Hydrolysis Of Raffinose Research Articles

Enzymatic properties of α-galactosidase from Trichoderma reesei in the hydrolysis of galactooligosaccharides

Enzymatic properties of the α-galactosidase (α-galactoside galactohydrolase, EC 3.2.1.22) from Trichoderma reesei in the hydrolysis of natural galactooligosaccharides and α -O-methyl D-galactopyranoside have been investigated in a wide range of substrate concentrations. The hydrolyses of α -O-methyl D-galactopyranoside and melibiose were inhibited by substrate at concentrations higher than 100 mM while in the hydrolysis of raffinose and stachyose such an effect was not observed. It was shown by 1H and 13C NMR spectroscopy and HPLC techniques that inhibition by the excess of α -O-methyl D-galactopyranoside or melibiose strongly correlated with formation of transglycosylation products. The product of autocondensation reaction with α -O-methyl D-galactopyranoside as substrate was found to be α -O-methyl galactopyranosyl-1,6-D-galactopyranoside. The stereochemical course of stachyose hydrolysis has been determined. The enzyme catalyses the hydrolysis with retention of anomeric configuration and is assumed to operate via a double displacement mechanism. Simultaneous hydrolysis of stachyose and raffinose effected by the α-D-galactosidase was studied by direct 1H NMR measurements. Cleavage of the terminal galactose residue of stachyose was found to be the rate-limiting step. Formation constants of enzyme-substrate complex for stachyose and raffinose were calculated. The suggested model can be used for simulating the two-substrate system and predicting the extent of stachyose hydrolysis.

Characterization of α-galactosidases from germinating soybean seed and their use for hydrolysis of oligosaccharides

Raffinose oligosaccharides (RO) are the major factors responsible for flatulence following ingestion of soybean derived products. Removal of RO from seeds or soymilk would then have a positive impact on the acceptance of soy-based foods. Enzymic hydrolysis of the RO is accomplished by α-galactosidase. While the content of RO decreases during seed germination, the activity of α-galactosidase increases substantially. Two α-galactosidases were isolated from germinating seeds by partition in an aqueous two-phase system followed by ion-exchange and affinity chromatography. One of the enzyme preparations (P1) showed a single protein with M r of 33 kDa, and the second (P2) had two proteins with M r of 31 and 33 kDa. Maximal activities against the synthetic substrate ρ-nitrophenyl-α- d-galactopyranoside ( ρNPGal) were detected at pH 5.0–5.5 and 45–50°C. Both enzymes were fairly stable at 40°C, but lost most of their activities after 30 min at 50°C. The K m values for hydrolysis of ρNPGal by the P1 and P2 enzymes were 1.55 and 0.76 mM, respectively. The K m values determined for hydrolysis of raffinose and melibiose by the P2 enzyme were 5.53 and 5.34 mM, respectively and galactose was a competitive inhibitor ( K i=0.65 mM). To different extents, both enzymes were sensitive to inhibition by galactose, melibiose, CuSO 4, and SDS. Sucrose and β-mercaptoethanol showed discrete inhibitory effects on both enzymes.

Carbohydrate metabolism during early fruit development of sweet melon (Cucumis melo)

In sink tissues of cucurbits, including sweet melon fruits, the galactosyl‐sucrose oligosaccharides, stachyose and raffinose, together with sucrose, are the major translocated carbohydrates. In the present study we investigated the carbohydrate metabolism of young melon (Cucumis melo L. cv. C‐8) fruit during the period of initial fruit set and development, from 3 days prior to anthesis until 20 days after anthesis (DAA), prior to the onset of sucrose accumulation. The enzymes assayed could be classified into two categories according to developmental patterns. Two of the enzymes, alkaline α‐galactosidase I [EC 3.2.1.22], which hydrolyzes both raffinose and stachyose, and acid invertase [EC 3.2.1.26] either increased or remained stable during the first 10 DAA. The remaining measured enzymes (the stachyose‐specific alkaline α‐galactosidase form II, acid α‐galactosidase, alkaline invertase, sucrose synthase [EC 2.4.1.13], galactokinase [EC 2.7.1.6], UDP‐Gal PPase [EC 2.7.7.10], UDP‐Glc‐4 epimerase [EC 5.1.3.2], UDP‐Glc PPase [EC 2.7.7.9], phosphoglucomutase [EC 5.4.2.2] and phosphoglucoisomerase [EC 5.3.1.9]) all showed a similar developmental pattern of steady decrease in activity following anthesis. We also compared the saccharide metabolism of pollinated and non‐pollinated ovaries during the initial days following anthesis. In the absence of pollination, ovary growth dramatically decreased by the first DAA and was accompanied by a sharp decrease in the activity of UDP‐Glc PPase. Other enzymes in the pathway, including the enzymes of stachyose and raffinose hydrolysis, did not decrease in activity until 2 or 4 DAA, after ovary growth was affected. These results provide information to assess the possible regulating enzymes in cucurbit ovary development and fruit set.

