In vitro production of pectolytic and cellulolytic enzymes byColletotrichum lindemuthianum isolated from soybean grown in Saudi Arabia

Colletotrichum lindemuthianum isolated from soybean in Saudi Arabia produced polygalacturonase, pectin methylesterase, pectin trans-eliminase and carboxymethylcellulasein vitro. Polygalacturonase showed maximaum activity at 30 to 35°C and pH 4.0 to 5.0. The absorption maximum for pectin trans-eliminase reaction products was at approximately 548 nm. The polygalacturonase and pectin trans-eliminase activities increased with culture age. The degradation of carboxymethylcellulose was also demonstrated.     

Effect of pectin on pectinases in autolysis of Botrytis cinerea

Pectic activity in autolyzed cultures of Botrytis cinerea in a medium with and without pectin was similar, but in the medium with pectin maximal activities occurred in younger cultures. The pectic activities found were polygalacturonase, polymethylgalacturonase, endo activity (pectin as substrate) and pectin lyase. The molecular weights of polygalacturonase, polymethylgalacturonase and endo activity (pectin as substrate) were 36000, 33000 and 30200 daltons respectively, and the molecular weight of pectin lyase was 18200 daltons. By gel electrophoresis four different pectic activities were detected, three in the top of the gel and one in the bottom. Two enzymes were characterized, the polygalacturonase activity (first band in the top) inhibited by Ca⁺⁺ and the pectin lyase activity (in the bottom) which was not inhibited by Ca⁺⁺. These enzymes are not induced by the presence of pectin in the medium during degradation of Botrytis cinerea.     

Zur Rolle des Pektins bei der Ausprägung sorten- und jahresbedingter Unterschiede der Naßfäuleanfälligkeit von Kartoffelknollen

The influence of soil moisture and mean day temperature of July to September on pectin structure and soft rot resistance of potato tubers was investigated. Tuber samples of middle early and middle late potato cultivars respectively were tested from 1976 to 1979. Cultivar characteristics as well as growth conditions effect the pectin structure. The tuber tissue of soft rot resistant cultivars contains relative high amounts of esterified pectin. The pectin esterification was promoted by drought during vegetation periods (1976, 1978). A late rise of metabolic activity caused by rainfall at the end of the vegetation period (1978) increased the amount of soluble pectin in the cell wall. This lack of pectin stabilization facilitates tissue maceration by soft rot bacteria. Increased pectin solubility was also caused by unfavorable storing conditions of potato tubers. A low degree of esterification and high solubility of the cell wall pectin affect the relative resistance of potato tubers against soft rot caused by Erwinia carotovora var. atro-septica (van Hall) Dye adversely.     

Relationship between Molecular Weights of Pectin and Hypocholesterolemic Effects in Rats

Hypocholesterolemic activities and other properties of three different molecular weight pectin were examined. The low-molecular-weight pectin (M_r ≒ 66,000) obtained by decomposition of original pectin (M_r≒ 750,000) had the properties of low viscosity and high solubility, but it lost hypocholesterolemic activities in rats. On the other hand, the medium-molecular-weight pectin (M_r ≒ 185,000) had characteristics of both low viscosity and hypocholesterolemic activities.     

Synthesis of sulfated pectins and their anticoagulant activity

The following pectins were sulfated: bergenan BC (the pectin of Bergenia crassifolia L), lemnan LM (the pectin of Lemna minor L), and galacturonan as a backbone of pectins. Pyridine monomethyl sulfate, pyridine sulfotrioxide, and chlorosulfonic acid were used as reagents for sulfation. Chlorosulfonic acid proved to be the optimal reagent for sulfation of galacturonan and other pectins. Galacturonan and pectin derivatives with different degrees of sulfation were synthesized and their anticoagulant activities were shown to depend on the quantity of sulfate groups in the pectin macromolecules.     

Microbial methanol formation: A major end product of pectin metabolism

Various pectinolytic strains ofClostridium, Erwinia, andPseudomonas species produced methanol as a major end product during growth on pectin but not on glucose or polygalacturonic acid. Pectin metabolism ofClostridium butyricum strain 4P1 correlated with a final product concentration of 16 mM at the end of growth, and a 1:1 stoichiometry for methanol production and percent initial substrate methoxylation. Growth on pectin was associated with high activity of pectin methylesterase and the absence of methanol consumption. The ecological significance of pectin metabolism and the establishment of microbial methylotrophic metabolism in nature is discussed.     

