The safety of a rhamnogalacturonan-I-enriched pectin extract (G3P-01) from pumpkin (Cucurbita moschata var. Dickinson) was evaluated for use as an ingredient in food and dietary supplements. G3P-01 was tested in a battery of genetic toxicity studies including reverse mutagenicity and in vitro micronucleus assay. In addition, Sprague-Dawley rats were randomized and orally dosed with G3P-01 incorporated in animal diet at concentrations of 0, 9000, 18,000, and 36,000 ppm daily for 13-weeks (n=10/sex/group) in line with OECD guidelines (TG 408). The results of the in vitro bacterial reverse mutation assay and micronucleus assay in TK6 cells demonstrated a lack of genotoxicity. The 13-week oral toxicity study in Sprague-Dawley rats demonstrated that the test article, G3P-01 was well tolerated; there were no mortalities and no adverse effects on clinical, gross pathology, hematology, blood chemistry, and histological evaluation of the essential organs of the animals. The present study demonstrates that G3P-01 is non-genotoxic and is safe when ingested in diet at concentrations up to 36, 000 ppm. The subchronic no-observed-adverse-effect level (NOAEL) for G3P-01 was concluded to be 36,000 ppm, equivalent to 1,899 and 2,361 mg/kg/day for male and female rats respectively.

lhe recently described rhamnogalacturonase B, which is able to degrade ramified hairy regions of pectin, was found to be a rhamnogalacturonan a-i-rhamnopyranosyl-( 1 +4)-a-~-galactopyrano-syluronide lyase. The cleavage site and mechanism differ from that of the previously described rhamnogalacturonase A, which is a hydrolase and can now be termed rhamnogalacturonan a-D-galac-topyranosyluronide-( 1 +2)-a-i-rhamnopyranosyl hydrolase.

The epoxide hydrolase from Rhodotorula glutinis was isolated and initially characterized. The enzyme was membrane associated and could be solubilized by Triton X-100. Purification yielded an enzyme with sp. act. of 66 μmol 1,2-epoxyhexane hydrolyzed min−1 mg−1 protein. The enzyme was not completely purified to homogeneity but, nevertheless, a major protein was isolated by SDS-PAGE for subsequential amino acid determination of peptide fragments. From sequence alignments to related enzymes, a high homology towards the active site sequences of other microsomal epoxide hydrolases was found. Molecular mass determinations indicated that the native enzyme exists as a homodimer, with a subunit molecular mass of about 45 kDa. Based upon these, this epoxide hydrolase is structurally related to other microsomal epoxide hydrolases.

Abstract A new enzyme, rhamnogalacturonan (RG) α-d-galactopyranosyluronohydrolase (RG-galacturonohydrolase), able to release a galacturonic acid residue from the nonreducing end of RG chains but not from homogalacturonan, was purified from an Aspergillus aculeatus enzyme preparation. RG-galacturonohydrolase acted with inversion of anomeric configuration, initially releasing β-d-galactopyranosyluronic acid. The enzyme cleaved smaller RG substrates with the highest catalytic efficiency. A Michaelis constant of 85 μm and a maximum reaction rate of 160 units mg−1 was found toward a linear RG fragment with a degree of polymerization of 6. RG-galacturonohydrolase had a molecular mass of 66 kD, an isoelectric point of 5.12, a pH optimum of 4.0, and a temperature optimum of 50°C. The enzyme was most stable between pH 3.0 and 6.0 (for 24 h at 40°C) and up to 60°C (for 3 h).