A novel alkaline alpha-galactosidase from melon fruit with a substrate preference for raffinose

The cucurbits translocate the galactosyl-sucrose oligosaccharides raffinose and stachyose, therefore, alpha-galactosidase (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) is expected to function as the initial enzyme of photoassimilate catabolism. However, the previously described alkaline alpha-galactosidase is specific for the tetrasaccharide stachyose, leaving raffinose catabolism in these tissues as an enigma. In this paper we report the partial purification and characterization of three alpha-galactosidases, including a novel alkaline alpha-galactosidase (form I) from melon (Cucumis melo) fruit tissue. The form I enzyme showed preferred activity with raffinose and significant activity with stachyose. Other unique characteristics of this enzyme, such as weak product inhibition by galactose (in contrast to the other alpha-galactosidases, which show stronger product inhibition), also impart physiological significance. Using raffinose and stachyose as substrates in the assays, the activities of the three alpha-galactosidases (alkaline form I, alkaline form II, and the acid form) were measured at different stages of fruit development. The form I enzyme activity increased during the early stages of ovary development and fruit set, in contrast to the other alpha-galactosidase enzymes, both of which declined in activity during this period. In the mature, sucrose-accumulating mesocarp, the alkaline form I enzyme was the major alpha-galactosidase present. We also observed hydrolysis of raffinose at alkaline conditions in enzyme extracts from other cucurbit sink tissues, as well as from young Coleus blumei leaves. Our results suggest different physiological roles for the alpha-galactosidase forms in the developing cucurbit fruit, and show that the newly discovered enzyme plays a physiologically significant role in photoassimilate partitioning in cucurbit sink tissue.

α-galactosidase activity in ingested seeds and in the midgut ofDysdercus peruvianus (Hemiptera: Pyrrhocoridae)

The midgut of Dysdercus peruvianus is divided into three main sections (V1-V3) and is linked through V4 to the hindgut. The distribution of α-galactosidase activity in the different gut segments of D. peruvianus females was studied. α-galactosidase hydrolyzes the trisaccharide raffinose, the major carbohydrate of cotton seeds, on which the insects live. In D. peruvianus midgut, α-galactosidase activity is mainly found in soluble fractions of V1 contents. However, a comparison between specific activities using different α-galactosidase substrates in cotton seed extracts, V1 tissue homogenate, and midgut contents suggested that the contribution of the enzymes from seeds may be very significant. Gel filtration on Sephacryl S-200 of samples from seed extracts, V1 tissue, and V1 contents revealed that in all samples raffinose hydrolysis is accomplished by α-galactosidases with similar Mr (30,000 ± 3,000) and does not involve the activity of a β-fructosidase. Thermal inactivation studies of extracts from the three sources suggested that there was only one molecular form of the insect α-galactosidase and that the activity found in V1 contents includes enzymes derived from the seed kernel. In insects fed with cotton seeds, the α-galactosidase activity increased in parallel with diet ingestion. In starved insects fed with tablets of sucrose plus raffinose, an increase in α-galactosidase activity was also observed, confirming that the insect is able to synthesize part of the gut enzyme. The results indicated that raffinose digestion starts in V1 utilizing α-galactosidases derived from the seed kernel and by an additional α-galactosidase synthesized by insect tissues. The action of α-galactosidases liberates galactose and sucrose, which are sequentially hydrolyzed by the major membrane-bound α-galactosidase releasing glucose and fructose in V1 and V2 lumina. Arch. Insect Biochem. Physiol. 34:443–460, 1997. © 1997 Wiley-Liss, Inc.

