Chemistry and Biosynthesis of the Poly(γ-d-glutamyl) Capsule in Bacillus licheniformis

Results of chemical, physical, and enzymatic analyses have established that the product of a cell envelope-associated polyglutamyl synthetase, isolated from an encapsulated strain of Bacillus licheniformis ATCC 9945A, is poly(γ-d-glutamic acid). Proof that the optical configuration of the glutamyl residues in the enzymatically synthesized polymer and the native capsular polymer is d, has been provided by (a) chromatographic separation of diastereoisomeric dipeptides obtained by derivatization with l-leucine-N-carboxyanhydride, and (b) gas-liquid chromatographic separation of N-trifluoroacetyl-l-prolylglutamyl dipeptide methyl esters. The amide linkage has been shown to be γ by (a) kinetics of mild acid catalyzed hydrolysis and (b) LiBH4 reduction of the methyl ester of the polymer followed by acid hydrolysis which yielded γ-amino-δ-hydroxyvaleric acid. Identical treatment of an authentic poly(α-d-glutamyl) polymer yielded α-amino-δ-hydroxyvaleric acid. Neither the enzymatically synthesized polymers nor the native capsular polymers synthesized in vivo contained any ester, anhydride, or lactone linkages. Detailed ultracentrifugal studies have established that the biosynthetic polymers are polydisperse with weight-average molecular weight distribution values ranging from 1.72 x 105 to 3.63 x 105. Similar studies carried out on native γ-d-glutamyl capsular polymers synthesized in vivo showed a much greater degree of polydispersity with apparent weight-average molecular weight distribution values ranging from 8.4 x 104 to 1.15 x 106. The biosynthetic polymers are susceptible to enzymatic hydrolysis by a poly(γ-d-glutamyl) depolymerase. This enzyme, which readily hydrolyzed the native capsular polymer, showed no activity against authentic α-l-glutamyl polymers. These data provide additional evidence that the product of the membrane-mediated reaction is structurally identical with the native capsular γ-d-glutamyl polymers synthesized in vivo.