Integral kinetics of α‐galactosidase purified from <i>Glycine max</i> for simultaneous hydrolysis of stachyose and raffinose

Porter, Jill E.; Herrmann, Klaus M.; Ladisch, Michael R.

doi:10.1002/bit.260350104

Cited by 22 publications

(16 citation statements)

References 18 publications

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“…This is very interesting for industrial purposes as the amount of stachyose in soybean products is higher than that of raffinose. Activities on aryl R-D-galactosides as well as on melibiose, raffinose, and stachyose are exhibited by several R-galactosidases, but most of these enzymes present a higher affinity for the trisaccharide raffinose than for the tetrasaccharide stachyose (42). Also, the K m values calculated for the D. hansenii UFV-1 extracellular R-galactosidase (11) with the same substrates (melibiose, raffinose and stachyose) were close to (3); iodoacetamide (4); AgNO 3 (5); NaCl (6); SDS (7); KCl (8); CuSO 4 (9); CaCl 2 (10); -mercaptoethanol (11); raffinose (12); maltose (13); sucrose (14); melibiose (15); D-glucose (16); D-galactose (17); D-mannose (18); lactose (19); stachyose (20) on D. hansenii UFV-1 intracellular R-galactosidase, and (1) without effectors.…”

Section: Resultsmentioning

confidence: 99%

Debaryomyces hansenii UFV-1 Intracellular α-Galactosidase Characterization and Comparative Studies with the Extracellular Enzyme

Viana

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et al. 2009

J. Agric. Food Chem.

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Debaryomyces hansenii cells cultivated on galactose produced extracellular and intracellular alpha-galactosidases, which showed 54.5 and 54.8 kDa molecular mass (MALDI-TOF), 60 and 61 kDa (SDS-PAGE) and 5.15 and 4.15 pI values, respectively. The extracellular and intracellular deglycosylated forms presented 36 and 40 kDa molecular mass, with 40 and 34% carbohydrate content, respectively. The N-terminal sequences of the alpha-galactosidases were identical. Intracellular alpha-galactosidase showed smaller thermostability when compared to the extracellular enzyme. D. hansenii UFV-1 extracellular alpha-galactosidase presented higher kcat than the intracellular enzyme (7.16 vs 3.29 s-1, respectively) for the p-nitrophenyl-alpha-D-galactopyranoside substrate. The Km for hydrolysis of pNPalphaGal, melibiose, stachyose, and raffinose were 0.32, 2.12, 10.8, and 32.8 mM, respectively. The intracellular enzyme was a competitively inhibited by galactose (Ki = 0.70 mM), and it was inactivated by Cu(II) and Ag(I). Enzyme incubation with soy milk for 6 h at 55 degrees C reduced stachyose and raffinose amounts by 100 and 73%, respectively.

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Section: Resultsmentioning

confidence: 99%

Debaryomyces hansenii UFV-1 Intracellular α-Galactosidase Characterization and Comparative Studies with the Extracellular Enzyme

Viana

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Passos

et al. 2009

J. Agric. Food Chem.

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“…Quemener and Brillouet (1983) reported that the in vitro hydrolysis rate of ciceritol by a--D-galactosidase is much lower than for raffinose, stachyo- se and verbascose. Porter et al (1990) indicated that hydrolysis of stachyose in soybean seeds is a rate-limiting step and soybean a-galactosidase simultaneously hydrolyzes two substrates: stachyose and raffinose. By analogy, it can be expected that hydrolysis of verbascose and trigalactosyl pinitol A in winter vetch seeds can be limited by the rate of hydrolysis of products: stachyose and ciceritol, respectively.…”

Section: Resultsmentioning

confidence: 99%

Inhibition of raffinose family oligosaccharides and galactosyl pinitols breakdown delays germination of winter vetch (Vicia villosa Roth.) seeds

Lahuta

Goszczyńska

2011

Acta Soc Bot Pol

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Beside RFOs, which are commonly present in legume seeds, seeds of some species contain galactosyl pinitols (GPs). These carbohydrates, like RFOs, have been hypothesized to constitute an important energy and carbon skeletal source during germination. To test this hypothesis we have applied a specific a-galactosidase inhibitor (1-deoxygalactonojirimycin, DGJ) to germinating winter vetch (Vicia villosa Roth.) seeds, containing more galactosyl pinitols than RFOs. The breakdown of RFOs but not that of GPs was completely blocked in both embryonic axes and cotyledons tissues, during the first 18 h of imbibition in DGJ. The inhibitor only decreased the rate of GPs degradation. The inhibitory effect of DGJ on GPs degradation was partially alleviated by addition of sucrose or galactose to DGJ solutions. After three days of germination in water, RFOs and GPs disappeared in axial tissues of seeds imbibed in water, galactose or sucrose. Eighteen-hour imbibition of seeds in DGJ drastically reduced germination, by ca 50%, during the first three days. The inhibitory effect of DGJ decreased during the next seven days of germination. The presence of galactose or sucrose in imbibition solution initially stimulated seed germination, but later this effect was not statistically significant. Our study provides clear evidence that galactosyl pinitols play an important role in early winter vetch seeds germination. Additionally, we suggest that galactosyl pinitols can replace RFOs as reserve material necessary for early germination.KEY WORDS: germination, galactosyl pinitols, raffinose family oligosaccharides, seed, winter vetch. _______________ Abbreviations:RFOs raffinose family of oligosaccharides; GPs a-D-galactosides of D-pinitol; GPA galactosyl pinitol A; GPB galactosyl pinitol B; DGPA di-galactosyl pinitol A (ciceritol); TGPA tri-galactosyl pinitol A; DGJ 1-deoxygalactonojirimycin; DGMI di-galactosyl-myo-inositol