The mode of action of RG-hydrolase and RG-lyase toward purified linear rhamnogalacturonan (RG) oligomers has been studied. Major tools in the characterization of the degradation products were the exo-acting RG-rhamnohydrolase and RG-galacturonohydrolase. They were used to prepare a series of standards of RG oligomers for HPAEC. 1H NMR spectroscopy confirmed the structure assignment made using HPAEC for a selection of isolated degradation products. Identification of degradation products from purified RG oligomers was then performed by comparing retention times of HPAEC peaks with those of standards. RG-hydrolase was able to cleave RG oligomers which contained five Rha units or more, i.e. DP 9 with a Rha unit at both nonreducing and reducing end. Its preferential cleavage site was at four units from the first nonreducing Rha. RG-lyase was active toward oligomers that contained at least six GalA units, i.e. DP 12 with a GalA at the nonreducing and a Rha at the reducing end. The preferential cleavage site was for the smaller oligomers four residues, and for the largest oligomer six residues from the reducing Rha. From the observed cleavage patterns it can be speculated that in hairy regions, the RG stretches have to be at least 13 residues long for RG-hydrolase and 16 residues long for RG-lyase in order to produce one tetramer.

The stereochemical course of hydrolysis catalysed by four Aspergillus aculeatus enzymes acting on alpha-L-rhamnosyl and alpha-D-galacturonosyl linkages in the hairy regions of pectins has been determined using 1H-NMR. Exogalacturonase acts with inversion of anomeric configuration (e-->a), shown by the initial release of beta-D-GalpA from the non-reducing end of polygalacturonic acid. Similarly, rhamnogalacturonan (RG) hydrolase also acts with inversion of anomeric configuration (e-->a) during hydrolysis of alpha-D-GalpA-(1-->2)-alpha-L-Rhap linkages in RG, initially releasing oligosaccharides with beta-D-GalpA at the reducing end. This result is consistent with the recently solved crystal structure of this enzyme, as well as its classification based on amino acid sequence similarity into glycosyl hydrolase family 28. alpha-L-Rhamnosidase and RG-rhamnohydrolase also act with inversion of configuration (a-->e), initially releasing beta-L-Rhap from p-nitrophenyl alpha-L-rhamnopyranoside and RG oligosaccharides, respectively. Thus, all four enzymes examined are inverting hydrolases which probably catalyse hydrolysis via single displacement mechanisms.

The stereochemical course of hydrolysis catalysed by fourAspergillus aculeatusenzymes acting on α-l-rhamnosyl and α-d-galacturonosyl linkages in the hairy regions of pectins has been determined using1H-NMR. Exogalacturonase acts with inversion of anomeric configuration (e→a), shown by the initial release of β-d-GalpA from the non-reducing end of polygalacturonic acid. Similarly, rhamnogalacturonan (RG) hydrolase also acts with inversion of anomeric configuration (e→a) during hydrolysis of α-d-GalpA-(1→2)-α-l-Rhaplinkages in RG, initially releasing oligosaccharides with β-d-GalpA at the reducing end. This result is consistent with the recently solved crystal structure of this enzyme, as well as its classification based on amino acid sequence similarity into glycosyl hydrolase family 28. α-l-Rhamnosidase and RG-rhamnohydrolase also act with inversion of configuration (a→e), initially releasing β-l-Rhapfromp-nitrophenyl α-l-rhamnopyranoside and RG oligosaccharides, respectively. Thus, all four enzymes examined are inverting hydrolases which probably catalyse hydrolysis via single displacement mechanisms.

The recently described rhamnogalacturonase B, which is able to degrade ramified hairy regions of pectin, was found to be a rhamnogalacturonan [alpha]-L-rhamnopyranosyl-(1->4)-[alpha]-D-galactopyranosyluronide lyase. The cleavage site and mechanism differ from that of the previously described rhamnogalacturonase A, which is a hydrolase and can now be termed rhamnogalacturonan [alpha]-D-galactopyranosyluronide-(1->2)-[alpha]-L-rhamnopyranosyl hydrolase.

