Extracellular pectin lyase produced by Neocallimastix sp. LM1, a rumen anaerobic fungus

A viscometric assay was used to assess the extracellular pectinolytic enzyme activity produced by Neocallimastix sp. LM1 during growth in a medium containing grass leaves as substrate. The highest activity was measured at pH 8.0, in the presence of CaCl₂. This anaerobic fungus apparently produced an endo-acting pectin lyase (EC 4.2.2.10), which was induced in the presence of pectin.     

Fermentation of pectin by a newly isolated Clostridium thermosaccharolyticum strain

Abstract By enrichment on pectin a thermophilic anaerobic bacterium was isolated. This strain, identified as Clostridium thermosaccharolyticum , was capable of fast growth on pectin (μ_max 0.58 h⁻¹) forming acetate, butyrate, hydrogen, carbon dioxide, methanol and traces of ethanol. The optimum temperature for growth was 58°C and the optimal pH was 6. The initial breakdown of pectin was catalysed by methylesterase and polygalacturonate hydrolase activity; no polygalacturonate lyase activity was found.     

Spirochaeta smaragdinae sp. nov., a new mesophilic strictly anaerobic spirochete from an oil field

An obligately anaerobic spirochete designated strain SEBR 4228^T (T = type strain) was isolated from an oil field of Congo, Central Africa. The strain grew optimally with a sodium chloride concentration of 5% (sodium chloride concentration growth range 1.0–10%) at 37°C (growth temperature range 20–40°C) and pH of 7.0–7.2 (pH growth range pH 5.5–8.0). Strain SEBR 4228^T grew on carbohydrates (glucose, fructose, ribose, d -xylose, galactose, mannitol and mannose), glycerol, fumarate, peptides and yeast extract. Yeast extract was required for growth and could not be replaced by vitamins. It reduced thiosulfate and sulfur, to H₂S. Glucose was oxidised to lactate, acetate, CO₂ and H₂S in the presence of thiosulfate but in its absence lactate, ethanol, CO₂ and H₂ were produced. Fumarate was fermented to acetate and succinate. The G+C content of strain SEBR 4228^T was 50%. Strain SEBR 4228^T was spiral shaped measuring 5–30 by 0.3–0.5 μm and was motile with a corkscrew-like motion. Electron microscopy revealed the presence of periplasmic flagella in a 1-2-1 arrangement. Strain SEBR 4228^T possessed features typical of the members of the genus Spirochaeta . 16S rRNA sequence analysis revealed that it was closely related to Spirochaeta bajacaliforniensis (similarity 98.6%). The lack of DNA homology with S. bajacaliforniensis (38%), together with other phenotypic differences, indicated that strain SEBR 4228^T is a new species, which we have designated Spirochaeta smaragdinae . The type strain is SEBR 4228^T (= DSM 11293).     

Purification and characterization of mitomycin C-induced pectin lyase of Erwinia carotovora subsp. atroseptica strain SCRI 1043

Erwinia carotovora supsp. atroseptica strain SCRI 1043 produces pectin lyase (PNL) which degrades highly methyl-esterified pectin by trans -elimination when induced by DNA damaging agents such as mitomycin C. Purification of the enzyme (66.5-fold) to homogeneity with 42.3% recovery was achieved by cation exchange chromatography on an S-Sepharose fast flow column equilibrated to pH 8.5 with 20 mmol 1^-1 Tris buffer, followed by elution of the bound proteins with a 1 mol^-1 NaCl gradient. SDS-PAGE and IEF-activity staining analyses showed that the mol. wt and pI of the enzymes were 31 kDa and 9.4 respectively and only one isomer was present. The optimum pH and temperature were 8.0 and 35°C respectively, and divalent cations, 1.37 mmol 1^-1 Ca₂₊ and 1.37 mmol 1_-1 Mg₂₊, although not essential, stimulated enzyme activity by about four and six times respectively. The endo mode of action of PNL was determined by viscometry. PNL induction by mitomycin C was determined spectrophotometrically and by ELISA, and was concomitant with bacteriocin production and bacterial cell lysis. The purified enzyme caused rapid maceration of potato tuber discs and IEF-activity staining of PNL in extracts of rotting tuber discs inoculated with strain SCRI 1043 showed that two isoenzymes were present, one corresponding to the enzyme produced in vitro and the other with a slightly higher pI.     

