Identification of thermostable β-xylosidase activities produced by <Emphasis Type="Italic">Aspergillus brasiliensis</Emphasis> and <Emphasis Type="Italic">Aspergillus niger</Emphasis>

Twenty Aspergillus strains were evaluated for production of extracellular cellulolytic and xylanolytic activities. Aspergillus brasiliensis, A. niger and A. japonicus produced the highest xylanase activities with the A. brasiliensis and A. niger strains producing thermostable β-xylosidases. The β-xylosidase activities of the A. brasiliensis and A. niger strains had similar temperature and pH optima at 75°C and pH 5 and retained 62% and 99%, respectively, of these activities over 1 h at 60°C. At 75°C, these values were 38 and 44%, respectively. Whereas A. niger is a well known enzyme producer, this is the first report of xylanase and thermostable β-xylosidase production from the newly identified, non-ochratoxin-producing species A. brasiliensis.     

Homologue expression of a β-xylosidase from native <Emphasis Type="Italic">Aspergillus niger</Emphasis>

Xylan constitutes the second most abundant source of renewable organic carbon on earth and is located in the cell walls of hardwood and softwood plants in the form of hemicellulose. Based on its availability, there is a growing interest in production of xylanolytic enzymes for industrial applications. β-1,4-xylan xylosidase (EC 3.2.1.37) hydrolyses from the nonreducing end of xylooligosaccharides arising from endo-1,4-β-xylanase activity. This work reports the partial characterization of a purified β-xylosidase from the native strain Aspergillus niger GS1 expressed by means of a fungal system. A gene encoding β-xylosidase, xlnD, was successfully cloned from a native A. niger GS1 strain. The recombinant enzyme was expressed in A. niger AB4.1 under control of A. nidulans gpdA promoter and trpC terminator. β-xylosidase was purified by affinity chromatography, with an apparent molecular weight of 90 kDa, and showed a maximum activity of 4,280 U mg protein⁻¹ at 70°C, pH 3.6. Half-life was 74 min at 70°C, activation energy was 58.9 kJ mol⁻¹, and at 50°C optimum stability was shown at pH 4.0–5.0. β-xylosidase kept residual activity >83% in the presence of dithiothreitol (DTT), β-mercaptoethanol, sodium dodecyl sulfate (SDS), ethylenediaminetetraacetate (EDTA), and Zn²⁺. Production of a hemicellulolytic free xylosidase showed some advantages in applications, such as animal feed, enzymatic synthesis, and the fruit-juice industry where the presence of certain compounds, high temperatures, and acid media is unavoidable in the juice-making process.     

Regeneration of peach (<Emphasis Type="Italic">Prunus persica</Emphasis> L. Batsch) cultivars and <Emphasis Type="Italic">Prunus persica</Emphasis> × <Emphasis Type="Italic">Prunus dulcis</Emphasis> rootstocks via organogenesis

Somatic peach plants were regenerated from callus derived from the base of stem explants of the scion cultivars ‘UFO-3’, ‘Maruja’, ‘Flariba’ and ‘Alice Bigi’, and the peach × almond rootstocks ‘Garnem’ and ‘GF677’. A protocol for organogenic plant regeneration was developed using three culture media containing different concentrations of 6-benzyladenine (BA) and indolebutyric acid to produce organogenic calli. Shoots were obtained from sliced calli after their transfer to a differentiation culture medium containing 2 mg l⁻¹ BA and 1 mg l⁻¹ α-naphthalene acetic acid. Using this procedure, up to 29 regenerated plants per callus were obtained. The highest regeneration rate was obtained with the peach × almond rootstocks. This work provides an effective protocol that could be utilized for peach transformation research.     

