Production of cell wall-degrading enzymes by a pH tolerant estuarine fungal isolate <Emphasis Type="Italic">Fusarium moniliforme</Emphasis> NCIM1276 in different culture conditions

A pH tolerant strain of Fusarium moniliforme NCIM1276 with a saprophytic mode of nutrition was isolated from a coastal estuarine environment. Under laboratory conditions, the fungus produced significant biomass between pH 3 and 9, and produced cell wall degrading enzymes such as pectinases (polygalacturonase and pectate lyase), carboxymethylcellulase, xylanase and amylase. The production of these enzymes by the isolate in liquid medium, semi-solid medium and in infected tomato and cauliflower plants tissue was investigated. In liquid medium, the production of cell wall-degrading enzymes was induced by appropriate substrates, whereas the organism secreted all enzymes constitutively on wheat bran. The production of polygalacturonase, pectate lyase, carboxymethylcellulase, xylanase and amylase was increased by 3, 2, 11, 10 and 4-fold respectively on semi-solid medium containing wheat bran and orange pulp. Moreover when the fungus was allowed to infect tomato and cauliflower plants, the fungus was localized in the cortical tissues of the plants and secreted pectinases, carboxymethylcellulase and xylanase enzymes in the infected host tissue.     

Production of cell wall dissolving enzymes by Erwinia carotovora subsp. atroseptica in vitro at 27°C and 30°5C

Cultures of Erwinia carotovora subsp. atroseptica , grown at 27°C and 30·°C in different liquid media were assayed for activities of pectate lyase, polygalacturonase and cellulase. Total production of both pectate lyase and of polygalacturonase was 3–6 times less at 30·5°C than at 27°C; secretion of pectate lyase was similarly affected. Cellulase was cell bound and its production was not affected by the temperatures investigated. Growth, protein synthesis and protease activity were similar at the two temperatures and production of enzyme activity at 27°C and 30·5°C was independent of the growth medium.     

Isolation and characterization of Tn5 insertion mutants of Erwinia chrysanthemi that are deficient in polygalacturonate catabolic enzymes oligogalacturonate lyase and 3-deoxy-D-glycero-2,5-hexodiulosonate dehydrogenase.

Mutants of Erwinia chrysanthemi EC16 deficient in the polygalacturonate catabolic enzymes oligogalacturonate lyase (Ogl-) and 3-deoxy-D-glycero-2,5-hexodiulosonate (ketodeoxyuronate) dehydrogenase (KduD-) were obtained by Tn5 mutagenesis using the R plasmid pJB4JI. Ogl- Exu+ (Exu+, D-galacturonate utilization) and KduD- Exu- strains macerated potato tuber tissue and utilized glucose, glycerol, and gluconate, but they did not utilize polygalacturonate, unsaturated digalacturonate, or saturated digalacturonate. Genetic and physical evidence indicated that the Ogl- mutants and a KduD- recombinant contained a single copy of Tn5 and that Tn5 (Kmr) was linked to the mutant phenotypes. In the Ogl+ parents, basal levels of oligogalacturonate lyase were present in glycerol-grown cells and induced levels were present with saturated or unsaturated digalacturonate, while oligogalacturonate lyase was undetectable under similar conditions in Ogl- strains. Pectate lyase, polygalacturonase, and ketodeoxyuronate dehydrogenase were induced in an Ogl- strain by 3-deoxy-D-glycero-2,5-hexodiulosonate and by the enzymatic products of unsaturated digalacturonate but not by the digalacturonates. The KduD- strains lacked the dehydrogenase activity but in the presence of the digalacturonates produced higher levels of pectate lyase, polygalacturonase, and oligogalacturonate lyase than the KduD+ parents did. In the KduD- strains, pectate lyase and oligogalacturonate lyase were induced by unsaturated digalacturonate in a "gratuitous" manner, suggesting an intracellular accumulation of the inducer(s). We conclude that an intermediate(s) of the ketodeoxyuronate pathway induces pectate lyase, polygalacturonase, oligogalacturonate lyase, and ketodeoxyuronate dehydrogenase in E. chrysanthemi.     

