Cx Enzyme Research Articles

Analysis of Rumen Degradation Characteristics, Attached Microbial Community, and Cellulase Activity Changes of Garlic Skin and Artemisia argyi Stalk.

The purpose of this study was to study the chemical composition, rumen degradation characteristics, surface attached microbial community and cellulase activity of garlic skin (GS) and Artemisia argyi stalk (AS), in order to explain their feeding value. Four 14-month-old healthy Min Dong male goats with permanent rumen fistula were selected as experimental animals. The rumen degradation characteristics of GS and AS were determined by using the nylon bag method, and the bacterial composition, cellulase activity and their relationship on the surface of the two groups were analyzed with high-throughput sequencing of 16S rRNA gene. The results showed that in GS and AS, the effective degradation rate (ED) values of dry matter (DM) were 42.53% and 37.12%, the ED values of crude protein (CP) were 37.19% and 43.38%, the ED values of neutral detergent fiber (NDF) were 36.83% and 36.23%, and the ED values of acid detergent fiber (ADF) were 33.81% and 34.77%. During rumen degradation, the richness and evenness of bacteria attached to the AS surface were higher. At the phylum level, Bacteroidetes and Firmicutes were always the main rumen bacteria in the two groups. At the genus level, fiber-degrading bacteria such as Prevotella, Treponema, and Ruminococcus showed higher levels in GS (p < 0.05). Compared with GS, the activity of β-glucosidase (BG enzyme), endo-β-1,4-glucanase (C1 enzyme), exo-β-1,4-glucanase (Cx enzyme) and neutral xylanase (NEX enzyme) attached to AS surface showed a higher trend. Correlation analysis showed that the relative abundance of Succinivibrio and Rikenellaceae_RC9_gut_group was positively correlated with the rumen degradability of nutrients in GS, and the relative abundance of Christensenellaceae R-7_group, Succinivibrio and Ruminococcus was positively correlated with the rumen degradability of nutrients in AS. The conclusion of this study shows that AS has more potential to become ruminant roughage than GS. In addition, this study also revealed the relationship between cellulase activity and bacteria, which provided new information for us to better analyze the effects of GS and AS on the rumen of ruminants and provided an important theoretical basis for the development and utilization of agricultural by-products.

English

The production of polygalacturonase (PG), polymethylgalacturonase (PMG) and cellulase (Cx) enzymes have been studied at different incubation period. The fruit rot pathogen of tomato Alternaria solani was cultured at 28&deg;C on semi ripe tomato fruit broth medium for 2, 4, 6, &nbsp;8, 10 and 12 days. Isolated pathogen A. solani has a capability of producing PG, PMG and Cx enzyme within short incubation period, that is, 2 days incubation. Production of these enzymes gradually increased by increasing the length of incubation period up to 6 days and further increase in length of incubation up to 12 days, did not show any effect on the production of PG, PMG and Cx. The 6 days incubation was found to be the best incubation period for the production of all these three enzymes. The production of PG and Cx was also more in comparison with PMG enzyme. The enzyme activity in healthy and diseased semi ripe tomato fruits was also assayed at different incubation periods and it was found to be maximum in diseased tomato fruits in comparison with healthy tomato fruits. It was also found that PG and PMG enzymes were produced in higher concentration than the Cx enzyme. Among six incubation periods, 6 days incubation period was found best for maximum production of all these three enzymes. &nbsp; Key words: Alternaria solani, semi ripe tomato fruit medium, semi ripe tomato fruit, incubation period. &nbsp

Cellulase activity of some phytopathogenic fungi isolated from diseased leaves of broad bean

Forty-five fungal species and two varieties belonging to the 23 genera were collected from 50 samples of diseased leaves of broad bean collected from Qena Governorate in Egypt on glucose-Czapek’s, dichloran-chloramphenicol-malt extract and dichloran-chloramphenicol-peptone agar at 28 °C. The most common genera were Alternaria, Aspergillus, Cladosporium, Fusarium, Mycosphaerella and Penicillium. The most prevalent species were Alternaria alternata, Aspergillus flavus, A. fumigatus, A. niger, Cladosporium cladosporioides, Fusarium merismoides, Mycosphaerella tassiana and Penicillium chrysogenum. Among the eight dematiaceous Hyphomycetes phytopathogenic fungi screened for their abilities to produce both exo- and endo-β-1,4-glucanase enzymes (C1 and Cx), five species had high C1 enzyme production and two species had high Cx enzyme production. However, two and three species were moderately active in producing C1 and Cx enzymes, respectively. The remaining species had low production of both C1 and Cx enzymes. The highest cellulase activity was recorded by Alternaria citri and Cochliobolus spicifer for C1 and by A alternata and A. citri for Cx enzyme. Maximum production of C1 enzyme by A. citri and C. spicifer was obtained after six days of incubation at 30 °C with an initial pH of 6 in a culture medium containing lactose and calcium nitrate as the carbon and nitrogen sources, respectively. The highest Cx enzyme production was by A. alternata and A. citri,which was recorded after eight days of incubation at 30 °C with an initial pH of 6 in a culture medium containing carboxymethyl cellulose and sodium or calcium nitrate as the carbon and nitrogen sources, respectively.