Purification and characterization studies of a thermostable β-xylanase from <Emphasis Type="Italic">Aspergillus awamori</Emphasis>

This study presents data on the production, purification, and properties of a thermostable β-xylanase produced by an Aspergillus awamori 2B.361 U2/1 submerged culture using wheat bran as carbon source. Fractionation of the culture filtrate by membrane ultrafiltration followed by Sephacryl S-200 and Q-Sepharose chromatography allowed for the isolation of a homogeneous xylanase (PXII-1), which was 32.87 kDa according to MS analysis. The enzyme-specific activity towards soluble oat spelt xylan, which was found to be 490 IU/mg under optimum reaction conditions (50°C and pH 5.0–5.5), was 17-fold higher than that measured in the culture supernatant. Xylan reaction products were identified as xylobiose, xylotriose, and xylotetraose. K _m values (mg ml⁻¹) for soluble oat spelt and birchwood xylan were 11.8 and 9.45, respectively. Although PXII-1 showed 85% activity retention upon incubation at 50°C and pH 5.0 for 20 days, incubation at pH 7.0 resulted in 50% activity loss within 3 days. PXII-1 stability at pH 7.0 was improved in the presence of 20 mM cysteine, which allowed for 85% activity retention for 25 days. This study on the production in high yields of a remarkably thermostable xylanase is of significance due to the central role that this class of biocatalyst shares, along with cellulases, for the much needed enzymatic hydrolysis of biomass. Furthermore, stable xylanases are important for the manufacture of paper, animal feed, and xylooligosaccharides.     

Purification and characterization of an intracellular β-glucosidase from the protoplast fusant of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> and <Emphasis Type="Italic">Aspergillus niger</Emphasis>

Protoplasts of Aspergillus oryzae 3.481 and Aspergillus niger 3.316 were prepared using cellulose and snail enzyme with 0.6 M NaCl as osmotic stabilizer. Protoplast fusion has been performed using 35% polyethylene glycol 4,000 with 0.01 mM CaCl₂. The fused protoplasts have been regenerated on regeneration medium and fusants were selected for further studies. An intracellular (β-glucosidase (EC 3.2.1.21) was purified from the protoplast fusant of Aspergillus oryzae 3.481 and Aspergillus niger 3.316 and characterized. The enzyme was purified 138.85-fold by ammonium sulphate precipitation, DE-22 ion exchange and Sephadex G-150 gel filtration chromatography with a specific activity of 297.14 U/mg of protein. The molecular mass of the purified enzyme was determined to be about 125 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme had an optimum pH of 5.4 and temperature of 65°C, respectively. This enzyme showed relatively high stability against pH and temperature and was stable in the pH range of 3.0–6.6. Na⁺, K⁺, Ca²⁺, Mg²⁺ and EDTA completely inhibited the enzyme activity at a concentration of 10 mM. The enzyme activity was accelerated by Fe³⁺. The enzyme activity was strongly inhibited by glucose, the end product of glucoside hydrolysis. The K _m and V _max values against salicin as substrate were 0.035 mM and 1.7215 μmol min⁻¹, respectively.     

Production of the <Emphasis Type="Italic">Aspergillus aculeatus</Emphasis> endo-1,4-β-mannanase in <Emphasis Type="Italic">A. niger</Emphasis>

The β-mannanase gene (man1) from Aspergillus aculeatus MRC11624 (Izuka) was patented for application in the coffee industry. For production of the enzyme, the gene was originally cloned and expressed in Saccharomyces cerevisiae. However the level of production was found to be economically unfeasible. Here we report a 13-fold increase in enzyme production through the successful expression of β-mannanase of Aspergillus aculeatus MRC11624 in Aspergillus niger under control of the A. niger glyceraldehyde-3-phosphate dehydrogenase promoter (gpd _P) and the A. awamori glucoamylase terminator (glaA_T). The effect of medium composition on mannanase production was evaluated, and it was found that the glucose concentration and the organic nitrogen source had an effect on both the volumetric enzyme activity and the specific enzyme activity. The highest mannanase activity levels of 16,596 nkat ml⁻¹ and 574 nkat mg⁻¹ dcw were obtained for A. niger D15[man1] when cultivated in a process-viable medium containing corn steep liquor as the organic nitrogen source and high glucose concentrations.     

