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Purification and characterization of proteases from Bacillus amyloliquefaciens isolated from traditional soybean

Seong-Jun Cho1, Sung-Hoon Oh, R David Pridmore

  • 1Graduate School of Biotechnology, Korea University, Seoul 136-701, Korea.

Journal of Agricultural and Food Chemistry
|December 11, 2003
PubMed

Insights

This study identifies two novel proteases, neutral metalloprotease (NPR68) and alkaline serine-protease (APR68), from Bacillus amyloliquefaciens FSE-68. These enzymes exhibit distinct specificities, suggesting they are natural variants of known proteases.

Area of Science:

  • Microbiology
  • Enzymology
  • Biochemistry

Background:

  • Bacillus amyloliquefaciens is a key microorganism in traditional Korean fermented foods like meju.
  • Proteases play crucial roles in food fermentation and biotechnological applications.
  • Characterization of novel enzymes from microbial sources is essential for discovering new biocatalysts.

Purpose of the Study:

  • To isolate and identify Bacillus amyloliquefaciens FSE-68 from meju.
  • To purify and characterize a neutral metalloprotease (NPR68) and an alkaline serine-protease (APR68) from the isolate.
  • To determine the substrate specificity and molecular weight of the purified proteases.

Main Methods:

  • Bacterial isolation and identification using biophysical tests and 16S rRNA gene sequencing.
  • Enzyme purification via ammonium sulfate precipitation and cation exchange chromatography.
  • Molecular weight determination using Electrospray Ionization Mass Spectrometry (ESI-MS).
  • Substrate specificity analysis using oxidized insulin chains.

Main Results:

  • Bacillus amyloliquefaciens FSE-68 was successfully isolated and identified.
  • Purification yielded a neutral metalloprotease (NPR68) and an alkaline serine-protease (APR68).
  • NPR68 (32743 Da) showed cleavage preference at Leu (P1') followed by Phe.
  • APR68 (27443 Da) exhibited broad specificity, favoring Leu at the P1 site.
  • The identified proteases were determined to be natural variants of subtilisin and bacillolysin.

Conclusions:

  • Bacillus amyloliquefaciens FSE-68 produces two distinct proteases with unique characteristics.
  • NPR68 and APR68 represent novel variants of subtilisin and bacillolysin, respectively.
  • These findings contribute to understanding microbial enzyme diversity and potential applications in biotechnology.

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