Culture conditions for the production of pectinolytic enzymes by the white-rot fungus Trametes trogii on a laboratory scale

Different cultural parameters that regulate pectinolytic enzyme production in vitro by Trametes trogii were studied. When grown in a medium containing pectin, T. trogii produced extracellular polymethylgalacturonase, polygalacturonase and pectin lyase but no pectate lyase activity. No significant differences in the maximum enzyme activities measured were observed with the addition of xylan, carboxymethylcellulose or both to the medium containing pectin. The addition of glucose to that medium considerably decreases all the activities studied, and in a medium with glucose as the sole carbon source no galacturonase activity could be measured, and pectin lyase activity was at its minimum. The low synthesis of pectin lyase in cultures containing glucose suggests that this enzyme is constitutive in contrast to the polygalacturonases that were not detected. The increase in pectin concentration stimulated growth and enzyme production. The highest specific activities were attained with the greatest concentration tested (15 g/l). Casamino acids were the best nitrogen source for enzyme production. Maximum growth was measured at pH 3.3; pH values of around 4.5 stimulated enzyme production, but high pectinase activities were also detected in media with more alkaline initial pH values (6.2 for galacturonases and 6.6 for lyases), probably owing to the specific induction of particular isoforms. In the range of 23 to 28°C, good results were obtained in growth as well as in enzyme production. The addition of Tween 80 promoted growth and gave the highest yield of polymethylgalacturonase and pectin lyase (0.37 and 36.2 E.U./ml, respectively). The highest polygalacturonase activity (1.1 E.U/ml) was achieved with polyethylene glycol. Tween 20 and Triton X-100 inhibited growth and pectinase production.     

Development and analysis of pectic screening media for use in the detection of pectinase mutants

Summary Two solid pectic media were devised for mutually exclusive detection of extracellular polygalacturonase and pectin lyase produced by fungi including the vascular parasite of tomato Verticillium albo-atrum. These media allowed detection of pectinase-defective mutants. Polygalacturonase detection medium contains non-methylated polygalacturonan (sodium polypectate) is buffered at pH 5.0 (Na citrate, 0.05 M) and is calcium-free. In contrast pectin lyase medium contains polymethylgalacturonan (pectin), is buffered at pH 8.0 (HEPES, 0.05 M) and contains calciumrich agar. When glucose was added to the media for selection of catabolite-resistant mutants, enzyme synthesis was still evident, whereas in comparable conditions in liquid culture production was almost completely repressed. This apparent discrepancy is discussed in terms of the influence of basal synthesis, colony biomass and accumulation of oligouronides which repress induced synthesis and activity.Abbreviations CR catabolite repression - CTAB cetyltrimethyl ammoniumbromide - GALA galacturonic acid - NAPP sodium polypectate - PG polygalacturonase - PL pectin lyase - TBA thiobarbituric acid - UGALA unsaturated galacturonic acid     

Studies in the Physiology of Parasitism: XVIII. Pectic Enzymes secreted by Bacterium aroideae

Cell-free filtrates from cultures of Bacterium aroideae on asimple synthetic medium contained an enzyme, provisionally termeddepolymerase, which rapidly reduced the viscosity of pectinsolutions, and protopectinase which macerated slices of potatotuber tissue. These filtrates had little pectin-esterase activity. The activity of depolymerase was directly proportional to enzymeconcentration; the activity of protopectinase was approximatelyproportional to the square root of the enzyme concentration.Crude solutions were partially purified by acetone or ethanolprecipitation; ammonium sulphate was less satisfactory as aprecipitating agent. Enzyme preparations which rapidly reduced the viscosity of pectinand pectate solutions were relatively inactive when assayedfor polygalacturonase. activity by measuring reducing groupsliberated. After prolonged incubation some 20 and 40 per cent.hydrolysis of solutions of pectin and pectate respectively wasobtained. The pH optimum for depolymerase activity was near 9.0, the enzymewas activated by Ca⁺⁺ but not by a number of other cations;the loss of activity following dialysis was largely restoredby adding Ca⁺⁺. The enzyme was rapidly inactivated at temperaturesabove 60° C. and at pH 2.7. The properties of protopectinase generally resembled those ofdepolymerase. Analysis of the breakdown products following enzyme degradationof pectin and pectate solutions by paper chromatography showedthat galacturonic acid was not produced but that a number ofother products were formed, including one of fairly low molecularweight. The differences between the pectic enzymes of B. aroideae andthose from other sources, and the possible identity of depolymerase,polygalacturonase, and protopectinase are discussed.     

