Detection of<i>γ</i>-Polyglutamic Acid (<i>γ</i>-PGA) by SDS-Page

Yamaguchi, Fumio; Ogawa, Yoshihiro; Kikuchi, Mamoru; Yuasa, Katsumi; Motai, Hiroshi

doi:10.1271/bbb.60.255

Cited by 39 publications

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“…The molecular weight of proteins was estimated by comparison with protein markers (Daiichi Pure Chemistry Co.). SDS-PAGE of PGA was also done using a 10% polyacrylamide gel by the method of Yamaguchi et al (45). The amount of PGA was determined by an amino acid analyzer after acid hydrolysis according to the method of Ogawa et al (30).…”

Section: Methodsmentioning

confidence: 99%

Characterization of the Bacillus subtilis ywsC Gene, Involved in γ-Polyglutamic Acid Production

2002

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The genes required for ␥-polyglutamic acid (PGA) production were cloned from Bacillus subtilis IFO16449, a strain isolated from fermented soybeans. There were four open reading frames in the cloned 4.2-kb DNA fragment, and they were almost identical to those in the ywsC and ywtABC genes of B. subtlis 168. Northern blot analysis showed that the four genes constitute an operon. Three genes, ywsC, ywtA, and ywtB, were disrupted to determine which gene plays a central role in PGA biosynthesis. No PGA was produced in ⌬ywsC and ⌬ywtA strains, indicating that both of these genes are essential for PGA production. To clarify the function of the YwsC protein, histidine-tagged YwsC (YwsC-His) was produced in the ⌬ywsC strain and purified from the lysozyme-treated lysate of the transformant by Ni-nitrilotriacetic acid affinity chromatography. Western blot analysis revealed that the YwsC-His protein consists of two subunits, the 44-kDa and 33-kDa proteins, which are encoded by in-phase overlapping in the ywsC gene.14 C-labeled PGA was synthesized by the purified proteins from L- Some Bacillus strains produce ␥-polyglutamic acid (PGA), an amino acid polymer that consists of only D-glutamic acid or D-and L-glutamic acid polymerized through ␥-glutamyl bonds, as a capsular or an extracellular viscous material (6). PGA was first discovered as a component of the capsule of Bacillus anthracis (19) and Bacillus mesentericus (18) and was isolated from the culture medium of Bacillus subtilis (7). Since then, a number of bacteria producing PGA, including B. subtilis (8,17,21,22), Bacillus licheniformis (9, 42), and Bacillus megaterium (13, 41), have been reported. PGA is a main constituent of the sticky material in natto, a Japanese traditional food made from soybeans that have been steamed and then fermented by B. subtilis (14).Concerning PGA biosynthesis, Makino et al. reported cloning of three genes, capBCA, responsible for capsular PGA biosynthesis from B. anthracis, and the gene products occurred together as membrane-associated proteins in the Escherichia coli transformant (26,27,43). The complete genome sequence of B. subtilis 168, in which ywsC and ywtAB were found to be highly homologous to the capBCA genes of B. anthracis, has been made available in databases (31). Recently, pgsBCA genes for PGA biosynthesis were also cloned from B. subtilis IFO3336, and their sequences were found to be the same as those of the ywsC and ywtAB genes of B. subtilis 168 (2). These three genes seem to be involved in PGA production; however, little is known about the function of each gene product in PGA biosynthesis.In this paper, we describe the cloning and gene disruption of the ywsC and ywtAB genes, which are responsible for PGA production in B. subtilis IFO16449, a strain isolated from natto, and we also describe the characterization of the YwsC 44-kDa and 33-kDa proteins, which catalyze the biosynthesis of PGA from L-glutamate, a crucial enzyme in PGA production. MATERIALS AND METHODSBacterial strains, plasmids, and media. Bacillus subtilis IF...

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Section: Methodsmentioning

confidence: 99%

Characterization of the Bacillus subtilis ywsC Gene, Involved in γ-Polyglutamic Acid Production

2002

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“…Each sample solution (20.uI) \vas put on a 4-15°;;) gradient acrylamide slab gel (Daiichi Pure Chemicals, Ltd.), and then electrophoresis was perfonned to separate the labeled y-PGA from free 14( -glucose or 14C-glutamic acid. The y-PGA was detected by staining with alcian blue as described by Yamaguchi et al,22) and the radioactivity of y-PGA was detected by autoradiography. The spots corresponding to y-PGA were cut from the gel and the radioactivity was counted in a toluene scintillation solution.…”

Section: L'+c-glucose and 14c-l-glutamic Acid Incol1)()}'(ltioll Intomentioning

confidence: 99%

Efficient Production of γ-Polyglutamic Acid byBacillus subtilis(natto) in Jar Fermenters

Ogawa

Yamaguchi

Yuasa

et al. 1997

Bioscience, Biotechnology, and Biochemistry

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“…Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of PGA was performed using 10% polyacrylamide gel by the method of Yamaguchi et al (1996). The amount of PGA in the culture was determined by an amino acid analyzer after acid hydrolysis according to the method of Ogawa et al (1997).…”

Section: Methodsmentioning

confidence: 99%

Phylogeny of γ-polyglutamic acid-producing Bacillus strains isolated from a fermented locust bean product manufactured in West Africa

Meerak

Yukphan

Miyashita

et al. 2008

J. Gen. Appl. Microbiol.

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Detection ofγ-Polyglutamic Acid (γ-PGA) by SDS-Page

Cited by 39 publications

References 0 publications

Characterization of the Bacillus subtilis ywsC Gene, Involved in γ-Polyglutamic Acid Production

Characterization of the Bacillus subtilis ywsC Gene, Involved in γ-Polyglutamic Acid Production

Efficient Production of γ-Polyglutamic Acid byBacillus subtilis(natto) in Jar Fermenters

Phylogeny of γ-polyglutamic acid-producing Bacillus strains isolated from a fermented locust bean product manufactured in West Africa

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