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Production of diamino propionic acid ammonia lyase by a new strain of Salmonella typhimurium PU011.

Rupesh KR, PremKumar PL, Shiva Kumar VV, Jayachandran SS - BMC Microbiol. (2002)

Bottom Line: 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 0.685 mM, calculated from a Line Weaver Burk plot.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biotechnology, Pondicherry University, Kalapet, Pondicherry 605014, India. krrupesh@yahoo.com

ABSTRACT

Background: 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.

Results: 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 0.685 mM, calculated from a Line Weaver Burk plot.

Conclusion: A new bacterial strain, S.typhimurium PU 011, which is capable of producing DAP ammonia lyase, was isolated.

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Effect of Temperature on the Stability of DAP ammonia lyase. The crude enzyme extract was incubated at various temperatures ranging between 20°C – 60°C in 10 mM phosphate buffer and the enzyme activity measured. The enzyme activity is expressed as (U/mg) × 10-3.
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Figure 4: Effect of Temperature on the Stability of DAP ammonia lyase. The crude enzyme extract was incubated at various temperatures ranging between 20°C – 60°C in 10 mM phosphate buffer and the enzyme activity measured. The enzyme activity is expressed as (U/mg) × 10-3.

Mentions: The enzyme activity was measured using spectrophotometer at temperatures ranging between 30°C – 60°C at increment of 5°C. Up to 45°C there was no decrease in enzyme activity; however, thereafter a decline in enzyme activity with increase in temperature was observed (Fig 4). The loss in DAP ammonia lyase enzyme activity with increase in temperature indicates the level of stability of the enzyme per se to different temperature regimes and not temperature activity relationship since the enzyme was not incubated along with the substrate. While 45°C was found to support maximum enzyme activity in Salmonella sp. (Nagasawa et al., 1988) [7], 40°C was found to support maximum activity in Pseudomonas (Vijiyalakshmi et al., 1975)[6]. The fact that maximum activity was scored at above ambient temperature is a good augury for the use of this enzyme in the field scale applications for detoxification of DAP in L. sativus in drought prone areas of the world where elevated temperatures and desiccation pose threats for inactivation of enzyme activity.


Production of diamino propionic acid ammonia lyase by a new strain of Salmonella typhimurium PU011.

Rupesh KR, PremKumar PL, Shiva Kumar VV, Jayachandran SS - BMC Microbiol. (2002)

Effect of Temperature on the Stability of DAP ammonia lyase. The crude enzyme extract was incubated at various temperatures ranging between 20°C – 60°C in 10 mM phosphate buffer and the enzyme activity measured. The enzyme activity is expressed as (U/mg) × 10-3.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC101404&req=5

Figure 4: Effect of Temperature on the Stability of DAP ammonia lyase. The crude enzyme extract was incubated at various temperatures ranging between 20°C – 60°C in 10 mM phosphate buffer and the enzyme activity measured. The enzyme activity is expressed as (U/mg) × 10-3.
Mentions: The enzyme activity was measured using spectrophotometer at temperatures ranging between 30°C – 60°C at increment of 5°C. Up to 45°C there was no decrease in enzyme activity; however, thereafter a decline in enzyme activity with increase in temperature was observed (Fig 4). The loss in DAP ammonia lyase enzyme activity with increase in temperature indicates the level of stability of the enzyme per se to different temperature regimes and not temperature activity relationship since the enzyme was not incubated along with the substrate. While 45°C was found to support maximum enzyme activity in Salmonella sp. (Nagasawa et al., 1988) [7], 40°C was found to support maximum activity in Pseudomonas (Vijiyalakshmi et al., 1975)[6]. The fact that maximum activity was scored at above ambient temperature is a good augury for the use of this enzyme in the field scale applications for detoxification of DAP in L. sativus in drought prone areas of the world where elevated temperatures and desiccation pose threats for inactivation of enzyme activity.

Bottom Line: 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 0.685 mM, calculated from a Line Weaver Burk plot.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biotechnology, Pondicherry University, Kalapet, Pondicherry 605014, India. krrupesh@yahoo.com

ABSTRACT

Background: 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.

Results: 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 0.685 mM, calculated from a Line Weaver Burk plot.

Conclusion: A new bacterial strain, S.typhimurium PU 011, which is capable of producing DAP ammonia lyase, was isolated.

Show MeSH
Related in: MedlinePlus