Locust Bean Gum Galactomannan Research Articles

Hemicellulose계열 올리고당 탐색 및 탄소원 대체에 의한 장내세균 생육활성용 신규 MRS배지의 조제

Purification and some properties of Xylogone sphaerospora -mannanase were reprevious previous paper. Locust bean gum galactomannan was hydrolyzed by the purified -mannanase, and then the hydrolysates was separated by activated carbon column chromatography. The main hydrolysates were composed of D.P. (Degree of Polymerization) 4 and 6 galactosyl mannooligosaccharides. For elucidate the structure of D.P 4 and 6 galactosyl mannooligosaccharides, sequential enzymatic action was performed. D.P 4 and 6 were identified as and . To investigate the effects of locust bean gum galactosyl mannooligosaccharides on in vitro growth of Bifidobacterium longum, B. bifidum, B. infantis, B. adolescentis, B. animalis, B. auglutum and B. breve. Bifidobacterium spp. were cultivated individually on the modified-MRS medium containing carbon source such as D.P. 4 and D.P. 6 galactosyl mannooligosaccharides, respectively. B. longum and B. bifidum grew up to-fold and 6.6-fold more effectively by the treatment of D.P. 6 galactosyl mannooligosaccharides, compared to those of standard MRS medium. Especially, D.P. 6 was more effective than D.P. 4 galactosyl mannooligosaccharide on the growth of Bifidobacterium spp.

Mannans as stabilizers of oil-in-water beverage emulsions

The stabilizing effect of spruce galactoglucomannan (GGM) on a model beverage emulsion system was studied and compared to that of guar gum and locust bean gum galactomannans, konjac glucomannan, and corn arabinoxylan. In addition, guar gum was enzymatically modified in order to examine the effect of the degree of polymerization and the degree of substitution of galactomannans on emulsion stability. Use of GGM increased the turbidity of emulsions both immediately after preparation and after storage of up to 14 days at room temperature. GGM emulsions had higher turbidity than the emulsions containing other mannans. The initial turbidity increased with increasing GGM content, but after 14 days storage at room temperature, the turbidity was the highest for GGM/oil ratio of 0.10:1 when ethanol-precipitated GGM was used. Increasing the storage temperature to +45 °C led to rapid emulsion breakdown, but a decrease in storage temperature increased emulsion stability after 14 days. Confocal microscopy showed that the average particle size in the bottom part of GGM emulsions stored for 14 days was smaller than 1 μm. A low degree of polymerization and a high degree of substitution of the modified galactomannans were associated with a decrease in emulsion turbidity.

Characterization of the Bacillus subtilis WL-3 mannanase from a recombinant Escherichia coli

A mannanase was purified from a cell-free extract of the recombinant Escherichia coli carrying a Bacillus subtilis WL-3 mannanase gene. The molecular mass of the purified mannanase was 38 kDa as estimated by SDS-PAGE. Optimal conditions for the purified enzyme occurred at pH 6.0 and 60 degrees C. The specific activity of the purified mannanase was 5,900 U/mg on locust bean gum (LBG) galactomannan at pH 6.0 and 50 degrees C. The activity of the enzyme was slightly inhibited by Mg(2+), Ca(2+), EDTA and SDS, and noticeably enhanced by Fe(2+). When the enzyme was incubated at 4 degrees C for one day in the presence of 3 mM Fe(2+), no residual activity of the mannanase was observed. The enzyme showed higher activity on LBG and konjac glucomannan than on guar gum galactomannan. Furthermore, it could hydrolyze xylans such as arabinoxylan, birchwood xylan and oat spelt xylan, while it did not exhibit any activities towards carboxymethylcellulose and para-nitrophenyl-beta-mannopyranoside. The predominant products resulting from the mannanase hydrolysis were mannose, mannobiose and mannotriose for LBG or mannooligosaccharides including mannotriose, mannotetraose, mannopentaose and mannohexaose. The enzyme could hydrolyze mannooligosaccharides larger than mannobiose.

