Production of Polysaccharidases in Different Carbon Sources by Leucoagaricus gongylophorus Möller (Singer), the Symbiotic Fungus of the Leaf-Cutting Ant Atta sexdens Linnaeus

Leucoagaricus gongylophorus, the fungus cultured by the leaf-cutting ant Atta sexdens, produces polysaccharidases that degrade leaf components by generating nutrients believed to be essential for ant nutrition. We evaluated pectinase, amylase, xylanase, and cellulase production by L. gongylophorus in laboratory cultures and found that polysaccharidases are produced during fungal growth on pectin, starch, cellulose, xylan, or glucose but not cellulase, whose production is inhibited during fungal growth on xylan. Pectin was the carbon source that best stimulated the production of enzymes, which showed that pectinase had the highest production activity of all of the carbon sources tested, indicating that the presence of pectin and the production of pectinase are key features for symbiotic nutrition on plant material. During growth on starch and cellulose, polysaccharidase production level was intermediate, although during growth on xylan and glucose, enzyme production was very low. We propose a possible profile of polysaccharide degradation inside the nest, where the fungus is cultured on the foliar substrate.     

Production of Pectinase and Cellulase by Cladosporium cucumerinum with Dissolved Carbohydrates and Isolated Cell Walls of Cucumber as Carbon Sources

The scab fungus Cladosporium cucumerinum can use pectins and polygalacturonic acid as sole sources of carbon. Cellulose and Ca-polygalacturonate are not available carbon sources for the fungus. When growing on sucrose or pectin, pectinase is produced. In these cases the production of cellulase is insignificant. On a mixture of pectin and carboxymethylcellulose also cellulase is produced. Both pectinase and cellulase are released into the culture filtrate when the fungus grows on cell walls without ionic proteins, whereas only cellulase is released when cell walls with ionic proteins are the carbon source. Pectinase produced by the pathogen can bind to isolated cell walls. The bound pectinase can be extracted with 1 M NaCl from cell walls without ionic proteins, but not from cell walls with ionic proteins. A water-extract or 1 M NaCl-extract of cucumber hypocotyls with visible disease symptoms contains cellulase but no pectinase activity. Lack of pectinase activity in the 1 M NaCl-extract may be due to inhibition by a component that could be extracted by NaCl from the cucumber cell walls.     

Relationship between conidial enzymes and germination of the apple blue mold,Penicillium expansum

The relationship between conidial enzymes of Penicillium expansum and spore germination was examined. The activities of xylanase and pectinase, but not of cellulase and amylase, were detected in the conidia. The levels of xylanase and pectinase were greatly enhanced by xylan and pectin as respective carbon sources in the basal medium. No conidia germinated in the basal medium without a carbon source. The type of carbon source and the enzyme levels of the conidia did not affect the rate of germination. However, a relationship was found between the enzyme levels and the elongation of the germ tubes.     

Fusion of yeast protoplasts and isolated nuclei of Fusarium moniliforme

Protoplasts of a xylose-fermenting yeast strain (a fusion product of Pachysolen tannophilus and Saccharomyces cerevisiae) were fused with isolated nuclei of the xylan degrading filamentous fungus Fusarium moniliforme. Polyethyleneglycol 4000 was used as the fusogenic agent. Fourteen stable hybrids showing xylanase activity were obtained. It can be assumed that this ability was acquired from the nuclear genome of the fungus, since the parental yeast strain did not show any xylanase activity. The enzymatic activity was determined quantitatively. The parental strain of the fungus reached its maximum xylanase activity of 796 nkat/ml at 96 h of growth. Four of the hybrids had a xylanase activity of between 211 and 297 nkat/l at 24 h of growth. Zymograms of these hybrids showed the presence of xylanases when grown on xylan as the sole carbon source. Using pulse field electrophoresis gels, no difference between the chromosome pattern of the fusion products and the parental yeast strain was observed.     

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.     

Effect of shear rate and culture pH on the production of xylanase by Thermomyces lanuginosus

Summary The pH-value and the stirrer speed during cultivation of the thermophilic fungus Thermomyces lanuginosus were found to have a pronounced influence on xylanase production using corn cobs as carbon source. The highest xylanase activity of 32500 nkat/ml was produced in labscale fermentation within 118 hours at a stirrer speed of 50 rpm and a controlled pH-value of 7.5.     