Expression of a codon-optimized Aspergillus niger pectin methylesterase gene in the methylotrophic yeast Candida boidinii

A codon-optimized Aspergillus niger pectin methylesterase (PME) gene was expressed in the methylotrophic yeast Canidia boidinii. The PME-producing strains showed better growth on pectin than the wild-type strains, suggesting that the PME-producing strains could efficiently utilize methyl ester moieties of pectin. On the other hand, overproduction of PME negatively affected the proliferation of C. boidinii on leaves of Arabidopsis thaliana.     

Competitive binding of pectin and xyloglucan with primary cell wall cellulose

Assemblies of pectin, xyloglucan and cellulose were studied in vitro using two ternary systems. In the first one, xyloglucan concentration varied, while pectin amount was kept constant. In the second one, pectin concentration varied, whereas xyloglucan amount was fixed. The use of ternary systems allowed to put forward the hypothesis that pectin/cellulose and xyloglucan/cellulose associations may exist together or separately, depending on the proportion of non-cellulosic polysaccharides in cell walls. It can be hypothesized that pectin plays a double role within primary cell walls: (i) pectin loosely bound to cellulose, in xyloglucan-rich cell walls, (ii) pectin associated with cellulose, in xyloglucan-poor cell walls.     

Pectin lyase production by Penicillium griseoroseum: Effect of tea extract, caffeine, yeast extract, and pectin

Summary Mycelial growth and production of extracellular pectin lyase by Penicillium griseoroseum at different concentrations of inducers were investigated. The fungus was cultured in mineral medium using sucrose as a carbon source and caffeine, yeast extract, tea extract or pectin as inducers. Caffeine, yeast extract and tea extract in the presence of sucrose, and tea extract alone were capable of inducing pectin lyase in P. griseoroseum, even at low concentrations.     

Production of pectin lyase by Penicillium griseoroseum as a function of the inoculum and culture conditions

Optimum activity of an extracellular pectin lyase produced by Penicillium griseoroseum in submerged culture was after 120 h using 0.1% (w/v) citrus pectin as substrate. Sucrose at 0.1% (w/v) stimulated enzyme production and citrus pectin gave the highest activity of enzyme per unit growth.     

Pectin secretion and distribution in the anther during pollen development in Lilium

Using the monoclonal antibodies JIM 5 and 7, pectin was immunolocalized and quantitatively assayed in three anther compartments of Lilium hybrida during pollen development. Pectin levels in both the anther wall and the loculus increased following meiosis, were maximal during the early microspore stages and declined during the remainder of pollen ontogenesis. In the microspores/pollen grains, pectin was detectable at low levels during the microspore stages but accumulated significantly during pollen maturation. During early microspore vacuolation, esterified pectin epitopes were detected both in the tapetum cytoplasm and vacuoles. In the anther loculus, the same epitopes were located simultaneously in undulations of the plasma membrane and in the locular fluid. At the end of microspore vacuolation, esterified pectin epitopes were present within the lipids of the pollenkitt, and released in the loculus at pollen mitosis. Unesterified pectin epitopes were hardly detectable in the cytoplasm of the young microspore but were as abundant in the primexine matrix as in the loculus. During pollen maturation, both unesterified and esterified pectin labelling accumulated in the cytoplasm of the vegetative cell, concurrently with starch degradation. In the mature pollen grain, unesterified pectin epitopes were located in the proximal intine whereas esterified pectin epitopes were deposited in the distal intine. These data suggest that during early microspore development, the tapetum secretes pectin, which is transferred to the primexine matrix via the locular fluid. Further, pectin is demonstrated to constitute a significant component of the pollen carbohydrate reserves in the mature grain of Lilium. Received: 3 July 2000 / Accepted: 19 October 2000     

Characterisation of a new thermoalkaliphilic bacterium for the production of high-quality hemp fibres, <Emphasis Type="Italic">Geobacillus thermoglucosidasius</Emphasis> strain PB94A