Physical properties of pectic polysaccharidases ofPseudomonas solanacearum from Nigeria

Pseudomonas solanacearum (obtained from Nigeria) produced certain pectic polysaccharidases when grown in aerobic batch cultures containing pectic substances. The pH optima of the enzymes were different. The optimum for polygalacturonase EC 3.2.2.15 was 5.5, and for pectate lyase EC 4.2.99.3 it was 8.5. The -1,4-glycosidic bonds between galacturonide units were cleaved at random, indicating the endo character of the enzymes. The pectic polysaccharidases were purified by (NH₄)₂SO₄ fractionation and by electrofocusing. Highest polygalacturonase activity and pectate lyase activity were obtained in the fractions at 41%–60% and 61%–80% (NH₄)₂SO₄ saturation, respectively. Polygalacturonase was resolved into two components with isoelectric points of 5.0 and 7.5; the isoelectric point of pectate lyase was 8.1.     

Comparative study of pectic activities from different formae speciales of Fusarium oxysporum

Pectic enzyme production by three formae speciales of Fusarium oxysporum , two of which (f. sp. niveum and melonis ) cause vascular wilts and one (f. sp. radicislycopersici ) a root rot, was studied in vitro. The two vascular wilt pathogens produced much higher levels of endo and lyase activities than the root rotting pathogen. Endo activity was greatest for the wilt fungi after 7 d during the growth phase when the pH of the medium was still acidic, but it declined thereafter. By contrast the lyase activity reached a peak after 20 d when growth was complete and the pH was alkaline. It then fell rapidly and was undetectable after 22 d. All the pectic activities degraded pectin and sodium polypectate and presented a similar profile during the incubation time studied. The preferred substrates were pectin for endo activity and polypectate for total and lyase activities. The behaviour of the forma specialis suggests that similar enzymes are produced by each taxon.     

Kinetic of the production of polygalacturonase and pectin lyase by two closely relatedFormae speciales ofFusarium oxysporum

The kinetic of thein vitro production of polygalacturonase and pectin lyase of two closely related fungi,Fusarium oxysporum f.sp.lycopersici andF. oxysporum f.sp.radicis-lycopersici, was examined under various culture conditions such as the source of carbon, the pH, and the age of cultures. Over a 5-day period, the production of these enzymes by various isolates of the sameforma specialis (f. sp.) ofF. oxysporum was not significantly different (P ≥ 0.05). However, the amount of the enzymes produced differed markedly between both f. sp. The different carbon sources added to the culture media, such as citrus pectin, apple pectin, tomato cell wall fragments, andd-galacturonic acid, proved to be higher pectinase inducible substrates than sucrose and glucose. For both fungi, polygalacturonase and pectin lyase activities were optimal at pH 5.0 and 8.0, respectively. Furthermore, pectin lyase production had a partial Ca²⁺ requirement in contrast to polygalacturonase production which was limited by Ca²⁺. In most experiments performed, the production of polygalacturonase appeared superior withF. oxysporum f.sp.radicislycopersici than withF. oxysporum f.sp.lycopersici. On the other hand, pectin lyase production ofF. oxysporum f.sp.lycopersici was approximately 10-fold greater than that byF. oxysporum f.sp.radicis-lycopersici in media supplemented withd-galacturonic acid.     

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.     