Production of polygalacturonase byByssochlamys fulva

Summary Byssochlamys fulva was grown in two fermentation media using shake flasks, stirred fermentor and disc fermentor under conditions to give maximum production of pectolytic enzymes. Only polygalacturonase activity was detected in the culture filtrates during all fermentations. In all production conditions studied, no evidence of pectin methylesterase, pectin lyase, cellulase or proteinase activities were found. The maximum polygalacturonase activity (4.5 units/ml) was achieved when the microorganism was grown on medium II in shake flasks at pH 4.0–4.5 and 30°C after 12 days of fermentation.     

An isoelectric focusing study of the effect of methyl-esterified pectic substances on the production of extracellular pectin isoenzymes by soft rot Erwinia spp.

The production of extracellular pectic isoenzymes by seven strains of soft rot bacteria, Erwinia carotovora subsp. carotovora, E.c. atroseptica and E. chrysanthemi , when grown in media containing four different pectic substances with different degrees of methylation or with potato tuber cell-wall extract was examined by isoelectric focusing activity staining. In addition to the isoenzymes of pectate lyase, polygalacturonase and pectin methyl esterase produced constitutively or following induction by polygalacturonic acid (PGA) and coded by known genes, between two and seven novel isoenzymes of the three enzymes with a wider pI range were apparently induced by the pectins and cell-wall extract. Pectin lyase, which is induced in vitro by DNA-damaging agents, was not produced in the absence of mitomycin C in a medium containing PGA but up to two isoenzymes were found with pectin or cell-wall extract. In contrast, cellulase isoenzyme production was not affected by pectin or cell-wall extract. A greater number of novel isoenzymes of all pectic enzymes except pectin lyase tended to be produced in media containing Link pectin, which is PGA methylated to 98%, than the other pectic substances and cell-wall extract. Pectate lyase and polygalacturonase were induced by pectin lyase-degraded products of highly methylated pectin but not by PGA in an E. chrysanthemi strain with all its known pei and peh genes mutated. The results suggest that the production of novel pectic isoenzymes could be related to the presence of CH⁺₃ groups and that their induction differs from that for isomers induced by PGA-degraded products and DNA-damaging agents or produced constitutively.     

Role of the pectinolytic enzyme in the lactic acid fermentation of potato pulp by<Emphasis Type="Italic"> Rhizopus oryzae</Emphasis>

Rhizopus oryzae strain NBRC 4707 produced lactic acid and ethanol more efficiently than strain NRRL 395 in potato pulp, an agricultural by-product of the starch industry. The two strains developed comparable activities of xylanase, cellulase, -amylase, and glucoamylase, while the polygalacturonase activity of strain NBRC 4707 was double that of strain NRRL 395. The addition of commercial pectinase enhanced the formation of metabolites, suggesting that the degradation of pectic substances determines the fermentation of potato pulp by R. oryzae. Orange and apple peel were more effective in the induction of polygalacturonase activity than potato pulp, sugarbeet pulp, or wheat bran when used as a principal carbon source for fungal growth in a solid-state culture. The fungal cells in both types of fruit peel stimulated the fermentation of potato pulp and increased the quantity of lactic acid and ethanol to higher levels than those in other agricultural by-products.     

Purification and characterization of an extracellular pectate lyase from an Amycolata sp.