Metabolism and physiological behaviour of Leuconostoc mesenteroides subsp. mesenteroides growing on raffinose

AbstractThe α‐galactosidase activities of 11 strains of Leuconostoc mesenteroides subsp. mesenteroides grown on glucose, lactose or raffinose were determined. The inductive effects of lactose and raffinose differed depending on the strain. In four of the strains, α‐galactosidase activity of cells grown on lactose was higher than that of cells grown on raffinose, while the reverse was true for one other strain. The study of two strains exhibiting a different behaviour was extended to all raffinose‐component sugars: melibiose, sucrose, glucose, galactose and fructose. The apparent fermentation yields with raffinose‐component sugars complied with heterofermentation theory, except for sucrose, where there was a 40% deficit in fermentation products. Raffinose hydrolysis enzymes (α‐galactosidase and β‐fructosidase) underwent a catabolic repression by glucose. Growth on raffinose was considerably stimulated if the inoculum was first grown on melibiose or raffinose (induction of α‐galactosidase).

Successive binding of raf repressor to adjacent raf operator sites in vitro

The raf repressor negatively regulates the transcription of the raf operon which encodes functions required for the uptake and hydrolysis of raffinose in Escherichia coli. Overexpression of the repressor gene under lac promoter control led to the formation of inclusion bodies. These were partially purified by centrifugation, solubilized in 0.1% SDS and reactivated by dilution. DNase I protection and gel retardation experiments demonstrated the specific binding of raf repressor to DNA fragments that contained the previously identified raf operator, an element comprising two 18 bp palindromic nucleotide sequences that flank the -35 raf promoter box. By using DNA fragments with one, two, or four copies of the 18 bp palindrome, these experiments revealed concentration dependent, successive occupation of all available binding sites by raf repressor. Melibiose released the repressor from the operator complexes, whereas raffinose and other alpha-galactosides did not, indicating that melibiose is the actual inducer in vivo. We suggest that successive occupation by repressor of two strategically located operator sites is a specific type of stepwise down-regulation of gene expression in response to repressor concentration.

Digestive physiology and food utilization of the larvae ofEarias vittella (Lepidoptera: Noctuidae) from its malvaceous host plant

The hydrogen-ion concentration of the contents of fore-, mid- and hindgut of the larva ofEarias vittella (F.) lays within a pH range of 8-8 and 9-6 These caterpillars possessed the ability to break down starch, raffmose, maltose, melibiose, sucrose and proteins. Synthesis of certain higher oligosaccharides indicating transglycosidic activity was also noticed during hydrolysis of raffinose, melibiose and sucrose inin vitro experiments. The activity of carbohydrases detected in the larval midgut was variably influenced by the hydrogen-ion concentration of the medium. Further, they differed from one another in their relative strength. Amylase was the most powerful and a-galactosidase was the weakest of all. An in vivo examination into the fate of fed starch, maltose, cellobiose and lactose within the gut revealed the physiological competency of the caterpillar to utilise only the first two carbohydrates (the former partly and the latter completely) through swiftly operating processes of digestion and absorption.

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

alpha-Galactosidase from soybean (Glycine max) was purified by a five-step procedure. The enzyme's natural substrates, raffinose and stachyose, have K(m)'s of 3. 0 mM and 4. 79 mM, respectively. The products, galactose and sucrose, were measured after separation by liquid chromatography. Galactose is a competitive product inhibitor of stachyose and raffinose hydrolysis with a K(i) of 0. 12 mM. We determined these parameters by an integral kinetic approach. Stachyose hydrolysis gives a nearly constant level of raffinose shortly after hydrolysis begins. Thus, cleavage of the first alpha-(1,6)-bond in the tetrasaccharide is the rate-limiting step. Since the stachyose hydrolysis yields raffinose, soybean alpha-galactosidase simultaneously hydrolyzes two substrates. We present a novel approach for analyzing simultaneous substrate hydrolysis with competitive product inhibition by a modified integral rate expression. The experimentally found kinetic parameters are confirmed by solving the simultaneous equations which describe the hydrolysis. This technique may be applicable to other hydrolytic enzymes with multiple substrates.