Pectinase production by a Brazilian thermophilic fungus <Emphasis Type="Italic">Thermomucor indicae-seudaticae</Emphasis> N31 in solid-state and submerged fermentation

Thermophilic organisms produce thermostable enzymes, which have a number of applications, justifying the interest in the isolation of new thermophilic strains and study of their enzymes. Thirty-four thermophilic and thermotolerant fungal strains were isolated from soil, organic compost, and an industrial waste pile based on their ability to grow at 45°C and in a liquid medium containing pectin as the only carbon source. Among these fungi, 50% were identified at the genus level as Thermomyces, Aspergillus, Monascus, Chaetomium, Neosartoria, Scopulariopsis, and Thermomucor. All isolated strains produced pectinase during solid-state fermentation (SSF). The highest polygalacturonase (PG) activity was obtained in the culture medium of thermophilic strain N31 identified as Thermomucor indicae-seudaticae. Under SSF conditions on media containing a mixture of wheat bran and orange bagasse (1 : 1) at 70% of initial moisture, this fungus produced the maximum of 120 U/ml of exo-PG, while in submerged fermentation (SmF) it produced 13.6 U/ml. The crude PG from SmF was more thermostable than that from SSF and exhibited higher stability in acidic pH.     

Production of extracellular enzymes by immobilizedTrichoderma reesei in shake flask cultures

Polyurethane foam and nylon-web carriers were compared for simultaneous production of endo-1, 4-β-glucanase and xylanase by immobilizedTrichoderma reesei on a medium based on lactose (27 g/l), cellulose (3 g/l) and sorbose (0.3 g/l). Nylon sheet with 1.2 cm² carrier surface/ml medium was superior to the others, and it was selected for further studies. The carbon source had a marked effect on enzyme production by the immobilized fungus. With pure cellulose (10 g/l) as substrate, the maximum endoglucanase activity was 690 nkat/ml and xylanase activity 4800 nkat/ml. Supplementation with 0.5 g/l of sorbose resulted in an increase in both endoglucanase and xylanase activities in all media studied. A more detailed study on the effect of sorbose on a lactose(7 g/l)-and cellulose(3 g/l)-based medium revealed a clear optimum sorbose concentration of 1.5 g/l, with a maximum endoglucanase activity of 660 nkat/ml, xylanase activity of 3670 nkat/ml, and filter-paper activity (overall cellulolytic activity) of 2.0 filter-paper units/ml. However, the addition of 1.5 g/l sorbose to the pure-cellulose(10 g/l)-based medium resulted in a slight decrease in the enzyme production.     

Pectinase production bySclerotium rolfsii: Effect of culture conditions

Production of pectinase bySclerotium rolfsii was studied under submerged conditions. A 7.1-fold increase in the production of pectinase was obtained by optimizing the culture conditions. Pectinase was obtained in good yields only when pectin was used as carbon source, best at initial pH between 6 and 7. The enzyme was not induced on sorbose, lactose, mannitol, glycerol, maltose, fructose or raffinose and growth was poor on these substrates. Incorporation of corn-steep liquor in the medium containing pectin increased the production of the enzyme by 45%. Maximum yield of pectinase obtained was 500 nkat/mL.     

Xylanase production by Trichoderma reesei Rut C-30 grown on L-arabinose-rich plant hydrolysates

The suitability of L-arabinose-rich plant hydrolysates as carbon sources and inducers of xylanase production in Trichoderma reesei Rut C-30 was tested. Significantly higher xylanase activities were obtained in cultures on oat husk and sugar beet pulp hydrolysates than on lactose. In batch culture with oat husk hydrolysate and lactose, the xylanase activity was about 9 times higher ( approximately 510 IU/ml) than in lactose ( approximately 60 IU/ml). Even higher xylanase activity ( approximately 630 IU/ml) was obtained when the batch cultivations were done on sugar beet pulp hydrolysate and lactose. In a fed-batch culture using oat husk hydrolysate-lactose the xylanase activity was as high as 1350 IU/ml in 4 days. The cellulase production clearly decreased when T. reesei was cultured on both hydrolysates compared to the cultivation on lactose. Moreover, the relative amounts of the xylanases I-III were similar regardless the used carbon source.     