The effect of rumen chitinolytic bacteria on cellulolytic anaerobic fungi

J. KOPEČNÝ, B. HODROVÁ AND C. S. STEWART. 1996. The polycentric anaerobic fungus Orpinomyces joyonii A4 was cultivated on microcrystalline cellulose alone and in association with the rumen chitinolytic bacterium Clostridium sp. strain ChK5, which shows strong phenotypic similarity to Clostridium tertium . The presence of strain ChK5 significantly depressed the solubilization of microcrystalline cellulose, the production of short-chain fatty acids (SCFA) and the release of endoglucanase by the fungus. Co-culture of the monocentric anaerobic fungus Neocallimastix frontalis strain RE1, Neocallimastix sp. strain G-1 and Caecomyces sp. strain SC2 with strain ChK5 also resulted in depressed fungal cellulolysis. Cell-free supernatant fluids from strain ChK5 inhibited the release of reducing sugars from carboxymethylcellulose by cell-free supernatant fluids from O. joyonii strain A4. Strain 007 of the cellulolytic anaerobe Ruminococcus flavefaciens was also shown to produce small amounts of soluble products upon incubation with colloidal chitin. Mixtures of culture supernates from this bacterium and from O. joyonii strain A4 showed cellulase activity that was less than that of the component cultures. It is suggested that the ability of some rumen bacteria to hydrolyse or transform chitin may be an important factor in the interactions between bacteria and fungi in the rumen.     

Protoplasts from pectinolytic fungi: isolation, regeneration and pectinolytic enzyme production

S. Solís, M.E. FLORES AND C. HUITRON. 1996. Protoplast release in pectinolytic strain mutants of Aspergillus sp. CH-Y-1043 (A13) and Aspergillus flavipes ATCC-16795 (F7) is described. Optimum yield of protoplasts A13 was obtained in a lapse of 1 h when commercially lytic enzymes of Trichoderma harzanium (2 mg ml⁻¹) were added in 0.05 mol 1⁻¹ citrate-phosphate buffer pH 5.0 containing 0.7 mol 1⁻¹ KCl and 10 mg ml⁻¹ BSA. Best results in F7 were obtained when the protoplasting system of A13 was supplemented with 10 mg ml⁻¹ Aureobasidium sp. lytic enzymes. Isolated protoplasts in A13 and F7 were capable of a high regeneration frequency of 87% and 53% when 0.7 mol 1⁻¹ KCl and sorbitol were used as osmotic stabilizers. Endo-P, Exo-P and pectin lyase production were not modified during the process of regeneration.     

Cultural conditions for production of glucoamylase from Lactobacillus amylovorus ATCC 33621

Lactobacillus amylovorus ATCC 33621 is an actively amylolytic bacterial strain which produces a cell-bound glucoamylase (EC 3.2.1.3). Conditions of growth and glucoamylase production were investigated using dextrose-free de Man-Rogosa-Sharpe (MRS) medium in a 1.5 I fermenter, with varying dextrin concentration (0.1–1.5% (w/v)), pH (4.5–6.5) and temperature (25–55°C). Cell extracts were prepared by subjecting cells to treatment with a French Pressure cell in order to release intracellular proteins. Glucoamylase activity was then assayed. The effects of pH (4.0–9.0), temperature (15–85°C) and substrate (dextrin and starch, 0–2% w/v) concentration on crude enzyme activity were investigated. Optimal growth was obtained in MRS medium containing 1% (w/v) dextrin, at pH 5.5 and 37°C. Glucoamylase production was maximal at the late logarithmic phase of growth, during 16–18 h. Crude enzyme had a pH optimum of 6.0 and temperature optimum of 60°C. With starch as the substrate, maximal activity was obtained at a concentration of 1.5% (w/v). The effects of ions and inhibitors on glucoamylase activity were also investigated. Enzyme activity was not significantly influenced by Ca²⁺ and EDTA at 1 mmol 1⁻¹ concentration; however Pb²⁺ and Co²⁺ were found to inhibit the activity at concentrations of 1 mmol 1⁻¹. The crude enzyme was found to be thermolabile when glucoamylase activity decreased after about 10 min exposure at 60°C. This property can be exploited in the brewing of low calorie beers where only mild pasteurization treatments are used to inactivate enzymes. The elimination of residual enzyme effect would prevent further maltodextrin degradation and sweetening during long-term storage, thus helping to stabilize the flavour of beer.     

