Cell Wall Polysaccharide-degrading Enzymes Research Articles

Characterization of Crude and Processed Pulp Cell Walls of Three Selected Mamey Accessions (Mammea americana L.).

Mamey (Mammea americana L.) is a tropical fleshy fruit native from the West Indies and northern South America. It is very appreciated for its flavor and color but has been little described. The present study investigates the composition and histochemistry of the pulp cell walls of three mamey accessions readily available in Martinique. The impact of pulp processing into puree on cell wall composition is evaluated. The histology and rheology of mamey puree are assessed considering these characterizations. Mamey pulp cell wall composition is dominated by highly methyl-esterified pectins (DM: 66.2-76.7%) of high molecular weight, and show few hemicelluloses, mainly xyloglucans. Processing reduced methyl-esterified uronic acid contents and gave purees with significantly different viscosities. Mamey puree was composed of polydisperse particles (20-2343 µm), which size distributions were different depending on the accession: Ti Jacques was dominated by smaller particles (50% had approximated diameters lower than 160 µm), Sonson's by larger particles (50% had approximated diameters higher than 900 µm), and Galion's had an intermediate profile. This new knowledge on mamey pulp is valuable for future works on mamey processing into new food products, even more so for those including cell wall polysaccharide-degrading enzymes.

Mechanism of ozone treatment in delayed softening of fresh-cut kiwifruit during storage

Firmness is one of the most important factors affecting the quality and commercial value of fresh-cut kiwifruits. The aim of this study was to investigate the effects of 1 mg/L of gaseous ozone treatment for 10 min on the texture of fresh-cut kiwifruit stored at 4 °C for 12 d. Ozone treatment relatively maintained the firmness of the fresh-cut fruit for 12 d compared with the untreated group. Laser scanning confocal microscopy (LSCM) showed that endogenous free Ca2+ was higher and more evenly distributed in ozone-treated fruits than in the untreated group, and correlation analysis indicated that the higher the Ca2+ concentration, the greater the formation of Ca-pectinate. Additionally, ozone treatment inhibited the activity of cell wall polysaccharide-degrading enzymes, suppressed electrolyte leakage and malondialdehyde and hydrogen peroxide contents, and maintained protopectin and cellulose levels, thereby preventing fruit softening. Overall, these results showed that ozone treatment delayed the softening of fresh-cut kiwifruit by increasing endogenous Ca2+ concentrations and preventing polysaccharide solubilization.

Optimization of xylanase production by filamentous fungi in solid-state fermentation and scale-up to horizontal tube bioreactor.

Five microorganisms, namely Aspergillus niger CECT 2700, A. niger CECT 2915, A. niger CECT 2088, Aspergillus terreus CECT 2808, and Rhizopus stolonifer CECT 2344, were grown on corncob to produce cell wall polysaccharide-degrading enzymes, mainly xylanases, by solid-state fermentation (SSF). A. niger CECT 2700 produced the highest amount of xylanases of 504 ± 7 U/g dry corncob (dcc) after 3 days of fermentation. The optimization of the culture broth (5.0 g/L NaNO3, 1.3 g/L (NH4)2SO4, 4.5 g/L KH2PO4, and 3 g/L yeast extract) and operational conditions (5 g of bed loading, using an initial substrate to moistening medium of 1:3.6 (w/v)) allowed increasing the predicted maximal xylanase activity up to 2,452.7 U/g dcc. However, different pretreatments of materials, including destarching, autoclaving, microwave, and alkaline treatments, were detrimental. Finally, the process was successfully established in a laboratory-scale horizontal tube bioreactor, achieving the highest xylanase activity (2,926 U/g dcc) at a flow rate of 0.2 L/min. The result showed an overall 5.8-fold increase in xylanase activity after optimization of culture media, operational conditions, and scale-up.

Production and seeding of protoplasts of Porphyra okhaensis (Bangiales, Rhodophyta) in laboratory culture

