Estudo da alfa amilase de Bacillus koreensis isolado de ecossistema aquático da Amazônia: clonagem molecular, expressão e caracterização

Abstract

The Amazon region has an immense Biodiversity of aquatic ecosystems of the planet and several biochemical pathways in these aquatic ecosystems, acting in the recycling of the organic carbon, such as the alpha amylase. For this, the objective of this work was to isolate amylolytic bacteria from Amazonian aquatic ecosystems, selection of a good alpha amylase producer, cloning and expression of the alpha amylase gene (Amy). In order to isolate the colony forming units (CFUs) and to identify the qualitative and quantitative amylolytic enzyme activity, besides cloning the AMYKOR gene of the alpha amylase and expressing it in E. coli. The water samples were collected in the Negro, Solimões and Mamori rivers, and later, clones with the ability to hydrolyze starch were identified. It was then extracted from the total DNA of the amylolytic bacteria for amplification of the Ribosomal 16 S RNA and alpha-amylase genes for molecular taxonomic identification. Sequence analysis showed that Bacillus koreensis was the best producer of alpha-amylase. Subsequently, sequences of the alpha amylase gene were sent to the chemical synthesis, which was subsequently cloned and expressed in E. coli. B. koreensis and the recombinant alpha-amylase producing E. coli clone were cultured in 50 ml LB medium with shaking at 30 °C and 37 °C respectively. Cells were removed by slow speed centrifugation and the enzymes from supernatants precipitated with 50% ethanol, recovering a total of 106 units of alpha amylase (recombinant) and 120 units of wild alpha amylase from Bacillus koreensis). The results of the biochemical characterization showed that both the natural and the recombinant enzyme presented optimum pH equal to 6 and optimum temperature of 60 °C, for both enzymes studied. We verified the parameters of apparent enzyme kinetics of Km of 4.8 mg / mL and the apparent maximum rate of 2.6 mg-1. mL-1. minute-1 of the recombinant alpha amylase, as well as apparent enzyme kinetics parameters of apparent Km of 4.0 mg / ml and the apparent maximum rate of 2.3 mg-1. mL-1. minute-1 of the wild-type alpha amylase from Bacillus koreensis. It can be seen that both natural and recombinant alpha amylase exhibited optimum temperatures, optimum pHs and similar kinetic parameters. These parameters indicate that the enzyme has potential for industrial use.