Mycelial Homogenates Research Articles

Peroxidase activity in mycelia of Aspergillus parasiticus that degrade aflatoxin

Extracts of 9-day-old mycelia of Aspergillus parasiticus NRRL 2999 were assayed for peroxidase activity and for their ability to degrade aflatoxin. A positive relationship existed between rates of aflatoxin degradation and amount of peroxidase activity in these extracts. The supernatant fluid of homogenates from mycelia grown under similar conditions varied in amount of peroxidase present (170 to 2215 U/g). The fraction obtained, by precipitation with (NH4)2SO4 at 45% of saturation, from six different homogenates prepared from three mycelial mats contained peroxidase and degraded aflatoxin. Rates of aflatoxin degradation by and amounts of peroxidase activity in each sample obtained from mycelial homogenates with (NH4)2SO4 at 60% of saturation varied; however, when increased amounts of peroxidase activity were present, more aflatoxin was degraded and vice versa. Relatively little peroxidase activity was present in the fraction obtained with (NH4)2SO4 at 30% of saturation and little or no aflatoxin was degraded by this precipitate. Trends for degradation of aflatoxin when more or less peroxidase activity was present in mycelial preparations suggest that the enzyme may be involved in degradation of aflatoxin by the Aspergillus.

Effect of methyl glycosides and oligosaccharides on cell death and browning of potato tuber discs induced by mycelial components of Phytophthora infestans

In the hypersensitive response of potato tuber tissue to an incompatible race of Phytophthora infestans the infected cells rapidly die and later turn brown. Cell-free extracts of sonicated mycelial homogenates of the fungus induce a hypersensitive-like response in tuber tissue from resistant and susceptible potato varieties. Cell death was assessed directly by the use of a vital stain, and indirectly by reflectance densitometric measurements of browned tuber tissue. A positive correlation was demonstrated between browning, measured in this way, and percentage cell death calculated from direct viability counts. A number of methyl glycosides and oligosaccharides were tested for their ability to affect sonicate-induced cell death and browning. Laminaribiose and methyl-β- d-glucopyranoside caused a significant inhibition of cell death and browning; cellobiose, gentiobiose, methyl-α- d-glucopyranoside and a number of other compounds were ineffective. The results suggest that macromolecules containing β-1,3-linked glucose residues play an active role in the interaction between mycelial components of P. infestans and the potato. Furthermore, these residues may be contained in the fungal elicitor and be specific for the active site of its receptor in the potato cell.

ULTRASTRUCTURAL AND ENZYMATIC EVIDENCE FOR THE PRESENCE OF MICROBODIES IN THE FUNGUS ACHLYA

Ultrastructural evidence for structures resembling microbodies is presented for the fungus Achlya ambisexualis Raper. These structures are DAB positive and thus presumably contain the enzyme catalase. Activities from mycelial homogenates for. the following enzymes are given: catalase, glycolate oxidase, uricase, isocitrate lyase, malate dehydrogenase, citrate synthetase, malate synthetase and glutamate: oxaloacetate transaminase. These results suggest that Achlya contains microbodies and that they may be of the glyoxysome type. The specific activity of catalase increases substantially following initiation of antheridial hyphae by the hormone antheridiol.

Properties and Cellular Localization of Chitin Synthetase in Phycomyces blakesleeanus

