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.     

Involvement of secondary metabolites and extracellular lytic enzymes produced by Pseudomonas fluorescens in inhibition of Rhizoctonia solani, the rice sheath blight pathogen

Fourteen strains of Pseudomonas fluorescens isolated from rhizosphere soil of rice were tested for their antagonistic effect towards Rhizoctonia solani, the rice sheath blight fungus. Among them, PfMDU2 was the most effective in inhibiting mycelial growth of R. solani in vitro. Production of chitinase, beta-1,3-glucanase, siderophores, salicylic acid (SA) and hydrogen cyanide (HCN) by P. fluorescens strains was evaluated. The highest beta-1,3-glucanase activity, siderophore production, SA production and HCN production were recorded with PfMDU2. A significant relationship between the antagonistic potential of P. fluorescens against R. solani and its level of beta-1,3-glucanase, SA and HCN was observed.     

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.     

Studies on ammonium-assimilating enzymes of Platymonas striata Butcher (Prasinophyceae)

Platymonas striata Butcher displays significant levels of glutamate synthase (GS) (EC 2.6.1.53) and glutamine synthetase (GOGAT) (EC 6.3.1.2.), but very low levels of glutamate dehydrogenase (GDH) (EC 1.4.1.4). This suggests that the GS/GOGAT pathway is important for nitrogen assimilation. The in vitro rates of enzyme activity can however only account for about 10% of the in vivo rates of nitrogen assimilation. Nitrogen-starvation reduced GS activity to undetectable levels. On nitrate or ammonium ion refeeding the cellular GS activity was rapidly restored, and reached levels of 56% and 91% greater than the unstarved values 24h after refeeding nitrate or ammonium respectively.Abbreviations NAR nitrate reductase - NIR nitrate reductase     

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.     

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.     

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.     

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.     

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.     

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     

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 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     

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.     

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).     

Mannanase production by the lettuce endosperm

Endo--mannanase (EC 3.2.1.78) is produced and secreted by the cells of the endosperm of lettuce (lactuca sativa L.) seeds (achenes). In imbibed intact seeds, production is prevented by inhibitors. If the endosperm is incubated alone, these inhibitors can be removed by leaching, allowing mannanase production. Abscisic acid, a component of lettuce seeds, inhibits the production of mannanase in the isolated endosperm, and may be involved in regulation of mannanase production in intact seeds. During germination the inhibition is removed, beginning 4–8 h after red-light irradiation, which was given 4 h from sowing. The cotyledons participate in this process, and are controlled by events occuring in the axis within 4 h from red-light irradiation. This control by the axis apparently depends on the exchange of diffusible substances. Both benzyladenine and gibberellic acid can replace the influence of the axis if the latter is removed, and may therefore be involved in the control by the axis of the rest of the seed.Abbreviations ABA abscisic acid - BA 6-benzyladenine - GA₃ gibberellic acid - IAA indol-3-yl acetic acid - MES 2-(N-morpholino)ethane sulfonic acid - R red light Part of this work was carried out by P. Halmer at the Department of Biology, Washington University, St. Louis, MO 63130, USA (his present address)