Novel thermophilic and alkaliphilic bacteria for the processing of bast fibres were isolated using hemp pectin as substrate. The strain PB94A, which showed the highest growth rate (μ = 0.5/h) was identified as Geobacillus thermoglucosidasius (DSM 21625). The strain grew optimally at 60°C and pH 8.5. During growth on citrus pectin, the strain produced pectinolytic lyases, which were excreted into the medium. In contrast to the commercially available pectinase Bioprep 3000 L, the enzymes from G. thermoglucosidasius PB94A converted pectin isolated from hemp fibres. In addition to hemp pectin, the culture supernatant also degraded citrus, sugar beet and apple pectin and polygalacturonic acid. When hemp fibres were incubated with the cell-free fermentation broth of G. thermoglucosidasius PB94A, the fineness of the fibres increased. The strain did not produce any cellulases, which is important in order to avoid damaging the fibres during incubation. Therefore, these bacteria or their enzymes can be used to produce fine high-quality hemp fibres.     

Pectinolytic Systems of Two Aerobic Sporogenous Bacterial Strains with High Activity on Pectin

Strains Paenibacillus sp. BP-23 and Bacillus sp. BP-7, previously isolated from soil from a rice field, secreted high levels of pectinase activity in media supplemented with pectin. Production of pectinases in strain Paenibacillus sp. BP-23 showed catabolite repression, while in Bacillus sp. BP-7 production of pectin degrading enzymes was not negatively affected by glucose. The two strains showed lyase activities as the predominant pectinases, while hydrolase activity was very low. Analysis of Paenibacillus sp. BP-23 in SDS–polyacrylamide gels and zymograms showed five pectinase activity bands. The strict requirement of Ca²⁺ for lyase activity of the strain indicates that correspond to pectate lyases. For Bacillus sp. BP-7, zymograms showed four bands of different size. The strain showed a Ca²⁺ requirement for lyase activity on pectate but not on pectin, indicating that the pectinolytic system of Bacillus sp. BP-7 is comprised of pectate lyases and pectin lyases. The results show differences in pectin degrading systems between the two aerobic sporogenous bacterial strains studied.     

A proteomic study of pectin‐degrading enzymes secreted by Botrytis cinerea grown in liquid culture

Botrytis cinerea is a pathogenic filamentous fungus, which infects more than 200 plant species. The enzymes secreted by B. cinerea play an important role in the successful colonization of a host plant. Some of the secreted enzymes are involved in the degradation of pectin, a major component of the plant cell wall. A total of 126 proteins secreted by B. cinerea were identified by growing the fungus on highly or partially esterified pectin, or on sucrose in liquid culture. Sixty‐seven common proteins were identified in each of the growth conditions, of which 50 proteins exhibited a SignalP motif. Thirteen B. cinerea proteins with functions related to pectin degradation were identified in both pectin growth conditions, while only four were identified in sucrose. Our results indicate it is unlikely that the activation of B. cinerea from the dormant state to active infection is solely dependent on changes in the degree of esterification of the pectin component of the plant cell wall. Further, these results suggest that future studies of the B. cinerea secretome in infections of ripe and unripe fruits will provide important information that will describe the mechanisms that the fungus employs to access nutrients and decompose tissues.     

Continuous-flow enrichment of a strain ofErwinia carotovora having specificity for highly methylated pectin

A pectinolytic bacterium was isolated from a mixed microbial population by means of a chemostat enrichment procedure. The bacterium, which was identified asErwinia carotovora, grew only on highly methylated pectin and produced a pectin lysase which released unsaturated monomer and dimer from 71% esterified citrus pectin. The pectin lyase was inducible only by pectins having a high methyl content and in pectin-limited chemostats its synthesis passed through a maximum at a dilution rate close to 0.04h^-1.     

Rheology of Mixed Pectin Solutions

The effects of sucrose (S) and pectin (P) concentrations and the ratio between two distinct pectins (R) on the rheological behavior of diluted pectin systems were evaluated simultaneously using the surface response methodology. The systems were composed of a mixture of two high methoxy pectins with different degree of methyl esterification values (HM1/HM2) and of a mixture of a high-methoxy with an amidated low-methoxy pectin (HM1/LMA). For the HM1/HM2 systems, the multivariate analysis showed that the sucrose and pectin concentrations exerted statistically significant (p < 0.05) linear effects on the consistency index k and viscosity, the influence of pectin being about five times higher than that of sucrose. The pectin concentration and the ratio between the different pectins were shown to be significant with respect to the rheological parameters of the HM1/LMA systems. Evaluating the influence of the ratio between the different pectins, a synergistic effect on the structure reinforcement was observed when mixing HM1 and LMA in similar proportions, indicating the importance of the presence of hydrophobic interactions between methyl ester groups in addition to the stronger hydrogen bonding in junction zone stabilization. In general, the conditions in which hydrogen bonds were favored in relation to hydrophobic interactions led to systems with higher pseudoplasticity.     

