CHAPTER I1

SOURCES OF L-ASPARAGINASE

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[121-124,1271. 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

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. 76°33'-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 1989. 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 3 5 O ~ for 5 days. The colour of the medium

changed from yellow to red around some colonies.

28

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], Strickland and

Parsons[l87] and Grasshoff[l88].

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 3 5 O ~ for

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 ( A R ) 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

Table 11.1.

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 02 13 05 - -

Aeromonas 46 07.0 M 01 20 04 - 01

Alkaliqenes 2 7 04.2 n - 06 08 05 - S 48 15 - - -

eacillus 127 19.8 n 47 17 - - -

Cvtophayafla- S 06 07 01 0 1 - vobacterium 34 05.3

M 06 11 02 - - Enterobacteri- S - - 05 0 9 0 7 aceae 54 08.4

n - 01 05 10 17

S 12 10 - - - ~icrococcus 4 7 07.3

M 14 11 - - -

Sarcina

Vibrio

...................................................................... Total No. in each 200 191 108 85 58 642 - group ...................................................................... Percentaye of 31.2 29.8 16.8 13.2 09.1 - 100% each group

*S - Sediment **Group I - 5.000-0.05 IU/mg of protein n - ~ussal Group II - 0.051-0.1 Iu/mq of protein

Group 111 - 0.101-0.15 IU/mq of protein Group IV - 0.151-0.2 IU/mg of protein Group V - >0.2 Iu/mq of protein

- 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.

(0.8185 2 0.01 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 - 291 Pseudomonas - 185 Vibrio - 268 Vibrio - 439 Enterobacteriaceae - 608 Enterobacteriaceae - 357 Aeromonas - 382

* Number indicates the strain number in the order of collection.

The generic composition of L-asparaginase positive

bacterial population isolated from estuarine sediments and

molluscs is also given in Table 11.1. 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).

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

(X ( p g 1-I) ( r g 1-I] (mg % ) 4

( X ~ O g-l) ( X 10' g-l)

Aug. 1988 04.80 0.65 05.90 0.20 Sept . 02.28 0.65 04.95 0.34

I Oct. 04.12 1.53 11.20 0.22 NOV . 10.30 1.71 07.85 0.26 Dec . 25.80 0.94 07.22 0.28 Jan. '89 27.00 0.75 07.87 0.22

Aug. 1988 04.80 0.63 05.50 0.28 Sept . 02.41 0.62 04.28 0.33

I1 Oct . 04.48 1.52 12.15 0.38 NOV . 10.40 1.62 04.95 0.40 Dec . 27.60 0.89 05.86 0.36 Jan. '89 28.00 0.67 07.48 0.29

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,

-0.69 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 = +0.56 and +0.66

respectively).

A profound effect was observed on the growth of total

heterotrophic population and percentage of L-asparaginase

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.

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[40169,70,88119311941. 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.

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 - Gram negative straight rods Motility - Motile Oxidase reaction - Positive Growth in medium containing - Positive bile salts

Sensitivity to vibriostatic - Negative agents, 0/129 (2,4 diamino- 6,7,diisopropyl pteridine)

Indole production in 1% peptone - Positive water

Growth in KCN broth

Nitrate reduction

- Positive - Positive

L-Arabinose utilization - Positive Fermentation of salicin - Positive Fermentation of mannitol - Positive Breakdown of inositol - Negative

Catalase - Positive Acetoin from glucose - Positive (Voges - Proskauer test) 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) - 1 g

Sodium chloride (HM) - 500 mg

Distilled water - 100 ml

b) Synthetic medium

Starch (HM) - 2 g

Sodium chloride (HM) - 500 mg

Ammonium dihydrogen - 1 9 phosphate (HM)

Potassium dihydrogen - 100 mg phosphate (AR)

Magnesium sulphate (AR)- 30 mg

Tap water - 100 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.

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 11.1.

-Growth *--- Activity I

Incubation time (hr.)

Figure: 11-1. Growth Curve of Aeromonas.

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 11.2.

- Growth ---.. Activity

Figure: 11-2. Effect of pH on growth and enzyme production in Aeromonas.

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 11.3.

0 10 20 30 40 50 6 0

Temperature t°C)

Figure: 11-3. Effect of Temperature on growth and enzyme production in Aeromonas.

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 11.4.

0 1.0 2.0 2 . 5

Concentration of sodium chloride(%)

I - Growth I I -*-- Enzyme activity

Figure: 11-4. Optimum concentration of Sodium Chloride for growth and enzyme production in Aerouonas.

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 11.4.

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

Activity/ml Specific

Salt activity

(IU m1-I 7

+ SEMI - (IU my-l protein) - -

Ammonium dihydrogen phosphate 0.581 + 0.025 - 0.262

Potassium iodide 0.581 - + 0.029 0.270

Magnesium sulphate 0.536 - + 0.030 0.260

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

Mercuric chloride

Mercuric sulphate

Cupric nitrate

0.533 - + 0.031

0.492 - + 0.028

0.469 - + 0.027

0.436 - + 0.024

0.434 - + 0.018

0.402 + - 0.019 0.402 - + 0.021

0.399 - + 0.018

0.398 5 0.024

0.335 + 0.014 - 0.224 - + 0.012

0.223 - + 0.011

0.045 - + 0.009

NGA*

NGA

NGA

NGA

* NGA - No growth and enzyme activity

The optimum concentration of substrates and products

was studied by supplementing asparagine (Sigma),

glutamine ( S R L ) , 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: 11-5. Optimum concentration of I.- Asparagine for growth and enzyme production in Aeromonas.

concentration of glutamine ( 8 )

Figure: 11-6. Optimum concentration of L- Glutamine for growth and enzyme production in Aeromonas.