High yields of viable protoplasts were produced from Porphyra okhaensis H. Joshi, Oza & Tewari following two-step enzymatic digestion (protease pretreatment and cell wall polysaccharides-degrading enzyme treatment) of the thallus. Pretreatment of the tissues with 1% Protease P6 at 20± 1 °C for 30 min prior to digestion with cell wall polysaccharide-degrading enzymes increased the protoplast yield two fold compared to tissues that were digested with polysaccharide-degrading enzyme mixture. The polysaccharide-degrading enzymes employed for protoplast isolation from P. okhaensis were Cellulase Onozuka R-10, Macerozyme R-10, abalone acetone powder and agarase. Suitable pH, temperature and duration of enzyme treatment for optimal production of viable protoplasts were pH 6, 20± 1 °C and 3 h, respectively. Mannitol (0.8 M) was found to be an excellent osmotic stabilizer. When the tissue of P. okhaensis pretreated with 1% protease solution was digested with commercial enzyme mixture consisting of 2% Cellulase Onozuka R-10, 2% Macerozyme R-10, 1% abalone acetone powder, 50 units of agarase and 0.8 M mannitol in 1% NaCl (adjusted to pH 6.0 with 25 mM MES buffer) with gentle agitation for 3 h at 20± 1 °C, 23.2± 0.24× 106 protoplasts g−1 fresh wt. were obtained. The regeneration rate of protoplasts isolated in the present study was found to be 79%. Protoplasts that regenerated cell walls underwent regular cell divisions and developed into leafy gametophytic thallus in the laboratory cultures. Further, the seeding of nylon threads with partially developed protoplasts of P. okhaensis was successful in the laboratory conditions and germlings as long as 3–4 cm were obtained from such seeded threads in one month period in aerated cultures.

Regulation of Aspergillus genes encoding plant cell wall polysaccharide-degrading enzymes; relevance for industrial production.

The genus Aspergillus is widely used for the production of plant cell wall polysaccharide-degrading enzymes. The range of enzymes purified from these fungi covers nearly every function required for the complete degradation of cellulose, xyloglucan, xylan, galacto(gluco)mannan and pectin. This paper describes the Aspergillus enzymes involved in the degradation of these polysaccharides and discusses the regulatory systems involved in the expression of the genes encoding these proteins. The latter is of major importance in the large-scale production of these enzymes for industrial applications.

Release of ferulic acid from agroindustrial by-products by the cell wall-degrading enzymes produced by Aspergillus niger I-1472

Aspergillus niger I-1472 was grown on sugar beet pulp to produce cell wall polysaccharide-degrading enzymes, including feruloyl esterases. Compared to enzymatic activities measured in commercially available mixtures previously used for the release of ferulic acid, the A. niger enzymes were more various. These enzymes were tested to release ferulic acid from sugar beet pulp, maize bran, or autoclaved maize bran. They were as efficient as the commercial mixture to release ferulic acid from sugar beet pulp. On the other hand, they were much more efficient to release ferulic acid from maize bran after autoclaving pretreatment, as 95% of ferulic acid ester were solubilized. Thus, A. niger enzymes exhibited a high interest in the release of ferulic acid from various agro-industrial by-products.

Aspergillus enzymes involved in degradation of plant cell wall polysaccharides.

Degradation of plant cell wall polysaccharides is of major importance in the food and feed, beverage, textile, and paper and pulp industries, as well as in several other industrial production processes. Enzymatic degradation of these polymers has received attention for many years and is becoming a more and more attractive alternative to chemical and mechanical processes. Over the past 15 years, much progress has been made in elucidating the structural characteristics of these polysaccharides and in characterizing the enzymes involved in their degradation and the genes of biotechnologically relevant microorganisms encoding these enzymes. The members of the fungal genus Aspergillus are commonly used for the production of polysaccharide-degrading enzymes. This genus produces a wide spectrum of cell wall-degrading enzymes, allowing not only complete degradation of the polysaccharides but also tailored modifications by using specific enzymes purified from these fungi. This review summarizes our current knowledge of the cell wall polysaccharide-degrading enzymes from aspergilli and the genes by which they are encoded.

Aspergillus niger I-1472 and Pycnoporus cinnabarinus MUCL39533, selected for the biotransformation of ferulic acid to vanillin, are also able to produce cell wall polysaccharide-degrading enzymes and feruloyl esterases.

The filamentous fungal strains Aspergillus niger I-1472 and Pycnoporus cinnabarinus MUCL39533, previously selected for the bioconversion of ferulic acid to vanillic acid and vanillin respectively, were grown on sugar beet pulp. A large spectrum of polysaccharide-degrading enzymes was produced by A. niger and very few levels of feruloyl esterases were found. In contrast, P. cinnabarinus culture filtrate contained low amount of polysaccharide-degrading enzymes and no feruloyl esterases. In order to enhance feruloyl esterases in A. niger cultures, feruloylated oligosaccharide-rich fractions were prepared from sugar beet pulp or cereal bran and used as carbon sources. Number of polysaccharide-degrading enzymes were induced. Feruloyl esterases were much higher in maize bran-based medium than in sugar beet pulp-based medium, demonstrating the ability of carbon sources originating from maize to induce the synthesis of feruloyl esterases. Thus, A. niger I-1472 could be interesting to release ferulic acid from sugar beet pulp or maize bran.