Abstract The response of Phycomyces sporangiophores to various stimuli show up as changes in the elongation rate of the cell wall, a structure largely composed of chitin fibrils. The enzyme chitin synthetase was chosen as the subject of this study for the possible role that regulation of its activity might play in the behavioral outputs. A simple assay for the chitin synthetase has been developed. Membrane preparations from the mycelia of Phycomyces were found to catalyze the synthesis of chitin from UDP-N-acetyl-d-glucosamine. Both Mg2+ and N-acetyl-d-glucosamine stimulate the enzyme activity. The pH optimum for the enzyme activity is about 6.5, and the temperature optimum is about 28°. The Km for UDP-N-acetyl-d-glucosamine is 0.6 mm. The antibiotic polyoxin D competitively inhibits the activity at levels which are comparable to those required for the inhibition of mycelial growth. In the presence of polyoxin D (0.1 mm), germinating spores of Phycomyces develop into protoplast-like structures. At concentrations up to 0.5 mm, ATP stimulates the enzyme activity and at 2 mm, cyclic 3' : 5'-AMP is slightly inhibitory. The enzyme activity was found in the Phycomyces mycelia of different ages as well as in the sporangiophores of each of the five developmental stages. Phycomyces mycelial homogenate was fractionated through a series of differential and isopynic centrifugations. Each fraction was studied for its (a) specific and total chitin synthetase activities; (b) morphology under the electron microscope; and (c) specific and total activities of the marker enzymes 5'-nucleotidase (plasma membrane), glucose 6-phosphatase (endoplasmic reticulum), succinic-2-(p-indophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium-reductase (mitochondria), catalase (peroxisomes) and acid phosphatase (lysosomes). The fraction which exhibited the highest specific activity contained essentially only membraneous structures. The distribution of the specific chitin synthetase activity coincided with that of 5'-nucleotidase activity and did not coincide with that of any of the other marker enzymes. These results suggest that the chitin synthetase is a plasma membrane-bound enzyme. Autoradiography studies showed that along the sporangiophore the growing zone (where cell elongation takes place) has higher chitin synthetase activity than the nongrowing zone.

Penicillium stoloniferum Virus: Large-Scale Concentration and Purification by Polyethylene Glycol

An experimental procedure developed to concentrate fungal viruses from large volumes of homogenized mycelia with water-soluble polymers, such as polyethylene glycol, possesses advantages over more conventional methods. Concentration of the Penicillium stoloniferum fast-moving virus from mycelial homogenates after addition of polyethylene glycol was rapid and produced large quantities of pure virus.

Studies on the α-L-Arabinofuranosidase Complex from Sclerotinia fructigena in Relation to Brown Rot of Apple

α-L-Arabinofuranosidase (AF) was detected in apple fruitlets experimentally infected by Sclerotinia fructigena. In extracts of such fruitlets, three AF isoenzymes were separated by preparative isoelectric focusing. When the fungus was grown in shake culture with different carbon sources, AF was detected in each culture filtrate and mycelial homogenate. Although fungus growth and total AF varied with the carbon source, the AF isoenzyme pattern was similar in each instance to that obtained when grown on sodium polypectate. Each of the partially purified AF isoenzymes behaved differently in substrate specificity and inhibitor studies; however, each showed a specificity for α-L-arabino-furanosides. The two extracellular AF isoenzymes released monomeric arabinose when incubated with araban or apple cell walls. External AF III (pI 6·5) was more active on a substrate of apple cell wall material than external AF I (pI 3·0). The latter form of the enzyme was less susceptible to inhibition by either oxidized or unoxidized apple juice. Two isolates of Sclerotinia fructigena with low growth rate in vivo secreted no AF III in vitro.

Extra- and Intra-cellular -L-Arabinofuranosidase of Sclerotinia fructigena

SUMMARY: The production of both intracellular and extracellular α-l-arabinofuranosidase (AF) by Sclerotinia fructigena was demonstrated. They had different kinetic parameters, pH stability and optimal pH values. Two peaks of activity were found on gel filtration: maximum activity was associated with the peak of lower molecular weight in the culture filtrate, but with the higher molecular weight peak in the mycelial homogenate. Isoelectric-focusing studies on culture filtrates revealed two peaks of AF activity with pI 3.0 and 6.5 respectively, maximum activity being associated with the latter. In the mycelial homogenate, most of the activity was associated with a third peak of activity with pI 4.5. Physical and kinetic data suggest that the two AF activities present externally in the culture filtrates are identical with the two internal ones, with corresponding pI.

Purification and properties of alliin lyase from the fungus Penicillium corymbiferum

Penicillium corymbiferum contains alliin lyase (cysteine sulfoxide lyase; alliin alkyl sulfenate-lyase, EC 4.4.1.4). This appears to be the first report of such an enzyme being isolated from a fungus. A 45-fold purification in two steps was effected by taking a 40–65% (NH 4) 2SO 4 cut from a mycelial homogenate and chromatographing it, after dialysis, on Sephadex G-200. The resulting preparation required pyridoxal phosphate, had an apparent K m using alliin ( S- allyl- l-cysteine sulfoxide ) as a substrate of 5.8 mM, did not utilize S-alkylcysteines, and had a pH optimum of 6.5. These properties are similar to those of alliin lyase from garlic. It differed from this higher plant enzyme in that it had relatively little activity at pH 8.0, and was not stimulated by divalent ions or EDTA.