Synthesis of sugar fatty acid esters using a β-xylosidase from <Emphasis Type="Italic">Aspergillus awamori</Emphasis> for transxylosylation

Aspergillus awamori K4 β-xylosidase has broad acceptor specificity. It has been used to synthesize a sugar fatty acid ester via its transxylosylation activity. One xylosyl residue was initially transferred to hexamethylene glycol as a linker with a yield of 0.36 g/g xylobiose. Linoleic acid was subsequently linked to one terminal hydroxyl side of the transfer product hydroxyhexyl xyloside through an esterification reaction catalyzed by a lipase. The synthesis of hexyl linoleoyl xyloside was confirmed by TOF-MS analysis. The binding with a linker improved the esterification reaction because of the hydrophobic hexamethylene chain and also prevented steric hindrance by the xylosyl residue. This sugar fatty acid ester synthesis method using transglycosylation should facilitate the production of emulsifiers or surfactants with various functions.     

Optimization of ethanol,citric acid,and α-amylase production from date wastes by strains of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>, <Emphasis Type="Italic">Aspergillus niger</Emphasis>, and <Emphasis Type="Italic">Candida guilliermondii</Emphasis>

The present study deals with submerged ethanol, citric acid, and α-amylase fermentation by Saccharomyces cerevisiae SDB, Aspergillus niger ANSS-B5, and Candida guilliermondii CGL-A10, using date wastes as the basal fermentation medium. The physical and chemical parameters influencing the production of these metabolites were optimized. As for the ethanol production, the optimum yield obtained was 136.00 ± 0.66 g/l under optimum conditions of an incubation period of 72 h, inoculum content of 4% (w/v), sugars concentration of 180.0 g/l, and ammonium phosphate concentration of 1.0 g/l. Concerning citric acid production, the cumulative effect of temperature (30°C), sugars concentration of 150.0 g/l, methanol concentration of 3.0%, initial pH of 3.5, ammonium nitrate concentration of 2.5 g/l, and potassium phosphate concentration of 2.5 g/l during the fermentation process of date wastes syrup did increase the citric acid production to 98.42 ± 1.41 g/l. For the production of α-amylase, the obtained result shows that the presence of starch strongly induces the production of α-amylase with a maximum at 5.0 g/l. Among the various nitrogen sources tested, urea at 5.0 g/l gave the maximum biomass and α-amylase estimated at 5.76 ± 0.56 g/l and 2,304.19 ± 31.08 μmol/l/min, respectively after 72 h incubation at 30°C, with an initial pH of 6.0 and potassium phosphate concentration of 6.0 g/l.     

Improved purification of α-L-rhamnosidase from <Emphasis Type="Italic">Aspergillus niger</Emphasis> naringinase

To achieve an efficient separation and purification of α-L-rhamnosidase (Rha) from naringinase (Nar) that was prepared from a fermented broth of Aspergillus niger, improved experimental methods were developed with aid of a chemical dithiothreitol (DTT) and a novel HPLC method. The addition of DTT did not negatively affect the purification of Rha and Nar, but greatly simplified the purification steps due to its strong capability of separating the Rha from Nar. The novel HPLC method enabled simultaneous measurement and differentiation of the Rha and the Nar with the naringin as the substrate. These improvements resulted in an efficient purification of the homogenous Rha with an estimated molecular weight of 87 kDa. Otherwise, Rha could not be extracted with enough purity even by the combination of sulphate precipitation, ion exchange chromatography and gel filtration chromatography. The modified methods have ensured efficient purification of the Rha from A. niger naringinase. This study provides a powerful and simple procedure to separate and purify the Rha of Nar, which will facilitate further more in-depth studies of these enzymes, as well as their industrial applications.     

Synthesis and transformations of a pyrazole containing <Emphasis Type="Italic">α</Emphasis>,<Emphasis Type="Italic">β</Emphasis>-didehydro-<Emphasis Type="Italic">α</Emphasis>-amino acid derivatives

Summary.  2H-Pyran-2-ones 1 were transformed with various hydrazines into (E)- or (Z)-α,β-didehydro-α-amino acid (DDAA) derivatives 4 (and 7) containing a highly substituted pyrazolyl moiety attached at the β-position. With heterocyclic hydrazines, the products 4 were accompanied also by decarboxylated enamines E-6. In order to separate (E/Z)-mixtures of acids, they were transformed to the corresponding methyl esters 9 and 10 by the application of diazomethane. Catalytic hydrogenation under high pressures with Pd/C as a catalyst resulted in the formation of racemic alanine derivatives 11. Received January 29, 2002 Accepted May 27, 2002 Published online December 18, 2002 RID="*" ID="*"  Dedicated with deep respect to Professor Waldemar Adam on the occasion of his 65^th birthday. Acknowledgements We thank the Ministry of Education, Science and Sport of the Republic of Slovenia for the financial support (P0-0503-103). Dr. B. Kralj and Dr. D. Žigon (Center for Mass Spectroscopy, “Jožef Stefan” Institute, Ljubljana, Slovenia) are gratefully acknowledged for the mass measurements. Authors' address: Prof. Marijan Kočevar, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Aškerčeva 5, SI-1000 Ljubljana, Slovenia, E-mail: marijan.kocevar@uni-lj.si     

