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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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SDS-PAGE analysis of DAP ammonia lyase produced by Salmonella typhimurium PU 011. The crude enzyme was purified and run on SDS-PAGE. Molecular weight marker (Lane a), Crude Enzyme Extract (Lane b), Enzyme obtained after passing through Ammonium Sulfate precipitation (Lane c), Sephadex G-100 (Lane d), CM-Sephadex (Lane e) and CM-Sephacel (Lane f).
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Figure 5: SDS-PAGE analysis of DAP ammonia lyase produced by Salmonella typhimurium PU 011. The crude enzyme was purified and run on SDS-PAGE. Molecular weight marker (Lane a), Crude Enzyme Extract (Lane b), Enzyme obtained after passing through Ammonium Sulfate precipitation (Lane c), Sephadex G-100 (Lane d), CM-Sephadex (Lane e) and CM-Sephacel (Lane f).

Mentions: According to the procedure described in the text, the enzyme was purified 68 fold (Table 2). When the purified enzyme was subjected to SDS-PAGE, a single band was observed corresponding to the molecular weight of 43 KDa (Fig 5). This is in conformity with the results of Vijiyalakshmi (1975) [6]. However, Nagasawa et al (1988) [7] reported that the relative molecular mass of DAP ammonia lyase produced by Salmonella typhimurium, estimated by ultra centrifugal equilibrium method, was 89,000 Da and the enzyme consisted of 2 subunits identical in Molecular mass.


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)

SDS-PAGE analysis of DAP ammonia lyase produced by Salmonella typhimurium PU 011. The crude enzyme was purified and run on SDS-PAGE. Molecular weight marker (Lane a), Crude Enzyme Extract (Lane b), Enzyme obtained after passing through Ammonium Sulfate precipitation (Lane c), Sephadex G-100 (Lane d), CM-Sephadex (Lane e) and CM-Sephacel (Lane f).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: SDS-PAGE analysis of DAP ammonia lyase produced by Salmonella typhimurium PU 011. The crude enzyme was purified and run on SDS-PAGE. Molecular weight marker (Lane a), Crude Enzyme Extract (Lane b), Enzyme obtained after passing through Ammonium Sulfate precipitation (Lane c), Sephadex G-100 (Lane d), CM-Sephadex (Lane e) and CM-Sephacel (Lane f).
Mentions: According to the procedure described in the text, the enzyme was purified 68 fold (Table 2). When the purified enzyme was subjected to SDS-PAGE, a single band was observed corresponding to the molecular weight of 43 KDa (Fig 5). This is in conformity with the results of Vijiyalakshmi (1975) [6]. However, Nagasawa et al (1988) [7] reported that the relative molecular mass of DAP ammonia lyase produced by Salmonella typhimurium, estimated by ultra centrifugal equilibrium method, was 89,000 Da and the enzyme consisted of 2 subunits identical in Molecular mass.

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