Pectin and pectinases: Production, characterization and industrial application of microbial pectinolytic enzymes

Pectinases are a big group of enzymes that break down pectic polysaccharides of plant tissues into simpler molecules like galacturonic acids. It has long been used to increase yields and clarity of fruit juices. Since pectic substances are a very complex macromolecule group, various pectinolytic enzymes are required to degrade it completely. These enzymes present differences in their cleavage mode and specificity being basically classified into two main groups that act on pectin smooth regions or on pectin hairy regions. Pectinases are one of the most widely distributed enzymes in bacteria, fungi and plants. This review describes the pectinolytic enzymes and their substrates, the microbial pectinase production and characterization, and the industrial application of these enzymes. © Pedrolli et al.; Licensee Bentham Open.

Borate-Rhamnogalacturonan II Bonding Reinforced by Ca2+ Retains Pectic Polysaccharides in Higher-Plant Cell Walls1

The extent of in vitro formation of the borate-dimeric-rhamnogalacturonan II (RG-II) complex was stimulated by Ca2+. The complex formed in the presence of Ca2+ was more stable than that without Ca2+. A naturally occurring boron (B)-RG-II complex isolated from radish (Raphanus sativus L. cv Aokubi-daikon) root contained equimolar amounts of Ca2+ and B. Removal of the Ca2+ by trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid induced cleavage of the complex into monomeric RG-II. These data suggest that Ca2+ is a normal component of the B-RG-II complex. Washing the crude cell walls of radish roots with a 1.5% (w/v) sodium dodecyl sulfate solution, pH 6.5, released 98% of the tissue Ca2+ but only 13% of the B and 22% of the pectic polysaccharides. The remaining Ca2+ was associated with RG-II. Extraction of the sodium dodecyl sulfate-washed cell walls with 50 mm trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid, pH 6.5, removed the remaining Ca2+, 78% of B, and 49% of pectic polysaccharides. These results suggest that not only Ca2+ but also borate and Ca2+ cross-linking in the RG-II region retain so-called chelator-soluble pectic polysaccharides in cell walls.

Characterization of Recombinant Rhamnogalacturonan α-l-Rhamnopyranosyl-(1,4)-α-d-Galactopyranosyluronide Lyase from Aspergillus aculeatus1 : An Enzyme That Fragments Rhamnogalacturonan I Regions of Pectin

The four major oligomeric reaction products from saponified modified hairy regions (MHR-S) from apple, produced by recombinant rhamnogalacturonan (RG) α-l-rhamnopyranosyl-(1,4)-α-d-galactopyranosyluronide lyase (rRG-lyase) from Aspergillus aculeatus, were isolated and characterized by 1H-nuclear magnetic resonance spectroscopy. They contain an alternating RG backbone with a degree of polymerization of 4, 6, 8, and 10 and with an α-Δ-(4,5)-unsaturated d-galactopyranosyluronic acid at the nonreducing end and an l-rhamnopyranose at the reducing end. l-Rhamnopyranose units are substituted at C-4 with β-galactose. The maximum reaction rate of rRG-lyase toward MHR-S at pH 6.0 and 31°C was 28 units mg−1. rRG-lyase and RG-hydrolase cleave the same alternating RG I subunit in MHR. Both of these enzymes fragment MHR by a multiple attack mechanism. The catalytic efficiency of rRG-lyase for MHR increases with decreasing degree of acetylation. Removal of arabinose side chains improves the action of rRG-lyase toward MHR-S. In contrast, removal of galactose side chains decreased the catalytic efficiency of rRG-lyase. Native RG-lyase was purified from A. aculeatus, characterized, and found to be similar to the rRG-lyase expressed in Aspergillus oryzae.

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).

In vitro determination of prebiotic properties of oligosaccharides derived from an orange juice manufacturing by-product stream

Fermentation properties of oligosaccharides derived from orange peel pectin were assessed in mixed fecal bacterial culture. The orange peel oligosaccharide fraction contained glucose in addition to rhamnogalacturonan and xylogalacturonan pectic oligosaccharides. Twenty-four-hour, temperature- and pH-controlled, stirred anaerobic fecal batch cultures were used to determine the effects that oligosaccharides derived from orange products had on the composition of the fecal microbiota. The effects were measured through fluorescent in situ hybridization to determine changes in bacterial populations, fermentation end products were analyzed by high-performance liquid chromatography to assess short-chain fatty acid concentrations, and subsequently, a prebiotic index (PI) was determined. Pectic oligosaccharides (POS) were able to increase the bifidobacterial and Eubacterium rectale numbers, albeit resulting in a lower prebiotic index than that from fructo-oligosaccharide metabolism. Orange albedo maintained the growth of most bacterial populations and gave a PI similar to that of soluble starch. Fermentation of POS resulted in an increase in the Eubacterium rectale numbers and concomitantly increased butyrate production. In conclusion, this study has shown that POS can have a beneficial effect on the fecal microflora; however, a classical prebiotic effect was not found. An increase in the Eubacterium rectale population was found, and butyrate levels increased, which is of potential benefit to the host.