Investigation of molecular weight distribution of LBG galactomannan for flours prepared from individual seeds, mixtures, and commercial samples

The molecular weight distribution has been determined for the galactomannan solubilized from three types of locust bean gum (LBG) flours: single carob seeds, mixtures, and a range of commercial products. To prepare crude endosperm flours from carob seeds with minimal galactomannan degradation, a new extraction and milling method was developed. The method consists of applying a brief thermal shock to the seeds, followed by an extended 3-day swelling period, and manual separation of endosperms; particle size reduction to a flour is accomplished on hydrated endosperms using a centrifugal mill. This method was optimized so that redissolved LBG flours produced solutions with the highest possible viscosity and the least amount of galactomannan degradation as determined by SEC. For the three samples types, the molecular weight distribution, w( M), was found to be unimodal, appearing as a sharply defined main peak ( M p≈1.1×10 6 g/mol) with a small high molecular weight tail (up to 2.0×10 6 g/mol) and broad low molecular weight tail (down to 0.01×10 6 g/mol); polydispersities ( M w/ M n ) were estimated to be 1.5–1.8. Variations in M w and [ η] for galactomannans extracted from individual seeds originating from the same carob tree were minimal and nearly indistinguishable from a bulk mixture (6 seeds, M w=0.96–1.1×10 6 g/mol, [ η]=14.2–15.1 dl/g). There was a higher variability in these molecular parameters for galactomannans solubilized from commercial LBG flours, which generally exhibited lower M w and [ η], broader distributions, and reduced solubilities ( M w=0.86–1.0×10 6 g/mol, [ η]=12.4–13.6 dl/g). These side-effects were attributed to damage caused by industrial scale seed processing. The near constancy of M p for the three sample types suggests that the average molecular size of LBG galactomannan varies only slightly due to natural or biological causes.

Effect of Polysaccharide Structure on Mechanical and Thermal Properties of Galactomannan-Based Films

Films were prepared from guar gum and locust bean gum galactomannans. In addition, enzymatic modification was applied to guar gum to obtain structurally different galactomannans. Cohesive and flexible films were formed from galactomannans plasticized with 20-60% (w/w of polymer) glycerol or sorbitol. Galactomannans with lower galactose content (locust bean gum, modified guar gum) produced films with higher elongation at break and tensile strength. The mechanical properties of films were improved statistically significantly by decreasing the degree of polymerization of guar gum with mannanase treatments (4 h) of 2 and 10 nkat/g, whereas 50 nkat/g produced films with low elongation at break and tensile strength. Galactomannans with approximately 6 galactose units per 10 mannose backbone units resulted in films with 2 peaks in loss modulus spectra, whereas films from galactomannans with approximately 2 galactose groups per 10 mannose units behaved as a single phase in dynamic mechanical analysis.

Global hydrodynamic analysis of the molecular flexibility of galactomannans

In the past, intrinsic viscosity and sedimentation velocity analyses have been used separately to assess the conformation and flexibility of guar and locust bean gum galactomannans based on worm-like chain and semi-flexible coil models. Publication of a new global method combining data sets of both intrinsic viscosity and sedimentation coefficient with molecular weight, and minimising a target (error) function now permits a more robust analysis. Using this approach, values for the persistence length of (10 ± 2) nm for guar and (7 ± 1) nm for locust bean gum are returned if the mass per unit length M L is floated as a variable. Using a fixed mass per unit length based on the known compositional data of each galactomannan yields a similar value for L p in both cases, (8 ± 1) nm for guar and (9 ± 1) nm for locust bean gum, with combined set of data yielding (9 ± 1) nm: within experimental error the flexibilities of both galactomannans are very similar.

A Barley Xyloglucan Xyloglucosyl Transferase Covalently Links Xyloglucan, Cellulosic Substrates, and (1,3;1,4)-β-D-Glucans

