Determination of Easily Hydrolyzable Fructose Units in Dextran Preparations

Wise, C. S.; Dimler, R. J.; Davis, H. A.; Rist, C. E.

doi:10.1021/ac60097a011

Cited by 215 publications

(88 citation statements)

References 14 publications

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“…Oligosaccharide standards of inulin series were prepared from tubers of H. tuberosus as described by Cairns & Pollock (1988). Ketoses were detected with urea-phosphoric acid reagent (Wise et al 1955). Measurement of enzyme activities -Samples of enzyme preparations (100 µL) were mixed with an equal volume of citrate phosphate (McIlvaine) buffer pH 5.4 containing 600 mM 1-kestose, 400 mM nystose or 10% (w/v) inulin purified from tuberous roots of V. discolor at vegetative phase (Isejima & Figueiredo-Ribeiro 1993).…”

Section: Methodsmentioning

confidence: 99%

Fructan degradation and hydrolytic activity in tuberous roots of Viguiera discolor Baker (Asteraceae), a herbaceous species from the cerrado

et al. 2003

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-(Fructan degradation and hydrolytic activity in tuberous roots of Viguiera discolor Baker (Asteraceae), a herbaceous species from the cerrado). Fructans of the inulin type are the major reserve carbohydrates in tuberous roots of Viguiera discolor, a perennial herb native to the cerrado. Changes in molecular mass of the polymer, followed by releasing free fructose suggested that hydrolysis could be related to the sprouting of the buds after the dormant period, when aerial parts of the plant are naturally absent. Excision of aerial parts resulted in the increase of fructan 1-exohydrolase (1-FEH) activity in tuberous roots after sprouting. 1-FEH was partially purified from this material by binding to ConA-Sepharose and the highest activity was detected at pH 5.4 and between 20 and 40 °C. Values of K m for V. discolor inulin could not be determined since no saturation was observed up to 10%. The study of the kinetics of the 1-FEH activity showed that it does not follow MichaelisMenten and apparently presents allosteric behaviour, as data fits in the Hill equation. The 1-FEH from V. discolor is a glycoprotein, more active on low molecular mass fructans than on high molecular mass inulin from the same species.Key words -Asteraceae, cerrado, fructan 1-exohydrolase, inulin RESUMO -(Degradação de frutanos e atividade hidrolítica em raízes tuberosas de Viguiera discolor Baker, uma espécie herbácea de Asteraceae do cerrado). Frutano do tipo inulina é o principal carboidrato de reserva das raízes tuberosas de Viguiera discolor, uma espécie herbácea nativa do cerrado. Variações na massa molecular do polímero, acompanhadas de altas proporções de frutose livre sugeriram que a hidrólise do mesmo poderia estar associada à brotação das plantas, após o período de dormência, quando toda a parte aérea é naturalmente eliminada. Após indução experimental de brotação e estímulo da atividade hidrolítica, por eliminação da parte aérea, o extrato enzimático foi parcialmente purificado por adsorção em Sepharose ConA, sendo a maior atividade detectada em pH 5,4 e entre 20 e 40 °C. Não foi possível determinar os valores de K m para a inulina, uma vez que até a concentração de 10% não houve a saturação do substrato. O estudo da cinética da atividade de frutano 1-exohidrolase mostrou que a mesma não corresponde à cinética de Michaelis-Menten, apresentando um comportamento aparentemente alostérico, uma vez que os dados se enquadram na equação de Hill. A 1-FEH de V. discolor é uma glicoproteína, mais ativa sobre oligofrutanos do que sobre inulina de alta massa molecular, como a encontrada em outras Asteráceas, e principalmente na própria espécie.Palavras-chave -Asteraceae, cerrado, frutano 1-hexohidrolase, inulina

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Section: Methodsmentioning

confidence: 99%

Fructan degradation and hydrolytic activity in tuberous roots of Viguiera discolor Baker (Asteraceae), a herbaceous species from the cerrado

et al. 2003

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“…After elution with 10 volumes distilled water, the eluate was concentrated under vacuum at 37 o C and the neutral sugars analyzed by ascending thin-layer chromatography (TLC) as described by Kanaya et al (1978). Carbohydrates were visualized by the ketose-specific urea-phosphoric acid reagent (Wise et al 1955). Components were identified by comparison with oligosaccharides of the inulin series from tubers of H. tuberosus chromatographed simultaneously.…”

Section: Methodsmentioning

confidence: 99%

Fructan variation in the rhizophores of Vernonia herbacea (Vell.) Rusby, as influenced by temperature

Dias-Tagliacozzo¹,

Figueiredo-Ribeiro²,

Carvalho³

et al. 1999

Rev. bras. Bot.