The extracellular pectate lyase (EC 4.2.2.2) of a nonsporulating Amycolata sp. was purified to homogeneity by anion- and cation-exchange chromatographies followed by hydrophobic interaction chromatography. The enzyme cleaved polygalacturonate but not highly esterified pectin in a random endolytic transeliminative mechanism that led to the formation of a wide range of 4,5-unsaturated oligogalacturonates. As shown by high-performance anion-exchange chromatography and pulsed amperometric detection, these unsaturated oligogalacturonates were further depolymerized by the enzyme to the unsaturated dimer and trimer as final products. The pectate lyase had a molecular weight of 31,000 determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a molecular mass of 30,000 Da determined by matrix-assisted laser desorption ionization mass spectrometry. The isoelectric point of the protein was 10. Maximum activity occurred at pH 10.25. Calcium was essential for activity, and EDTA inactivated the enzyme under standard assay conditions. Interestingly, EDTA did not inhibit the ability of the enzyme to cleave the native pectin (protopectin) of ramie (Boehmeria nivea) fibers. The Km value with sodium polygalacturonate as the substrate was 0.019 g liter-1. The purified enzyme lost its activity after a 1-h incubation at 50 degrees C but was stabilized by calcium or polygalacturonate. The N-terminal sequence showed high similarity within a stretch of 13 amino acids to the N-terminal sequences of pectate lyases PLa and PLe from Erwinia chrysanthemi. The Amycolata sp. did not produce additional isozymes of pectate lyase but produced further activities of pectinesterase, xylanase, and carboxymethyl cellulase when grown in a medium with decorticated bast fibers from ramie as the sole carbon source.     

Production of cell wall-degrading enzymes by Aspergillus nidulans: a model system for fungal pathogenesis of plants.

The cell wall-degrading enzymes polygalacturonase and pectate lyase have been suggested to be crucial for penetration and colonization of plant tissues by some fungal pathogens. We have found that Aspergillus nidulans (= Emericella nidulans), a saprophytic Ascomycete, produces levels of these enzymes equal to those produced by soft-rotting Erwinia species. Induction of polygacturonase and pectate lyase in A. nidulans requires substrate and is completely repressed by glucose. Surprisingly, inoculation of excised plant tissues with A. nidulans conidia leads to formation of necrotic, water-soaked lesions within which the organism sporulates. Thus, A. nidulans has phytopathogenic potential. The release of glucose and other sugars from wounded tissues may repress pectolytic enzyme production and limit disease development. Therefore, we tested creA204, a mutation that relieves glucose repression of some A. nidulans carbon utilization enzymes, for its effect on production of pectolytic enzymes. creA204 failed to relieve catabolite repression of polygalacturonase or pectate lyase and had no effect on disease severity.     

Simultaneous production and induction of cellulolytic and xylanolytic enzymes in aStreptomyces sp.

Extracellular cellulolytic and xylanolytic enzymes ofStreptomyces sp. EC22 were produced during submerged fermentation. The cell-free culture supernatant of the streptomycete grown on microcrystalline cellulose contained enzymes able to depolymerize both crystalline and soluble celluloses and xylans. Higher cellulase and xylanase activities were found in the cell-free culture supernatant of the strain when grown on microcrystalline cellulose than when grown on xylan. Total cellulase and endoglucanase [carboxymethyl-cellulase (CMCase)] activities reached maxima after 72 h and xylanase activity was maximal after 60h. Temperature and pH optima were 55°C and 5.0 for CMCase activity and 60°C and 5.5 for total crystalline cellulase and xylanase activities. At 80°C, approximate half-lives of the enzymes were 37, 81 and 51 min for CMCase, crystalline cellulose depolymerization and xylanase, respectively.     

Xylanases of anaerobic fungus <Emphasis Type="Italic">Anaeromyces mucronatus</Emphasis>

The anaerobic fungus Anaeromyces mucronatus KF8 grown in batch culture on M10 medium with rumen fluid and microcrystalline cellulose as carbon source produced a broad range of enzymes requisite for degradation of plant structural and storage saccharides including cellulase, endoglucanase, xylanase, α-xylosidase, β-xylosidase, α-glucosidase, β-glucosidase, β-galactosidase, mannosidase, cellobiohydrolase, amylase, laminarinase, pectinase and pectate lyase. These enzymes were detected in both the intra- and extracellular fractions, but production into the medium was prevalent with the exception of intracellular β-xylosidase, chitinases, N-acetylglucosaminidase, and lipase. Xylanase activity was predominant among the polysaccharide hydrolases. Extracellular production of xylanase was stimulated by the presence of cellobiose and oat spelt xylan. Zymogram of xylanases of strain KF8 grown on different carbon sources revealed several isoforms of xylanases with approximate molar masses ranging from 26 to 130 kDa.     