Molecular interactions between wall polysaccharides, which include cellulose and a range of noncellulosic polysaccharides such as xyloglucans and (1,3;1,4)-beta-D-glucans, are fundamental to cell wall properties. These interactions have been assumed to be noncovalent in nature in most cases. Here we show that a highly purified barley xyloglucan xyloglucosyl transferase HvXET5 (EC 2.4.1.207), a member of the GH16 group of glycoside hydrolases, catalyzes the in vitro formation of covalent linkages between xyloglucans and cellulosic substrates and between xyloglucans and (1,3;1,4)-beta-D-glucans. The rate of covalent bond formation catalyzed by HvXET5 with hydroxyethylcellulose (HEC) is comparable with that on tamarind xyloglucan, whereas that with (1,3; 1,4)-beta-D-glucan is significant but slower. Matrix-assisted laser desorption ionization time-of-flight mass spectrometric analyses showed that oligosaccharides released from the fluorescent HEC:xyloglucan conjugate by a specific (1,4)-beta-D-glucan endohydrolase consisted of xyloglucan substrate with one, two, or three glucosyl residues attached. Ancillary peaks contained hydroxyethyl substituents (m/z 45) and confirmed that the parent material consisted of HEC covalently linked with xyloglucan. Similarly, partial hydrolysis of the (1,3;1,4)-beta-D-glucan:xyloglucan conjugate by a specific (1,3;1,4)-beta-D-glucan endohydrolase revealed the presence of a series of fluorescent oligosaccharides that consisted of the fluorescent xyloglucan acceptor substrate linked covalently with 2-6 glucosyl residues. These findings raise the possibility that xyloglucan endo-transglucosylases could link different polysaccharides in vivo and hence influence cell wall strength, flexibility, and porosity.

A cellulose-binding module of the Trichoderma reesei β-mannanase Man5A increases the mannan-hydrolysis of complex substrates

Endo-β-1,4- d-mannanases (β-mannanase; EC 3.2.1.78) are endohydrolases that participate in the degradation of hemicellulose, which is closely associated with cellulose in plant cell walls. The β-mannanase from Trichoderma reesei (Man5A) is composed of an N-terminal catalytic module and a C-terminal carbohydrate-binding module (CBM). In order to study the properties of the CBM, a construct encoding a mutant of Man5A lacking the part encoding the CBM (Man5AΔCBM), was expressed in T. reesei under the regulation of the Aspergillus nidulans gpdA promoter. The wild-type enzyme was expressed in the same way and both proteins were purified to electrophoretic homogeneity using ion-exchange chromatography. Both enzymes hydrolysed mannopentaose, soluble locust bean gum galactomannan and insoluble ivory nut mannan with similar rates. With a mannan/cellulose complex, however, the deletion mutant lacking the CBM showed a significant decrease in hydrolysis. Binding experiments using activity detection of Man5A and Man5AΔCBM suggests that the CBM binds to cellulose but not to mannan. Moreover, the binding of Man5A to cellulose was compared with that of an endoglucanase (Cel7B) from T. reesei.

Multiple α-galactosidases from Aspergillus niger: purification, characterization and substrate specificities

Enzymes with α-galactosidase activity are produced by many organisms, often in multiple forms. Here we compare the biochemical and hydrolytic properties of four major α-galactosidase forms (α-gal I-IV) that were purified from the culture filtrate of Aspergillus niger. α-Gal II, III and IV appear to be isoforms of the same enzyme, and N-terminal amino acid sequence data suggest that they are closely related or identical to A. niger AglB in family 27 of the glycosyl hydrolases. α-Gal I is a completely different enzyme that belongs to family 36. α-Gal I had an isoelectric point of 4.15 and appears to be a tetramer composed of four 94-kDa subunits. α-Gal II, III and IV were dimers with monomeric molecular masses of 64 kDa and isoelectric points of 4.5, 4.7 and 4.8, respectively. α-Gal II-IV were stable when incubated for 17 h at 50°C and pH 2–5, whereas α-gal I was most stable at pH 5–6. All enzymes had maximal catalytic activity at pH 4.5 and 60°C, and hydrolyzed melibiose, raffinose and stachyose. α-Gal II-IV also degraded galactomanno-oligosaccharides and released 66% of the galactose side groups from polymeric locust bean gum galactomannan. α-Gal I released galactose from locust bean gum only in combination with A. niger β-mannosidase. Kinetic experiments showed that α-gal I hydrolyzed p-nitrophenyl-α- d-galactopyranoside and melibiose more efficiently than α-gal II-IV. The distinct hydrolytic and biochemical properties of α-gal I and α-gal II-IV further signifies the difference between α-galactosidases of family 27 and 36.

Effects of xanthan and galactomannan on the freeze/thaw properties of starch gels.

Three starches (maize, rice and wheat), and the two non-starch polysaccharides xanthan and locust bean gum galactomannan (LBG) were examined in gel and dough systems for texture and stability properties during freezing and low temperature storage. Xanthan and LBG were found to confer increased resistance to freeze/thaw cycling on rice starch gels but the non-starch polysaccharides had little effect on the performance of maize and wheat starch gels or on wheat dough.

