Production of Diamino propionic acid ammonia lyase by a new strain of Salmonella typhimurium PU011
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Seeds of the legume plant Lathyrus sativus, which is grown in arid and semi arid tropical regions, contain Diamino Propionic acid (DAP). DAP is a neurotoxin, which, when consumed, causes a disease called Lathyrism. Lathryrism may manifest as Neurolathyrism or Osteolathyrism, in which the nervous system, and bone formation respectively, are affected. DAP ammonia lyase is produced by a few microorganisms such as Salmonella typhi, Salmonella typhimurium and Pseudomonas, and is capable of detoxifying DAP.
S. typhimurium PU011, a non-virulent bacterial strain isolated in our lab, was found to produce DAP ammonia lyase enzyme when grown in minimal medium containing DAP. There was a direct correlation between biomass yield and enzyme activity, until 16 h post inoculation in minimal medium containing DAP. Following ammonium sulphate precipitation and passing through Sephadex G100, CM-Sephadex and DEAE-Sephacel for crude enzyme extract preparation, about 68-fold enzyme purity was obtained. The purified enzyme gave maximum activity at pH 8.0 and was stable up to 45 degrees C. The Km value for the substrate was found to be 0.685mM, calculated from a Line Weaver Burk plot.
A new bacterial strain, S.typhimurium PU 011, which is capable of producing DAP ammonia lyase, was isolated.
KeywordsAmmonium Sulfate Post Inoculation Crude Enzyme Extract Maximum Enzyme Activity Virulence Assay
Lathyrus sativus, belonging to the family of leguminoceae, is a popular drought resistant legume plant whose grains serve as food in the drought prone areas of Asia and Africa. The 2,3 α-β-Diamino propionic acid (DAP) present in L. sativus seeds when consumed causes Lathyrism in humans, affecting either the nervous system (Neurolathyrism) or bone formation (Osteolathyrism), . Medical scientists evince keen interest in understanding the causes of neurolathyrism as a model for neurodegenerative diseases striking the more affluent sections of the human population. The scourge of neurolathyrism, known since ancient times, mainly affects the poor rural classes in developing countries. Though different methods such as boiling in water and development of DAP free cultivars , had been tried, as yet no effective method has been developed for safe consumption by the famine hit, poverty ridden, malnourished hungry millions in the drought prone areas in central India and Africa.
Rajagopal et al. (1970)  have studied the metabolism of DAP in Pseudomonas and demonstrated that it is metabolized to pyruvic acid through Ammonia lyase reaction. Very scant information is available on the physicochemical and catalytic properties of diamino propionic acid ammonia lyase. In this communication we report the production of DAP ammonia lyase by S. typhimurium PU011 and describe a few of its properties.
Results and Discussion
Virulence of S. typhimurium in an orally infected typhoid mouse model
Clinical Symptoms of liver and spleen
S. typhimurium PU011
Enlarged, inflamed & edematous
Control (Mice with PBS only)
Further, the mice injected with virulent S. typhimurium showed the clinical symptom of diarrohea prior to death, which was conspicuously absent in mice that were inoculated with S. typhimurium PU011. The clinical examination of sacrificed mice injected with virulent S. typhimurium revealed typical symptoms of enlarged, inflamed and edematous liver and spleen, which were absent in mice, injected with S. typhimurium PU011 strain. Conclusively this is evident enough to show that S. typhimurium PU011 strain employed in our studies is a non-virulent strain. To the best of our knowledge this is the first report about the production of DAP ammonia lyase by a non-virulent S. typhimurium with wider ramifications.
Biomass and Enzyme production
As to whether any carbon source other than DAP in the growth medium could support growth and enzyme production, it was observed that none other than DAP could substitute any of the carbon sources (data not shown). Since S. typhimurium PU011 employed in the present study was found to synthesize DAP ammonia lyase, it was thought fit to employ this enzyme for detoxification of DAP present in L. sativus seeds prior to consumption. Therefore, the characteristics of this enzyme were studied in detail.
Effect of pH
Effect of temperature
Purification of DAP ammonia lyase from S. typhimurium PU011
Purification Table of Diamino propionic acid ammonia lyase
Total Protein (mg)
Total Activity (U)
Specific Activity (U/mg)
Ammonium Sulphate precipitated
Passed through Sephadex – G100
Passed through CM – Sephadex
Passed through DEAE – Sephacel
The discovery of non-virulent S. typhimurium PU011 capable of producing DAP ammonia lyase has opened new vistas in the application of this enzyme in the tribal tracts of arid and semi-arid regions of Central India. For commercial exploitation of this enzyme, attempts are underway to clone and overexpress the gene coding for DAP ammonia lyase for applicative purposes in field.
