CHAPTER I1 SOURCES OF L-ASPARAGINASE

Transcription

1 CHAPTER I1 SOURCES OF L-ASPARAGINASE

2 11. A. SCREENING OF MICRO ORGANISMS FOR L-ASPARAGINASE Although enzymes are abundant in nature, bacteria are the proven potential sources for the clinically important enzymes[57,85]. Not only the quantity of the material available from this source could be increased as needed to meet the increasing demands but also the quality of the product could be ensured by controlled production[57]. It has been established that repeated administration of L-asparaginase to blood stream causes hypersensitivity, ranging from mild allergic reactions to anaphylactic shock, in 5-30% of the patients[ ,1271. Therefore L-asparaginase with similar antitumor property but with different antigenic structures are needed for clinical trials. Since the enzyme isolated from different species have different physiological, pharmacological and serological properties[l23], it would be imperative to screen some of the common bacteria for production of L-asparaginase with optimal physiological and pharmacological actions and with less immunoloyical complications. So far, Erwinia carotovora L-aspasaginase has been shown to be useful in clinical trials as an alternative to that of E. coli[121,123]. Estuarine bacteria were found to be one of the best sources of L-asparayinase[57] and the halophilic nature

3 of the bacteria can be exploited for the industrial production. So we have made an attempt to screen the L-asparaginase producing micro organisms from estuarine sediments and molluscs. We have also studied the influence of various ecological parameters on bacterial population and also formulated the optimum cultural conditions for the production of the enzyme industrially. MATERIALS AND METHODS Sediments and che bivalve mollusc Villorita cyprinoids were collected from two different stations in Ashtamudi estuary (long '-76034'~, lat. 8O56'- 8 57'~) - Station I: Kakathuruthu, a mangrove region having sandy sediment with gravels; and Station 11: Peruman, a coconut husk retting zone with clay like sediments. collections were made monthly from August 1988 to January Shells of the molluscs were washed with sterile 50% sea water, flesh removed and chopped. The chopped flesh (5 g) was homogenated with 20 ml sterile saline. Serial dilutions of homogenized flesh and sediments were prepared separately with sterile saline. These were then pour plated using ZoBell's 2216 e mafine agar medium (HM) incorporated with 0.2% L-asparagine (SRL) and a few drops of phenol red indicator. The plates were incubated at 35O~ for 5 days. The colour of the medium changed from yellow to red around some colonies. 28

4 Since no change in colour was observed in medium without L-asparagine, this colour change would be due to the production of ammonia from asparagine by the action of L-asparaginase, around the positive strains. These colonies were grouped on the basis of morphological characters and were stocked in nutrient ayar (HM) slants. The bacteria were identified upto the generic level following the scheme of Simidu & Aiso[185]. Salinity, phosphate and nitrate content of the overlying water as well as organic content of the sediment were determined by the procedures described by Martin[l86], Parsons[l87] and Grasshoff[l88]. Strickland and For estimating the enzyme activity, L-asparaginase positive bacteria were grown in nutrient broth (HM) for 24 hrs and harvested by centrifugation at 5,000 rpm for 10' in a Remi Research Centrifuge R-24. The cells were washed with deionized water and then suspended in 2 ml cold distilled water. This was then subjected to freezing and thawing and the same was used as crude enzyme preparation. Assay of the enzyme activity was done following the method of Wriston[l89]. To 0.25 ml of crude enzyme preparation, 1.25 ml of 0.2 M borate buffer (ph 8.6) was added. Then 0.5 ml of 0.04 M L-asgarayine (Siyma) in borate buffer was added and incubated at 35O~ for

5 30'. The reaction was stopped by the addition of 0.5 ml of 15% TCA (SRL) and the assay mixture was centrifuged at 6000 rpm for 10'. The supernatant (1 ml) was mixed with 4 ml distilled water free from ammonia. To this, 0.5 ml Nessler's reagent (EM) was added and the colour intensity was read in a photoelectric calorimeter at 425 nm. The ammonia content was estimated using standard ammonium chloride (AR) solution. Since L-aspartase activity was found to be negligible at the assay ph, the ammonia liberated was only from asparagine by L-asparaginase. Protein content of the enzyme preparation was estimated by the method of Lowry et a1.[190]. L-asparaginase activity is expressed in ~nternational Units (IU). "One IU is the amount of enzyme which will liberate one micromole of ammonia per minute under experimental conditions". Specific activity is expressed as IU mg protein which denotes the amount of enzyme activity shown by one mg protein of the enzyme preparation. Correlation and regression were studied using,the method of Aiswas[l91]. RESULTS AND DISCUSSION Altogether 642 L-asparayinase positive strains were isolated from the estuarine sediments and molluscs. Based on the specific activity, these were classified into 5 groups (Table 11.1). Majority of isolates showed