Comparative in situ analyses of cell wall matrix polysaccharide dynamics in developing rice and wheat grain

Cell wall polysaccharides of wheat and rice endosperm are an important source of dietary fibre. Monoclonal antibodies specific to cell wall polysaccharides were used to determine polysaccharide dynamics during the development of both wheat and rice grain. Wheat and rice grain present near synchronous developmental processes and significantly different endosperm cell wall compositions, allowing the localisation of these polysaccharides to be related to developmental changes. Arabinoxylan (AX) and mixed-linkage glucan (MLG) have analogous cellular locations in both species, with deposition of AX and MLG coinciding with the start of grain filling. A glucuronoxylan (GUX) epitope was detected in rice, but not wheat endosperm cell walls. Callose has been reported to be associated with the formation of cell wall outgrowths during endosperm cellularisation and xyloglucan is here shown to be a component of these anticlinal extensions, occurring transiently in both species. Pectic homogalacturonan (HG) was abundant in cell walls of maternal tissues of wheat and rice grain, but only detected in endosperm cell walls of rice in an unesterified HG form. A rhamnogalacturonan-I (RG-I) backbone epitope was observed to be temporally regulated in both species, detected in endosperm cell walls from 12 DAA in rice and 20 DAA in wheat grain. Detection of the LM5 galactan epitope showed a clear distinction between wheat and rice, being detected at the earliest stages of development in rice endosperm cell walls, but not detected in wheat endosperm cell walls, only in maternal tissues. In contrast, the LM6 arabinan epitope was detected in both species around 8 DAA and was transient in wheat grain, but persisted in rice until maturity.

Tissue-specific expression of esterases in Triatoma infestans (Triatominae, Heteroptera)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Extracellular matrix of the charophycean green algae

A comprehensive knowledge of cell wallstructure and function throughout the plant kingdom is essential to understanding cell wall evolution. The fundamental understanding of the charophycean green algal cell wall is broadening. The similarities and differences that exist between land plant and algal cell walls provide opportunities to understand plant evolution. A variety of polymers previously associated with higher plants were discovered in the charophycean green algae (CGA), including homogalacturonans, cross-linking glycans, arabinogalactan protein, β-glucans, and cellulose. The cellulose content of CGA cell walls ranged from 6% to 43%, with the higher valuescomparable to that found in the primary cell wall of land plants (20-30%). (1,3)β-glucans were found in the unicellular Chlorokybus atmophyticus, Penium margaritaceum, and Cosmarium turpini, the unbranched filamentous Klebsormidium flaccidum, and the multicellular Chara corallina. The discovery of homogalacturonan in Penium margaritaceum representsthe first confirmation of land plant-type pectinsin desmids and the second rigorous characterization of a pectin polymer from the charophycean algae. Homogalacturonan was also indicated from the basal species Chlorokybus atmophyticus and Klebsormidium flaccidum. There is evidence of branched pectins in Cosmarium turpini and linkage analysis suggests the presence of type I rhamnogalacturonan (RGI). Cross-linking β-glucans are associated with cellulose microfibrils during land plant cell growth, and were found in the cell wall of CGA. The evidence of mixed-linkage glucan (MLG) in the 11 charophytesis both suprising and significant given that MLG was once thought to be specific to some grasses. The organization and structure of Cosmarium turpini and Chara corallina MLG was found to be similar to that of Equisetumspp., whereas the basal species of the CGA, Chlorokybus atmophyticus and Klebsormidium flaccidum, have unique organization of alternating of 3- and 4-linkages. The significance of this result on the evolution of the MLG synthetic pathway has yet to be determined. The extracellular matrix (ECM) of Chlorokybus atmophyticus, Klebsormidium flaccidum, and Spirogyra spp. exhibits significant biochemical diversity, ranging from distinct “land plant” polymers to polysaccharides unique to these algae. The neutral sugar composition of Chlorokybus atmophyticus hot water extract and Spirogyra extracellular polymeric substance (EPS), combined with antibody labeling results, revealed the distinct possibility of an arabinogalactan protein in these organisms. Polysaccharide analysis of Zygnematales (desmid) EPS, indicated a probable range of different EPS backbones and substitution patterns upon the core portions of the molecules. Desmid EPS is predominately composed of a complex matrix of branched, uronic acid containing polysaccharides with ester sulfate substitutions and, as such, has an almost infinite capacity for various hydrogen bonding, hydrophobic interaction and ionic cross-bridging motifs, which characterize their unique function in biofilms. My observations support the hypothesis that members of the CGA represent the phylogenetic line that gave rise to vascular plants and that the primary cell wall of vascular plants many have evolved directly from structures typical of the cell wall of filamentous green algae found in the charophycean green algae.