At 16 h post inoculation S. typhimurium PU011 produced maximum biomass yield and maximum enzyme activity in minimal medium containing DAP. Interestingly no carbon source other than DAP supported DAP ammonia lyase synthesis in the medium effectively. With respect to specific enzyme activity of DAP ammonia lyase at different pH regimes, the enzyme had a sharp activity at pH 8.0 in phosphate buffer and at any pH less than 6 or more than 11 a drastic decrease in enzyme activity was observed. Up to 45°C there was no decrease in enzyme activity; however, thereafter there was decline in enzyme activity with increase in temperature. Based on SDS-PAGE analysis of the DAP ammonia lyase enzyme, the molecular weight was determined to be 43 KDa having a Km value of 0.685 mM.
Materials and Methods
Culture and media
Salmonella typhimurium PU011 employed in the present study was isolated in our lab and further characterized by standard microbiological, serological and biochemical tests . Cells were grown at 30°C in minimal medium (0.05% Yeast Extract, 500 μl Mineral salt solution and 2.0% Agar) supplemented with DL – DAP (0.3%) for production of DAP ammonia lyase by the bacterium.
Virulent S. typhimurium (obtained from the Department of Microbiology, Jawaharlal Institute of Post Graduate Medical Education and Research (JIPMER), Pondicherry, India) and S. typhimurium PU011 suspensions were grown at 37C in LB broth overnight, with shaking at 200 rpm. The next day, bacteria were diluted 1:100 into fresh LB broth and incubated with shaking. After 4 h, bacteria were washed once with PBS and resuspended in PBS. BALB/c female mice, aged 6–10 weeks, were inoculated orally with bacterial suspensions at 2.5 × 106 CFU per mouse, which is approximately twice the reported 50% Lethal Dose (LD50) , after being deprived of water for 4 h. Control mice were given PBS only. The liver and spleen of experimental mice that died on different days after administration of S. typhimurium were harvested, homogenized and the resultant slurry was spread on Mac Conkey plates to obtain bacterial counts. After sacrifice, the mice were critically examined for the presence of typical symptoms of enlarged, inflamed and edematous liver and spleen. Mice surviving after 28 days post inoculation were indicative of the non-virulence nature of S. typhimurium employed.
Determination of Biomass and Enzyme activity
Cells were grown in minimal medium supplemented with 0.3% DAP at 30°C on a rotary shaker (180 rpm) as per the method outlined by Premkumar  and Rupesh . For estimation of biomass, at various intervals of time 10 ml of culture was withdrawn, centrifuged at 8000 g for 10 min, washed once with distilled water, dried at 105°C and weighed. However, for estimation of enzyme activity 25 ml of culture was withdrawn, centrifuged at 8000 g for 10 min at 4°C, suspended in 0.5 ml of extraction buffer (10 mM Potassium phosphate buffer pH 8.0, 1 mM EDTA, 1 mM β – Mercaptoethanol, 100 μM Pyridoxal-5-Phosphate and 5% Glycerol) and sonicated for 10 min using Branson Sonifier 250 (10 min bursts at 40% duty cycle with intermittent cooling every 2 min in order to maintain the suspension below 5°C). After sonication, the lysate was centrifuged at 12,000 g for 10 min at 4°C and the supernatant served as the crude extract for the estimation of enzyme activity. The DAP ammonia lyase activity was measured as per the method of Vijiyalakhsmi et al . Protein estimation was done as per the method of Lowry et al .
Enzyme Assay (Vijayalakshmi et al., 1975)
DAP ammonia lyase was assayed spectrophotometrically, using a coupled assay system, utilizing the following reaction.
Sum: DAP + NADH + H2O + H+ → Lactate + NAD + 2NH3
The reduction in absorbance at 340 nm was measured in Hitachi U-2000 spectrophotometer. The assay system contained in a total volume of 3.0 ml in a 1 cm light path cuvette: 1 mmol Potassium phosphate buffer pH 8.0, 20 mmol DL-DAP, 0.3 μmol PLP, 15 Units of LDH, 1 μmol NADH and appropriate amount of enzyme. The reaction was studied at 28°C, and was started by the addition of DAP. The decrease in absorbance was recorded at 30 s intervals for a period of 2 min. One Unit of enzyme was defined as the amount of enzyme required for the conversion of 1 μmol of NADH to NAD+ in one minute. Specific activity is defined as Units per mg of protein.