6 Table Generic cmposition of L-asparaginase positive bacteria Source' E. of isolates in activity Total Percent- Bacteria groups" NO. of age of I I1 I11 IV V isolates yenera (1)... S Aeromonas M Alkaliqenes n S eacillus n Cvtophayafla- S vobacterium M Enterobacteri- S aceae n S ~icrococcus M Sarcina Vibrio... Total No. in each group... Percentaye of % each group *S Sediment **Group I IU/mg of protein n - ~ussal Group II Iu/mq of protein Group IU/mq of protein Group IV IU/mg of protein Group V - >0.2 Iu/mq of protein

7 - 1 minimum activity below 0.05 IU mg protein, and 9.05% of -1 the isolates showed an activity above 0.2 IU mg.. protein. Of the 642 strains, 8 showed higher activity above 0.25 IU mg-i protein (Table 11.2). Maximum specific activity was shown by an Aeromonas Spp. ( IU mg-' protein). Since high activity was shown by Aeromonas and no information is available on L-asparaginase of Aeromonas, this strain was used for further studies. Table 11.2 Specific activity of bacteria with high L-asparaginase activity Bacteria Specific activity (IU mg-' of protein) Pseudomonas - 542* Pseudomonas Pseudomonas Vibrio Vibrio Enterobacteriaceae Enterobacteriaceae Aeromonas * Number indicates the strain number in the order of collection.

8 The generic composition of L-asparaginase positive bacterial population isolated from estuarine sediments and molluscs is also given in Table Pseudomonas was the dominating group followed by Bacillus and Vibrio. Members belonging to Group IV and V were obtained largely from mussels. Bacillus, Micrococcus and Flavobacterium which predominantly come under Group I were present in both sediments and molluscs, where as Sarcina, Corynebacterium and Aeromonas which predominantly come under Group I1 & 111 were obtained mainly from molluscs. Strains of Pseudomonas, Vibrio and members of Enterobacteriaceae which predominantly come under Group IV and V were present in both sediments and molluscs. The specific activity of bacteria isolated from molluscs was higher when compared to that from the sediments. But the total percentage of L-asparayinase positive population was slightly higher in sediments than in molluscs. The low percentage of L-asparayinase positive population in molluscs may be due to the presence of some inhibitory factors present in molluscs as suggested by Selvakumar[S71. Seasonal variations in the population of total bacteria and L-asparaginase producing strains were also studied in both the Stations (Table 11.3).

9 Table 11.3 Variation in different parameters of study area Organic cont- Total het- L-asparagi- % of +Ve Station Month Salinity PO4-P NO -N ent of dry erotrophic nase +ve strains 3 sediment population population 4 (X ( pg 1-I) ( rg 1-I] (mg %) (X~O g-l) (X 10' g-l) Aug Sept I Oct NOV Dec Jan. ' Aug Sept I1 Oct NOV Dec Jan. '

10 In Station I total microbial population increased during September and decreased during October. NO appreciable change was observed during August, November and December, whereas, L-asparaginase positive population increased during January and decreased during October. In Station I1 total microbial population increased during December and November and decreased during August and January. L-asparaginase positive population increased during December and decreased during January. However, the percentage of L-asparaginase positive population was hiyher during October in both the stations. This fluctuation correlates with fluctuations in nitrate, phosphate and organic contents of the sediment. A negative correlation was observed between nitrate and phosphate and total heterotrophic population and L-asparaginase positive population ( = -0.76, -0.55, and -0.5 respectively). But in Station 11, there were no significant alterations both in total population and in L-asparaginase positive population with nitrate content. A positive correlation was observed between phosphate and total microbial population and L-asparaginase positive population ( Y = and respectively). A profound effect was observed on the growth of total heterotrophic population and percentage of L-asparaginase

11 positive population with organic carbon. A positive correlation (.'Y = +0.65) was observed on the growth of total heterotrophic population as reported by Aayykkannu[l92], while percentage of L-asparaginase positive population decreased considerably with increase of organic carbon in the sediments (Table 11.3). This effect was pronounced in samples collected from Station 11. Since this station is a coconut husk retting zone this effect may be due to the organic components and some other probable inhibitory factors such as tannins and phenolic compounds that might have come from the coconut husk retting process. The present study establishes the superior quality of Aeromonas isolated from estuarine mollusc, in the production of L-asparaginase and may be recommended for studying the industrial production of the enzyme to meet the increasing demand of the enzyme for therapeutic purpose. This study also establishes the role of estuarine bacteria in the nitrification process by releasing ammonia from asparagine/glutamine accumulated due to the decay of different organisms.