Pectin lyase from <Emphasis Type="Italic">Aspergillus giganteus</Emphasis>: Comparative study of productivity of submerged fermentation on citrus pectin and orange waste

The aim of this study was to investigate some of the factors affecting pectin lyase (PL) production by an Aspergillus giganteus strain, and to characterize this pectinolytic activity excreted into the medium. The highest activities were obtained with orange waste, citrus pectin and galacturonic acid as carbon sources. The highest activity, using citrus pectin as carbon source, was obtained in 11-day-old standing cultures, but the highest specific activity was obtained in 6.5-day-old shaken cultures, at pH 6.5 and 35°C. Using orange waste as carbon source, the highest activity was observed in 8-day-old standing cultures, at pH 7.0 and 30°C. Optimal assay conditions were pH 8.5–9.0 and 50°C. The PL activity showed thermal stability, with half-lives of 30 and 27 min when incubated at 45 and 50°C, respectively. High stability was observed at room temperature from pH 6.0 to 10.0; more than 85% of enzyme activity was preserved in this pH range. Under optimum conditions, the highest pectin lyase activity in the medium was 470 U/ml, with orange waste as carbon source.     

Sorghum straw for xylanase hyper-production by Thermomyces lanuginosus (D2W3) under solid-state fermentation

This paper reports the production of very high levels of cellulase free xylanase and associated hemicellulases by an indigenous thermophilic isolate of Thermomyces lanuginosus (D(2)W(3)) using solid-state fermentation. Sorghum straw, an inexpensive and abundant source of carbon supported maximal xylanase activity (11,855 units/g dry substrate). Culturing T. lanuginosus D(2)W(3) on sorghum straw and optimizing other culture conditions (media types, particle size of carbon source, inoculum level, inoculum age and additives), yielded increased levels of xylanase (39,726 units/g dry substrate). Further optimization of enzyme production was carried out using Box-Behnken design of experiments with three independent variables (inoculum level, glycerol and ammonium sulphate concentrations) which resulted in very high levels of xylanase, 48,000+/-1774 units/g dry substrate, and 2.6+/-0.2, 13.4+/-0.56, 68+/-1.7, 1.4+/-0.08, 1.2+/-0.05 (units/g dry substrate) of beta-xylosidase, alpha-galactosidase, pectinase, beta-mannosidase and alpha-L-arabinofuranosidase, respectively.     

Production and characterization of thermostable xylanase and pectinase from Streptomyces sp. QG-11-3

Streptomyces sp. QG-11-3, which produces a cellulase-free thermostable xylanase (96 IU ml⁻¹) and a pectinase (46 IU ml⁻¹), was isolated on Horikoshi medium supplemented with 1% w/v wheat bran. Carbon sources that favored xylanase production were rice bran (82 IU ml⁻¹) and birch-wood xylan (81 IU ml⁻¹); pectinase production was also stimulated by pectin and cotton seed cake (34 IU ml⁻¹ each). The partially purified xylanase and pectinase were optimally active at 60°C. Both enzymes were 100% stable at 50°C for more than 24 h. The half-lives of xylanase and pectinase at 70, 75 and 80°C were 90, 75 and 9 min, and 90, 53 and 7 min, respectively. The optimum pH values for xylanase and pectinase were 8.6 and 3.0, respectively, at 60°C. Xylanase and pectinase were stable over a broad pH range between 5.4 and 9.4 and 2.0 to 9.0, respectively, retaining more than 85% of their activity. Ca²⁺ stimulated the activity of both enzymes up to 7%, whereas Cd²⁺, Co²⁺, Cr³⁺, iodoacetic acid and iodoacetamide inhibited xylanase up to 35% and pectinase up to 63%; at 1 mM, Hg²⁺ inhibited both enzymes completely. Journal of Industrial Microbiology & Biotechnology (2000) 24, 396–402. Received 29 September 1999/ Accepted in revised form 02 February 2000     

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.     

High-level of xylanase production by the thermophilic Paecilomyces themophila J18 on wheat straw in solid-state fermentation

The production of extracellular xylanase by a newly isolated thermophilic fungus, Paecilomyces themophila J18, on the lignocellulosic materials was studied in solid-state fermentation (SSF). The strain grew well at 50 degrees C and produced a high-level of xylanase activity using the selected lignocellulosic materials, especially wheat straw. Production of xylanase by P. themophila J18 on wheat straw was enhanced by optimizing the particle size of wheat straw, nitrogen source, initial moisture level, growth temperature and initial pH of the culture medium. Under the optimized conditions, yield as high as 18,580 Ug(-1) of carbon source of xylanase was achieved. No CMCase activity was observed. The xylanase exhibited remarkable stability and retained more than 50% of its original activity at 70 degrees C for 4h at pH 7.0-8.0. Therefore, P. themophila J18 could to be a promising microorganism for thermostable, cellulase-free xylanase production in SSF.     