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(recebido em 07/11/98; aceito em 24/03/99) ABSTRACT -(Fructan variation in the rhizophores of Vernonia herbacea (Vell.) Rusby, as influenced by temperature). The influence of climatic variations on fructan content in tropical regions is not well known. The present study deals with the effects of temperature on fructan contents in rhizophores of plants of Vernonia herbacea, a native species from the Brazilian cerrado vegetation. Intact plants and fragmented rhizophores were subjected to different temperatures under natural and controlled environmental conditions. Rhizophores of plants in pre-dormant stage (aerial parts showing some yellowish leaves) presented higher fructan content at 5 o C than those kept at 25 o C, whereas in dormant plants (aerial parts absent) temperature treatments did not affect fructan contents. Fragmented rhizophores obtained from dormant plants presented higher levels of fructo-polysaccharides at the end of the experiment than at the beginning of the treatment, regardless of the temperature they were stored, whereas fragments obtained from vegetative plants showed a decrease in fructan content under the same treatments. It was concluded that variations observed in fructan contents are related to the phenological state of the plants prior to the treatment rather than to extraneous temperatures they are subjected to during this stage. RESUMO -(Variações nos frutanos de rizóforos deVernonia herbacea (Vell.) Rusby, em função da temperatura). As relações entre variação nos conteúdos de frutanos e a variação nas condições climáticas e edáficas em regiões tropicais são pouco conhecidas. O objetivo deste trabalho foi investigar o efeito da temperatura no conteúdo e composição dos frutanos presentes em rizóforos de Vernonia herbacea, uma planta nativa do cerrado brasileiro. Plantas intactas e fragmentos de rizóforos foram submetidos a diferentes temperaturas. Ao final do tratamento de frio (5 o C) plantas intactas em início de dormência (parte aérea presente) apresentaram conteúdo de frutanos maior que o das plantas mantidas a 25 o C. Plantas completamente dormentes (sem a parte aérea) apresentaram poucas diferenças no conteúdo de frutanos quando mantidas a 5 ou 25 o C. Independentemente da temperatura utilizada, fragmentos de rizóforos, isolados de plantas em fase de dormência, apresentaram ao final do experimento um aumento no conteúdo de polifrutanos em relação ao início do tratamento; no entanto, fragmentos isolados de plantas em fase de brotação apresentaram diminuição no conteúdo de polifrutanos. Concluiu-se que as variações observadas estão mais relacionadas com o estádio fenológico da planta do que com o tratamento de temperatura.

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“…Samples (10 μl) of the reaction mixture (see above) or post-culture medium were spotted on silica gel plates (Kavalier, Czechoslovakia and Merck, Germany) and developed three times using an n-butanol-isopropanol-acetic acid-water (35:25:20:10) mixture (v/v) according to Lüscher et al (1993) or four times in an ethanol-butanol-water (5:5:3) mixture (Kozumi et al, 1985). Plates were visualized with a ketose-specifi c urea/ phosphoric acid reagent (Wise et al, 1955). Fructose and sucrose were used as standards.…”

Section: End Product Identifi Cationmentioning

confidence: 99%

Digestion and utilization of fructose polymers by the rumen bacterium <i>Treponema</i> sp. strain T

Kasperowicz¹,

Michałowski²

2007

J. Anim. Feed Sci.

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In this experiment, strain T of ruminal treponemes was studied regarding the ability to utilize β-2,6-and β-2,1-fructose polymers as well as sucrose and fructose. Bacteria were able to grow on laboratory-prepared timothy grass fructan and commercial inulin, but not on sucrose or fructose. The optical density of cultures growing on inulin and timothy grass fructan was 0.5 and 1.1 absorbance units, respectively. Bacteria utilized up to 80% of the timothy grass fructan and only 30% of inulin during a 24 h incubation period. It was found that bacteria utilized only the β-2,1-inulooligosaccharides present in the commercial inulin while the long-chain polysaccharides remained undigested. Enzymatic studies showed that the bacterial cell-free extract degraded the timothy grass fructan, inulin and sucrose, releasing 13.4-19.1, 11.1-14.9 and 42.4-52.9 μM fructose/mg protein/h, respectively. It is suggested that enzymes involved in the degradation of fructose polymers could form a complex of which the enzyme specifi cally digesting the timothy grass fructan is associated with the outer membrane and acts on cell surfaces, while unspecifi c β-fructofuranosidase occupies an intracellular space and digests only the carbohydrates inside the bacterial cells. It is also suggested that such a localization of enzymes could be responsible for the inability of bacteria to utilize long-chain inulin. It is supposed that the examined bacteria are lacking in mechanism(s) transporting sucrose and fructose from the growth medium to the intracellular space of these microorganisms.

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Determination of Easily Hydrolyzable Fructose Units in Dextran Preparations

Cited by 215 publications

References 14 publications

Fructan degradation and hydrolytic activity in tuberous roots of Viguiera discolor Baker (Asteraceae), a herbaceous species from the cerrado

Fructan degradation and hydrolytic activity in tuberous roots of Viguiera discolor Baker (Asteraceae), a herbaceous species from the cerrado

Fructan variation in the rhizophores of Vernonia herbacea (Vell.) Rusby, as influenced by temperature

Digestion and utilization of fructose polymers by the rumen bacterium <i>Treponema</i> sp. strain T

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