12 11. B. OPTIMIZATION OF CULTURAL CONDITIONS FOR L-ASPARAGINASE PRODUCTION BY AEROMONAS Not only the best source of the enzyme but also the optimum cultural conditions are important factors for the production of the enzyme industrially[57]. There are a number of reports about the various factors stimulating or affecting the synthesis of L-asparaginase in various hacteria[40169,70, Interestingly each strain exhibits a distinct pattern of enzyme regulation and poses special problems. An understanding of the mechanisms of induction of enzymes in micro organisms is important for designing techniques to obtain maximum yield. It includes manipulation of the medium constituents and optimization of physico-chemical factors which in turn can influence enzyme synthesis and cell yield. Only a few organisms have been exploited for the large scale production of enzymes. Hence a detailed investigation was carried out to find out the optimum cultural conditions and medium composition as well as to study the effect. of various chemicals and biochemicals which would affect the growth of the Aeromonas and the production of L-asparaginase.

13 MRTERIALS AND METHODS Bacterial strain The estuarine Aeromonas, which showed maximum L-asparaginase activity, isolated from estuarine mollusc Villorita cyprinoids was used for the study. Identification of the Bacterial strain The bacterial strain was identified as Aeromonas according to the guidelines of Bergey's manual of Systematic Bacteriology[l95]. It showed the following characters on cultural, morphological and biochemical examinations. Morphology and Gram's strain Motility Oxidase reaction Growth in medium containing bile salts - Gram negative straight rods - Motile - Positive - Positive Sensitivity to vibriostatic agents, 0/129 (2,4 diamino- 6,7,diisopropyl pteridine) - Negative Indole production in 1% peptone - Positive water

14 Growth in KCN broth Nitrate reduction L-Arabinose utilization Fermentation of salicin Fermentation of mannitol Breakdown of inositol Catalase Acetoin from glucose (Voges - Proskauer test) - Positive - Positive - Positive - Positive - Positive - Negative - Positive - Positive Gas from glucose - Positive Hydrogen sulphide from cysteine - Positive Maintenance of Aeromonas Aeromonas strain was maintained on Trypticase Soya agar (HM) slants. It was incubated overnight to allow good growth and was kept in refrigerator at ~OC. In this condition it could be stored for about one month. Culture media a) Peptone water media Peptone (HM) - 1g Sodium chloride (HM) mg Distilled water ml

15 b) Synthetic medium Starch (HM) Sodium chloride (HM) - 2g mg Ammonium dihydrogen phosphate (HM) Potassium dihydrogen mg phosphate (AR) Magnesium sulphate (AR)- 30 mg Tap water ml 0 The media were sterilized by autoclaviny at 121 C for 15' at 15 lbs. Note: Peptone water medium was used to study the physical factors affecting growth and enzyme production. Synthetic medium was used to study the effect of various chemicals, biochemicals, inorganic salts, amino acids etc. on enzyme production; ph was adjusted to 7.2. Growth and enzyme activity were estimated as mentioned earlier. 25 ml culture media were inoculated with the Aeromonas and after 20 hrs, the cells were harvested by centrifugation. This was then washed twice with deionized water and subjected to freezing and thawing. This was used for the estimation of enzyme activity and protein content.

16 RESULTS AND DISCUSSION The growth curve of Aeromonas prepared by estimating the enzyme activity and protein of the culture at an interval of 1 hr, for 24 hrs is given in Figure Growth *--- Activity I Incubation time (hr.) Figure: Growth Curve of Aeromonas.

17 Effect of ph of the fermentation medium was studied by adjusting the ph with sodium hydroxide and acetic acid. The results are given in Figure Growth Activity Figure: Effect of ph on growth and enzyme production in Aeromonas.

18 Effect of temperature was studied by incubating 25 ml peptone water inoculated with 2.5 ml seed culture, at various temperatures and the protein content was determined. The enzyme activity was measured as mentioned earlier. The results are given in Figure Temperature t C) Figure: Effect of Temperature on growth and enzyme production in Aeromonas.