OPTIMIZATION OF PROCESS PARAMETERS FOR CELLULASE PRODUCTION BY NOVEL THERMOTOLERANT YEAST

The goal of the present study was to investigate production of cellulase in low cost medium by thermotolerant yeast. After screening, an efficient yeast isolate having capability of C1 (exo-gluconase) and Cx (endo-gluconase) production was isolated and designated as strain R-1. Maximum enzyme production was achieved at 50 °C, pH 5.5 in the medium containing bagasse powder 4% (w/v), and ammonium sulphate 0.1% (w/v) after 72 hours of incubation. The composition containing bagasse powder, 4% (w/v); ammonium sulphate, 0.5 % (w/v); and glucose, 0.5% (w/v) achieved better production after complete medium optimization. The yeast isolate was able to tolerate wide ranges of temperature, pH, and substrate concentration for higher enzyme production. The isolated yeast was able to produce C1 (exo-gluconase) and Cx (endo-gluconase) enzymes in appropriate concentrations on a crude cellulosic substrate. Therefore, yeast may be used to power alcohol production.

Effects of culture ages on the production and activities of polygalacturonase and cellulase (CX) enzymes produced by &lt;i&gt;Pythium aphanidermatum&lt;/i&gt; (Edson Fitzpat.) Isolated from soft stem rot disease of cowpea.

Effect of culture ages was investigated on the production and activities of polygalacturonase (PG) and Cx cellulase enzymes. Pythium aphanidermatum was isolated from cowpea seedlings infected by soft stem rot disease and prepared into pure culture through successive culturing. Two discs (2 mm each) of two-day-old culture of P. aphanidermatum were put into 21 Erlenmeyer flasks containing 50 ml of Reese and Levinson’s solution, consisting of Sodium Polypectate (NaPP) as carbon source, and incubated for 7 days at 38oC. At one-day interval, content of 3 flasks was independently centrifuged at 10,000g for 30 min. The PG enzyme activity was determined viscometrically and expressed as the Relative Viscometric Unit (RVU). Data collected were analysed using analysis of variance (Anova) and the means separated by the Duncan Multiple Range Test. Activity of Cx cellulase enzyme was also evaluated by the same method used for PG enzyme but using, carboxylmethyl cellulose (CMC) as the carbon source. The results showed that there was significant difference in the production and activities of PG and Cx enzymes in the various days of the incubation. The PG and Cx enzymes were produced progressively from the 1st day of incubation and reached their peak on the 5th day for PG (RVU 105) and 4th day for Cx (RVU 49), after which, production and activities of the enzymes started declining. Results suggested that soft stem rot disease in cowpea is a monocyclic disease, whereby any attacked plant that is able to withstand the disease is capable of surviving after the peak of the infection. Key words: Pythium aphanidermatum, polygalacturonase, Cx cellulose,enzymes, viscometric, rot, disease.

Production of cellulase enzymes and their activities in cultures of Xanthomonas campestris pv. malvacearum (Smith) Dowson

Summary Cellulase enzyme production was differentially sensitized by varied carbon sources in the culture of Xanthomonas campestris pv. malvacearum, being best in carboxymethyl cellulose (CMC) and pectin as carbon sources. Next to it were glucose, casamino acid, and maltose. 60min of incubation of the enzyme-substrate complex at 30°C effected optimum Cx enzyme activities, except for casamino acid that needed 90min of incubation. A better cellulolytic breakdown, leading to good accumulation of glucose and cellobiose, was induced by 24-h-old culture of this pathogen after 24h of incubation of the enzyme-substrate complex with pectin, glucose, CMC, and maltose. The least effective of the 5 carbon sources was casamino acid.

