γ-PGA Synthesis Research Articles

Mutations Suppressing the Loss of DegQ Function in Bacillus subtilis (natto) Poly-γ-Glutamate Synthesis

The degQ gene of Bacillus subtilis (natto), encoding a small peptide of 46 amino acids, is essential for the synthesis of extracellular poly-gamma-glutamate (γPGA). To elucidate the role of DegQ in γPGA synthesis, we knocked out the degQ gene in Bacillus subtilis (natto) and screened for suppressor mutations that restored γPGA synthesis in the absence of DegQ. Suppressor mutations were found in degS, the receptor kinase gene of the DegS-DegU two-component system. Recombinant DegS-His(6) mutant proteins were expressed in Escherichia coli cells and subjected to an in vitro phosphorylation assay. Compared with the wild type, mutant DegS-His(6) proteins showed higher levels of autophosphorylation (R208Q, M195I, L248F, and D250N), reduced autodephosphorylation (D250N), reduced phosphatase activity toward DegU, or a reduced ability to stimulate the autodephosphorylation activity of DegU (R208Q, D249G, M195I, L248F, and D250N) and stabilized DegU in the phosphorylated form. These mutant DegS proteins mimic the effect of DegQ on wild-type DegSU in vitro. Interestingly, DegQ stabilizes phosphorylated DegS only in the presence of DegU, indicating a complex interaction of these three proteins.

Loss of poly-.GAMMA.-glutamic Acid Synthesis of Bacillus subtilis (natto) Due to IS4Bsu1 Translocation to swrA Gene

The mobile genetic element IS4Bsu1 of Bacillus subtilis (natto), the commercial strain used for natto fermentation, was found to spontaneously translocate to the swrA gene, which led to a defect in poly-γ-glutamic acid (γPGA) synthesis. The γPGA defect of NAFIS1 (swrA::IS4Bsu1) was restored by a plasmid harboring the swrA gene. The insertion of IS4Bsu1 to the swrA gene appeared not to interfere with minJ gene expression, located downstream of the swrA gene, because a defect in cell motility exhibited by NAF16 (minJ::Spcr) was not observed in NAFIS1 (swrA::IS4Bsu1). We found that the minJ gene is essential for γPGA synthesis.

Biosynthesis of Polyglutamic Acid and its Application on Agriculture

Poly γ-glutamic acid (γ-PGA) is a kind of water soluble and biodegradable polymer, which is nontoxic toward humans and the environment. Therefore, the potential applications of γ-PGA and its derivatives have been of interest in a broad range such as food, cosmetics, medicine, water treatment and agriculture. This paper summarizes γ-PGA synthesis pathways in microorganisms, producing strains, fermentation parameters, as well as separation and purification methods. In addition, the prospect of γ-PGA application on agriculture was introduced, pointing out the problems and the future trends in research and application of γ-PGA.

Depressed Biofilm Production in Bacillus amyloliquefaciens C06 Causes γ-Polyglutamic Acid (γ-PGA) Overproduction

Bacillus amyloliquefaciens C06, a potential agent in biological preservation of post-harvest fruit, was found to secrete extra-cellular γ-polyglutamic acid (γ-PGA) in liquid culture. In this work, M306, a transposon mutant of B. amyloliquefaciens C06, defective in forming structured colony and displaying enhanced ability of producing γ-PGA, was obtained. Inverse PCR and quantitative reverse transcription PCR (qRT-PCR) analysis demonstrated that the defective phenotype in M306 was associated with an ORF showing high similarity to RBAM_034550 from B. amyloliquefaciens FZB42. In this paper, the ORF was designated pbrA, standing for γ-PGA production and biofilm formation regulatory factor. qRT-PCR analysis also indicated that pbrA down-regulated mRNA expression of epsD and yqxM, the crucial genes involved in biofilm formation, but affected little on expression of ywtB, the gene directing γ-PGA synthesis. Evaluations in γ-PGA productivity of wild-type C06 and its mutants C06ΔepsA and C06ΔtasA, respectively, deficient in producing exopolysaccharides (EPS) and TasA, revealed that γ-PGA overproduction in M306 was probably due to the redistributed metabolic flux caused by defective production of EPS.

Purification and Properties ofγ-Glutamyltranspeptidase fromBacillus subtilis(natta)

To understand the mechanism by which γ-poly glutamic acid (γ-PGA) in the sticky material of natto was synthesized, we purified the γ-glutamyltranspeptidase (γ-GTP) (EC 2.3.2.2) from the culture broth of Bacillus subtilis (natto) to homogeneity. γ-GTP was composed of two subunits with molecular weight of 45,000 and 22,000. The N-terminal amino acid sequence of light subunit was homologous with that of γ-GTP from Escherichia coli. The optimum pH and temperature of activity were 8.5 and 60°C. The enzyme was inactivated by incubation for 15 min at pH 8.0 and 55°C, but little loss of the activity was detected at 40°C. γ-GTP used glutamine as a γ-glutamyl donor and acceptor for γ-PGA synthesis. Dipeptides were better γ-glutamyl acceptors than free amino acids.

Purification and Properties of .GAMMA.-Glutamyltranspeptidase from Bacillus subtilis (natto).

To understand the mechanism by which γ-polyglutamic acid (γ-PGA) in the sticky material of natto was synthesized, we purified the γ-glutamyltranspeptidase (γ-GTP) (EC 2.3.2.2) from the culture broth of Bacillus subtilis (natto) to homogeneity. γ-GTP was composed of two subunits with molecular weight of 45, 000 and 22, 000. The N-terminal amino acid sequence of light subunit was homologous with that of γ-GTP from Escherichia coli. The optimum pH and temperature of activity were 8.5 and 60°C. The enzyme was inactivated by incubation for 15 min at pH 8.0 and 55°C, but little loss of the activity was detected at 40°C. γ-GTP used glutamine as a γ-glutamyl donor and acceptor for γ-PGA synthesis. Dipeptides were better γ-glutamyl acceptors than free amino acids.