Rhamnogalacturonan α-d-Galactopyranosyluronohydrolase1 : An Enzyme That Specifically Removes the Terminal Nonreducing Galacturonosyl Residue in Rhamnogalacturonan Regions of Pectin1

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).

Abundance of copepods from multinet samples during POLARSTERN cruise ANT-XX/1

Abundance and species composition of copepods were studied during the expedition ANT XXI/1 on a latitudinal transect in the eastern Atlantic from 34°49.5' N to 27°28.1' S between 2-20 November 2002. Stratified zooplankton tows were carried out at 19 stations with a multiple opening-closing net between 300 m water depth and the surface. Cyclopoid and calanoid copepods showed similar patterns of distribution and abundance. Oithona was the most abundant cyclopoid genus, followed by Oncaea. A total of 149 calanoid copepod species were identified. Clausocalanus was by far the most abundant genus, comprising on average about 45% of all calanoids, followed by Calocalanus (13%), Delibus (9%), Paracalanus (6%), and Pleuromamma (5%). All other genera comprised on average less than 5% each, with 40 genera less than 1%. The calanoid copepod communities were distinguished broadly in accordance with sea surface temperature, separating the subtropical from the tropical stations, and were largely determined by variation in species composition and species abundance. Nine Clausocalanus species were identified. The most numerous Clausocalanus species was C. furcatus, which on average comprised half of all adult of this genus. C. pergens, C. paululus, and C. jobei, contributed an average of 19%, 9%, and 9%, respectively. The Clausocalanus species differed markedly in their horizontal and vertical distributions: C. furcatus, C. jobei, and C. mastigophorus had widespread distributions and inhabited the upper water layers. Major differences between the species were found in abundance. C. paululus and C. arcuicornis were biantitropical and were absent or occurred in very low numbers in the equatorial zone. C. parapergens was found at all stations and showed a bimodal distribution pattern with maxima in the subtropics. C. pergens occurred in higher numbers only at the southern stations, where it replaced C. furcatus in dominance. In contrast to the widespread species, the bulk of the C. paululus, C. arcuicornis, C. parapergens, and C. pergens populations was concentrated in the colder, deeper water layers below the thermocline, thereby avoiding the warm surface waters. C. lividus was found only at the most northern and C. ingens only at the most southern stations. Both species were found almost exclusively in the upper 50 m. The distinct differences in abundance and horizontal and vertical distribution suggest a strong ecological differentiation among the Clausocalanus species.

Unravelling the nanostructure of strawberry fruit pectins by atomic force microscopy

Atomic force microscopy (AFM) allows the analysis of individual polymers at nanostructural level with a minimal sample preparation. This technique has been used to analyse the pectin disassembly process during the ripening and postharvest storage of several fleshy fruits. In general, pectins analysed by AFM are usually visualized as isolated chains, unbranched or with a low number of branchs and, occasionally, as large aggregates. However, the exact nature of these structures is unknown. It has been suggested that pectin aggregates represent a mixture of rhamnonogalacturonan I and homogalacturonan, while isolated chains and their branches are mainly composed by polygalacturonic acid. In order to gain insight into the nature of these structures, sodium carbonate soluble pectins from ripe strawberry (Fragaria x ananassa, Duch.) fruits were subjected to enzymatic digestion with endo-Polygalacturonase M2 from Aspergillus aculeatus, and the samples visualized by AFM at different time intervals. Pectins isolated from control, non-transformed plants, and two transgenic genotypes with low level of expression of ripening-induced pectinase genes encoding a polygalacturonase (APG) or a pectate lyase (APEL) were also included in this study. Before digestion, isolated pectin chains from control were shorter than those from transgenic fruits, showing number-average (LN) contour length values of 73.2 nm vs. 95.9 nm and 91.4 nm in APG and APEL, respectively. The percentage of branched polymers was significantly higher in APG polyuronides than in the remaining genotypes, 33% in APG vs. 6% in control and APEL. As a result of the endo-PG treatment, a gradual decrease in the main backbone length of isolated chains was observed in the three samples. The minimum LN value was reached after 8 h of digestion, being similar in the three genotypes, 22 nm. By contrast, the branches were not visible after 1.5-2 h of digestion. LN values were plotted against digestion time and the data fitted to a first-order exponential decay curve, obtaining R2 values higher than 0.9. The half digestion time calculated with these equations were similar for control and APG pectins, 1.7 h, but significantly higher in APEL, 2.5 h, indicating that these polymer chains were more resistant to endo-PG digestion. Regarding the pectin aggregates, their volumes were estimated and used to calculate LN molecular weights. Before digestion, control and APEL samples showed complexes of similar molecular weights, 1722 kDa, and slightly higher than those observed in APG samples. After endo-PG digestion, size of complexes diminished significantly, reaching similar values in the three pectin samples, around 650 kDa. These results suggest that isolated polymer chains visualized by AFM are formed by a HG domain linked to a shorter polymer resistant to endo-PG digestion, maybe xylogalacturonan or RG-I. The silencing of the pectate lyase gene slightly modified the structure and/or chemical composition of polymer chains making these polyuronides more resistant to enzymatic degradation. Similarly, polygalacturonic acid is one of the main component of the aggregates.