Cellulolytic enzymes associated with the fruit rots of Citrus sinensis caused by Aspergillus aculeatus and Botryodiplodia theobromae

Botryodiplodia theobromae and Aspergillus aculeatus were inoculated in carboxymethylcellulose (CMC) medium and on filter papers. Hydrolysis of the CMC medium and degradation of the filter papers were observed, indicating the production of C1 and Cx cellulases by the two rot pathogens. The C1 and Cx enzymes were also detected in filtrates of rotted orange fruits obtained by infection with the two pathogens. The cellulases could not induce rot development on their own. However, when they were added to pectinases in an enzyme inoculum, the incubation period for inducing rot development was shorter, thus establishing a secondary role for the cellulases in the rot development. Optimum conditions for the action of the cellulases included a neutral pH and temperature ranging from 25 to 30 degrees C.

Cellulolytic enzymes associated with the fruit rots of Citrus sinensis caused by Aspergillus aculeatus and Botryodiplodia theobromae

Botryodiplodia theobromae and Aspergillus aculeatus were inoculated on carboxymethylcellulose (CMC) medium and filter papers. The hydrolysis of the CMC medium and degradation of the filter papers were observed, indicating the production of the Cl and Cx cellulases by the two rot pathogens. The Cl and Cx enzymes were also detected in filtrates of rotted orange fruits incited by the two rot pathogens. The cellulases could not induce rot development on their own. However, when they were added to pectinases in an enzyme inoculum, the incubation period for inducing rot development was shorter; thus establishing a secondary role for the cellulases in the rot development. This secondary role of the cellulases produced by the two fungi was found to be at peak at pH 7 and a temperature range of 25°–30 °C in the two fungi.

Production of Cellulolytic Enzymes by the Fungi Associated with the Spoilage of Copra

Among the fungi isolated from spoiled copra, Botryodiplodia theobromae, Aspergillus flavus and, A. niger produced more cellulolytic enzyme (C1) while Curvularia senegalensis and penicillium citrinum produced more Cx enzyme. The enzyme activity increased with the period of incubation and was maximum on 16th day. In vivo preparations showed very little activity.

The Production of Cellulase byTalaromyces Emersonii

The ability of Talaromyces emersonii to synthesize extracellular cellulase has been studied in stationary liquid medium. While this organism could not degrade filter paper, its cell-free filtrate hydrolyzed soluble carboxymethyl cellulose to reducing sugars. Hence it was suggested that T. emersonii was able to synthesize the C,, but not the C1 enzyme. The optimum temperature for synthesis of the Cx enzyme was found to be 45 C, while that for its activity was 50 C. The best pH for the enzyme activity was 6, and enzyme production was found to be affected by the concentration of cellulose in the medium. The probable participation of T. emersonii in the degradation of insoluble cellulose in the natural habitat and in the biodeterioration of stored agricultural products is discussed.

Cellulases from Chaetomium thermophile var. dissitum.

When cultivated in a liquid medium with cellulose, Chaetomium thermophile var. dissitum produced extracellular cellulolytic enzymes. By concentration of the culture filtrate, followed by ion‐exchange chromatography on DEAE‐Sephadex A‐50 and gel filtration on Biogel P‐100, three electrophoretically pure components were obtained. Of these, one was a typical Cx enzyme (endoglucanase) causing rapid decrease of the viscosity of carboxymethylcellulose solutions, while showing low effect on native cellulose.The other was active towards native cellulose, but had little effect on the viscosity of carboxymethylcellulose. It is concluded that this enzyme is an exoglucanase (C1 enzyme), possibly a cellobiohydrolase.The third component showed only cellobiase (β‐glucosidase) activity, and had no effect on cotton or carboxymethylcellulose. The three components, when mixed, showed synergistic effects on highly ordered cellulose. The endo‐ and exoglucanases were characterized with regard to molecular size and iso‐electric point (pI). Both cellulases had pI near 4.55, but their molecular weights were different: 67 000 (exoglucanase) and 41 000 (endoglucanase). The effect of temperature on the activity of the cellulases was examined with both cotton and carboxymethylcellulose as substrate. Arrhenius activation energies, Q10, and temperature optima for the different reactions were determined.

Studies on cellulases of phytopathogenic fungus, Pyricularia oryzae Cavara. I. Number and interrelation of components of CX enzyme from Pyricularia oryzae.