19 The optimum concentration of sodium chloride on growth and enzyme production was studied by adding different quantities of sodium chloride to the medium. The results are shown in Figure I - Growth I I -*-- Enzyme activity Concentration of sodium chloride(%) Figure: Optimum concentration of Sodium Chloride for growth and enzyme production in Aerouonas.

20 The effect of various salts on growth and enzyme production was studied by supplementing various mineral salts (AR) to the peptone water medium to a final concentration of 0.25%. The results are given in Table Table 11.4 Effect of salts on production of L-asparaginase in Aeromonas - - Salt Activity/ml Specific + SEM) - (IU mg-' protein) Peptone water medium (control) Potassium acetate Ammonium sulphate Ammonium oxalate Potassium dihydrogen phosphate Ferric nitrate Ammonium chloride Elanganous sulphate Sodium dihydrogen phosphate Calcium carbonate Sodium acetate Disodium hydrogen phosphate

21 Salt Activity/ml Specific activity (IU m1-i SEMI (IU my-l protein) - - Ammonium dihydrogen phosphate Potassium iodide Magnesium sulphate Potassium nitrite Potassium oxalate Ferric chloride Potassium nitrate Potassium chloride Zinc sulphate Cupric acetate Ammonium nitrate Barium chloride Sodium nitrate Magnesium chloride Calcium chloride Sodium sulphate Mercuric nitrate NGA* Mercuric chloride Mercuric sulphate Cupric nitrate NGA NGA NGA * NGA - No growth and enzyme activity

22 The optimum concentration of substrates and products was studied by supplementing asparagine (Sigma), glutamine (SRL), aspartic acid (SRL) and glutamic acid (SRL) to the synthetic medium. The results are given in Figures 11.5, 11.6, 11.7 and 11.8 respectively. Concentration of asparaqine ( %I Figure: Optimum concentration of I.- Asparagine for growth and enzyme production in Aeromonas.

23 - 4 I rl E c.r( $4.0- C $ L, w Growth -- Activity,--*---- * I - - d I 4 E H h -0.6 u.d >.rl u -0.4 o d concentration of glutamine (8) Figure: Optimum concentration of L- Glutamine for growth and enzyme production in Aeromonas.

24 ,-I I rl E - Growth Activity Concentration of aspartic acid (%) Figure: Optimum concentration of L- Aspartic acid for growth and enzyme production in Aeromnas.

25 Concentration of glumatic acid (O) Figure: Optimum concentration of Glutamic acid for growth and enzyme production in Aeromonas.

26 Since lactate was reported to have a stimulatory effect on L-asparaginase production in microbes, by many workers, the optimum concentration for the production of the enzyme by the estuarine Aeromonas was also worked out using different concentrations of lactate (HM) added to the synthetic medium. The results are given in Figure Grcwth -t-r. Activity Concentration of lactate (8) Figure: Optimum concentration of Lactate for growth and enzyme production in Aeroeonas.

27 The effect of various growth substances and trace elements on growth and enzyme production was also studied using various concentrations of yeast extract (HM) added to the synthetic medium. The results aye given in Figure Growth ---- Enzyme activity Concentration of yeast extract (8) Pigure: Optimum concentration of Yeast Extract for growth and enzyme production in Aeromonas. Aeromonas is capable of utilizing a variety of carbon and nitrogen sources. So the ability of the estuarine Aeromonas to utilize various carbohydrates (HM) as carbon source was studied and the results are given in Table

28 Table 11.5 Effect of carbon sources on growth and enzyme production in Aeromonas Activity/ml Specific activity Carbon source (IU ml-i - + SEM) ( IU mg-l protein) Sucrose Starch Mannitol Mannose Maltose Glucose Lactose Lactate Pyruvate Inulin Citrate Alpha ketoglutarate Basal medium: Sodium chloride (Ht4) 0.5 g Ammonium dihydrogen phosphate (HM) 1 g Potassium dihydroyen phosphate (AR) 100 mg Magnesium sulphate (AR) 30 mg Tap water ml To this, various carbon compounds (HM) were added to a final concentration of 1% and the ph was adjusted to 7.5.

