CHAPTER I1 SOURCES OF...
Transcript of CHAPTER I1 SOURCES OF...
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.
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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.