Studies on cutin-esterase II. Characteristics of cutin-esterase from Botrytis cinerea and its activity on tomato-cutin

The activity of cutin-esterase, cutinase, was detected in the mycelial homogenate of Botrytis cinerea cultured in a peptone-sucrose medium at 25°C for 7 days. The crude enzyme solution was prepared from the homogenate by centrifugation at 106,600×g, treatment with (NH4)2SO4 at 70% saturation, and dialysis against 0.01 M phosphate buffer. The optimum pH, temperature and assay duration for enzyme activity were 5.0, 25°C and 18 hr, respectively. Specific activity was 255 mµmoles/mg protein/18 hr as palmitate under optimum conditions. 83% of the activity was lost by heating the enzyme solution (pH 7.6) for 4 min at 95°C. Palmitic, stearic, oleic, 9, 10-dihydroxystearic or linoleic, dihydroxyeicosanoic and octadecanedioic acids were recognized in the enzymic hydrolysate of tomato-cutin using gas-liquid chromatography. Among these fatty acids, palmitic, oleic and octadecanedioic acids were readily liberated by the enzyme, but dihydroxyeicosanoic acid, the major component of tomato-cutin, was isolated only in small amounts. The enzyme is, therefore, an exo-type cutinase which hydrolyses minor side chains of fatty acids bound to the major structure of cutin. Cutinesterase may facilitate cuticular invasion by fungi as a result of reduction in mechanical strength of the cuticle by the enzyme

Enzymatic activity in basidiomycetes. I. Submerged growth and oxidation-reduction activity of Oudemansiella mucida.

The oxidation-reduction activity of the basidiomyceteOudemansiella mucida was studied in relation to its growth in a laboratory fermentor. Cytochromes were detected in the mycelium destroyed by sonication, flavin diaphorase, polyphenoloxidase, peroxidase and catalase were found to be present in the mycelial homogenate from Waring-blendor. The obtained values of the enzymatic activity were dependent on the method of preparation of the mycelium. Homogenization in a Waring-blendor was the most appropriate. Certain interrelationships between the appearance and activity of the enzymes followed and the phase of submerged growth of the fungus were shown. The results document the succession of cytochromes, flavins and polyphenoloxidase and the specific time differences of the activity of catalase and that of peroxidase.

Influence of clay minerals on microorganisms. IV. Montmorillonite and kaolinites on fungi.

The respiration of mycelial homogenates of 27 fungal species representing four classes was generally not affected by pH nor by montmorillonite or kaolinite at concentrations below 4%. Respiration was markedly inhibited by montmorillonite at concentrations of 4% and above, but comparable inhibition by kaolinite occurred only at concentrations above 40%. The inhibition was greater as the metabolic activity of the mycelium increased but was not caused by a limitation in carbon substrate. The inhibition was related to the viscosity of the systems, which, in turn, presumably influenced the rate of O2 diffusion. The viscosities of systems containing montmorillonite were vastly greater than of those containing kaolinite. These results were in contrast to those previously obtained with bacteria, which revealed that montmorillonite greatly stimulated respiration.

Peptidases of Dermatophytes

The normal sites of dermatophyte infection are the keratin-containing tissues, hair, nail and epidermis; and these fungi are commonly considered to be keratin digesting organisms. However a keratin degrading system has never been isolated from them and little is known of the properties of their proteolytic enzymes. Micro-fungi which have been studied in more detail (e.g. Johnson and Peterson, (1), McConnell (2), Crewther and Lennox (3)), show the production of varying mixtures of endo- and exo-peptidases with activities spread over a wide range of pH values, and recorded observations on dermatophytes accord in general with this pattern. Fujii (4) showed peptidase activity in Trichophyton mentagrophytes between pH 5-9 while Cruickshank and Trotter (5) showed such activity to be maximal at about pH 7 and 10 in this organism and also in Trichophyton rubrum. The present paper presents a more detailed study of the variation of proteolytic activity with pH and substrate in representative strains of some dermatophytes together with attempts to separate and characterise various components of the crude extracted proteolytic system from T. rubrum and Trichophyton verrucosum var.dis-coides. MATERIALS AND METHODS Cultivation of Mycelium All strains were fresh isolates provided either by Dr. Jacqueline Walker or Dr. C. J. La Touche; they were maintained on peptone agar. Growth for purposes of enzyme extraction was in stationary culture in Sabouraud broth, with the addition of 0.1% Difco yeast extract for the growth of T. verrucosum var.discoides, the inoculum being a small amount of a mycelial homogenate. The mycelial mats were filtered on to Buchner funnels and well washed with distilled water. Large batches of mycelium were grown by Dr. J. C. Belton at the Ministry of Supply, Microbiological Research Station, Port on, Wilts., under the above conditions and then freeze dried before being sent to Leeds.

