (1977). The results of the purification of L-asparaginase (Table 3.1) are in good agreement with the proposed goal.

7 L-asparaginase Activity of Crude Extract The crude enzyme prepared from strain EPD 27 yielded mg of protein and IU of L-asparaginase. It was found to have good specific activity of the enzyme about 0.89 IU/mg of protein while compared to recombinant E. coli W3110 L-asparaginase which showed only 0.73 IU/mg of protein (Youssef et al 2008). This suggests that the crude extract preparation was suitable for the subsequent purification of enzyme. Table 3.1 Purification of L-asparaginase from strain EPD 27 Purification steps Protein (mg) Enzyme (IU) S.A. (IU/mg) Fold purification Yield (%) Crude extract Ethanol fractionation DEAE cellulose CM-Sepharose Sephadex G Purification of L-asparaginase from Crude Enzyme A modified procedure has been developed for the purification of L-asparaginase which includes alcohol fractionation followed by subsequent chromatographic separation of enzyme. Alcohol fractionation of crude enzyme yielded 320 mg of protein and 843 IU of enzyme with an increased specific activity to 2.63 IU/mg of protein from 0.89 IU/mg. It also yielded 2.95 fold purification and 89.3% recovery of the enzyme. It was found to be a higher yield and relatively pure material for subsequent chromatography. This suggests that alcohol fractionation is found to be advantageous to obtain 90 % recovery of the enzyme. Alcohol is the superior precipitating agent than the others since alcohol precipitation is easy to scale up, and requires less

8 50 manipulation. Most of the previous studies have used ammonium sulphate precipitation in the initial step in the purification of L-asparaginase (Boyd and Phillips 1971, Novak and Phillips 1974, Distasio and Niederman 1976, Mesas et al 1990, Manna et al 1995, Bessoumy et al 2004, Kotzia and Labrou 2007, Youssef et al 2008). Ammonium sulphate precipitation affects the purification of L-asparaginase by yielding an impure material for subsequent chromatography (Bessoumy et al 2004). DEAE column was used as a first column in second step of purification L-asparaginase. A typical elution profile of enzyme from the DEAE cellulose anion exchange column (Figure 3.1) indicates a distinct peak of L-asparaginase. 3 Total protein (absorbance at Protein Enzyme 2 1 Enzyme activity (at 450 nm) Fraction number Figure 3.1 Elution profile of L-asparaginase from DEAE cellulose chromatography The high active fractions of DEAE column were pooled together which contained 8.7 mg of protein and 520 IU of enzyme with a remarkable

9 51 specific activity of IU/mg protein from 2.63 IU/mg protein. DEAE column was found to yield fold purification and 55.1% recovery of the enzyme. The specific activity and fold purification was found to be higher than the recombinant E.coli W3110 L-asparaginase (Youssef et al 2008) which showed only 2.57 IU/mg of protein and 3.5 % of recovery of the enzyme respectively. Previous studies have used DEAE column in the final step of purification. (Roberts et al 1968, Boyd and Phillips 1971, Novak and Phillips 1974, Kitto et al 1979, Mesas et al 1990, Manna et al 1995 and Bessoumy et al 2004). Analysing the above studies, DEAE column is found to be important in distinguishing L-asparaginase associated glutaminase activity and free L-glutaminase in crude extract. The distinct differences between L-asparaginase associated L-glutaminase and free L-glutaminase can also be shown. The free L-glutaminase was precipitated at ph 5 whereas L-asparaginase associated L-glutaminase remained soluble. Free glutaminase was not eluted from DEAE-cellulose until a concentration of 0.25 M KCl reached whereas L-asparaginase associated L-glutaminase was eluted at 0.1 M KCl. Hence DEAE column was intentionally used as a first column in the purification of strain EPD 27 L-asparaginase to eliminate the possibility of incomplete separation L-glutaminase and L-asparaginase. DEAE column was found to be an effective column in the initial step of purification of L-asparaginase. Further the enzyme preparation obtained was found to be stable after purifying in DEAE column. The pooled high active fractions of DEAE column were applied on the second column of CM sepharose affinity column. The specific elution profile of L-asparaginase on CM Sepharose affinity column (Figure 3.2) indicates only one distinct peak corresponding to L-asparaginase.