Gradient ammonium sulphate fractionation of galactomannans

Water-soluble locust bean and guar gum galactomannans were fractionated by a graded ammonium sulphate precipitation technique. Locust bean gum galactomannans yielded five fractions obtained at 20, 30, 45, 80 and 100% saturation with (NH 4) 2SO 4. The yields of the fractions based on the total material recovered after fractionation) were 3.0, 60.0, 23.0, 9.0 and 5.0, respectively. Guar gum galactomannans yielded three fractions obtained at 70, 80 and 100% saturation with (NH 4) 2SO 4. The yields were 31.0, 39.0 and 30.0%, respectively. Monosaccharide and [ 13C]-NMR analyses revealed differences in structural characteristics among samples. Fractions precipitated at relatively low saturation of (NH 4) 2SO 4 had a low degree of galactose substitution and a high frequency of unsubstituted mannose residue pairs. They also showed high limiting viscosity values. Fractions which required much higher concentration of (NH 4) 2SO 4 for precipitation were highly substituted, had low frequency of unsubstituted mannose pairs and relatively low limiting viscosity values.

Isolation of mannan-utilizing bacteria and the culture conditions for mannanase production

A locally isolated strain, Bacillus subtilis NM-39, was selected as an active mannan-utilizing bacterium based on high saccharifying activities on coconut residue and locust bean gum galactomannan. The optimal pH and temperature ranges for activity of the crude enzyme were 5.0 to 6.0 and 50 to 60°C, respectively. The organism gave maximum mannanase activity when grown in liquid mineral salts medium containing 1% (w/v) each of coconut residue and soybean flour, as carbon and nitrogen sources, respectively, at pH 7.0 and in aerobic growth for 28 h at 37°C. High saccharifying activity on coconut mannan was also observed.

Hydrolytic properties of two cellulases of Trichoderma reesei expressed in yeast.

Two cellulases of the filamentous fungus Trichoderma reesei, cellobiohydrolase II (CBHII, EC 3.2.1.91) and endoglucanase I (EGI, EC 3.2.1.4), produced in recombinant strains of the yeast Saccharomyces cerevisiae, were tested in the hydrolysis of cellulose, xylan and other polymeric substrates. Both enzymes were active against unsubstituted, insoluble cellulose. CBHII had greater activity than EGI against crystalline cellulose, whereas in the case of amorphous substrate the order was reversed. Evidence for synergism was obtained when mixtures of the two enzymes were used with a constant total protein dosage. The EGI was also active against soluble substituted cellulose derivatives, whereas the activity of CBHII against these substrates was insignificant. Both enzymes were active against barley (1-->3,1-->4)-beta-glucan, but were inactive against (1-->3,1-->6)-beta-glucan (laminarin). An apparent low mannan-degrading activity of EGI against locust-bean (Ceratonia siliqua) gum galactomannan was not confirmed when homopolymeric mannan was used as substrate in a prolonged hydrolysis test. EGI exhibited considerably greater activity against insoluble, unsubstituted hardwood xylan than against amorphous cellulose. Soluble 4-O-methyl-glucuronoxylan was also attacked by EGI, although to a somewhat lesser extent than the unsubstituted xylan. By comparison with two purified xylanases of T. reesei, EGI produced xylo-oligosaccharides with a longer mean chain length when acting on both substituted and unsubstituted xylan substrates. CBHII was inactive against xylan.

Isolation and characterization of alpha-galactosidase from Lens esculanta.

α‐Galactosidase from Lens esculanta (red lentils) was purified 4090‐fold through a multistep process involving acidification, ammonium sulphate fractionation, Sephadex gel filtration, DEAE‐cellulose and CM‐cellulose chromatography. The purified enzyme is stable at 4°C for nearly six months with only a small loss of activity. It showed a pH optimum of 5.5 with raffinose as the substrate and 6.0 with p‐nitrophenyl‐α‐d‐galactoside. The Km for the former substrate was 40 mM and for the latter, 0.26 mM.The enzyme failed to liberate galactose from guar gum, tara gum and locust bean gum galactomannans. A study of the hydrolyzability of α‐d‐galactoside, α‐d‐fucoside and β‐l‐arabinoside showed that the presence of primary alcoholic group in the sugar was important for faster hydrolysis and a stronger binding. The substrate specificity studies indicated the absence of any related glycosidase activity in the α‐galactosidase preparation.