The number of extracellular and intracellular Cx enzyme components and their interrelations were studied using enzymes from strains, N-1 (H 373) and C-3 (N 87), of P. oryzae. When P. oryzae was grown on carboxymethyl cellulose (CMC) as the carbon source, high activities were detected in Cx enzyme and β-glucosidase, with essentially no C, enzyme activity. Purification of extracellular and intracellular Cx enzymes showed that the former consisted of three components (Ex-I, Ex-II and Ex-III) and the latter of two components (In-I and In-II). When the culture filtrate was incubated for 11 days at 28°C under sterile condition, the amount of component Ex-III, which was undetectable within the cell, increased with the concomitant decrease of that of component Ex-II. Thus, component Ex-II was suggested as converting to component Ex-III.

The mechanism of enzymatic cellulose degradation. Purification and some properties of two different 1,4beta-glucan glucanohydrolases from Trichoderma viride.

1. A low-molecular-weight and a high-molecular-weight 1,4beta-glucan glucanohydrolase (Cx enzyme) have been isolated from a commercial cellulase preparation derived from culture filtrates of the fungus Trichoderma viride. 2. The purification method for the isolation of the low-molecular-weight enzyme is a three-step procedure including chromatography on Bio-Gel P-10, chromatography on a dipolar adsorbent (arginine-Sepharose 6 B) and isoelectric focusing. 3. The starting material for the isolation of the high-molecular-weight enzyme was pre-fractionated by chromatography on Bio-Gel P-10, by DEAE-Sephadex chromatography and by SE-Sephadex chromatography as described previously by us. Further fractionation of this material was achieved by affinity chromatography and repeated isoelectric focusing. 4. Free zone electrophoresis of the low-molecular-weight enzyme indicated a homogeneous protein. The high-molecular-weight enzyme was homogenous in sedimentation equilibrium analysis. 5. The molecular weights of the enzymes were 12 500 and 50 000 +/- 2000 respectively. The former value was determined by chromatography on a calibrated column of Bio-Gel P-100 and the latter value by sedimentation equilibrium analysis. 6. The low-molecular-weight enzyme was isoelectric at pH 4.60 (10 degrees C) and contained 21% carbohydrate. The corresponding values for the high-molecular-weight enzyme were pH 3.39 and 12%. 7. Both enzymes were active in releasing free fibers from filter-paper. The low-molecular-weight enzyme was estimated to be about twice as effective as the high-molecular-weight enzyme in this regard.

Role of Pectic and Cellulolytic Enzymes in Pathogenesis of Pythium aphanidermatum in Tomato Seedlings

An appreciable reduction in pectin and cellulose content was observed in P. aphanidermatum infected tomato hypocotyls: Only in the infected tissues ando- PG, PGTE and macerating enzymes were detected while PME and Cx enzymes were found both in healthy and infected tissues. The pathogen produced endo PG, PGTE, Cx and macerating enzymes in vitro

Occurrence and Some Properties of Cellulase in the Filtrates of Conidiospores and Mycelia of Pyricularia oryzae Cavara

Occurrence of cellulase activity was demonstrated in the filtrates of germinating conidiospores and growing mycelia of P. oryzae. Activity and some properties of cellulase in the filtrate of mycelia grown on rice plant powder as carbon source were compared among various strains.Cellulase activity (C1 and Cx enzymes; cellulose and carboxymethylcellulose as substrates, respectively) in the filtrate of germinating conidiospores was detected in the pathogenic T–l (Ken 53–33) strain as well as nonpathogenic 0 (THU 3 × 1) strain of P. oryzae. The activity was higher in the former than the latter strains. Cellulase activity (Cx enzyme) in the filtrate of growing mycelia was detected in the four strains used, T–l (Ken 53–33), C–3 (N 87), N–1 (H373), and 0 (THU 3 × 1). Cellulase activity (Cx enzyme) in the filtrate of mycelia was optimal at pH 5.0 and 40°C, and stable up to 40°C. Their properties did not differ significantly except for the pH-activity curve at alkaline side among various strains; but cellulase act...