Isolation and identification of oligosaccharides produced from raffinose by transgalactosylation reaction of thermostable .ALPHA.-galactosidase from Pycnoporus cinnabarinus.

The thermostable α-galactosidase purified from Pycnoporus cinnabarinus catalyzed the galac-tosyltransfer reaction in the hydrolysis of raffinose and produced several kinds of transfer products. The main transfer products were isolated and the structures of the six kinds of transfer products (oligosaccharides A, B, C, D, E, and F) were investigated by acid and enzymatic hydrolysis, and methylation analysis. Oligosaccharides A, B, C, D, E, and F were identified as O-α-d-galactopyranosyl-(1→3)-O-α-d-glucopyranosyl-(1→2)-β-d-fructofuranoside, O-α-d-galactopyranosyl-(1→3)-O-α-d-galactopyranosyl-(1→6)-O-α-d-glucopyranosyl-(1→2)-β-d-fructofuranoside, stachyose, O-α-d-galactopyranosyl-(1→3)-O-α-d-galactopyranosyl-(1→6)-O-α-d-galactopyranosyl-(1→6)-O-α-d-glucopyranosyl-(1→2)-β-d-fructofuranoside, verbascose, and ajugose, respectively.

Hydrolysis of raffinose by immobilized cells of saccharomyces oleaceus

The production, extraction, purification and characterization of α-galactosidase (melibiase) from Saccharomyces cereuisiae var. oleaceus (N.R.R.L. Y 12056) are described. The yeast cells were immobilized by entrapment into cellulose diacetate beads. The removal of raffinose from molasses using this catalyst was studied under different reactor configurations.

Construction of a Stable α-Galactosidase-Producing Baker's Yeast Strain

Molasses is widely used as a substrate for commercial yeast production. The complete hydrolysis of raffinose, which is present in beet molasses, by Saccharomyces strains requires the secretion of alpha-galactosidase, in addition to the secretion of invertase. Raffinose is not completely utilized by commercially available yeast strains used for baking, which are Mel. In this study we integrated the yeast MEL1 gene, which codes for alpha-galactosidase, into a commercial mel baker's yeast strain. The Mel phenotype of the new strain was stable. The MEL1 gene was expressed when the new Mel baker's yeast was grown in molasses medium under conditions similar to those used for baker's yeast production at commercial factories. The alpha-galactosidase produced by this novel baker's yeast strain hydrolyzed all the melibiose that normally accumulates in the growth medium. As a consequence, additional carbohydrate was available to the yeasts for growth. The new strain also produced considerably more alpha-galactosidase than did a wild-type Mel strain and may prove useful for commercial production of alpha-galactosidase.

Immobilization of thermostable α-galactosidase from Pycnoporus cinnabarinus on chitosan beads and its application to the hydrolysis of raffinose in beet sugar molasses

α-Galactosidase (EC 3.2.1.22) from Pycnoporus cinnabarinus was immobilized on chitosan beads, BCW 1000, and crosslinked chitosan beads, BCW 3000 and 3500, of three different sizes, which were untreated or previously treated with glutaraldehyde. The activity yields of the immobilized enzymes were between 25 to 45%, except for glutaraldehyde-untreated B BCW 1000. Leakage of the enzyme with increasing ionic strength was observed in glutaraldehyde-untreated BCW 1000 and 3000. The α-galactosidases immobilized on glutaraldehyde-treated BCW 3000 and 3500 were active at pH 3–6 and at 70–80°C, and stable between pH 3 and 9, and below 70°C. The immobilized α-galactosidase was continuously used for 30 days to hydrolyze raffinose in beet sugar molases.