Production of extracellular pectinase enzymes by a mutant (Pol6) of Penicillium occitanis

Summary Penicillium occitanis strain Pol6, a mutant developed for hyperproduction of cellulase and pectinase enzymes was used for the study of extracellular pectinase production when pectins from different sources (apple and citrus) and with varying degree of esterification (DE) were used as inducers. Highly esterified citrus pectins were found to be suitable substrates for polygalacturonase, pectinase and pectin methyl esterase production, while low esterified citrus pectin favoured pectin lyase (PL) production. Apple pectins induced other hydrolytic enzymes (e.g., -1,3-glucanase, -glucosidase, -galactosidase), in addition to pectolytic enzymes. Moreover, the combination of high and low esterified citrus pectins induced the production of a complete pectinase complex. The extent of degradation of the substrate and the affinity for PL decreased with decreasing DE irrespective of the source. There was no evidence of PL activity in this strain. No significant effect of cations (Ca⁺⁺, Mn⁺⁺, Na⁺) on PL activity was observed. However, EDTA (100 mm) inhibited 50% of the activity, when tested on highly esterified (rapid set citrus) pectin. Offprint requests to: S. Jain     

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.     

Growth conditions of Aspergillus sp. ATHUM-3482 for polygalacturonase production

A wild type of Aspergillus sp. ATHUM-3482 produced extracellular polygalacturonase when grown in liquid medium containing citrus pectin as sole carbon source. A number of factors affecting enzyme activity were investigated. Polygalacturonase activities as high as␣4.3 U␣ml⁻¹(reducing-group-releasing activity) and 17␣U␣ml⁻¹ (viscosity-diminishing activity) were obtained under optimum growth conditions. With sugar-beet as sole carbon source the respective activities were 6.5 U␣ml⁻¹ and 40 U ml⁻¹, the highest achieved in this work. Under these conditions no pectin lyase or pectinesterase activity was detected. The above yields of polygalacturonase activity compare favourably with those reported for fungi grown under similar growth conditions. Received: 5 March 1996 / Received last revision: 29 October 1996 / Accepted: 2 November 1996     

Role of uronic acids present in phytopathogenic fungi as inducers of polygalacturonases during autolysis

The presence of uronic acids in the culture fluid and mycelium of the fungi: Alternaria alternata, Botrytis cinerea, Drechslera halodes, Fusarium culmorum, Fusarium oxysporum, Monilinia fructigena, Mucor mucedo, Rhizopus stolonifer and Trichoderma hamatum was detected and quantified. In these fungi the concentration of uronic acids increased during the growth phase and the maximal concentrations were found at the end of the growth phase or onset of autolysis both in the mycelium as well as in the culture fluid. The uronic acids were metabolized during the first days of autolysis decreasing to constant levels until the end of the autolytic period studied.The variations in the activity of polygalacturonase and polymethylgalacturonase present in the culture fluid were determined at the onset and during autolysis in these fungi. These enzymic activities were found in the culture fluid of these fungi, with exception of M. rouxii, and they showed an increasing activity in the first days of autolysis and later a slight increase or decrease was observed. The presence of uronic acids in these phytopathogenic or saprophytic fungi and the low levels detected during autolysis could be related to the induction of pectic enzymes and the pathogenicity of these fungi.     

Pectic polysaccharide breakdown of cell walls in cucumber roots grown with calcium starvation

