RESUMO
Two rapid and easy-to-scale-up methods for the purification of cyclodextrin glycosyltransferase (CGTase) from Bacillus circulans were developed: affinity precipitation with starch and aqueous two-phase partition. The first method, optimised by a factorial design, gave an 80% CGTase adsorption at 11% starch and 1.6% ammonium sulphate, and a 65% recovery after elution with 10 mM alpha-cyclodextrin. The purification factor was 17. Aqueous two-phase partition yielded a 72% CGTase recovery in a two-step procedure; CGTase was obtained in the bottom phase with a purification factor of 37.
Assuntos
Bacillus/enzimologia , Cromatografia de Afinidade/métodos , Glucosiltransferases/química , Glucosiltransferases/isolamento & purificação , Amido/química , Bacillus/classificação , Glucosiltransferases/biossíntese , Especificidade da EspécieRESUMO
From wild type inoculum of Bacillus circulans DF 9 which produce cyclomaltodextrin-glucanotransferase (EC 2.4.1.19, CGTase), two different kinds of colonies were isolated, which correspond to the classical S-R variation. From the culture medium of both colonies grown together a CGTase was purified about 50 fold with a yield of 54% in two steps. From pure R-cell culture the enzyme was purified by about 38 folds with a yield of 79% in only one step, showing a complete homogeneity as judged by a native PAGE analysis.
Assuntos
Bacillus/classificação , Bacillus/enzimologia , Glucosiltransferases/isolamento & purificação , Bacillus/isolamento & purificação , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Especificidade da EspécieRESUMO
The cyclomaltodextrin-glucanotransferase (EC2.4.1.19, CGTase) which was purified to homogeneity from Bacillus circulans strain DF 9, R type, showed a pI of 5.3 determined by disc-isoelectric focusing, a Mw of 78 kDa estimated by SDS-PAGE with a range of pH of optimal enzymatic activity rather large (4.5-7.5). The thermal stability of the enzyme at 55 degrees C was increased 4-5 times when calcium ion (10 to 100 mM) or alpha-cyclodextrins (10 mM) were added to the preincubation mixtures. The alpha: beta: gamma ratio determined by HPLC was about 1:0.9:0.4 and the maximal conversion to cyclodextrins with 5% soluble starch was about 36%.
Assuntos
Bacillus/enzimologia , Glucosiltransferases/química , alfa-Ciclodextrinas , Bacillus/classificação , Cálcio/farmacologia , Ciclodextrinas/biossíntese , Ciclodextrinas/farmacologia , Estabilidade Enzimática/efeitos dos fármacos , Glucosiltransferases/isolamento & purificação , Glucosiltransferases/metabolismo , Concentração de Íons de Hidrogênio , Ponto Isoelétrico , Peso Molecular , Solubilidade , Amido/metabolismo , TemperaturaRESUMO
A lethal product (BPG) produced by a glycerol kinase mutant of Escherichia coli was purified, and its mode of action on E. coli was studied. At concentrations where BPG strongly inhibits in vivo deoxyribonucleic acid, ribonucleic acid, and protein synthesis, it produces small effects on other functions: slight inhibition of respiration and small changes in intracellular pools of substrates, nucleic acids degradation, and adenosine triphosphate levels. BPG also inhibits in vitro protein synthesis and produces inactivation of bacteriophage T4. The bactericidal product has been identified in another laboratory as methylglyoxal (MG). By comparing BPG and MG, we confirmed this observation and concluded that the activity found in our BPG preparation is due to its MG content. We also observed that MG is able to react with guanosine triphosphate. According to these results, it is interpreted that MG could act directly on macromolecular synthesis by reacting with the guanine residues of nucleic acids and its precursors.
Assuntos
Aldeídos , Antibacterianos , Escherichia coli/metabolismo , Mutação , Trifosfato de Adenosina/metabolismo , Aldeídos/biossíntese , Aldeídos/isolamento & purificação , Aldeídos/farmacologia , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Proteínas de Bactérias/biossíntese , Isótopos de Carbono , Colífagos/efeitos dos fármacos , Colorimetria , DNA Bacteriano/biossíntese , Escherichia coli/enzimologia , Genética Microbiana , Glicerol , Guanosina Trifosfato/metabolismo , Temperatura Alta , Isoleucina/metabolismo , Desnaturação de Ácido Nucleico , Consumo de Oxigênio , Fosfotransferases/metabolismo , RNA Bacteriano/biossíntese , Espectrofotometria , Timidina/metabolismo , Uracila/metabolismoRESUMO
The problem of whether isolated mitochondria are able to synthesize specific proteins was investigated, particular consideration being paid to the possible contribution of micro-organisms to this activity. With ox heart mitochondria it was shown that: (1) The medium used for the incubations inhibits the exponential phase of bacterial growth for at least 8h either in the absence or the presence of fresh mitochondria, but the inhibition disappears after 4h when mitochondria damaged by freezing and thawing are used. (2) The incorporation of [(14)C]leucine into total proteins is linear up to at least 8h, although part of the radioactivity at the later periods might be due to some incorporation by resting-phase bacteria. (3) A contamination by as little as 800 cells/mg of mitochondrial protein is enough to contribute substantially to the total radioactivity incorporated by the mitochondrial preparations. (4) Purified cytochrome b and cytochrome oxidase are labelled even under conditions of minimal contamination by micro-organisms (less than 60 cells/mg of mitochondrial protein) and the contribution of bacterial proteins to the radioactivity found in cytochromes is negligible, as shown by double-labelling experiments. (5) At 4h the specific radioactivities of cytochrome b and cytochrome oxidase are seven- and 16-fold lower respectively than that of a structural protein-rich fraction, suggesting that the labelling of cytochromes is due to a residual contamination by these proteins.