In vitro growth response of <Emphasis Type="Italic">Phytophthora cactorum</Emphasis>, <Emphasis Type="Italic">P. nicotianae</Emphasis> and <Emphasis Type="Italic">P.</Emphasis> × <Emphasis Type="Italic">pelgrandis</Emphasis> to antibiotics and fungicides

The reactions of isolates of Phytophthora cactorum, P. nicotianae and P. × pelgrandis to metalaxyl, mancozeb, dimethomorph, streptomycin and chloramphenicol were tested to obtain information about the variability of resistance in these pathogens. Distinct genetic groups showed significant differences in resistance to all tested substances except streptomycin. In response to streptomycin, the growth inhibition rates of distinct groups did not differ significantly. The most remarkable differences were detected in the reactions to chloramphenicol and metalaxyl. Discriminant analysis evaluating the effect of all substances confirmed the differences among the groups, which are in agreement with the differences revealed by earlier DNA analyses.     

Improved productivity of <Emphasis Type="Italic">β</Emphasis>-fructofuranosidase by a derepressed mutant of <Emphasis Type="Italic">Aspergillus niger</Emphasis> from conventional and non-conventional substrates

Summary Wild-type cultures of Aspergillus niger produced a basal level of β-fructofuranosidase on glucose of 1 IU l⁻¹ h⁻¹. In contrast, a catabolite-derepressed mutant strain of the same organism produced a markedly higher level (25 IU l⁻¹ h⁻¹) of this enzyme when grown on the same carbon source. Wheat bran induced both the wild type (252 IU l⁻¹ h⁻¹) and the mutant strain (516 IU l⁻¹ h⁻¹) to produce 252- to 516-fold higher levels of this enzyme than was observed with the wild-type grown on glucose and was the best carbon source. When corn steep liquor served as a nitrogen source, the wild-type organism showed a higher activity of enzyme on monosaccharides and disaccharides comparable to that produced by corncobs in the basal medium and that mutant was a potentially improved (> 2-fold) organism for the production of β-fructofuranosidase on all carbon sources. Enhanced substrate consumption and product formation kinetic parameters suggest that the mutant organism may be exploited for bulk production of this useful enzyme.     

Differential expression of <Emphasis Type="Italic">Gnrh2</Emphasis>, <Emphasis Type="Italic">Gthβ</Emphasis>, and <Emphasis Type="Italic">Gthr</Emphasis> genes in sterile triploids and fertile tetraploids

Gonadotropin-releasing hormone (GnRH), gonadotropin hormone (GTH), and gonadotropin hormone receptor (GTHR) are the pivotal signal molecules of the hypothalamic-pituitary-gonad (HPG) axis, which plays a crucial role in regulating gonadal development in vertebrate. In this study, we comparatively analyze the expression characteristics of Gnrh2, Gthβ, and Gthr in red crucian carp diploids, triploids, and allotetraploids. The expression patterns of these genes are similar in the three fish ploidy types: the Gnrh2 gene is expressed in midbrains, pituitaries, and gonads; the Gthβ gene is expressed in pituitaries; the Gthr gene is mainly expressed in gonads. These results indicate that the three genes participate in the regulation of gonadal development. By real-time polymerase chain reaction and in situ hybridization, we find that, among these three fish ploidy types, the expression level of Gthr in the gonads of triploids is lower than those of diploids and tetraploids; this weakens the combination of GTHR with GTH released from the pituitary and leads to the sterility of triploids, since the gonad cannot produce enough sex steroids. In addition, the low expression of Gthr in triploids may affect the down-regulation of Gthβ, which then affects the down-regulation of Gnrh2; hence, the expression levels of Gnrh2 and Gthβ genes in triploids are the highest after the breeding season. In conclusion, the differential expression of Gnrh2, Gthβ, and Gthr in triploids and tetraploids is related to their sterility and bisexual fertility, respectively.     

