Purification and some properties of the mannanases fromThielavia terrestris

Summary Thielavia terrestris NRRL 8126 cell free supernatants contained mannanase and -mannosidase when cultured on a complex media containing locust bean gum. Using acetone precipitation, SP-Sephadex C50 ion exchange chromatography and preparative gel electrophoresis, the crude enzyme was resolved into one -d-mannosidase and four -d-mannanase components. -d-mannosidase had a specific activity of 0.02 (U/mg) onp-nitrophenyl--d-mannopyranoside substrate. Mannanase components M1, M2, M3 and M4 had specific activities of 28.2, 38.7, 52.8 and 4.17 (U/mg) respectively on purified locust bean galactomannan substrate. pH optima for the enzymes were in the range 4.5–5.5. Mannanase component M4 manifested the greatest thermostability, retaining full activity for 3 h at 60°C. Molecular weights determined by SDS-PAGE were 72 000 for -mannosidase and 52 000, 30 000, 55 000 and 89 000 for M1, M2, M3 and M4 respectively. Carbohydrate contents of the enzymes ranged from 6–36%. Preliminary studies indicate that enzyme components hydrolyse the mannan substrate in a synergistic manner.     

Cereal straw and pure cellulose as carbon sources for growth and production of plant cell-wall degrading enzymes by Sporotrichum thermophile

Sporotrichum thermophile grew well and produced plant cell-wall degrading enzymes on straw (barley and wheat) of different particle sizes and Avicel as carbon sources. Comparable activities of endoglucanase, Avicelase and cellobiase were produced on each substrate. In contrast, activities of xylanase, aryl--glucosidase, -xylosidase, esterase and -l-arabinofuranosidase were higher on straw (either wheat or barley) than on Avicel. The enzyme systems produced on barley straw of different particle sizes degraded finely milled barley straw in vitro more rapidly and to a greater extent than those produced on Avicel. In contrast, the enzyme systems produced on Avicel and very coarse barley straw hydrolysed Avicel to about the same extent while that produced on fine barley straw was slightly less effective. The main hydrolysis product in all cases was glucose. Isoelectric focusing revealed that the plant cell-wall degrading enzyme system produced by S. thermophile on barley straw was qualitatively and quantitatively superior to that produced on Avicel.C. Sugden was and M.K. Bhat is with the Department of Protein Engineering, Institute of Food Research, Reading Laboratory, Earley Gate, Whiteknights Road, Reading RG6 2EF, UK; C. Sugden is now with the Department of Biochemistry, University of York, Heslington, York YO1 5DD, UK.     

Pathogenicity of Sporotrichum pruinosum and Cladosporium oxysporum,isolated from the bronchial secretions of a patient,for laboratory mice

In this study we have demonstrated the occurrence of Sporotrichum pruinosum and Cladosporium oxysporum in the bronchial secretions of a patient with a presumptive diagnosis of tuberculosis. This observation coupled with the ability of both fungi to cause infection and elicit tissue responses in experimentally infected mice supported a probable etiologic relationship with the patient which could not be confirmed in the absence of histologic evidence. In vitro some antimycotics were tested against S. pruinosum and C. oxysporum by the agar dilution method. Oxiconazole with a minimum inhibitory concentration of 0.1 g/ml^–1 after 72 h and amorolfine at a concentration of 0.001 g/ml^–1 after 72 h were the most active ones against S. pruinosum and C. oxysporum respectively. It is suggested that the isolation of S. pruinosum and C. oxysporum from patients with bronchopulmonary disorders should be viewed with caution. Clinical and laboratory evaluation of such patients should be done critically before arriving at a firm diagnosis.     

Ligninolytic activity and levels of ammonia assimilating enzymes in Sporotrichum pulverulentum

Levels of ammonia-assimilating enzymes (glutamate dehydrogenase, glutamine synthetase, glutamate synthase) were determined in extracts of Sporotrichum pulverulentum grown under different conditions with respect to both nitrogen source and concentration. Evolution of ¹⁴CO₂ from ¹⁴C-synthetic lignin by fungal cultures grown under parallel conditions was also determined as a measure of lignin decomposition and the suppressive effect of nitrogen on ligninolysis confirmed. Under low nitrogen conditions, fungal extracts exhibited relatively high levels of NADP-dependent glutamate dehydrogenase and glutamine synthetase dehydrogenase. Conversely, in high nitrogen extracts, lower levels of NADP-dependent glutamate dehydrogenase and glutamine synthetase activity, and higher levels of NAD-dependent glutamate dehydrogenase, were recorded. Possible effects of enzyme activities on intracellular pool concentrations of glutamate/glutamine, and the implications for the regulation of lignin metabolism, are discussed.A preliminary report was presented at The Ekman Days 1981, International Symposium on Wood and Pulping Chemistry, Stockholm, Sweden, June 9–12, 1981.     