Effect of air flow rate on scleroglucan synthesis by Sclerotium glucanicum in an airlift bioreactor with an internal loop

The effects of several plant cell wall polysaccharides degrading enzymes on sugar beet pulps pressing were studied. Study was carried out using three two level fractional factorial experiment designs. With only 36 experiments, the effects of the presence of pectin methylesterase, pectin lyase, polygalacturonase, cellulase, arabinase, xylanase and two rhamnogalacturonases on pressing were examined. Pectin lyase, pectin methylesterase and cellulase had a negative effect and caused the decrease of sugar beet pulp pressability. On the contrary, the presence of polygalacturonase, arabinase and xylanase increased pressing efficiency. When increasing enzymes concentrations, these effects varied and positive interactions between xylanase and polygalacturonase appeared. The presence of each of the two rhamnogalacturonases improved pressability despite their antagonistic effects. These enzymes had a complex effect and strongly interacted with polygalacturonase, arabinase and xylanase.     

Comparison of pectic enzymes produced by Erwinia chrysanthemi, Erwinia carotovora subsp. carotovora, and Erwinia carotovora subsp. atroseptica

Erwinia spp. that cause soft-rot diseases in plants produce a variety of extracellular pectic enzymes. To assess the correlation between patterns of pectic enzyme production and taxonomic classification, we compared the enzymes from representative strains. Supernatants obtained from polygalacturonate-grown cultures of nine strains of Erwinia chrysanthemi, three strains of E. carotovora subsp. carotovora, and three strains of E. carotovora subsp. atroseptica were concentrated and subjected to ultrathin-layer polyacrylamide gel isoelectric focusing. Pectate lyase, polygalacturonase, and exo-poly-alpha-D-galacturonosidase activities were visualized by staining diagnostically buffered pectate-agarose overlays with ruthenium red after incubation of the overlays with the isoelectric focusing gels. The isoelectric focusing profiles of pectate lyase and polygalacturonase were nearly identical for strains of E. carotovora subsp. carotovora and E. carotovora subsp. atroseptica, showing three pectate lyase isozymes with isoelectric points higher than 8.7 and a polygalacturonase with pI of ca. 10.2. Isoelectric focusing profiles of the E. chrysanthemi pectic enzymes were substantially different. Although there was considerable intraspecific heterogeneity, all strains produced at least four isozymes of pectate lyase, which could be divided into three groups: basic (pI, ca. 9.0 to 10.0), slightly basic (pI, ca. 7.0 to 8.5), and acidic (pI, ca. 4.0 to 5.0). Several strains of E. chrysanthemi also produced a single form of exo-poly-alpha-D-galacturonosidase (pI, ca. 8.0).(ABSTRACT TRUNCATED AT 250 WORDS)     

Colletotrichum gloeosporioides pelB is an important virulence factor in avocado fruit-fungus interaction

Colletotrichum gloeosporioides is an important pathogen of tropical and subtropical fruits. The C. gloeosporioides pelB gene was disrupted in the fungus via homologous recombination. Three independent isolates, GD-14, GD-23, and GD-29, did not produce or secrete pectate lyase B (PLB) and exhibited 25% lower pectate lyase (PL) and pectin lyase (PNL) activities and 15% higher polygalacturonase (PG) activity than the wild type. The PLB mutants exhibited no growth reduction on glucose, Na polypectate, or pectin as the sole carbon source at pH 3.8 or 6.0, except for a 15% reduction on pectin at pH 6.0. When pelB mutants were inoculated onto avocado fruits, however, a 36 to 45% reduction in estimated decay diameter was observed compared with the two controls, the wild type and undisrupted transformed isolate. In addition, these pelB mutants induced a significantly higher host phenylalanine ammonia lyase activity as well as the antifungal diene, which is indicative of higher host resistance. These results suggest that PLB is an important factor in the attack of C. gloeosporioides on avocado fruit, probably as a result of its virulence factor and role in the induction of host defense mechanisms.     