Anti-Helicobacter pylori activity of Lactobacillus delbrueckii subsp. bulgaricus strains: preliminary report

Aims:  To evaluate the activities of six Lactobacillus delbrueckii subsp. bulgaricus (LB) strains against 30 Helicobacter pylori strains by agar-well diffusion method.
Methods and Results:  LB cultures [4 × 10⁸–4 × 10⁹ CFU ml⁻¹) either were prepared in milk at their native pH, 3·8–5·0, or were adjusted to pH 6·4–7·7. At low and neutralized pH, LB strains inhibited the growth by 40–86·7% and 16·7–66·7% of H. pylori strains, respectively. LB activity was strain-dependent. At low and neutralized pH, one and five H. pylori strains, respectively, were not inhibited by any LB strain. LB2 and LB3, taken together, were active against most metronidazole and clarithromycin resistant strains.
Conclusions:  All LB strains inhibited a number of H. pylori strains, including also antibiotic resistant strains. LB activity was strain-dependent and better at low pH. At low pH values, the most active LB strains were LB1, LB2 and LB3, inhibiting 86·7% of H. pylori strains, while at neutralized pH values, the most active LB strains were LB2 and LB3, inhibiting 53·3 and 66·7% of H. pylori strains, respectively.
Significance and Impact of the Study:  LB could be utilized in the treatment or prophylaxis of H. pylori infection and warrants clinical investigations.     

The content of prostaglandins and their precursors in Mortierella and Cunninghamella species

Five strains of filamentous fungi belonging to the genera Mortierella and Cunninghamella were examined for the content of dihomo-γ-linolenic, arachidonic, eicosapentaenoic acids and prostaglandins (type E₂ and F _2α). Prostaglandins were detected using an ELISA method in mycelia of all tested strains (range 50–4800 ng g⁻¹ of PGE₂ and 6–30 ng g⁻¹ of PG F _2α). Several micro-organisms also produced prostaglandins in the culture medium (2·2–137·6 μg l⁻¹ for PGE₂ and 0·4–7·8 μg l⁻¹ for PG F _2α).     

Anaerobic degradation of pectin by mixed consortia and optimization of fermentation parameters for higher pectinase activity

Samples from biogas digesters, sewage ponds, animal house effluents and food processing wastes were used in enrichment systems seeking anaerobic bacteria producing pectinases. Among the 46 anaerobic consortia developed from various samples, four showed high pectinase activity under static anaerobic conditions. Investigation of fermentation variables showed the optimum conditions for pectinase activity were pH 7.0, 45°C and 72 h of growth with 0.5% pectin in the cultivation medium. A 1.4- to 1.6-fold increase in the pectinase activity was achieved under these conditions. The maximum yield of enzymes (62.72 U ml-1 of pectinase, 4.74 U ml^-1 of polygalacturonase, 113.30 U ml^-1 of pectin lyase, 2.10 U ml^-1 of pectinesterase, 0.75 U ml^-1 of total cellulase and 9.27 U ml^-1 of xylanase) was recorded with the consortia C-S2 developed from decomposed plant samples collected from a pond.     