10 52 Total protein (absorbance at 280 nm) Fraction number Protein Enzyme Enzyme activity (at 450 nm) Figure 3.2 Elution profile of L-asparaginase from CM-sepharose affinity chromatography High active fractions of affinity column were pooled together which contained 2.2 mg of protein and IU of L-asparaginase with a notable specific activity of the enzyme IU/mg of protein. It was found to be higher specific activity when compared to Erwinia chrysanthemi L-asparaginase which showed IU/mg of protein (Kotzia and Labrou 2007) and recombinant E. coli W3110 L-asparaginase shows IU/mg of protein (Youssef et al 2008). Affinity column enriched the enzyme immediately after DEAE column as expected. It yielded fold purification and 51.9% recovery of the enzyme. This was found to be higher when compared with recombinant E. coli W3110 L-asparaginase which showed the fold purification of and 20 % recovery (Youssef et al 2008). Further the affinity column removed major contaminants (Figure 3.2) there by making it vital for further chromatographic separation and was found to be an ideal column. This suggests that affinity chromatography should be second or intermediate column to achieve homogeneity of the enzyme L-asparaginase.

11 53 The enzyme preparation obtained from CM sepharose column was precipitated with ethyl alcohol and was centrifuged. The protein pellet was collected and was dialyzed against 0.01 M Tris buffer (ph 8.6) for 24 h. Ethyl alcohol precipitation of high active fractions of affinity column was found to concentrate the enzyme from very dilute protein solutions. Further dialysis in later stage of purification was advantageous which yields relatively pure material for subsequent gel filtration chromatography. In general, antitumour study of L-asparaginase demands milligram level of protein contained considerable amount of enzyme and their specific activity. While analysing previous procedures, dialysis was used as second step immediately after the ammonium sulphate precipitation which failed to give sufficient yield. The dialyzed protein was applied finally on sephadex G 200 gel filtration chromatography. The result of purification of enzyme rich fractions of Sephadex G 200 column is shown in Figure 3.3. Total protein (absorbance at 280 nm) Protein Enzyme Fraction number 4.5 Protein Enzyme Enzyme activity (at 450 nm) Figure 3.3 Elution profile of L-asparaginase from Sephadex G 200 gel filtration chromatography

12 54 The high active fractions of sephadex G 200 column were pooled together. It contained 1.1 mg of protein, IU of L-asparaginase with specific activity of IU/ mg of protein. It was found to be increased when compared to Pseudomonas aeruginosa L-asparaginase which showed only IU/mg of protein (Bessoumy et al 2004). Sephadex G 200 column yielded fold purified enzyme and this was found to be higher than recombinant E. coli W3110 L-asparaginase which showed only fold. (Youssef et al 2008). It also yielded 48.4% recovery of the enzyme as expected. The specific activity and purity of enzyme increased with every step of purification with minimum loss in quantity giving a final recovery of almost 50%. This was found to be a good recovery of enzyme reported so far. Figure 3.4 clearly indicates a sharp distinctive peak of L-asparaginase and the homogenous form of L-asparaginase. It was found that gel filtration chromatography is highly essential in achieving complete purification of L-asparaginase. The pooled high active fraction was of sephadex G 200 was lyophilized and used for further analysis of the enzyme. Confirmation of the homogeneity of L-asparaginase is obligatory to study the antitumour study (Bessoumy et al 2004 and Kample et al 2006). The purity of the L-asparaginase was confirmed in HPLC. While lyophilized enzyme was analysed in HPLC, a clear peak of L-asparaginase (retention time min) was observed in the profile which is shown in Figure 3.4. It reveals that the purified L-asparaginase was found to be 100% pure and ultimately in evidence of the homogenous form of the strain EPD 27 L-asparaginase. The procedure adopted for purification of L- asparaginase was proved to be very effective and vital for attaining a homogenous form of enzyme. It highly refined the enzyme for further evaluation of antitumour activity. The purified L-asparaginase was stored at 4 ºC for subsequent characterization.

13 55 Figure 3.4 HPLC analysis of L-asparaginase from strain EPD CONCLUSION This work suggests that alcohol fractionation was found to be highly effective rather than other precipitation agents. DEAE column was found to be important for separation of L-asparaginase and L-glutaminase and was very effective in initial steps. Affinity and gel filtration chromatography were found to be suitable in final step of purification. The modified procedure developed for the purification of L-asparaginase simplified purification difficulties and yielded sufficient quantity of enzyme to study the antitumour activity of the enzyme. By this modified procedure, overall recovery of 48.4% of yield and a homogenous form of L-asparaginase with specific activity of IU/ mg protein was obtained.