RESUMO
Cyclodextrin glucanotransferase (CGTase) from Bacillus circulans (ATCC 21783) was immobilised on a silica-based support: purified seasand. Although adsorption of 98% was achieved, considerable desorption was encountered. This problem was minimised by crosslinking the adsorbed enzyme with glutaraldehyde. The immobilised enzyme after crosslinking could be used repeatedly for cyclodextrin (CD) production in a batch process. The activity retention was 80% at the end of the eighth cycle. The immobilised enzyme showed a shift in the pH optimum towards the alkaline side and also an improvement in the pH stability compared to the free enzyme. It catalysed the formation of beta-CD as a major product. A significant amount of alpha-CD production was also observed on prolonged incubation.
Assuntos
Bacillus/enzimologia , Enzimas Imobilizadas , Glucosiltransferases/farmacocinética , Dióxido de Silício , Adsorção , Biotecnologia/métodos , Reagentes de Ligações Cruzadas , Ciclodextrinas/metabolismo , Estabilidade Enzimática , Enzimas Imobilizadas/farmacocinética , Glucosiltransferases/química , GlutaralRESUMO
Genes encoding three isoforms of sucrose synthase (Sus1, Sus2, and Sus3) have been cloned from pea (Pisum sativum). The genes have distinct patterns of expression in different organs of the plant, and during organ development. Studies of the isoforms expressed as recombinant proteins in Escherichia coli show that they differ in kinetic properties. Although not of great magnitude, the differences in properties are consistent with some differentiation of physiological function between the isoforms. Evidence for differentiation of function in vivo comes from the phenotypes of rug4 mutants of pea, which carry mutations in the gene encoding Sus1. One mutant line (rug4-c) lacks detectable Sus1 protein in both the soluble and membrane-associated fractions of the embryo, and Sus activity in the embryo is reduced by 95%. The starch content of the embryo is reduced by 30%, but the cellulose content is unaffected. The results imply that different isoforms of Sus may channel carbon from sucrose towards different metabolic fates within the cell.
Assuntos
Glucosiltransferases/genética , Isoenzimas/genética , Pisum sativum/enzimologia , Carbono/metabolismo , Celulose/biossíntese , Clonagem Molecular , DNA Complementar , Escherichia coli , Regulação Bacteriana da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Glucosiltransferases/classificação , Glucosiltransferases/farmacocinética , Isoenzimas/classificação , Isoenzimas/farmacocinética , Modelos Biológicos , Mutação , Especificidade de Órgãos , Pisum sativum/genética , Filogenia , Plasmídeos , Sementes/enzimologia , Sementes/genética , Amido/biossíntese , Sacarose/metabolismoRESUMO
Cyclodextrin glycosyltransferase (CGTase) was released into the culture fluid by Bacillus macerans predominantly in the late stationary phase of growth and during autolysis in the presence of either glucose or starch as a carbon source. In both cases significant soluble intracellular enzyme activity could be observed in the early stationary phase, and a low non-soluble intracellular CGTase activity could be demonstrated also in the exponential growth phase in the presence of starch. At the end of the exponential phase the non-soluble specific intracellular enzyme activity was found to be constant with a value of 0.63 +/- 0.06 nkat/10(9) viable cells. Since amylase activity could not be detected in any intracellular or extracellular sample taken at any culture time, we conclude that cellbound CGTase is the only starch-digesting enzyme in growing B. macerans and, hence, may be fully responsible for the degradation of starch in the culture fluid.