Modulation of gut microbiota from obese individuals by in vitro fermentation of citrus pectin in combination with Bifidobacterium longum BB-46

This study aimed to evaluate the effects of three treatments, i.e., Bifidobacterium longum BB-46 (T1), B. longum BB-46 combined with the pectin (T2), and harsh extracted pectin from lemon (T3) on obesity-related microbiota using the Simulator of the Human Intestinal Microbial Ecosystem (SHIME®). The effects of the treatments were assessed by the analysis of the intestinal microbial composition (using 16S rRNA gene amplicon sequencing) and the levels of short-chain fatty acids (SCFAs) and ammonium ions (NH₄⁺). Treatments T2 and T3 stimulated members of the Ruminococcaceae and Succinivibrionaceae families, which were positively correlated with an increase in butyric and acetic acids. Proteolytic bacteria were reduced by the two treatments, concurrently with a decrease in NH₄⁺. Treatment T1 stimulated the production of butyric acid in the simulated transverse and descending colon, reduction of NH₄⁺ as well as the growth of genera Lactobacillus, Megamonas, and members of Lachnospiracea. The results indicate that both B. longum BB-46 and pectin can modulate the obesity-related microbiota; however, when the pectin is combined with B. longum BB-46, the predominant effect of the pectin can be observed. This study showed that the citric pectin is able to stimulate butyrate-producing bacteria as well as genera related with anti-inflammatory effects. However, prospective clinical studies are necessary to evaluate the anti/pro-obesogenic and inflammatory effects of this pectin for future prevention of obesity.

     

Partial purification and properties of pectin lyase from Penicillium expansum

A pectin lyase, poly(methoxygalacturonide) lyase, EC 4.2.2.10, from a culture filtrate of Penicillium expansum was partially purified 33-fold with 7.3% yield. The enzyme was monomeric with a molecular mass of 36.5 kDa. The enzyme did not contain pectate lyase activity and degraded citrus and apple pectin best at pH 7.0 and 40 to 45°C. The K _m for citrus pectin was 9 mg ml^-1.     

Isolation and properties of pectinases from the fungus Aspergillus japonicus

Using anion-exchange chromatography on different carriers and phenyl-Sepharose hydrophobic chromatography, five pectolytic enzymes were isolated from the culture liquid of a mutant strain of Aspergillus japonicus: two endo-polygalacturonases (I and II, 38 and 65 kD, pI5.6 and 3.3), pectin lyase (50 kD, pI3.8), and two pectinesterases (I and II) with similar molecular weights (46 and 47 kD) and the same pI(3.8). The pectinesterases apparently represent two isoforms of the same enzyme. All purified enzymes were homogenous according to SDS-PAGE and polyacrylamide gel-IEF, except for endo-polygalacturonase II that gave two bands on isoelectric focusing, but one band on electrophoresis. All enzymes had maximal activity in an acid medium (at pH 4.0-5.5). The pectin lyase and pectinesterase were stable at 40-50°C. The thermal stability of both endo-polygalacturonases was much lower (after 3 h of incubation at 30°C, endo-polygalacturonases I and II lost 40 and 10% of the activity, respectively). The activity of endo-polygalacturonases I and II towards polygalacturonic acid strongly depended on NaCl concentration (optimal concentration of the salt was 0.1-0.2 M); the enzymes were also capable of reducing the viscosity of pectin solution, but rather slowly. The pectin lyase had no activity towards polygalacturonic acid. The activity of the pectin lyase increased with increasing degree of methylation of pectins. Both endo-polygalacturonases demonstrated synergism with the pectinesterase during the hydrolysis of highly methylated pectin. On the contrary, in the mixture of pectin lyase and pectinesterase an antagonism between the two enzymes was observed.