Enzymatic synthesis of fructooligosaccharides from date by-products using an immobilized crude enzyme preparation of <Emphasis Type="Italic">β</Emphasis>-D-fructofuranosidase from <Emphasis Type="Italic">Aspergillus awamori</Emphasis> NBRC 4033

Aqueous extracts from date by-products of the sucrose-rich variety “Deglet Nour” were used as a starting substrate to achieve the enzymatic synthesis of fructooligosaccharides (FOS) commonly used as prebiotics. A crude β-fructofuranosidase (Ffase) preparation from Aspergillus awamori NBRC4033 was immobilized on chitosan by covalent binding through glutaraldehyde linkages (Yi = 88%, Ya = 54%), and used for this purpose. The effect of water-extraction volume on the FOS synthesis by transfructosylation was studied. It was found that 150 mL/100 g of date by-products gave the best FOS concentration and productivity (123 g/L and 18.5 g/h/100 g respectively), related to an optimal sucrose conversion of 53.26%. The main FOS product was purified via a biogel-P2 gel filtration column. Its structure was determined as 1-kestose: α-Dglucopyranosyl-( 1→2)-β-D-fructofuranosyl-(2→1)-β-Dfructofuranoside by combination of ¹H, ¹³C and 2D-NMR techniques. Our results provide new insights into the enzymatic synthesis of FOS from an alternative source of sucrose, namely date by-products.     

Characteristics of β-lactamases and their genes (<Emphasis Type="Italic">blaA</Emphasis> and <Emphasis Type="Italic">blaB</Emphasis>) in <Emphasis Type="Italic">Yersinia intermedia</Emphasis> and <Emphasis Type="Italic">Y. frederiksenii</Emphasis>

Background  

The presence of β-lactamases in Y. enterocolitica has been reported to vary with serovars, biovars and geographical origin of the isolates. An understanding of the β-lactamases in other related species is important for an overall perception of antibiotic resistance in yersiniae. The objective of this work was to study the characteristics of β-lactamases and their genes in strains of Y. intermedia and Y. frederiksenii, isolated from clinical and non-clinical sources in India.     

Isolation and properties of extracellular β-xylosidases from fungi <Emphasis Type="Italic">Aspergillus japonicus</Emphasis> and <Emphasis Type="Italic">Trichoderma reesei</Emphasis>

Homogeneous β-xylosidases with molecular mass values 120 and 80 kDa (as shown by SDS-PAGE), belonging to the third family of glycosyl hydrolases, were isolated by anion-exchange, hydrophobic, and gel-penetrating chromatography from enzyme preparations based on the fungi Aspergillus japonicus and Trichoderma reesei, respectively. The enzymes exhibit maximal activity in acidic media (pH 3.5–4.0), and temperature activity optimum was 70°C for the β-xylosidase of A. japonicus and 60°C for the β-xylosidase of T. reesei. Kinetic parameters of p-nitrophenyl β-xylopyranoside and xylooligosaccharide hydrolysis by the purified enzymes were determined, which showed that β-xylosidase of A. japonicus was more specific towards low molecular weight substrates, while β-xylosidase of T. reesei preferred high molecular weight substrates. The competitive type of inhibition by reaction product (xylose) was found for both enzymes. The interaction of the enzymes of different specificity upon hydrolysis of glucurono- and arabinoxylans was found. The β-xylosidases exhibit synergism with endoxylanase upon hydrolysis of glucuronoxylan as well as with α-L-arabinofuranosidase and endoxylanase upon hydrolysis of arabinoxylan. Addition of β-xylosidases increased efficiency of hydrolysis of plant raw materials with high hemicellulose content (maize cobs) by the enzymic preparation Celloviridine G20x depleted of its own β-xylosidase.     

