Production, purification and biochemical characterization of cyclodextrin glucanotrans-ferase from bacillus cereus N1

Cyclodextrin glucanotransferase [CGTase], [EC .2.4.1.19] producing bacteria were isolated from different sources of soils and identified as Bacillus cereus N1 and the best source was the soil of the National Research Centre. The maximum production of the crude CGTase enzyme was observed after 48hr of incubation at 37°C producing CGTase activity of 3.5 U/ml. The effect of nutritional requirements on the CGTasc production were carried out. Soluble starch and yeast extracts were found to be the best carbon and nitrogen sources, respectively. The enzyme was successively purified by ammonium sulphate precipitation, DEAE-cellulose and sephadex G-100 column chromatography and the final specific activity of CGTasc enzyme was increased by 24 fold. The SDS-PAGE showed that the purified CGTase enzyme was homogenous and the molecular weight of the purified enzyme was about 75 kDa. The characterization of the enzyme exhibited optimum pH and temperature at 6.0 and 40°C, respectively. The enzyme was stable at pH 6.5 to 8.0 and retained its high activity up to 45°C

Metodologia de seleção de cepas para produção etodologia da ciclodextrina glicosiltransferase e para purificação da enzima / Strains selection methodology for cyclodextrin glycosyltransferase production and enzyme purification

As ciclodextrinas (CDs) são maltooligossacarídeos, produzidas a partir do amido, pela enzima ciclodextrina glicosiltransferase (CGTase). Esta pesquisa teve por objetivo estabelecer metodologias de seleção de cepas para produção de CGTase e para purificação da enzima. Os microrganismos foram selecionados a partir de 53 análises de solos de cultura de amido, em placas contendo meio de cultivo específico, para seleção de cepas produtoras de CGTase. As enzimas foram obtidas com cultivo destes microrganismos em meio líquido. As atividades enzimáticas das CGTases foram determinadas pelos métodos espectrofotométricos e precipitação com tricloroetileno. A cepa isolada do solo de aveia foi a que apresentou maior atividade [0,1864 µmol de alfa-CD (min mL)-1]. Esta cepa foi utilizada para a produção da enzima em escala laboratorial e purificação em cromatografia de afinidade bioespecífica. A cepa selecionada nesta pesquisa abre novas perspectivas para produção de enzima e CDs em escala industrial. Palavras-chave: ciclodextrina, glicosiltransferase, CGTase.

Cyclodextrins (CDs) are maltooligosaccharides produced from starch by cyclodextrin glycosyltransferase (CGTase) enzyme. This research aimed at establishing method for strains selection for CGTase production and enzyme purification. The microorganisms were selected from 53 analyses of starch cultures soils on plates containing specific culture medium for strains selection that produce CGTase. The enzymes were obtained by culturing these microorganisms in liquid medium. The enzyme activity was determined with photospectrometric methods and precipitation with trichloroethylene. The strain isolated from oat soil was the one that showed the highest activity [0.1864 µmol of alpha-CD (min mL)-1]. This strain was used for enzyme production in laboratory scale and purification by biospecific affinity chromatography. The strain selected in this research opens new perspectives for enzymes production and CDs in industrial scale.