Pectinase Production by Bacteria Associated with Improved Preservative Permeability in Sitka Spruce: Synthesis and Secretion of Polygalacturonate Lyase by Cytophaga johnsonii

Cytophaga johnsonii synthesized a polygalacturonate lyase which produced random cleavage of galacturonic acid polymers. No pectin methyl-esterase or hydrolytic pectinase activities could be detected in cultures of the organism. Polygalacturonate lyase synthesis was inducible and also subject to repression by glucose and other compounds. Galacturonic acid was the most effective inducer; lower activities were obtained with citrus pectin, polygalacturonic and polypectic acids. Glucose repression of lyase synthesis was not alleviated by 5 mM-adenosine-3'.5'-cyclic-monophosphate. Enzyme production was growth-linked and ceased when batch cultures entered the stationary phase. In steady-state chemostat cultures lyase activity was maximal at a dilution rate ( D ) of 0.19 h^-1. Polygalacturonate lyase was both cell-bound and free in the supernatant medium. The proportion of free enzyme increased throughout the batch growth cycle and in chemostat cultures over 70% of the activity was cell free at dilution rates below 0.05 h^-1.     

Interaction of the rumen fungus Orpinomyces joyonii with Megasphaera elsdenii and Eubacterium limosum

The degradation and fermentation of microcrystalline cellulose were studied in monoculture of the polycentric anaerobic fungus Orpinomyces joyonii and in co-cultures with the rumen bacteria Megasphaera elsdenii and Eubacterium limosum. More than 25% of cellulose hydrolysis products (glucose and cellodextrins) were released by the fungus into the medium after 8 d of cultivation. These products were metabolized by bacteria in mixed cultures. In co-culture with the fungus M. elsdenii and E. limosum . increased the extent of microcrystalline cellulose degradation by 10·12% and 7·96%, respectively. Biomass yield in co-cultures was increased by 89·9% and 59·4% for M. elsdenii and E. limosum . Y_cellulose for fungus alone was 52·29 g dry matter mol^-1 glucose. These values were 64·93 and 55·92 g mol^-1 glucose unit in co-culture with M. elsdenii and E. limosum , respectively.     

Mobilization of CS fimbriae-associated plasmids of enterotoxigenic Escherichia coli of serotype O6: K15: H16 or H- into various wild-type hosts

Abstract CS fimbriae-associated plasmids of two enterotoxigenic Escherichia coli strains of serotype O6: K15: H16 or H- (biotypes A and F) with M _r values of 51 × 10⁶ and 72 × 10⁶, respectively, were mobilized into various alternative host bacteria. Expression of CS1 or CS2 fimbriae was obtained when either of the CS fimbriae-associated plasmids was introduced into CS Fim⁻, O6: K15: H16 or H- recipients with rhamnose-negative and rhamnose-positive fermentation phenotypes, respectively, whereas CS3 fimbriae were expressed irrespective of the biotype of the recipient. On transfer into a CS Fim⁻ variant of an enterotoxigenic O8: H9 strain and into two K-12 strains, a CS3-fimbriae-only phenotype was conferred by the presence of either of the plasmids. When a CS Fim⁻ variant of a Rha⁺ CS2-fimbriae-only strain of serotype O6: K15: H16 harboured either of the plasmids, both CS2 and CS3 fimbriae were expressed, indicating that the rare CS2-fimbriae-only wild-type phenotype is probably due to the presence of a defective plasmid in such strains. Mobilization of the 51 MDa CS fimbriae-associated plasmid into five non-enterotoxigenic Rha⁺ porcine isolates of E. coli with O6 serotypes other than O6: K15: H16 or H- yielded CS3-fimbriae-only transconjugants. Thus the correlation between a Rha⁺ fermentation phenotype and expression of CS2 fimbriae does not hold in general for O-group 6 strains.     

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.     