Synthesis of <Emphasis Type="Italic">N</Emphasis>-<Emphasis Type="Italic">Z</Emphasis>, <Emphasis Type="Italic">N</Emphasis>′-Formyl α-Amino Acid Derived Gem-Diamines

A variety of N-carbobenzoxy, N′-formyl gem-diaminoalkyl derivatives have been obtained through Goldsmith-Wick reaction of Z-α-amino acid/peptide acid derived isocyanates with 96% HCOOH in presence of 4-dimethylaminopyridine (DMAP) as catalyst. The reaction proceeds to completion within 2–4 h and results in good yields of the products isolated as stable solids.     

Retiolitid graptolites from the collection of Hermann Jaeger II: <Emphasis Type="Italic">Cometograptus</Emphasis>, <Emphasis Type="Italic">Spinograptus</Emphasis> and <Emphasis Type="Italic">Plectograptus</Emphasis>

Graptolites from the Jaeger collection at the Museum für Naturkunde (Berlin, Germany) provide important information on structural details of Silurian (Wenlock–Ludlow) retiolitids as well as for the biostratigraphic and biogeographic distribution of these magnificent graptolites. Species of the genera Cometograptus, Spinograptus and Plectograptus are described from isolated glacial boulder material, collected in northern Germany and from shale specimens found in the Lower Graptolite Shale of Thuringia. The biostratigraphic placement of material derived from glacial erratic boulders, however, is far from being precise. The fauna associated with the neotype of Plectograptus macilentus in the ‘Unterer Graptolithenschiefer’ of Thuringia is discussed and illustrated. Cometograptus alfeisenacki from the Cyrtograptus lundgreni Biozone is recognized as a new species. The genus is discovered for the first time in North German glacial erratic boulders.     

A Synthesis of Heteroaromatic <Emphasis Type="Italic">S</Emphasis>- and <Emphasis Type="Italic">N</Emphasis>-β-Glycosides of <Emphasis Type="Italic">N</Emphasis>-Acetylglucosamine under Phase-Transfer Conditions

The use of crown ethers for a phase transfer-catalyzed synthesis of heteroaromatic glycosides of N-acetylglucosamine was studied. The solid-liquid system and catalysis by 15-crown-5 were found to provide for both the 100% conversion of α-D-glucosaminyl chloride peracetate and a high reaction rate. The interaction of α-D-glucosaminyl chloride peracetate and oxadiazole and triazole mercapto derivatives capable of thiol-thione tautomerism carried out at room temperature in acetonitrile in the presence of anhydrous potassium carbonate and crown ethers was shown to lead to both S- and N-glucosides. The structures of the compounds synthesized were confirmed by X-ray analysis and ¹³C and ¹H NMR spectroscopy.     

Heterologous expression and characterization of a glucose-stimulated β-glucosidase from the termite <Emphasis Type="Italic">Neotermes koshunensis</Emphasis> in <Emphasis Type="Italic">Aspergillus oryzae</Emphasis>

Neotermes koshunensis is a lower termite that secretes endogenous β-glucosidase in the salivary glands. This β-glucosidase (G1NkBG) was successfully expressed in Aspergillus oryzae. G1NkBG was purified to homogeneity from the culture supernatant through ammonium sulfate precipitation and anion exchange, hydrophobic, and gel filtration chromatographies with a 48-fold increase in purity. The molecular mass of the native enzyme appeared as a single band at 60 kDa after gel filtration analysis, indicating that G1NkBG is a monomeric protein. Maximum activity was observed at 50 °C with an optimum pH at 5.0. G1NkBG retained 80% of its maximum activity at temperatures up to 45 °C and lost its activity at temperatures above 55 °C. The enzyme was stable from pH 5.0 to 9.0. G1NkBG was most active towards laminaribiose and p-nitrophenyl-β-d-fucopyranoside. Cellobiose, as well as cello-oligosaccharides, was also well hydrolyzed. The enzyme activity was slightly stimulated by Mn²⁺ and glycerol. The K _m and V _max values were 0.77 mM and 16 U/mg, respectively, against p-nitrophenyl-β-d-glucopyranoside. An unusual finding was that G1NkBG was stimulated by 1.3-fold when glucose was present in the reaction mixture at a concentration of 200 mM. These characteristics, particularly the stimulation of enzyme activity by glucose, make G1NkBG of great interest for biotechnological applications, especially for bioethanol production.