Production of pectolytic enzymes fromErwinia grown on different carbon sources

A quantitative analysis of pectolytic enzymes (polygalacturonase (PG), pectin methyl esterase (PME) and six isoenzymes of pectate lyase (PL)) produced byErwinia bacteria in the presence of diverse carbon sources was made by preparative electrophoresis. Synthesis of each of these enzymes was regulated independently; different induction and repression ratios (about 10- to 1000-fold) were observed for diverse PL isoenzymes, PG and PME. The possibility of using specially constructed media for the production of pectinase complexes with a specific spectra of pectolytic enzymes has been demonstrated.     

Studies on transvection at the bithorax complex in Drosophila melanogaster

Summary We have studied the influence of some mutations in the bithorax complex on the observed synapsis dependent phenotype of the genotypes Cbx ¹Ubx¹/+ and bx ^34e/Ubx¹. The effect of these mutations is similar to that introduced by disruption of pairing or by the z ^a mutation. Among the bx mutations, we find that bx ⁸ behaves differently from most other bx mutations in its influence on the synapsis dependent phenotype. This observation induced us to map the position of bx ⁸ with respect to other bx mutations; we find that it maps between bx ^34e and bx ³. We show how some of the observations reported here can be fitted into a model of activation of the bithorax complex proposed by one of us.     

Production and partial characterization of xylanase by Sporotrichum thermophile under solid-state fermentation

A number of factors affecting production of xylanase, by the thermophilic fungus Sporotrichum thermophile under solid state fermentation (SSF) were investigated. Initial moisture content and type of carbon source were consecutively optimized. Solid state fermentation in a laboratory horizontal bioreactor using the optimized medium allowed the production of 320 U g^–1 of carbon source which compared favourably with those reported for other microorganisms. Optimal xylanase activity was observed at pH 5 and 70 °C. Chromogenic (fluorogenic) 4-methylumbelliferyl -glycoside of xylobiose (MUX₂) was used to characterize the xylanase multienzyme component, after separation by isoelectric focusing and native PAGE electrophoresis. The zymograms indicated one major xylanase fraction exhibiting pI and molecular mass values 4 and 90–120 kDa, respectively.     

Purification and properties of xylanase fromAureobasidium

Summary The yeast-like fungusAureobasidium is a promising source of xylanase (EC 3.2.1.8) with an exceptionally high specific activity. For enzyme production in volumes of several liters, xylose was the preferred carbon source and inducer. Xylanase in clarified cultures was concentrated by reversible adsorption to cation-exchange matrix to 5% of the initial volume, and recovered at nearly 2 million IU/1. Selective conditions permitted 97% recovery of xylanase with a 1.8-fold enrichment in specific activity, to 70% of purity. The predominant xylanase species (20 kDa) was subsequently purified to >99% of homogeneity by gel filtration chromatography. Purified enzyme exhibited an isoelectric point of 8.5, and specific activity of 2100 IU/mg under optimal conditions, determined to be pH 4.5 and 45°C. The activity of purified enzyme was specific for polymeric xylan.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Dept. of Agirculture over other firms or similar products not mentioned.     

Effect of allopolyploidy on the activity of selected enzymes inHibiscus

Mature seeds of diploid and tetraploidHibiscus species were analyzed for enzyme activity (alcohol dehydrogenase, malate dehydrogenase, leucine aminopeptidase), total protein content, DNA amount and dry weight. The recently formed tetraploid,H. radiatus, generally had enzyme and protein levels very similar to the sum of its progenitors, while the more ancient speciesH. acetosella had several lower levels. This difference may reflect the greater amount of timeH. acetosella has had to evolve dosage compensations.Michigan Agricultural Experiment Station Journal Article 9665.A part of this research was used to satisfy the requirements ofA. Hoisington for a M.S. degree at the University of South Carolina.     

Morphological aspects of the galactomannan formation in the endosperm ofTrigonella foenum-graecum L. (Leguminosae)

The mode of deposition (secretion) of galactomannan in the cells of the seed endosperm ofTrigonella foenum-graecum has been studied by electron microscopy. In cells which are just beginning to secrete galactomannan there are stacks of rough endoplasmic reticulum (ER). The intracisternal space (containing the enchylema) of the rough ER then swells, becomes vacuolated and forms a voluminous network, with pockets of cytoplasm entrapped within poculiform rough ER. The enchylema contains material which reacts with periodate-thiocarbohydrazidesilver proteinate in a very similar manner to the galactomannan already deposited in the cell wall. It appears that the galactomannan is formed in the intracisternal space of the rough endoplasmic reticulum and then expelled outside the plasmalemma. This mode of deposition contrasts with that of other plant cell wall polysaccharides whose secretion is mediated by Golgi vesicles.Abbreviation ER endoplasmic reticulum This is part six in a series of papers dealing with galactomannan metabolism. Part five: Planta133, 219–222 (1977)     