29 Various nitrogen sources (HM) were used to check the ability of this organism to utilize various nitrogenous compounds. The results are given in Table Table 11.6 Effect of nitrogen sources on growth and enzyme production in Aeromonas Nitrogenous compounds Peptone Ammonium dihydrogen phosphate Beef Extract 4mmonium sulphate Lab-Lemco Ammonium nitrate Casein Urea Ammonium oxalate Ammonium chloride Creatine Activity/ml Specific activity (TU ml-i - + SEM) (IU mg-i protein ) No growth & activity Basal medium: Starch (HM) 2 g Sodium chloride (HM) g Potassium dihydrogen Phosphate (HM) 100 mg Magnesium sulphate (AR) 30 mg Tap water ml To this, various nitrogenous compounds (HM) were added to a final concentration of 1% and the ph was adjusted to

30 The effect of various amino acids on growth and enzyme production in Aeromonas was studied by supplementing various amino acids (SRL) to a final concentration of 0.25%, to the synthetic medium. The results are given in Table Table 11.7 Effect Of amino acids on growth and enzyme production in Aeromonas Amino acids Activity/ml Specific activity SEM) (IU mg-a protein) Synthetic medium (control) Aspartic acid Asparayine Glutamine Proline Ary inine Glutamic acid Tryptophan Lysine Histidine Serine (Contd...) 5 5

31 Amino acids Activity/ml Specific activity (IU ml-i - + SEM) ( IU mg-l protein) Leucine Hydroxy proline Ornithine Phenyl alanine Glycine Alanine Valine Methionine Threonine Tyrosine Cystine Cysteine Growth inhibited Basal medium: Starch (HM) 2 9 Ammonium dihydrogen phosphate (HM) - 1 g Sodium chloride (HM) g Potassium dihydroyen phosphate (AR) mg Magnesium sulphate (AR) 30 mg Tap water ml Amino acids(srl) were added to the above medium to a final concentration of 0.25% and the ph was adjusted to 7.5. Optimum aeration was provided by shaking the culture on a rotary shaker.

32 From the growth curve it can be seen that after a lag period of 5 hrs, there is a log phase of 10 hrs and after that a stationary phase of growth is achieved. The enzyme production increased with the growth of the organism and reached an optimum level when it has approached the stationary phase. Similar trend was reported in Arthrobacter citreus[l93] and marine Vibrio[57]. In -- E. coli, the enzyme yield per ml of the culture was highest when the culture was within of the maximum exponential yrowth[86,1961. But in the yeast Candida guilliermondii (BKM-Y-421, higher activity was detected at the early logarithmic phase and the enzyme activity decreased to a minimum at the stationary phase[45]. However in estuarine Aeromonas the enzyme activity was maximum at 18th hour of incubation, i.e., during the stationary phase and hence the cells have been harvested during this period. The optimum ph for growth and enzyme production in Aeromonas was found to be between 6.5 and 8.0. In -- E. coli. the optimum ph was 7.8 and was reported to be between 7 and 8 [ The. optimum ph for Thermoactinomyces vulgaris was reported to be [88] Oc was found to be the optimum temperature for E. coli[86,87]. But in Thermoactinomyces vulyaris, the

33 optimum temperature reported was 55Oc[88] and in Achromobacteraceae the optimum temperature for enzyme production was reported to be between 15-20~~[771. The optimum temperature for growth and enzyme production in Aeromonas was found to be 35Oc as reported by Selvakumar in marine Vibriot571. A low concentration of sodium chloride (about 0.1 g%) was necessary for growth and enzyme production in Aeromonas, but higher concentrations of sodium chloride (above 2.5%) was found to inhibit growth and enzyme production contrary to that one would expect in estuarine bacteria. It was found that sucrose, starch and mannitol were the best stimulators of enzyme production. Alpha ketoglutarate, citrate, inulin, pyruvate etc. showed inhibition of enzyme production. Glucose was reported to have an inhibitory effect on L-asparaginase production in -- E. coli[85-87,1961. Pseudomonas[l971. Achromobacteraceae[72], Arthrobacter citreus[l93] and Vibrio cholerae[69]. Similar trend was observed in Aeromonas also. Some of the salts like potassium acetate, ammonium sulphate, potassium dihydrogen phosphate, ferric nitrate,

34 ammonium oxalate, ammonium chloride, calcium carbonate etc. were found to induce enzyme production significantly whereas, mercuric salts, copper salts and nitrates were inhibitors. Among the inducers acetates, sulphates, and phosphates were found to have the maximum effect. It was found that growth and enzyme activity increased with increasing concentrations of asparagine, aspartit acid, glutamine and ylutamic acid, as these were reported to have an activating effect in -- E. coli[86,87,196] and Erwinia carotovora[40]. Asparagine and aspartic acid were reported to have stimulator~ effect in -- E. coli[86,196], Arthrobacter citreus[l93], Vibrio proteus[70] and Candida[451. Lactic acid has been reported to have an inducing effect in E. coli[84,86,196] and - V. proteus[70]. The present study indicates that lactic acid is an activator only up to a level of 0.5% and is inhibitory above that level, contrary to other reports. The salinity tolerance of estuarine Aeromonas was studied in starch median. Sodium chloride was essential for growth and enzyme production but beyond the level of 0.75%, it was found to be inhibitory. No growth was observed when sodium chloride concentration was at or above 2.5%.

