Produção do lipopeptídeo surfactina a partir de uma cepa de Bacillus subtilis com o operon srfA sob controle do promotor regulado Pgrac / Production of lipopeptide surfactin from a Bacillus subtilis strain with srfA operon under the control of the Pgrac promoter

AUTOR(ES)
FONTE

IBICT - Instituto Brasileiro de Informação em Ciência e Tecnologia

DATA DE PUBLICAÇÃO

29/11/2010

RESUMO

The cyclic lipoheptapeptide surfactin is produced by different Bacillus subtilis strains. At a concentration of 20µM, this biosusrfactant is able to reduce the surface tension of water from 72 mN/m to 27 mN/m. This makes surfactin one the most powerful biosurfactants known so far. The surfactin synthetase is a large multienzyme complex consisting of three enzymatic subunits, SrfA (402 kDa), SrfB (401 kDa), and SrfC (144 kDa), comprising of a total of seven modules. The corresponding NRPS genes are organized in the srfA operon. Surfactin production from Bacillus strains is usually low and production of such large proteins in a heterologous system such as E. coli is not viable. One option to improve surfactin production would be to use Bacillus subtilis itself as an expression system and to place the srfA operon under the control of a strong promoter that can function independently of endogenous negative feedback regulation. For this purpose, the native promoter of the surfactin operon in B.subtilis LB5a was replaced by the promoter Pgrac, which can be induced by lactose analogs. This expression system is based on a strong promoter and is not expected to be inhibited by endogenous factors and, should provide a new tool for producing higher levels of surfactin in Bacillus subtilis.The efficacy of the lipopeptide produced for the recombinant B.subtilis pGrac-SrfA was demonstrated using tests of hemolytic activity and evaluation of the surface tension of the medium. HPLC peaks of surfactin gotten of the genetically modified strain showed patterns of lipopeptides similar to those of the wild-type strain, and mass spectrometry analysis confirmed the presence of a lipopeptide homologous serie with variations in the hydrocarbon chain length, known as surfactin. Was verified the predominance of a homologue with a molecular mass of 1000.18 u. The surfactin production of the recombinant bacterium was up to about 57% greater than that of wild-type strain when it was induced by isopropyl b-D-1-thiogalactopyranoside

ASSUNTO(S)

biossurfactantes bacillus subtilis surfactina atividade antimicrobiana biosurfactantes surfactin antimicrobial activity bacillus subtilis

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