Studies on ectomycorrhiza

Summary Plants ofPicea abies (L.) Karst were grown in mycorrhizal association withTelephora terrestris (Pers. ex Fr.) andPisolithus tinctorius (Mich. ex Pers.) Coker and Couch on sphagnum peat in petri dishes or Perspex chambers. After 1 yearT. terrestris had formed prominent rhizomorphs which were characterized by light microscopy and investigated for³²P-orthophosphate uptake. The absorbed phosphate was transported to sinks throughout the rhizomorphal system as well as into the plant. The calculated translocation velocity and flux rate in the rhizomorph were in the range of 1–3 cm/h and 0.5–4.0 × 10^-10 mol cm^-2 s^-1, respectively. Label was observed to accumulate in the needles 2–3 days after application. Feeding a non-mycorrhized root with³²P-orthophosphate led to an accumulation of label in needles within 1 h, but no radioactivity appeared in the associatedT. terrestris rhizomorphs. The rhizomorphs ofP. tinctorius revealed a higher structural differentiation than those ofT. terrestris. Translocation of labelled phosphorus through rhizomorphs ofP. tinctorius into spruce needles was also demonstrated.     

Studies on the morphology,cytology, and distribution ofLeucojum subgenusRuminia

The following is a summary of part of a lecture held on february 13th 1974 at the Pädagogische Hochschule Berlin. A more detailed publication is under preparation.The following note has been based on studies parallel to those ofDamboldt & Phitos (cf. p. 119 of this issue). Results are complementary or corroborate each other. Our understanding of the interesting relationships between MediterraneanLeucojum taxa is thus improved. This justifies the parallel publication of the two articles in one issue of this journal (Editor).     

Cellulolytic enzymes of a thermotolerantAspergillus terreus strain and their action on cellulosic substrates

A thermotolerant fungal strainAspergillus terreus produced high activities of cellulolytic enzymes when grown in shake flasks for 8 days at 40°C or 14 days at 28°C in medium containing 2.5% (w/v) cellulose powder and 1% (w/v) wheat bran. There was little difference between the final activities of endo-(1,4)--glucanase (ca. 14.4 U/ml); filter paper activity (ca. 1.3 U/ml) and -glucosidase (ca. 10 U/ml). Endoglucanase had maximum activity at 60°C and pH 3.8; the other two enzymes were optimal at 60°C and pH 4.8. The maximum hydrolysis of different cellulosic substrates (about 50%) was obtained within 48 h when 1.1 U/ml of filter paper cellulase activity were employed to saccharify 100 mg alkali-treated cotton, filter paper, bagasse, and rice straw at 50°C and pH 4.8. The major end-product, glucose, was produced from all substrates, with traces of cellobiose and other larger oligosaccharides being present in rice straw hydrolysates.     

Studies on the early floral development inCleomoideae (Capparaceae) with emphasis on the androecial development

Floral ontogeny ofCleome spinosa, Cleome violacea andPolanisia dodecandra subsp.trachysperma was studied in the context of the question whether the fascicled androecium ofReseda andCapparis (with fused fascicles) or the 2 + 4-pattern of theBrassicaceae is primitive in theCapparales. InPolanisia dodecandra, the 9–18 stamens show unidirectional initiation from the adaxial side toward the abaxial side of the flower. InCleome violacea, the six stamens also are formed in an unidirectional order, but development starts abaxially and a zigzag-like pattern is superimposed. InCleome spinosa, two stamen primordia in transversal (lateral) position are followed by four stamens which arise on a somewhat higher level in two pairs in front of the median sepals. It is assumed that the evolutionary steps in the androecial development proceed fromReseda viaCapparis andPolanisia/Cleome toBrassicaceae. This interpretation is supported byrbcL-studies (Chase & al. 1993,Rodman & al. 1993).Dedicated to emer. Univ.-Prof. DrFriedrich Ehrendorfer on the occasion of his 70th birthday     

Experiences with the on-line measurement of culture fluorescence during cultivation of Bacillus subtilis, Escherichia coli and Sporotrichum thermophile

Bacillus subtilis and Escherichia coli K12 (both transformed for human leukocyte interferon production) and Escherichia coli B/r and Sporotrichum thermophile (a deuteromycete) were cultivated in submersed culture and the culture fluorescence recorded on-line using a fluorometer. During the cultivation of B. subtilis the signal from the fluorometer correlated with cell density and interferon production and thus could be used for process control (interferon production). However, the culture fluorescence of the other organisms did not increase (S. thermophile), was too weak to be measured with the fluorometer used (E. coli transformed for interferon production), or the signal from the fluorometer was not an accurate measure of the culture fluorescence because of the accumulation of a fluorophor in the culture medium (E. coli B/r).     