- d

I 4 E

4 - 8 3 H - h

- 0 . 6 u .d > .rl u

- 0 . 4 o d

- 0 . 2

- 4 I rl E c .r(

$ 4 . 0 -

0 0.5 1.0 1.5 2.0

- -Growth -- Activity

,--*---- * - - - - - - I - C $2.0. 0 L, w 1.0-

- Growth ,-I I A c t i v i t y rl E

Concentration of aspartic acid ( % )

Figure: 11-7. Optimum concentration of L- Aspartic acid for growth and enzyme production in Aeromnas.

Concentration of glumatic acid ( O )

Figure: 11-8. Optimum concentration of Glutamic acid for growth and enzyme production in Aeromonas .

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 11.9.

- Grcwth -t-r. Activity

0 0 . 5 1.0 1.5 2 . 0

Concentration of lactate ( 8 )

Figure: 11-9. Optimum concentration of Lactate for growth and enzyme production in Aeroeonas .

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 11.10.

- Growth ---- Enzyme activity

-

Concentration of yeast extract ( 8 )

Pigure: 11-10. 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 11.5.

52

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 - 100 ml

To this, various carbon compounds (HM) were added to a final concentration of 1% and the pH was adjusted to 7.5.

Various nitrogen sources (HM) were used to check the

ability of this organism to utilize various nitrogenous

compounds. The results are given in Table 11.6.

Table 11.6

Effect of nitrogen sources on growth and enzyme production in Aeromonas

Nitrogenous compounds

Activity/ml Specific activity

(TU ml-I + SEM) - (IU mg-I

protein )

Peptone

Ammonium dihydrogen phosphate

Beef Extract

4mmonium sulphate

Lab-Lemco

Ammonium nitrate

Casein

Urea

Ammonium oxalate

Ammonium chloride

Creatine

0.737 + 0.038 - 0.217

0.483 + 0.019 - 0.288

0.476 + - 0.020 0.182

0.474 - + 0.021 0.300

0.469 - + 0.018 0.186

0.431 - + 0.015 0.255

0.407 - + 0.014 0.145

0.383 - + 0.017 0.446

0.241 - + 0.008 0.127

0.239 + 0.011 - 0.165

No growth & activity

Basal medium:

Starch (HM) - 2 g Sodium chloride (HM) - 0.5 g Potassium dihydrogen

Phosphate (HM) - 100 mg Magnesium sulphate ( A R ) - 30 mg Tap water - 100 ml

To this, various nitrogenous compounds (HM) were added to a final concentration of 1% and the pH was adjusted to 7.5.

54

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 11.7.

Table 11.7

Effect Of amino acids on growth and enzyme production in Aeromonas

Amino acids

Activity/ml Specific activity

7

+ SEM) - (IU mg-A protein)

Synthetic medium (control) 0.483 + 0.019 - 0.288

Aspartic acid 0.877 - + 0.038 0.383

Asparayine

Glutamine

Proline 0.804 - + 0.036 0.391

Ary inine 0.790 + 0.034 - 0.398

Glutamic acid 0.744 + 0.032 - 0.381

Tryptophan 0.729 - + 0.029 0.536

Lysine 0.730 2 0.030 0.380

Histidine 0.672 - + 0.026 0.315

Serine 0.669 + 0.027 - 0.369

(Contd...)

5 5

Amino acids

Activity/ml Specific activity

(IU ml-I + SEM) - ( IU mg-l

protein)

Leucine

Hydroxy proline 0.627 + 0.024 - 0.324

Ornithine

Phenyl alanine

Glycine 0.581 + 0.028 - 0.235

Alanine

Valine

Methionine 0.462 + 0.024 - 0.274

Threonine 0.457 + 0.023 - 0.249

Tyrosine 0.446 + 0.024 - 0.243

Cystine 0.241 + 0.023 - 0.204

Cysteine Growth inhibited

Basal medium:

Starch (HM) - 2 9 Ammonium dihydrogen phosphate (HM) - 1 g Sodium chloride (HM) - 0.5 g Potassium dihydroyen phosphate (AR) - 100 mg Magnesium sulphate ( A R ) - 30 mg Tap water - 100 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.

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 50-708 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 [85-871. The. optimum pH for

Thermoactinomyces vulgaris was reported to be 8-8.2 [88].

37Oc was found to be the optimum temperature for

E. coli[86,87]. But in Thermoactinomyces vulyaris, the --

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,

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%.

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

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 0.554 and

0.483 IU/ml whereas in Yeast Extract and Beef Extract

media it was found to be 0.541 and 0.471 IU/ml

respectively. Similarly specific activity in the above

media were 0.228, 0.205, 0.314 and 0.182 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.

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) - 2 9

Sodium chloride (HM) - 0.5 g

Ammonium dihydrogen phosphate (HM) - 1cj 0.554 - + 0.018 0.205

Potassium dihydrogen phosphate (AR) - 100 my

Magnesium sulphate (AR) - 30 mg

Tap water - 100 ml

2. Starch medium

Starch (HM) - 2 g

Sodium chloride (HM) - 0.5 g

Ammonium dihydrogen phosphate (HM) - 1 g 0.483 - + 0.019 0.288

Potassium dihydrogen phosphate (AR) - 100 mg

Magnesium sulphate (AR) - 30 mg

Tap water - 100 ml

pH of the medium was adjusted to 7.5

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.