Isolation of alkalophilic CGTase-producing bacteria and characterization of cyclodextrin-glycosyltransferase

• Main Authors: lma Hiroko Higuti, Simone Wichert Grande, Roberta Sacco, Aguinaldo José do Nascimento
• Format: Article
• Language: English
• Published: Instituto de Tecnologia do Paraná (Tecpar) 2003-03-01
• Series: Brazilian Archives of Biology and Technology
• Subjects: Cyclodextrin glycosyltransferase; Bacillus firmus; characterization
• Online Access: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1516-89132003000200007

One hundred and twenty five soil samples were collected from the regions of roots of corn, cassava, potato, bean, sugar cane, soya, and pumpkin. From these, 75 strains were isolated that produced a yellowish halo surrounding the colonies, due to a phenolphtalein-cyclodextrin (CD) complex, and these were selected as alkalophilic CGTase-producing bacteria. All the 75 strains were identified as Bacillus firmus by microscopy and biochemical tests. The activity of the CGTase's varied from 2² to 2(10) dilutions,when assayed by CD-trichloroethylene (TCE)-complex precipitation. Strain 31 that produced the enzyme at the higher level was selected, and its enzyme was partially purified by starch adsorption (x 17) in a yield of 51%. Maximum enzyme activity occurred at pH 5.5 and 8.5. At pH 5.5, the optimum temperature was 60°C. On increased from 30°C to 85°C, the thermodynamic parameter for activation energy was 8.27 kcal.mol-1. The enzyme was inhibited by Ca2+, Mg2+, Fe2+, Cu2+, Mn2+, and Zn2+.<br>Foram coletadas 125 amostras de solo de raízes de milho, mandioca, batata, feijão, cana-de-açúcar, soja e abóbora. Destas, 75 cepas foram isoladas por desenvolverem um halo amarelo ao redor das colônias, devido a formação do complexo fenoftaleína-ciclodextrina (CD) sendo selecionadas como bactérias alcalofílicas produtoras de CGTase (EC 2.4.1.19).Todas as 75 cepas foram identificadas como Bacillus firmus por microscopia e testes bioquímicos. Quando ensaiada por precipitação com tricloroetileno a atividade da CGTase variou de diluição de 2² a 2(10). A cepa 31 foi selecionada devido a elevada produção de enzima, sendo esta parcialmente purificada por adsorção em amido (17 vezes) com uma recuperação de 51%. A máxima atividade enzimática ocorreu em pH 5,5 e 8,5. Em pH 5,5, a temperatura ótima foi de 60° C. Sob o aumento de 30° C a 85° C, o parâmetro de energia de ativação foi de 8,27 kcal.mol-1. A enzima foi inibida por Ca2+, Mg2+, Fe2+, Cu2+, Mn2+ e Zn2+.