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“…Os valores de K M ap para a rafinose (Tabela 4) foram próximos daqueles determinados para α-galactosidase purificada de sementes de soja madura (PORTER et al, 1990), mas menores do que aqueles descritos para a rafinose e as enzimas de Aspergillus fumigatus (De REZENDE e FELIX, 1999) Tanto a C1 quanto a C2 não hidrolisaram substratos sintéticos contendo resíduos de açúcar diferentes de galactose ou contendo galactose na posição β (Tabela 6).…”

Section: Resultsunclassified

Purificação e caracterização de alfa-galactosidases de sementes de Platymiscium pubescens Micheli

et al. 2005

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2RESUMO -Este trabalho objetivou foi determinar a composição bioquímica de sementes de espécies florestais e caracterizar a enzima α-galactosidase de sementes germinadas de Platymiscium pubescens. Os maiores teores de lipídios foram determinados em sementes de Chorisia speciosa, Caesalpinia peltophoroides, Tabebuia serratifolia e Tabebuia velanedae, enquanto sementes de Enterolobium contortisiliquum, Schizolobium parahyba e Cassia grandis apresentaram os maiores teores protéicos. A α-galactosidase catalisa a hidrólise dos oligossacarídeos de rafinose, em sementes de leguminosas, durante a germinação. A maior atividade da α-galactosidase foi detectada em sementes de Platymiscium pubescens após 72 h de embebição. Duas formas de α-galactosidases, C1 e C2, foram purificadas de sementes germinadas de P. pubescens, usando-se fracionamento com sulfato de amônio e cromatografias de filtração em gel e de afinidade. Essas enzimas apresentaram atividade máxima em pH 5,5 e a 50-55 °C. Os valores de K m ap das formas C1 e C2, para o substrato ρ-nitrofenil-α-D-galactopiranosídeo, foram de 0,54 mM e 0,78 mM, e para a rafinose, de 4,64 mM e 5,09 mM, respectivamente. Essas enzimas exibiram estabilidade térmica moderada, mantendo 70% da atividade original após 3 h de incubação a 45 °C. A atividade enzimática da C1 e C2 foi totalmente perdida na presença de CuSO 4 e dodecil sulfato de sódio (SDS). Tais enzimas também hidrolisaram melibiose, rafinose e estaquiose, indicando potencial para aplicações biotecnológicas.Palavras-chave: Platymiscium pubescens, α-galactosidase, sementes, germinação e purificação. PURIFICATION AND CHARACTERIZATION OF α α α α α-GALACTOSIDASES FROM Platymiscium pubescens Micheli SEEDSABSTRACT -The objective of this work was to determine seed biochemical composition of forest species and to characterize α-galactosidase enzyme of germinated seeds of Platymiscium pubescens. The highest lipid levels were found in seeds of Chorisia speciosa, Caesalpinia peltophoroides, Tabebuia serratifolia and Tabebuia velanedae, whereas seeds of Enterolobium contortisiliquum, Schizolobium parahyba and Cassia grandis showed the highest protein levels. α-galactosidase catalyzes the hydrolyzis of raffinose oligossacarides in legume seeds during germination. The highest activity of α-galactosidase was found in seeds of Platymiscium pubescens after 72 h of soaking in the water. Two forms of α-galactosidases, C1 and C2, were purified from germinated seeds of P. pubescens, using partition with ammonium sulfate, and gel filtration and affinity chromatographies. These enzymes presented maximum activity at pH 5.5, 50-55°C. K m ap values in the C1 and C2 forms for ρ-nitrophenyl-α-D-galactopyranoside substrate were 0.54 mM and 0.78 mM, and 4.64

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Integral kinetics of α‐galactosidase purified from Glycine max for simultaneous hydrolysis of stachyose and raffinose

Cited by 22 publications

References 18 publications

Debaryomyces hansenii UFV-1 Intracellular α-Galactosidase Characterization and Comparative Studies with the Extracellular Enzyme

Debaryomyces hansenii UFV-1 Intracellular α-Galactosidase Characterization and Comparative Studies with the Extracellular Enzyme

Inhibition of raffinose family oligosaccharides and galactosyl pinitols breakdown delays germination of winter vetch (Vicia villosa Roth.) seeds

Purificação e caracterização de alfa-galactosidases de sementes de Platymiscium pubescens Micheli

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