A new enzyme, rhamnogalacturonan (RG) alpha-D-galactopyranosyluronohydrolase (RG-galacturonohydrolase), able to release a galacturonic acid residue from the nonreducing end of RG chains but not from homogalacturonan, was purified from an Aspergillus aculeatus enzyme preparation. RG-galacturonohydrolase acted with inversion of anomeric configuration, initially releasing beta-D-galactopyranosyluronic acid. The enzyme cleaved smaller RG substrates with the highest catalytic efficiency. A Michaelis constant of 85 mu M and a maximum reaction rate of 160 units mg(-1) was found toward a linear RG fragment with a degree of polymerization of 6. RG-galacturonohydrolase had a molecular mass of 66 kD, an isoelectric point of 5.12, a pH optimum of 4.0, and a temperature optimum of 50 degrees C. The enzyme was most stable between pH 3.0 and 6.0 (for 24 h at 40 degrees C) and up to 60 degrees C (for 3 h).

Controlled acid hydrolysis was applied to a deesterified beet pulp and the resulting soluble fraction was fractionated on a Biorad AG 1X8 column eluted by ammonium acetate pH 6 from 0.05 to 2 M. Eight retained fractions were obtained, containing almost exclusively GalA and Rha. Three types of oligomers could be identified: homogalacturonans, of which mono-,di- and tri-GalA were isolated as individual components, and two series of rhamnogalacturonan (RG) oligomers. One RG oligomer, isolated after ion-exchange chromatography, was identified as alpha-D-GalAp-(1-->2)-alpha-L-Rhap-(1-->4)-alpha-D-GalA p-(1-->2)-L-Rhap. The major peak contained oligomers of dp 6 to more than 20, of which dp 6 to 16 could be isolated on Bio-Gel P-6 + P-4. NMR of the oligomers of dp 6 to 10 showed the following structure: alpha-D-GalAp-(1[-->2)-alpha-L-Rhap-(1-->4)-alpha-D-GalA p-(1]n-->2)-L-Rhap. A second, quantitatively minor, series of RG oligomers eluted at higher ionic strength. These oligomers, which could be hydrolysed by RG-hydrolase and RG-lyase, were based on the alternating RG structure. Their non-reducing end was GalA, susceptible to hydrolysis by RG-galacturonohydrolase, and their reducing end might have more than one consecutive GalA.

Microbial pectinases have been used for fruit and vegetable processing for already more than half a decade. With respect to application as well as to fundamental research, most attention has been paid to those enzymes acting towards the 'smooth' homogalacturonan part of the pectin molecule (i.e. polygalacturonase, pectin lyase, pectate lyase, pectin methyl esterase). More recently, enzymes active towards the 'hairy' rhamnogalacturonan part of pectin gained attention, since it was found that in juice processing those structures foul the ultrafiltration membranes used in a final clarification step. Two different rhamnogalacturonases (RGases A and B) were identified and purified from an Aspergillus aculeatus preparation, using apple pectic hairy regions (MHR) as substrate. Based on the structure of the products, RGase A was identified as a hydrolase, splitting the alpha-GalA(p)-(1-2)-alpha-Rha(p) linkage in rhamnogalacturonan, while RGase B appeared to be a lyase splitting the alpha-Rha(p) alpha-(1-4)-GalA(p) linkage by beta-elimination. Rhamnogalacturonan oligosaccharides were used to identify and purify two other novel enzymes with high specificity towards rhamnogalacturonan fragments: a rhamnogalacturonan rhamnohydrolase acid a rhamnogalacturonan galacturonohydrolase. As an accessory enzyme for the RGases, rhamnogalacturonan acetyl esterase (RGAE) was discovered in the same A. aculeatus preparation. This enzyme appeared to be specific for the de-acetylation of MHR and essential for the degradation of MHR by RGases A and B. The same enzyme (RGAE) could be purified from A. niger, together with two other esterases: a feruloyl esterase (FAE) and an acetyl esterase (PAE) specific for the removal of one type of acetyl group present in the 'smooth' regions of sugar-beet pectin. Finally, the A. aculeatus preparation was found to contain an enzyme releasing the dimer beta-Xyl(p)-(1-3)-GalA(p) from a soluble soy cell wall polysaccharide. The enzyme was partially purified and appeared to be active towards saponified MHR and gum tragacanth as well. It was concluded that this enzyme degraded the xylogalacturonan part in MHR by an exo-fashion.