Water stress and galactomannan breakdown in germinated fenugreek seeds. Stress affects the production and the activities in vivo of galactomannan-hydrolysing enzymes

Imposition of water stress on germinated fenugreek (Trigonella foenum-graecum L.) seeds and isolated fenugreek endosperms after the beginning of galactomannan mobilisation caused a reduction in the rate of breakdown of the polysaccharide relative to unstressed controls. The activities, measured in vitro, of the three hydrolytic enzymes involved in the breakdown process (-d-galactosidase, EC 3.2.1.22;endo--d-mannanase, EC 3.2.1.78;exo--d-mannanase, EC 3.2.1.25) were not decreased. Although there was some accumulation of galactomannan-hydrolysis products in endosperms under stress, there was no clear correlation between sugar levels and the inhibition of galactomannan breakdown. When water stress was applied to fenugreek seeds after germination but before the beginning of galactomannan hydrolysis, both galactomannan breakdown and the development of the hydrolytic enzyme activities were inhibited. Washing of newly germinated seeds for 2 h in water prior to the imposition of stress gave partial relief of the inhibition of galactomannan mobilisation, partial recovery ofendo--d-mannanase levels, and full recovery of -d-galactosidase levels. It is argued: 1) that water stress after germination but before the beginning of galactomannan hydrolysis inhibits the production of hydrolytic enzymes in the endosperm, probably via decreased removal at lowered water content of diffusible inhibitory substances; and 2) that water stress after the beginning of galactomannan hydrolysis decreases the rate of galactomannan breakdown in vivo principally via decreased diffusion at lowered water content of enzymes from the aleurone layer through the storage tissue of the endosperm.Abbreviation PEG polyethyleneglycol     

Quantitative estimation of the surface carbohydrates on the infection structures of rust fungi with enzymes and lectins

Lectins of Triticum vulgaris (WGA), Concanavalia ensiformis (ConA), Phaseolus vulgaris (PHA), Lotus tetragonolobus (LTA), Arachis hypogaea (PNA), Ricinus communis (RCA I), Griffonia simplicifolia (GSA II) and the enzymes endo-(13)--D-glucanase, exo-(13)--D-glucanase and laminarinase were tested for binding to the infection structures of Puccinia coronata and Uromyces appendiculatus. The enzymes and lectins were labeled with fluorescein and the fluorescence was measured with a microscope photometer. GSA II and ConA bound to all parts of the two rust fungi to a certain extent. The germ tubes of P. coronata bound at least two times more WGA than did the germ tubes of U. appendiculatus. The appressoria of both rust fungi additionally bound exo-(13)--glucanase, endo-(13)--glucanase and laminarinase. The substomatal vesicle and the infection hypha of both rust fungi mainly bound the glucanases. Furthermore, the substomatal vesicle of U. appendiculatus bound PHA. No obvious binding with LTA, RCA I and PNA was observed. Binding generally could be inhibited by appropriate haptens. Binding to uredospores generally appeared unspecific. The results indicate that the germ tubes have chitin on their outer surfaces, the appressoria chitin and glucans and the substomatal vesicles and infection hyphae mainly glucans. Compared to P. coronata, U. appendiculatus has more terminal linked glucose residues or the glucan has more (13)--linkages. Also, U. appendiculatus has N-acetylgalactosamine or a similar sugar on the surface of the substomatal vesicle.Abbreviations ConA Concanavalia ensiformis agglutinin - FITC fluorescein isothiocyanate - GSA II Griffonia simplicifolic agglutimin II - LTA Lotus tetragonolobus agglutinin - PBS phosphate buffered saline - PNA Peanut agglutinin - RCA I Ricinus communis agglutinin I - PHA Phaseolus vulgaris agglutinin - WGA Wheat germ agglutinin     

Extracellular mannanases and galactanases from selected fungi

Summary Eight thermophilic fungi were tested for production of mannanases and galactanases. Highest mannanase activities were produced byTalaromyces byssochlamydoides andTalaromyces emersonii. Mannanases from all strains tested were induced by locust bean gum except in the case ofThermoascus aurantiacus, where mannose had a greater inducing effect. Locust bean gum was also the best inducer of -mannosidase and galactanase except in the case ofT. emersonii where galactose was a better inducer of both these enzymes. Highest mannanase activity was produced byTalaromyces species when peptone was used as nitrogen source whereas sodium nitrate promoted maximum production of this enzyme byThielavia terrestris andT. aurantiacus. The pH optima of mannanases from the thermophilic fungi were in the range 5.0–6.6 and contrasted with the low pH optimum (3.2) of the enzyme fromAspergillus niger. Galactanases had pH optima in the range 4.3–5.8. The mannanase fromT. emersonii and the galactanase fromT. terrestris were most thermostable, each retaining 100% activity for 3 h at 60°C.