Influence of sampling site on concentrations and carbohydrate-degrading enzyme activities of protozoa and bacteria in the rumen.

Four ruminally cannulated cows were used to assess the distribution of the concentrations and carbohydrate-degrading enzyme activities of the liquid-associated protozoa (LAP) and solid-associated bacteria (SAB) in the rumen. The cows were fed diets (7 kg of DM/d) of 100% hay (Diet F) or 60% barley plus 40% hay (Diet C) in a 2 x 2 crossover experimental design. Samples of ruminal digesta were collected successively from the dorsal (DS), ventral (VS), and anterior (AS) sacs 1h before and 3 h after the morning feeding on two sampling days with a 2-d interval. Irrespective of diet and sampling time, the greatest proportion of entodiniomorphs, representing the main population of protozoa, was found in the DS (40% mean; P < .05). Low pH values observed in the DS (P < .05) indicated higher fermentative activity in this site than in the other parts of the rumen. Protozoa may contribute more than previously thought to the high digestive potential present at the top of the rumen that has classically been attributed to bacteria. The specific activity of plant cell wall polysaccharide-degrading enzymes in the LAP was correspondingly greater (P < .05) for DS than for VS or AS. For the two diets and sampling times, specific activity of fibrolytic enzymes in the SAB tended to be less in the upper than in the lower parts of the rumen, and less in the VS than in the AS. This tendency became significant (P < .05) for total fibrolytic enzyme activity. Differences in bacterial colonization of particles among the three sampling sites may explain such differences in fibrolytic activity of the SAB. Data suggest a spatio-temporal complementary action of the bacteria and protozoa in ruminal plant cell wall degradation, at least with the barley diet, for which the number of protozoa was highest.

Cell wall polysaccharide-degrading enzymes produced by isolates of Mycocentrospora acerina differing in aggressiveness on carrot

Plant cell wall polysaccharide-degrading enzymes have been reported to be a determining factor in fungal pathogenesis because of their ability to macerate plant tissue and also to release endogenous elicitors of plant defence responses. Four isolates of Mycocentrospora acerina , a necrotrophic fungus, were characterized for aggressiveness and studied for the production of these enzymes. Induction of polygalacturonase and β -1,4-glucanase in M. acerina required substrate and was repressed by glucose. When grown in liquid culture with carrot cell walls as a source of carbon, the fungus secreted pectin methylesterase, polygalacturonase, pectate lyase, β -1,4-glucanase; all activities were positively correlated with isolate aggressiveness. Time-course experiments showed that aggressive isolates produced polygalacturonases and pectin methylesterases more rapidly than hypoaggressive ones, but the patterns of pectin methylesterase and polygalacturonase isozymes were identical for all the tested isolates. The rate at which carrot cell walls were solubilized and depolymerized was also related to isolate aggressiveness, suggesting a strong contribution of cell wall polysaccharide-degrading enzymes in M. acerina aggressiveness. On the other hand, analysis of polygalacturonic acid hydrolysis by isolates indicated that the release of oligogalacturonides was delayed and prolonged with hypoaggressive isolates.

Postprandial variations in the activity of polysaccharide-degrading enzymes of fluid- and particle-associated ruminal microbial populations

The distribution and specific activities of polysaccharide-degrading enzymes were monitored during the postprandial period in the liquid-associated bacteria (LAB), liquid-associated protozoa (LAP), and solid-associated microbes (SBFP) isolated from ruminal contents of cattle fed a high-cereal diet. Polysaccharide depolymerase activities were highest in the SBFP and the LAP populations. The postprandial variations in the specific activity of amylase were similar within the subpopulations. However, carboxymethylcellulase and xylanase activities increased in the first 5 h after feeding in the LAP, but were highest at the end of the postprandial period in the SBFP. Glycosidases involved in the fermentation of soluble carbohydrates increased significantly immediately after feeding in the liquid-associated microbes. β-d-Glucosidase and β-d-xylosidase were most active in the SBFP and were maximal 23 h after feeding. The activities of the plant cell wall polysaccharide-degrading enzymes and glycosidases in the SBFP were inversely related to ruminal pH; however, the activity of enzymes in the liquid-associated populations were highest in the immediate post-feed period when ruminal pH was lowest.