4376167 Hydrolysis of raffinose by alpha-galactoxidase: Vincenza Vitobello, Paolo Branduzzi, Nadia Cimini, Rome, Italy, assigned to E N I Ente Nazionale Idrocarburi

4376167 Hydrolysis of raffinose by alpha-galactoxidase: Vincenza Vitobello, Paolo Branduzzi, Nadia Cimini, Rome, Italy, assigned to E N I Ente Nazionale Idrocarburi

Alkaline α-galactosidase in leaves of Cucurbita pepo

A fourth molecular form of α-galactosidase has been discovered in leaves of Cucurbita pepo and a 6-fold purification achieved by ammonium sulphate precipitation and ion exchange chromatography. The enzyme designated L IV has the unique property among α-galactosidases of possessing an alkaline pH optimum at 7.5 with p-nitrophenyl- α-D-galactopyranoside (PNPG) or stachyose as substrates. Raffinose hydrolysis was optimal at pH 6.5–7.0. A K m-value of 0.60 mM for PNPG hydrolysis was obtained. In immature importing leaves L IV activity was about 3-fold higher than the total acid α-galactosidase activity. Through the transition phase, during which the leaf changes from an importing to an exporting organ, a 6-fold decline in L IV activity occured. In mature leaves L IV activity was about 60% of the total acid α-galactosidase activity. It is suggested that L IV may play a major role in the hydrolysis of stachyose imported into immature leaves.

Hydrolysis of raffinose in a hollow‐fiber reactor using an unrefined mixture of α‐galactosidase and invertase

AbstractRaffinose was converted enzymatically in a hollow‐fiber reactor to melibiose, sucrose, galactose, glucose and fructose. The enzymes were a crude extract of α‐galactosidase and invertase produced by Aspergillus awamori NRRL 4869 on a solid substrate, Wheat bran. With a concentration of raffinose, C0, entering the reactor at a flow rate Q, and with C being the concentration of raffinose exiting the reactor, the conversion (C/C0), was studied as a function of Q at two level of C0. The data could be fairly well fitted using the analysis of Waterland et al. even though a mixed crude enzyme system was being investigated. It was found empirically that In (C/C0) was linear in Q−1, with the absolute value of the slope decreasing with increasing C0. The linearity of such plots were predicted by Lewis and Middleman from Waterland et al. for a single enzyme system obeying first‐order kinetics, the slope being independent of C0. Although the assumptions involved in this approximate analytical solution are not valid, the observed linearity of the In (C/C0) vs. Q−1 plots is excellent and should prove useful in reactor design considerations.

Expression of cryptic beta-fructofuranosidase in Saccharomyces rouxii.

Raffinose hydrolysis was studied in Saccharomyces rouxii. The responsible enzyme was identified as a beta-fructofuranosidase (EC 3.2.1.26), which has a pH optimum of 5.5 and a K(m) of 83 mM for raffinose. This enzyme was cryptic in cells from a 3-day culture. A 2-min treatment with 0.1 volume of ethyl acetate in sodium acetate buffer (pH 6) gave complete expression of the enzyme, which was still retained by the cell. Ghosts were prepared by modifying membrane structure with small basic proteins in distilled water, and after washing they showed the full complement of enzymatic activity. The enzyme remained cryptic in osmotically protected spheroplasts; however, after lysis (by dilution) release, as well as expression, was effected. Mechanical disruption of fresh cells revealed and released all of the enzyme. Cells in early stationary phase had all of their beta-fructofuranosidase in a cryptic state. Aging yielded fractional expression of activity; initially this was proportional to cell death, but later the degree of expression exceeded the death rate. Media from aged cultures or cell-free extracts of aged cells were not effective in revealing the cryptic enzyme of younger cells. S. rouxii beta-fructofuranosidase has a different autolytic-release pattern from its counterpart in S. cerevisiae. Also, high concentrations of glucose do not repress the S. rouxii enzyme. The beta-fructofuranosidase in young cells of S. rouxii must be enclosed by the protoplasmic membrane or a special vesicular structure. This system was compared with other Saccharomyces species in connection with the translocation of enzymes across the protoplasmic membrane.