Partial Purification of Extracellular Cellulase from Pyricularia oryzae and Comparison of the Elution Patterns among Various Strains

In order to explain the difference in extracellular cellulase activities (C1 and Cx enzyme activities) among various strains of P. oryzae, the elution patterns from the column were compared among various strains, following each step of the partial purification.The crude enzymes, prepared by ammonium sulfate fractionation (0.2~0.8 sat.) from the culture filtrates, which were obtained from various strains of P. oryzae cultured on rice plant powder as the carbon source, were fractionated by DEAE-Sephadex A–50 chromatography into two components; the passing-through fraction (I) and the fraction (II) adsorbed and eluted from the column with 0.5 M NaCl The percentage of the enzyme activity (Cx enzyme activity) in fraction I to that of the crude extract was found to vary chracteristically according to the strain, and the variation was in a good correlation to that of the extracellular cellulase activities.Fractions I and II were then separated by Sephadex G–100 into two (peaks a and b) and at least five (peaks c...

The Cellulolytic Activity of Thermophilic Fungi and Actinomycetes

SUMMARYThe thermophilic molds showed greater cellulolytic ability than did the thermophilic actinomycetes. Chaetomium thermophile var. coprophilc. C. thermophile var. dissitum, Himicola grisea var. thermoidea, H. insolens. Myriococcum albomyees, Sporotrichum thermophile, and Torula thermophila were able to degrade filter paper and utilize soluble carboxymethyl cellulose (CMC). Malbranchea pulchella var. sulfurea, Stilbella thermimephila and Talaromyees thermophilus could not degrade filter paper, but their cell-free filtrates contained the Cx enzyme that could hydrolyze soluble carboxymethyl cellulose to reducing sugars. Humicola lanuginosa, H. stellata, Mucor mechei, M. pusillus and Thermoascus aurantiacus could not degrade filter paper nor hydrolyze CMC. Streptomyces thermovielaceus var. pingens was the only actinomycete to significantly degrade filter paper. Thermomonospora curvata caused a slight loss in weight. Cell-free filtrates from these two, plus filtrates from Pseudonocardia thermophila, Streptomyces rectus, and S. thermovulgaris were able to hydrolyze CMC. Micromonospora chalcea, Streptomyees violaceoruber. Thermoactinomyees glaucus, T. vulgaris and Thermomonospora viridis could not degrade filter paper or hydrolyze CMC. The Cx enzyme is an induced enzyme, since none of the microorganisms produced it in a medium lacking cellulose.

Studies on the cellulolytic enzymes of phytopathogenic bacteria

(1) Cellulase (Cx) of phytopathogenic bacteria, twenty-eight species and one variety belonging to five genera, was detected when they were cultured on CMC-gel medium prepared by dissolving 2 percent of carboxymethyl-cellulose (CMC) into various basal media (Table 1). Cx enzyme was commonly produced by xanthomonads and all strains of Erwinia carotovora, serologically identified by the authors, though they show various degrees of activity according to each bacterium, while most pseudomonads, except P. solanacearum and P. panici, did not show the production of cellulase (Cx) on any medium tested. Erwinia milletiae and Corynebacterium sepedonicum produced the enzyme on only potato-CMC-gel medium, the most suitable one for the detection of cellulase (Cx), prepared by adding CMC into potato decoction (see Table 2).(2) The cellulase (Cx) activity of phytopathogenic bacteria was represented by percent loss in viscosity of one percent solution of CMC. It reacts with the reaction of linear equation, since reaction time necessary for reaching to fifty percent-loss in viscosity of CMC solution did not connect with the concentration of the substrate (see Table 3).(3) Change of cellulase (Cx) activity in filtrate of 10 days culture in bouillon or potato decoction with or without CMC, was different corresponding to the bacterial species or strains tested (see Fig. 1).When the velocity constant k of the linear equation is adopted as the standard of the enzyme activity, cellulase (Cx) in culture filtrate of bouillon or potato decoction containing 0.2 percent CMC increased its activity in a straight line with the culture duration in the case of Erwinia carotovora strain chrysanthemi. In the case of Xanth. campestris, however, the conspicuous decrease of activity followed its very rapid increase within 3-day-incubation period (see Fig. 2). Although the probable production of a proteinase is responsible to such prompt decrease of cellulase activity, its presence may be made evident elsewhere. In the case of Pseud. solanacearum the activity was observed to increase in a straight line during five-day incubation, but then stabilized at this level until 10 days. This stabilization of enzyme activity of P. solanacearum is supposed to be caused by the rapid loss in vitality of this bacterium usually occurring in bouillon or potato decoction medium. Cellulase (Cx) was produced under the cultural conditions without CMC (β-1.4 glucosidic linkage), though the activity was relatively low as compared with that under its presence.