The effect of citric acid on growth of proteolytic strains of Clostridium botulinum

In strictly anaerobic conditions in a culture medium adjusted to pH 5·2 with HCl and incubated at 30°C, inocula containing < 10 vegetative bacteria of Clostridium botulinum ZK3 (type A) multiplied to give > 10⁸ bacteria per ml in 3 d. Growth from an inoculum of between 10 and 100 spores occurred after a delay of 10–20 weeks. Citric acid concentrations of 10–50 mmol/l at pH 5·2 inhibited growth from both vegetative bacteria and spore inocula, a concentration of 50 mmol/l increasing the number of vegetative bacteria or of spores required to produce growth by a factor of approximately 10⁶. The citric acid also reduced the concentration of free Ca²⁺ in the medium. The inhibitory effect of citric acid on vegetative bacteria at pH 5·2 could be prevented by the addition of Ca²⁺ or Mg²⁺ and greatly reduced by Fe²⁺ and Mn²⁺. The addition of Ca²⁺, but not of the remaining divalent metal ions, restored the concentration of free Ca²⁺ in the medium to that in the citrate-free medium. The inhibitory effect of citric acid on growth from a spore inoculum was only partially prevented by Ca²⁺. Citric acid (50 mmol/l) did not inhibit growth of strain ZK3 at pH 6 despite the greater chelating activity of citrate at pH 6 than at pH 5·2. The effect of citric acid and Ca²⁺ at pH 5·2 on vegetative bacteria of strains VL1 (type A) and 2346 and B6 (proteolytic type B) was similar to that on strain ZK3.     

Antibacterial activity of bovine lactoferrin and its peptides against enterohaemorrhagic Escherichia coli O157:H7

The antimicrobial activities of bovine lactoferrin (bLF), its pepsin hydrolysate (bLFH) and the active peptide lactoferricin^® B (LFcinB) against four clinical isolates of enterohaemorrhagic Escherichia coli O157:H7 were studied. The MICs against these isolates were 3 mg ml⁻¹ for bLF, 0·1–0·2 mg ml⁻¹ for bLFH and 8–10 μg ml⁻¹ for LFcinB in 1% Bactopeptone broth. LFcinB killed these bacteria within 3 h at concentrations above 10 μg ml⁻¹. Transmission electron microscopy findings suggested that LFcinB acts on the bacterial surface and affects cytoplasmic contents. LFcinB was shown to influence the levels of verotoxins in the culture supernatant fluid of an E. coli 0157:H7 strain. These results demonstrate that E. coli O157:H7 strains are susceptible to the antimicrobial effects of bLF and its peptides.     

Effect of acetate on growth and ammonium uptake in the microalga Scenedesmus obliquus

The effect of acetate on growth and rate of ammonium uptake in Scenedesmus obliquus (UTEX 78) was investigated under light-limiting conditions. Addition of acetate to autotrophic cells with a growth constant of 0.71 day⁻¹ resulted in an increase in the growth rate (mixotrophy, k = 1.3 day⁻¹), and in the presence of acetate, growth occurred in the dark (heterophy, k = 0.44 day⁻¹). The rate of ammonium uptake in autotrophy (17.8 amol cell⁻¹ min⁻¹) was similar to that in heterotrophy (17.4 amol cell⁻¹ min⁻¹) but was 3.7 times lower than that in mixotrophy (65.9 amol cell⁻¹ min⁻¹). In general, mixotrophic cells showed optimum ammonium uptake at the acetate concentration at which they were grown. In autotrophy, uptake of ammonium leveled off at about 12.5 μ M while no saturation was observed in mixotrophic cells. An increase in the rate of uptake of ammonium was observed in autotrophic cells within 1 h after the addition of acetate. The activity of isocitrate lyase (EC 4.1.3.1), a key enzyme for the regulation of the glyoxylate cycle responsible for acetate catabolism, showed a 3.9-fold increase in activity after 24 h in the dark in the presence of acetate. The level of isocitrate lyase activity in cells grown for 24 h in the dark in the presence of 0–20 m M acetate also increased as a function of acetate concentration.     