35 When sucrose was supplemented to the synthetic medium, growth and enzyme production was found to be maximum followed by starch and mannitol. The readily fermentable sugars like glucose showed an appreciable growth but the enzyme activity decreased considerably. Enzyme production was decreased when lactose, pyruvate~ inulin, alpha ketoglutarate, citrate etc. were used as carbon sources. Maximum yrowth and enzyme activity were obtained when peptone was added to the medium as nitrogen source. Among the inorganic nitrogen sources, ammonium dihydrogen phosphate was found to have more inducing effect followed by ammonium sulphate. The amino acids like aspartic acid, asparagine, glutaminei glutamic acid, proline, aryininet lysiner tryptophan, histidine etc. were found to increase growth and enzyme production considerably. Asparagine, aspartic acid, glutamine and glutamic acid were found to increase the enzyme production suggesting that the enzyme is not regulated by feed back inhibition. The production of enzyme in the absence of substrate indicates that the enzyme is constitutive. The sulphur containing amino acids (methionine, cystine and cysteine) were inhibitors in Aeromonas contrary to the report that methionine is essential for the production of E. coli L-asparaginase[84, 85,86,196]. 60

36 It was found that the growth and enzyme activity increased with increasing concentrations of yeast extract. Aeration was reported to be essential for obtaining maximum yield of L-asparaginase in marine Vibrio[57] and -- E. coli[86,196]. Aeration inhibited the production of enzyme in Vibrio proteus[70]. In the present study, it was found that shake cultures yielded maximum amount of the enzyme and proper aeration was provided by shaking the culture. It would be advantageous if we suggest a cheap culture medium, since industrial production o f L-asparaginase is essential, because of its increasing demands for therapeutic purpose and no attempt has been made in this direction. Both sucrose and starch media were found to be very good for the enzyme production in neromonas and is less expensive. The enzyme activity in Sucrose and Starch media were found to be and IU/ml whereas in Yeast Extract and Beef Extract media it was found to be and IU/ml respectively. Similarly specific activity in the above media were 0.228, 0.205, and respectively. It would be possible to reduce the cost of the medium by more than 75%. The composition of the media is given in Table 11.8.

37 Table 11.8 L-asparaginase production by Aeromonas in synthetic media Media Activity/ml Specific activity ( IU ml-i - + SEM) (IU mg-l protein) 1. Sucrose medium Sucrose (HM) Sodium chloride (HM) g Ammonium dihydrogen phosphate (HM) - 1cj Potassium dihydrogen phosphate (AR) my Magnesium sulphate (AR) - 30 mg Tap water ml 2. Starch medium Starch (HM) - 2 g Sodium chloride (HM) g Ammonium dihydrogen phosphate (HM) - 1 g Potassium dihydrogen phosphate (AR) mg Magnesium sulphate (AR) - 30 mg Tap water ml ph of the medium was adjusted to 7.5

38 Campbell et al. demonstrated the existence of E. coli L-asparaginase in two forms, EC1 and EC2[821. Only EC2 shows antitumor property. The production of EC2 enzyme in E. coli was induced by its substrates and products at a ph optimum between 7.0 and 7.8. Absence of readily fermentable sugars like glucose and presence of lactate, sodium chloride and amino acid, methionine, were the other conditions. Since E. coli and Aeromonas are related and the above conditions for EC2 L-asparaginase production in E. coli are comparable to that of Aeromonas L-asparaginase, it suggests that, L-asparaginase of Aeromonas do have the properties similar to E. coli EC2 which has been established in our studies (Chapter IV). Considering the great demand of the enzyme in the treatment of acute lymphatic leukaemia, the selection of a new strain, which can produce substantial amount of the enzyme in a cheap medium, with improved physiological and pharmacological properties as well as with serological properties quite different from the one used currently, will be worth for the production of the enzyme industrially.