Production of polyglutamic acid-like mucilage protein by Peribacillus simplex strain 8h.

Polyglutamic acid (PGA), a protein in the mucilage of PGA-producing Bacillus spp., has expected applications in medical and biotechnological industries. Although the Bacillaceae family contains over 100 genera, research on bacterial PGA has exclusively focused on the genus Bacillus, especially B. subtilis var. natto and B. licheniformis. In the present study, indigenous Bacillaceae family strains were isolated from withered leaves and soil samples and screened for PGA production. As a result of the screening, the strain 8h was found to produce a mucilage possessing greater viscosity than PGA of B. subtilis var. natto (natto PGA). Biochemical analyses revealed that the 8h mucilage contains 63% protein and 37% polysaccharide, while mucilage of B. subtilis var. natto is composed of 61% protein and 39% polysaccharide. The most plentiful amino acid in 8h mucilage protein was glutamate (43%, mol/mol), which is similar to that of natto PGA, suggesting that it possesses characteristics of PGA. Although natto mucilage contains fructan, glucan was found as the polysaccharide of 8h mucilage. While phylogenetic studies indicated that the strain 8h belongs to Peribacillus simplex, the yield of the viscous mucilage by strain 8h was significantly higher than P. simplex type strain, suggesting that 8h is a mucilage-overproducing strain of P. simplex. Interestingly, 8h mucilage protein was found to contain more hydrophobic amino acid residues than natto PGA, suggesting that its amphiphilicity is suitable as a drug carrier and adjuvant. The present study is the first report of viscous mucilage and PGA-like protein produced by the genus Peribacillus.

Enzymatic characteristics of D-mannose 2-epimerase, a new member of the acylglucosamine 2-epimerase superfamily.

Carbohydrate epimerases and isomerases are essential for the metabolism and synthesis of carbohydrates. In this study, Runella slithyformis Runsl_4512 and Dyadobacter fermentans Dfer_5652 were characterized from a cluster of uncharacterized proteins of the acylglucosamine 2-epimerase (AGE) superfamily. These proteins catalyzed the intramolecular conversion of D-mannose to D-glucose, whereas they did not act on ß-(1 → 4)-mannobiose, N-acetyl-D-glucosamine, and D-fructose, which are substrates of known AGE superfamily members. The kcat/Km values of Runsl_4512 and Dfer_5652 for D-mannose epimerization were 3.89 and 3.51 min-1 mM-1, respectively. Monitoring the Runsl_4512 reaction through 1H-NMR showed the formation of ß-D-glucose and ß-D-mannose from D-mannose and D-glucose, respectively. In the reaction with ß-D-glucose, ß-D-mannose was produced at the initial stage of the reaction, but not in the reaction with α-D-glucose. These results indicate that Runsl_4512 catalyzed the 2-epimerization of the ß-anomer substrate with a net retention of the anomeric configuration. Since 2H was obviously detected at the 2-C position of D-mannose and D-glucose in the equilibrated reaction mixture produced by Runsl_4512 in 2H2O, this enzyme abstracts 2-H from the substrate and adds another proton to the intermediate. This mechanism is in accordance with the mechanism proposed for the reactions of other epimerases of the AGE superfamily, that is, AGE and cellobiose 2-epimerase. Upon reaction with 500 g/L D-glucose at 50 °C and pH 8.0, Runsl_4512 and Dfer_5652 produced D-mannose with a 24.4 and 22.8% yield, respectively. These D-mannose yields are higher than those of other enzyme systems, and ME acts as an efficient biocatalyst for producing D-mannose.