A note on the pectinolytic enzymes of Lachnospira multiparus

The identification of pectinolytic enzymes from two strains of Lachnospira multiparus grown on media containing pectin with differing degrees of methylation as the carbon source was investigated. Both pectinesterase and lyase activity were determined but there was no evidence of polygalacturonase being present as either an extracellular or intracellular enzyme.     

A methylotrophic pathway participates in pectin utilization by Candida boidinii

The methylotrophic yeast Candida boidinii S2 was found to be able to grow on pectin or polygalacturonate as a carbon source. When cells were grown on 1% (wt/vol) pectin, C. boidinii exhibited induced levels of the pectin-depolymerizing enzymes pectin methylesterase (208 mU/mg of protein), pectin lyase (673 mU/mg), pectate lyase (673 mU/mg), and polygalacturonase (3.45 U/mg) and two methanol-metabolizing peroxisomal enzymes, alcohol oxidase (0.26 U/mg) and dihydroxyacetone synthase (94 mU/mg). The numbers of peroxisomes also increased ca. two- to threefold in cells grown on these pectic compounds (3.34 and 2.76 peroxisomes/cell for cells grown on pectin and polygalacturonate, respectively) compared to the numbers in cells grown on glucose (1.29 peroxisomes/cell). The cell density obtained with pectin increased as the degree of methyl esterification of pectic compounds increased, and it decreased in strains from which genes encoding alcohol oxidase and dihydroxyacetone synthase were deleted and in a peroxisome assembly mutant. Our study showed that methanol metabolism and peroxisome assembly play important roles in the degradation of pectin, especially in the utilization of its methyl ester moieties.     

Cell wall degrading enzymes in Orobanche aegyptiaca and its host Brassica campestris

Activities of the cell-wall degrading enzymes cellulase (EC 3. 2. 1. 4). poly-galacturonase (EC 3. 2. 1. 15) and xylanase (EC 3. 2. 1. 8), and also that of protease (EC 3. 4. 21. 14) have been determined in the parasite Orobanche aegyiptiaca Pers. and in the roots of infected and control host plants of Brassica campestris L. var. rapa Hartm. Shoots, roots and root-freed tubers of Orobanche showed pronounced activities of these enzymes. When expressed in terms of specific activity, protease, cellulase and polygalacturonase activities were higher and xylanase lower in all parts of Orobanche than in the roots of infected or control host plants. Orobanche roots had higher activities of cellulase, polygalacturonase and protease than tuber or shoot. While xylanase activity was higher in the shoot than in roots or tuber. Infection by Orobanche significantly enhanced the cellulase activity of host roots. Cellulase and polygalacturonase in the Orobanche roots appear as the main enzymes involved in establishing haustorial connection with the host root. Protease might be involved in degrading proteins and lipoproteins of the cell walls and membranes of the host tissues.     

Polygalacturonase, Pectate Lyase and Pectin Methylesterase Activity in Pathogenic Strains of Phytophthora capsici Incubated under Different Conditions

Pectolytic enzymes are found mainly in fungi and bacteria. The most widely occurring enzymes are polygalacturonase (PGs), pectin methylesterase (PMEs) and pectate lyase (PLs) produced during the infection process and during culturing. The secretion of these enzymes results in the disorganization of the plant cell walls, which is responsible for the pathogenicity of the pathogens. These enzymes degrade the pectin of plants causing maceration of plant tissues and the enzyme activity increases under favourable environmental conditions. We have found that Phytophthora capsici , a pathogenic oomycete, produces levels of these three enzymes equal to those produced by soft-rotting Erwinia chrysanthemi . The activity of PGs, PLs and PMEs was investigated at the optimum temperature, pH and ionic strength in highly pathogenic P. capsici strains cultivated in two kinds of liquid medium containing either crude pepper extracts plus pectin or pectin as the carbon source. Virulence tests and enzymes activity showed that there was a high correlation between the enzyme activity and the pathogenicity of P. capsici . The effects of different carbon sources on the enzyme activity showed that pepper extract plus pectin was the best source for the carbon source.