Pectic polysaccharides from the roots of cucumber (Cucumis sativus L.) grown in liquid culture medium with or without calcium (1 mm CaCl₂) were studied after extraction successively by hot water and Na hexametaphosphate solution. The Ca²⁺ starvation-treatment caused a striking reduction in content of extracted pectic polysaccharide; from an equivalent weight of cell walls, only 33.1% of the control level was extracted from root cell walls of plants cultured under Ca²⁺ deficiency. The extracted pectic polysaccharides were fractionated into neutral and acidic polymers by a DEAE-Sephadex column. The acidic polymers, which represented more than 76% of the yield, appeared to be a major fraction of extracted pectic polysaccharides. The changes of molecular size and glycosyl residue composition of this fraction were compared for the control and Ca²⁺-deprived samples. The results indicate that Ca²⁺ deficiency caused structural changes which could involve both branching pattern and extent of contiguous galacturonosyl units in the water-solubilized pectic polysaccharides. Ca²⁺ starvation also led to a notable decrease in molecular size of the hexametaphosphate-solubilized polysaccharides and, to a lesser extent, of the water-solubilized fraction as well. In addition, polygalacturonase activity in tissue homogenates increased remarkably with the Ca²⁺ deficiency, whereas β-galactosidase activity did not undergo a change. Thus, it appears that one major effect of Ca²⁺ deprivation was to stimulate polygalacturonase activity, an effect which could be involved in the control of the breakdown of pectic polysaccharides in the cell walls.     

解淀粉芽孢杆菌B15抑菌物质对葡萄灰霉病灰葡萄孢的抑菌机理

【目的】利用荧光显微镜和激光共聚焦扫描显微镜技术初步探讨解淀粉芽孢杆菌(Bacillus amyloquefaciens)B15菌株发酵液中的抑菌混合物质伊枯草菌素A(iturin A)和芬芥素(fengycin)对葡萄灰霉病病原菌灰葡萄孢(Botrytis cinerea)的抑菌机理。【方法】采用琼脂稀释法讨论解淀粉芽孢杆菌B15发酵液对灰葡萄孢的抑菌活性。利用台盼蓝(trypan blue)染色、4′,6-二脒基-2-苯基吲哚(DAPI)、双氢罗丹明123(DHR123)、钙离子探针fluo-3/am和Annexin V-PI探针染色来观察解淀粉芽孢杆菌B15发酵液对灰葡萄孢细胞膜和菌丝形态、细胞核、活性氧、钙离子和磷脂酰丝氨酸层的影响。【结果】抑菌活性实验发现解淀粉芽孢杆菌B15发酵液对灰葡萄孢具有良好抑菌效果。荧光显微镜台盼蓝染色观察发现,经B15发酵液处理过的灰葡萄孢出现菌丝畸形、菌丝体粗大、尖端肿胀并被染成蓝色和明显的液泡化现象。同时未在处理组中观察到细胞内容物泄漏,说明处理组菌丝细胞膜未发生破损。该结果表明在此次试验中,B15发酵液中的抑菌有效物质不以破损细胞膜的方式直接导致灰葡萄孢的死亡。激光共聚焦显微镜观察结果发现,处理组的灰葡萄孢菌丝出现典型的细胞凋亡现象、染色质固缩、细胞核裂解、磷脂酰丝氨酸层外翻、活性氧和钙离子积累。【结论】该实验表明解淀粉芽孢杆菌B15发酵液以诱导细胞凋亡的形式来抑制灰葡萄孢菌丝的生长。     

Histochemical and cytochemical demonstration of Ca++-ATPase activity in the stellate cells of the adenohypophysis of the guinea pig

Summary The histo- and cytochemical localization of Ca⁺⁺-ATPase activity in the adenohypophysis of the guinea pig was studied utilizing a newly developed method (Ando et al. 1981). An intense reaction was observed in the wall of the blood vessels and between non-secretory cells (stellate cells) and endocrine cells of the pars distalis. Under the electron microscope the Ca⁺⁺-ATPase reaction product was located extracellularly in relation to the plasmalemma of the stellate cells. This reaction was dependent on Ca⁺⁺ and the substrate, ATP, and reduced by the addition of 0,1 mM quercetin to the standard incubation medium. Preheating of the sections before incubation completely inhibited the enzyme activity. When Mg⁺⁺ in different concentrations were substituted for Ca⁺⁺ in the incubation medium the reaction was always reduced. Both Ca⁺⁺ and Mg⁺⁺ in the incubation medium also reduced the reaction. The plasmalemma of the endocrine cells contains no demonstrable amount of Ca⁺⁺-ATPase activity. The function of the Ca⁺⁺-ATPase activity is discussed in relation to the regulation of the extracellular Ca⁺⁺ concentration which seems to be important with respect not only to the secretory process of the endocrine cells but also to the metabolism of the adenohypophysis.     