Linear rhamnogalacturonan (RG) fragments (RGO's) isolated from an acid hydrolysate of saponified sugar beet pulp were treated with the enzymes RG-hydrolase and RG-lyase. Major tools in the characterisation of the formed degradation products were the exo-acting enzymes RG-rhamnohydrolase and RG-galacturonohydrolase. These exo-enzymes were used to prepare a series of standards of RG oligomers and furthermore to confirm structure assignments, made using high-performance anion-exchange chromatography (HPAEC). The RG-hydrolase was active toward RGO's when the degree of polymerisation (DP) was 12 or higher, while the RG-lyase was only active when the DP was 14 or higher. The alternating RG sequences have to be at least 16 to 18 units long to produce similar oligomers as RG-hydrolase and RG-lyase liberate from apple modified hairy regions (MHR).

The recently described rhamnogalacturonase B (RGase B), which is able to degrade hairy regions of pectin, was found to be a rhamnogalacturonan (RG) alpha-L-rhamnopyranosyl(1-->4)-alpha-D-galactopyranosyluronide lyase. The cleavage site and mechanism are different from that of the previously described rhamnogalacturonase A (RGase A), which is a hydrolase, and can now be termed RG alpha-D-galactopyranosyluronide-(1-->2)-alpha-L-rhamnopyranosyl hydrolase.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Sugar-beet pulp was saponified and then hydrolysed with 0.1 M HCI at 80°C for 72 h, and a rhamnogalacturonan fraction was isolated by ion-exchange chromatography on AG 1X8 resin. Four individual oligomers, and a mixture of oligomers with higher degrees of polymerization, were obtained by chromatography on BioGel P-4. They all presented the α-d-GalAp-(1 [→2)-α-l-Rhap-(1 → 4)-α-d-GalAp-(1]n→2)-l-Rhap structure (with n ⩾2) The five fractions were submitted to hydrolysis with rhamnogalacturonase. The enzyme was active on oligomers with degrees of polymerization f10, and gave as main products α-l-Rhap-(1 → 4)-α-d-GalAp-(1 → 2)-α-l-Rhap(1 → 4)-d-GalAp and α-d-GalAp-(1 → 2)-α-l- Rhap- (1 → 4) -α-d- GalAp- (1 → 2) -α-l-Rhap-(1 → 4)-d-GalAp.

Two [alpha]-L-rhamnohydrolases with different substrate specificities were isolated from a commercial preparation produced by Aspergillus aculeatus. The first rhamnohydrolase was active toward p-nitrophenyl-[alpha]-L-rhamnopyranoside, naringin, and hesperidin and was termed p-nitrophenyl-[alpha]-L-rhamnopyranohydrolase (pnp-rhamnohydrolase). From the data collected, the enzyme seemed specific for the [alpha]-1,2- or [alpha]-1,6-linkage to [beta]-D-glucose. The pnp-rhamnohydrolase had a molecular mass of 87 kD (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), a pH optimum of 5.5 to 6, a temperature optimum of 60[deg]C, and a specific activity toward pnp-[alpha]-L-rhamnopyranoside (pnp-Rha) of 13 units mg-1 protein. The second rhamnohydrolase, on the contrary, was active toward rhamnogalacturonan (RG) fragments, releasing Rha, and was therefore termed RG-rhamnohydrolase. The RG-rhamnohydrolase had a molecular mass of 84 kD, a pH optimum of 4, a temperature optimum of 60[deg]C, and a specific activity toward RG oligomers of 60 units mg-1 protein. The RG-rhamnohydrolase liberated Rha from the nonreducing end of the RG chain and appeared specific for the [alpha]-1,4-linkage to [alpha]-D-galacturonic acid. The enzyme was hindered when this terminal Rha residue was substituted at the 4-position by a [beta]-D-galactose. The results so far obtained did not indicate particular preference of the enzyme for low or high molecular mass RG fragments. From the results it can be concluded that a new enzyme, an RG [beta]-L-rhamnopyranohydrolase, has been isolated with high specificity toward RG regions of pectin.