The Anaerobic Fungus Neocallimastix sp. Strain L2: Growth and Production of (Hemi)Cellulolytic Enzymes on a Range of Carbohydrate Substrates

The anaerobic fungus Neocallimastix sp. strain L2, isolated from the feces of a llama, was tested for growth on a range of soluble and insoluble carbohydrate substrates. The fungus was able to ferment glucose, cellobiose, fructose, lactose, maltose, sucrose, soluble starch, inulin, filter paper cellulose, and Avicel. No growth was observed on arabinose, galactose, mannose, ribose, xylose, sorbitol, pectin, xylan, glycerol, citrate, soya, and wheat bran. The fermentation products after growth were hydrogen, formate, acetate, ethanol, and lactate. The fermentation pattern was dependent on the carbon source. In general, higher hydrogen production resulted in decreased formation of lactate and ethanol. Recovery of the fermented carbon in products at the end of growth ranged from 50% to 80%. (Hemi)cellulolytic enzyme activities were affected by the carbon source. Highest activities were found in filtrates from cultures grown on cellulose. Growing the fungus on inulin and lactose yielded the lowest cellulolytic activities. Highest specific activities for avicelase, endoglucanase, β-glucosidase, and xylanase were obtained with Avicel as the substrate for growth (0.29, 5.9, 0.57, and 13 IU · mg⁻¹ protein, respectively). Endoglucanase activity banding patterns after SDS-PAGE were very similar for all substrates. Minor differences indicated that enzyme activities may in part be the result of secretion of different sets of isoenzymes. Received: 10 July 1996 / Accepted: 22 July 1996     

Characterization of major hydrolytic enzymes secreted by Pythium myriotylum, causative agent for soft rot disease

Pythium myriotylum, an oomycetous necrotroph is the causal agent of soft rot disease affecting several crops. Successful colonization by necrotrophs depends on their secretion of a diverse array of plant cell wall degrading enzymes (CWDEs). The induction dynamics of CWDEs secreted by P. myriotylum was analysed as little information is available for this pathogen. Activities of CWDEs that included pectinase, cellulase, xylanase and protease were detected using radial diffusion assay and differential staining. In Czapek Dox minimal medium supplemented with respective substrates as carbon source, the increase in CWDE activities was observed till 8 days of incubation after which a gradual decline in enzymatic activities was observed. With sucrose as sole carbon source, all the enzymes studied showed increase in activity with fungal growth while with cell wall material derived from ginger rhizome as sole carbon source, an initial spurt in cellulase, xylanase and pectinase activities was observed 3 days post incubation while protease activity increased from three days of incubation and reached maximum at 13 days of incubation. To further evaluate the role of CWDEs in pathogenicity, UV-induced mutants (pmN14uv1) were generated wherein significant reduction in cellulase, pectinase and protease activities were observed while that of xylanase remained unchanged compared to wild type isolate (RGCBN14). Bioassays indicated changes in infection potential of pmN14uv1 thereby suggesting the crucial role played by P. myriotylum CWDEs in initiating the rotting process. Hence appropriate strategies that target the production/activity of these secretory hydrolytic enzymes will help in reducing disease incidence/pathogen virulence.     

亚麻微生物脱胶菌种的筛选与鉴定

在研究天然水沤法脱胶的过程中,通过初筛、复筛,从沤麻主生物期的沤麻液中筛选出两株茵落周围产生透明圈较大、脱胶酶活较高的菌株。通过形态观察,并对其多项生理、生化指标进行了分析研究,初步鉴定并命名为枯草芽孢杆菌A1和B1。初步加茵脱胶实验表明：枯草芽孢杆菌A1产生果胶酶、木聚糖酶,而不产生纤维素酶,脱胶周期为72小时;枯草芽孢杆茵B1产生果胶酶、木聚糖酶和纤维素酶,脱胶周期为50小时。     

Production,purification and characterization of pectinase from a Bacillus sp. DT7

A soil isolate, Bacillus sp. DT7 has been found to produce significant amounts of an extracellular pectinase subsequently characterized as pectin lyase (EC 4.2.2.10). By optimizing growth conditions, Bacillus sp. DT7 produced higher amount of pectin lyase (53 units/ml) than that has been reported in the literature. Using gel filtration and ion exchange chromatography, this enzyme was purified and found to have a molecular mass of 106 kDa. The purified enzyme exhibited maximal activity at a temperature of 60 C and pH 8.0. The presence of 100 mM concentrations of CaCl₂ and mercaptoethanol significantly enhanced pectinase activity of the purified enzyme. This pectinase has tremendous applications in textile industry, plant tissue maceration and fruit juice wastewater treatments.