Purification and properties of an endopolygalacturonase produced byRhizopus stolonifer

Summary A polygalacturonase from culture filtrates of a strain ofRhizopus stolonifer was purified about 80 fold by ethanol precipitation, followed by ion exchange chromatography (CM-Sepharose 6B) and gel filtration (Sephadex G-100). The purified preparation was homogeneous when examined by PAGE. The enzyme is an endopolygalacturonase with an optimum catalytic activity at pH 5.0 and 45°C, and a molecular weight of 57,000±500 daltons. The activity was stimulated by Fe⁺⁺⁺, Mg⁺⁺, Co⁺⁺, and inhibited by Mn⁺⁺ and Zn⁺⁺. The enzyme was stable in the pH range of 3.0 to 5.0. The purified enzyme was specific for nonmethoxylate polygalacturonic acid, with K_m and V_max values respectively of 0.19 mg/ml and 1.3 mol/g/min. In addition, this enzymatic preparation degraded pectic substances in organge peel.     

Pectic Enzymes and Phenolic Substances in Apples Rotted by Fungi

The activities of pectic enzymes in extracts from sound applesand from apples rotted by different fungi are described. Sclerotiniafructigenaand Botrytis cinerea rots had little or no polygalacturonaseor macerating enzyme activity, but Penicillium expansum rotswere very active in these respects. Extracts from each of therots had very high pectinesterase activity, and contained galacturonicacid. None of the rots had any cellulase activity. Each of thefungi produced polygalacturonase, macerating enzymes, and pectinesterasein liquid media. The effects of adding extracts of apples tothese media are described. Filtrates from cultures of S. fructigenaand P. expansum liberated galacturonic acid from apple fruitfibre which had been thoroughly extracted with cold water. The phenolic jsubstances present in healthy and rotted tisueswere estimated. B. cinerea and S. fructigena rots containedvery little, but P. expansum rots contained as much as healthytissue which had been allowed to brown. An extract of healthyapple tissue reduced the activity of the polygalacturonase ina culture filtrate of S. fructigena. The substances responsiblefor this were tentatively identified as leuco-anthocyanins whichhad been changed to other compunds following the action of polyphenoloxidase.Thej significance of these results is discussed.     

Application of benzothiadiazole and Trichoderma harzianum to control faba bean chocolate spot disease and their effect on some physiological and biochemical traits

Chocolate spot disease is the most prevalent and important disease in the major faba bean growing regions in the world. Different concentrations of the abiotic inducer (0.3 and 0.5 mM benzothiadiazole) and the biotic inducer (1 × 10⁷ and 2 × 10⁷ spore/ml Trichoderma harzianum) were used alone or in combination to study their efficiency against faba bean chocolate spot disease caused by Botrytis fabae and Botrytis cinerea and their effect on some chemical analyses (phenylalanine ammonia lyase activity, total flavonoids and peroxidase isozymes, pectin and lignin content and total chlorophyll content). Application of the tested inducers as foliar treatment significantly reduced the severity of chocolate spot disease as compared with untreated infected plants. The reduction in disease severity was associated with a gradual increase in phenylalanine ammonia lyase activity. Maximum increase was recorded at 72 h after inoculation with B. fabae and B. cinerea. In addition, the levels of flavonoids in induced infected leaves recorded a sharp increase at 24 h after inoculation with B. fabae or B. cinerea. Also, pectin and lignin contents in the cell wall of induced infected plants were significantly increased as compared with untreated infected plants. Beside the induction of resistance, the tested inducers markedly increased total chlorophyll content in treated infected plants as compared with untreated infected plants. Isozymes analysis revealed that new peroxidase bands were induced only in treated faba bean leaves in response to infection with B. fabae or B. cinerea.