Characterization of alpha-galactosidase from Lactobacillus fermentum

α-Galactosidase activity was studied in Lactobacillus fermentum strains. The optimum temperature was found to be 45°C. The enzyme was inactivated at temperatures higher than 55°C, but remained active during storage at low temperatures (0, -30 and -70°C) for 5 months. Enzyme activity was observed within a 5.0–6.5 pH range, while optimum pH was dependent on the particular strain assayed. The addition of Zn²⁺ to the reaction buffer exerted a slight negative effect upon the activity, while Hg²⁺ and p -chloromercuribenzoate produced a strong inhibition. These results would indicate the presence of -SH groups in the catalytic site of the enzyme.     

Elevation of rat intestinal permeability by enterotoxigenic Escherichia coli in comparison to non-toxigenic E. coli and Salmonella typhimurium. Application of fluorescent dextran 3000 as permeability probe

Abstract The effect of bacterial enterotoxins on rat intestinal permeability properties was studied by comparing the effect of toxin-positive and toxin-negative Escherichia coli and Salmonella typhimurium inoculated into a segment of rat small intestine. Fluoresceinated dextran 3000 (FITC-D3; M _r 3000) was applied as permeability marker. The E. coli strain C922a-1 producing heat-labile (LT) and heat-stable (ST) enterotoxins and colonising factor CFA/II increased the transmural passage of the dextran probe into portal blood. In contrast, its plasmid-negative variant, a non-toxin producer lacking CFA, caused permeability changes indistinguishable from the bacteria-free nutrient broth control. Another pair of enterotoxigenic E. coli strains, 1628–14 (LT⁺, ST⁺, CFA/I⁺) and 1628–15 (LT⁺, ST⁻ and CFA/I⁻) both increased the intestinal permeability. The observations indicate that the LT⁺-only E. coli strain 1628–15 has the ability to promote permeability of rat intestine. The toxin-negative, rough S. typhimurium 395MR10 bacteria had a very small effect on the permeability, which was also achieved with culture filtrate only.
It is concluded that enterotoxigenic E. coli (ETEC) can alter the properties of the mucosal barrier towards intermediate-sized molecules that could be of antigenic significance, or which could play a crucial role in the nutritional status of the host organism.     

Purification and characterization of a feruloyl esterase from the fungus Penicilliumexpansum

An extracellular phenolic acid esterase produced by the fungus Penicillium expansum in solid state culture released ferulic and ρ-coumaric acid from methyl esters of theacids, and from the phenolic-carbohydrate esters O-[5-O-(trans-feruloyl)-α- l -arabinofuranosyl]-(1 → 3)-O-β- d -xylopyranosyl-(1 → 4)- d -xylopyranose (FAXX) and O-[5-O-((E)-ρ-coumaroyl)-α- l -arabinofuranosyl]-(1 → 3)-O-β- d -xylopyranosyl-(1 → 4)- d -xylopyranose(PAXX). The esterase was purified 360-fold in successive stepsinvolving ultrafiltration and column chromatography by gel filtration, anion exchange andhydrophobic interaction. These chromatographic methods separated the phenolic acid esterasefrom α- l -arabinofuranosidase, pectate and pectin lyase, polygalacturonase,xylanase and β- d -xylosidase activities. The phenolic acid esterase had an apparentmass of 65 kDa under non-denaturing conditions and a mass of 57·5 kDa underdenaturing conditions. Optimal pH and temperature were 5·6 and 37 °C,respectively and the metal ions Cu2+ and Fe³+ atconcentrations of 5 mmol l⁻¹ inhibited feruloyl esterase activity by 95% and44%, respectively, at the optimum pH and temperature. The apparent K_m and V_max of the purified feruloyl esterase for methyl ferulate at pH 5·6 and 37 °Cwere 2·6 mmol l⁻¹ and 27·1 μmol min⁻¹ mg⁻¹. The corresponding constants of ρ-coumaroylesterase for methyl coumarate were 2·9 mmol l⁻¹ and 18·6μmol min−1 mg⁻¹.