Identification and Partial Characterization of the Pectin Methyltransferase “Homogalacturonan-Methyltransferase” from Membranes of Tobacco Cell Suspensions1

A membrane preparation from tobacco (Nicotiana tabacum L.) cells contains at least one enzyme that is capable of transferring the methyl group from S-adenosyl-methionine (SAM) to the C6 carboxyl of homogalacturonan present in the membranes. This enzyme is named homogalacturonan-methyltransferase (HGA-MT) to distinguish it from methyltransferases that catalyze methyletherification of the pectic polysaccharides rhamnogalacturonan I or rhamnogalacturonan II. A trichloroacetic acid precipitation assay was used to measure HGA-MT activity, because published procedures to recover pectic polysaccharides via ethanol or chloroform:methanol precipitation lead to high and variable background radioactivity in the product pellet. Attempts to reduce the incorporation of the 14C-methyl group from SAM into pectin by the addition of the alternative methyl donor 5-methyltetrahydrofolate were unsuccessful, supporting the role of SAM as the authentic methyl donor for HGA-MT. The pH optimum for HGA-MT in membranes was 7.8, the apparent Michaelis constant for SAM was 38 μm, and the maximum initial velocity was 0.81 pkat mg−1 protein. At least 59% of the radiolabeled product was judged to be methylesterified homogalacturonan, based on the release of radioactivity from the product after a mild base treatment and via enzymatic hydrolysis by a purified pectin methylesterase. The released radioactivity eluted with a retention time identical to that of methanol upon fractionation over an organic acid column. Cleavage of the radiolabeled product by endopolygalacturonase into fragments that migrated as small oligomers of HGA during thin-layer chromatography, and the fact that HGA-MT activity in the membranes is stimulated by uridine 5′-diphosphate galacturonic acid, a substrate for HGA synthesis, confirms that the bulk of the product recovered from tobacco membranes incubated with SAM is methylesterified HGA.

DEVELOPMENT OF INTEGRATED BIOPROCESSING TECHNOLOGIES FOR THE PRODUCTION OF PECTIC OLIGOSACCHARIDES (POS) FROM AGRO-PROCESSING RESIDUES

This research deals with the production of pectic oligosaccharides (POS) from agro-industrial residues, with specific focus on development of continuous cross flow enzyme membrane reactor. Pectic oligosaccharides have recently gained attention due to their prebiotic activity. Lack of information on the continuous production of POS from agro-industrial residues formed the basis for the present study. Four residues i.e sugar beet pulp, onion hulls, pressed pumpkin cake and berry pomace were taken to study their pectin content. Based on the presence of higher galacturonic acid and arabinose (both homogalacturonan and rhamnogalacturonan) in sugar beet pulp and galacturonic acid (only homogalacturonan) in onion hulls, further optimization of different extraction methods of pectin (causing minimum damage to pectic chain) from these residues were done. The most suitable extractant for sugar beet pulp and onion hulls were nitric acid and sodium hexametaphosphate respectively. Further the experiments on the continuous production of POS from sugar beet pulp in an enzyme membrane reactor was initiated. Several optimization experiments indicated the optimum enzyme (Viscozyme) as well as feed concentration (25 g/L) to be used for producing POS from sugar beet pulp in an enzyme membrane reactor. The results highlighted that steady state POS production with volumetric and specific productivity of 22g/L/h and 11 g/gE/h respectively could be achieved by continuous cross flow filtration of sugar beet pulp pectic extract over 10 kDa membrane at residence time of 20 min. The POS yield of about 80% could be achieved using above conditions. Also, in this thesis preliminary experiments on the production and characterization of POS from onion hulls were conducted. The results revelaed that the most suitable enzyme for POS production from onion hulls is endo-polygalacturonase M2. The POS produced from onion hulls were present in the form of DP1 -DP10 in substituted as well as unsubstituted forms. This study clearly demonstrates that continuous production of POS from pectin rich sources can be achieved by using cross flow continuous enzyme membrane reactor.

Mineral stress response of Vitis cell wall transcriptome: identification, characterization and expression of genes from key families involved in wall biosynthesis and modification

Doutoramento em Engenharia Agronómica - Instituto Superior de Agronomia - UL