Phosphoglucomutase activity in Aspergillus niger.

1. Some of the properties of the phosphoglucomutase of Aspergillus niger have been studied in crude extracts of the mycelium. The results obtained have been compared with the data reported by previous workers for crude and for purified enzyme preparations from animal and plant sources and from yeast. 2. The incorporation of trace elements in a basal synthetic culture medium did not have any marked effect on the specific phosphoglucomutase activity of mycelium. 3. Differential centrifugation of mycelial homogenates prepared in buffered sucrose medium showed that phosphoglucomutase activity is localized in the supernatant from centrifugation at 15,000xg. 4. Spores of Aspergillus niger contain a powerful inhibitor of phosphoglucomutase.

Some observations of the metabolism of Streptomyces scabies.

In the presence of the dye 2,6-dichlorophenol indophenol crude extracts of alumina-ground or of sonically disrupted mycelium of Streptomyces scabies catalyzed dehydrogenase reactions with the substrates succinate, citrate, malate, and glutamic acid. These extracts or mycelial homogenates formed glutamic acid in transaminase reactions with α-ketoglutarate and the amino acids aspartic, leucine, and valine. Other amino acids, lysine, threonine, glycine, histidine, and tyrosine, failed to act as amino donors. No evidence could be obtained for direct transamination with α-ketoglutarate and glutamine or asparagine; with the latter, analyses suggested that glutamic acid formation occurred by preliminary deamination and subsequent transamination from the aspartic acid produced.

Inhibition of TTC reduction of the mycelial homogenates of the rice-blast fungus by fungicides.

A study has been made on the TTC reduction of the mycelial homogenate of rice-blast fungus, Piricularia oryzae Cav., particulaurly on its inhibition by several fungicides. The mycelia grown on Tochinai's synthetic media containing potato decoction at 27°C for 72 hours in shaken culture, were homogenized and fractionated according to the scheme shown in fig. 1, and the supernatant obtained was poured into Thunberg tube for test. After 2 hours of incubation at 28°C, the produced TPF (triphenyl formazan) was measured by an electrophotometer (filter S 48). As a relatively marked TTC reduction of the homogenates was noted at a rate of 20g mycelia per 100ml homogeaate, this concentration was used throughout the experiments.A considerable portion of the activity of TTC reduction was retained in the supernatant fraction after the centrifugation of mycelial homogenates (Table 3). TTC reductivity of the homogenate was lost by heat treatment at 50°C for 10 minutes, and by addition of malonate at a concentration of 0.02M, while reduction was accelerated by adding 0.02M succinate (Tables 4 and 5). Therfore it seems to be reasonable to assume that the TTC reduction above mentioned is probably attributable to succinodehydrogenase system of the present fungus. As shown in the figures 2 and 3, TTC reduction was retarded by various fungicides, especially by those belonging to mercuric group, among which MEMC and EMP exhibited a strong inhibition at concentrations of 10-5-10-7M. The grade of inhibition decreased in the following order; PMA, MC 10-4-10-6M; PMTS, EMTS 10-4-10-5 M; copper sulfate, thiram and zineb 10-3-10-5 M; DNPTC, TCDNB 10-3-10-4M; PCNB did not inhibit at all. These results are consistent with those obtained in the author's another experiment on the vapor action of fungicides and the direct fungitoxicity to spore germination. Cysteine at concentrations of 10-2-10-4M increased the rate of TTC reduction, and reversed the inhibition by the mercuric compounds such as PMA, MC and EMP.