RESUMO
By gel filtration of a crude extract of Bacillus subtilis ATCC 21332 and OKB 105, the multienzyme system that forms the lipoheptapeptide surfactin was separated into three enzyme fractions, E1, E2, and E3. E1, which appeared near the exclusion limit of the column, activates all amino acid components of surfactin as aminoacyladenylates and thioesters according to the thioester mechanism. In addition, a leucine-activating enzyme (E2) and an acyltransferase (E3) were detected that show molecular masses of approximately 160 and 40 kDa, respectively. The surfactin synthetase multienzyme system was reconstituted by complementation of all three enzyme fractions, yielding high rates of lipopeptide formation. E1 is composed of two multifunctional polypeptides (E1A and E1B) with molecular masses of 460 and 435 kDa, respectively, that can be separated by high-resolution anion-exchange chromatography on Pharmacia Mono Q. E1A binds L-Glu and L-Leu in a molar ratio of 1:2, whereas E1B incorporates L-Val, L-Asp, and L-Leu in a molar ratio of 1:1:1. The hydroxy fatty acid moiety is contributed by the acyltransferase accepting the hydroxy fatty acid coenzyme A thioester as substrate. The transfer of the hydroxy fatty acid to E1A and the formation of the hydroxyacyl-L-glutamate intermediate are the initiation steps in the biosynthesis of surfactin. The amino acid-activating enzyme components E1A, E1B, and E2 have been highly purified and partially characterized.