SOYBEAN FACTORS RELATING TO GAS PRODUCTION BY INTESTINAL BACTERIA

SUMMARYAn in vitro assay showed that toasted, dehulled, defatted soybean meal contains a gas‐producing factor and a gas‐inhibiting factor. The oligosaccharides—sucrose, raffinose and stachyose—are associated with the gas‐producing factor when incubated in thioglycollate media with anaerobic bacteria of the intestinal tract of dogs. The phenolic acids in soybeans—syringic and ferulic acid—are effective gas inhibitors in vitro and in intestinal segments of dogs. The lipids, proteins and water‐insoluble polysaccharides of soybean meal have no gas activity. During fermentation, gas production parallels the formation of monosaccharides by enzymatic hydrolysis of raffinose and stachyose. The amount and composition of gas produced from cottonseed and peanut meal were comparable to soybean meal.

Hydrolysis of Raffinose and Separation of its Hydrolytic Products Using a Cation Exchange Membrane Loaded with H+ Ions

AbstractThe hydrolysis of raffinose catalyzed by cation exchange membranes loaded with H+ ions is in vestigated in aqueous solution. The reaction taking place only inside the membranes follows first order kinetics. The hydrolytic products are equimolar quantities of fructose and melezitose. The reaction rate constant (k) and the diffusion coefficient of raffinose (Dr) at 20°C have values of k = 2.1 · 10−3 min−1 and Dr = 5.1 · 10−7 cm2 · séc−1.If the volume of one of the outer phases of the membrane is large compared with the other, the system reaches a quasi stationary state in which a separation of fructose and melezitose in the smaller compartment is observed (Cmelezitose/Cfructose = 1.45).

Título en español.

Se llevaron a cabo estudios in vitro de los patrones de acción de la invertasa en presencia del silicio. Se sabe que este elemento inhibe la enzima tanto en los tejidos vivientes como en las preparaciones de los tejidos liofilizados. Los substratos incluían sacarosa, rafinosa, estaquiosa y turanosa. Se preparó invertasa ácida de caña de azúcar de tejidos reservantes inmaduros liofolizados, purificándose con una fraccionación de sal, diálisis y filtración de gela. Se estudiaron productos enzimáticos mediante la cromatografía sobre papel. Se obtuvieron con rapidez de la sacarosa, dos productos en grandes cantidades: fructosa y glucosa. La reacción con la rafinosa fue más lenta, pero produjo cuatro distintos productos. Este hecho sugiere un fuerte potencial por parte de la invertasa de la caña, ya que una hidrólisis ácida completa de la rafinosa sólo produce tres. Se propone que los enlaces del 1—> 6 y del 1 —> 2 quedan rotos. La fructosa aparece por hidrólisis directa de la rafinosa y mediante ataques secundarios contra el producto intermedio, o sea, la sacarosa. El uso de la estaquiosa como un substrato presentó evidencia adicional de la hidrólisis del enlace del 1 —> 6. Se obtuvieron tres productos en presencia de un solo enlace, el 1 —> 2 fructosídico. Al añadir Si a los digestos de invertasa terminaron abruptamente las reacciones con la sacarosa, la rafinosa y la estaquiosa. La concentración efectiva de Si excedió ligeramente 3 µmoles de silicio por mililitro de digesto. Se menciona el significado que tiene el efecto inhibidor de la invertasa tanto desde el punto de vista del aumento que genera en el rendimiento de la sacarosa como de su valor como indicador analítico del contenido de ácido silícico en los tejidos de las plantas. Cuando se usó el disacárido turanosa como substrato, el aumento en los niveles de Si produjo otros productos adicionales más bien que una sus pensión de la acción enzimática. Aparecieron cuatro productos en presencia de 9 µmoles de Si. Los substratos sacarosa y rafinosa produjeron conjuntos de productos teñibles en grados variables a niveles en exceso de 27 µmoles por mililitro. Dichos productos eran de una baja movilidad cromatográfica y semejaban fragmentos de almidón hidrolizado. Para explicar la acción del Si sobre la invertasa se propone la formación de una gela de ácido silícico alrededor de la enzima y en proporción directa a la concentración del Si. La inhibición de la hidrólisis de la sacarosa no resulta de los cambios estructurales severos de la proteína. Más bien, la enzima hipotética encerrada en la gela pudiera dejar de funcionar contra un sustrato, reteniendo o aumentando, sin embargo, su capacidad para atacar a otros.