Effect of pH
Optimum pH at which a maximum specific activity of DAP ammonia lyase could be obtained was determined by employing different buffer systems at various pH as indicated in parenthesis. The buffer systems employed were: Glycine-HCl buffer (pH 3), Sodium acetate buffer (pH 5), Potassium phosphate buffer (pH 7), Potassium phosphate buffer (pH 8), Glycine-NaOH (pH 9), Glycine-NaOH (pH 11) and KCl-NaOH (pH 13). The crude enzyme extract was taken and incubated with the above buffers for 30 min. After incubation, the crude extract was precipitated with ammonium sulfate (80% saturation) and the precipitate was resuspended in minimal volume of 10 mM potassium phosphate buffer. The effect of pH was studied by measuring DAP ammonia lyase activity as per the procedure described earlier.
Effect of Temperature
The crude enzyme extract in 10 mM Potassium phosphate buffer pH 8.0 was incubated at various temperatures ranging from 0°C – 70°C. After 30 min of incubation, the crude extract was analyzed for DAP ammonia lyase activity.
Purification of DAP ammonia lyase
Preparation of Cell free crude extract
Cell free crude extract of S. typhimurium PU011 was prepared by sonicating the pellet in 0.5 ml extraction buffer for 10 min using Branson Sonifier 250. The sonicate was then centrifuged at 12,000 g for 10 min at 4°C and the supernatant served as the crude enzyme extract.
Ammonium sulfate fractionation
The enzyme was precipitated by addition of appropriate amounts of ammonium sulfate (35–65% saturation) as per the method of Nagasawa et al, (1988) . The precipitate was collected by centrifugation and dissolved in 1 ml of extraction buffer and dialyzed against the same buffer for 24 h.
Chromatography on Sephadex G-100
The crude enzyme preparation was subjected to gel filtration on sephadex G-100 (1.6 × 20 cm) column chromatography. The dialyzed enzyme solution was applied to sephadex G-100 column, which had been equilibrated with 10 mM phosphate buffer. The active fractions that showed absorption at 280 nm were collected separately and DAP ammonia lyase in each fraction was quantified. The fractions that showed maximum enzyme activity were pooled and passed through CM-Sephadex column.
Chromatography on CM-Sephadex
Chromatography on CM-Sephadex column was performed on column (2 × 12.5 cm) of the resin equilibrated with 10 mM phosphate buffer pH 8.0, 1 mM β-mercaptoethanol and 1 mM pyridoxal-5-phosphate. The unbound fraction containing DAP ammonia lyase activity was reloaded onto DEAE-Sephacel column.
Chromatography on DEAE-Sephacel
The active enzyme fraction was applied to a DEAE-Sephacel column (2 × 20 cm), which had been equilibrated with 10 mM phosphate buffer pH 8.0, 1 mM β-mercaptoethanol and 1 mM pyridoxal-5-phosphate. The enzyme fractions were eluted using 0 – 1 M KCl gradient.
All the column chromatography purifications were carried out in BIORAD Econo system. The active fractions were precipitated with 80% ammonium sulfate. The precipitate was resuspended in a minimum amount of 10 mM phosphate buffer and dialyzed against the same buffer for 24 h with three changes under continuous stirring at 4°C. The progress of purification of DAP ammonia lyase was monitored both by enzyme activity measurement and on 12% SDS-PAGE.
SDS-Polyacrylamide Gel Electrophoresis (SDA-PAGE) (Laemmli, 1970) 
Routinely 0.75 mm thick gels were cast in a protein gel apparatus using a separating gel (12 %) and stacking gel (5%). The separating gel solution was first poured in between a sealed clean glass plate cassette leaving 3 to 4 cm space at the top. Water saturated n-butanol was layered gently on the top of the separating gel to make an even surface. After the separating gel got polymerized, the upper layer was removed completely and rinsed with water. Water was removed completely using a blotting paper. Stacking gel solution was poured onto the separating gel up to the top. A suitable comb was immediately inserted into the cassette and polymerization was allowed to take place. The slots were cleaned by rinsing with electrophoresis buffer. Protein samples were loaded into the slots and the slots were filled with 1 × electrophoresis buffer. The gel cassette was then fixed with the upper buffer tank and then fixed onto the lower buffer tank. Both the tanks were filled with 1 × electrophoresis buffer and electrophoresis was carried out at a constant current of 15 mA.
After electrophoresis was over, the gel cassette was dismantled and the gel was placed in a tray containing Coomassiae Brilliant Blue staining solution (0.1 % Coomassie Brilliant Blue (R 250), 45 % Methanol, 10 % Glacial Acetic Acid and 45 % Distilled water) and the gel was agitated slowly for 4 h to overnight. The staining solution was poured out and replaced with destaining solution (45 % Methanol, 10 % Glacial Acetic Acid and 45 % Distilled water). Destaining was carried out for a minimum of 2 h with gentle agitation.
The assistance rendered by M. K. Natarajan, JIPMER, Pondicherry, India in carrying out the microbiology work is gratefully acknowledged.
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