Molecular characterisation and expression analysis of the first hemicellulase gene (bxl1) encoding beta-xylosidase from the thermophilic fungus Talaromyces emersonii

The gene coding for beta-xylosidase, bxl1, has been cloned from the thermophilic filamentous fungus, Talaromyces emersonii. This is the first report of a hemicellulase gene from this novel source. At the genomic level, bxl1 consists of an open reading frame of 2388 nucleotides with no introns that encodes a putative protein of 796 amino acids. The bxl1 translation product contains a signal peptide of 21 amino acids that yields a mature protein of 775 amino acids, with a predicted molecular mass of 86.8 kDa. The deduced amino acid sequence of bxl1 exhibits considerable homology with the primary structures of the Aspergillus niger, Aspergillus nidulans, Aspergillus oryzae, and Trichoderma reesei beta-xylosidase gene products, and with some beta-glucosidases, all of which have been classified as Family 3 glycosyl hydrolases. Northern blot analysis of the bxl1 gene indicates that it is induced by xylan and methyl-beta-D-xylopyranoside. D-Xylose induced expression of bxl1 but was shown to repress induction of the gene at high concentrations. The presence of six CreA binding sites in the upstream regulatory sequence (URS) of the bxl1 gene indicates that the observed repression by D-glucose may be mediated, at least partly, by this catabolite repressor.     

Characterisation of a Talaromyces emersonii thermostable enzyme cocktail with applications in wheat dough rheology

In this paper, we report new sequence data for secreted thermostable fungal enzymes from the un-sequenced xylanolytic filamentous fungus Talaromyces emersonii and reveal novel insights on the potential role of enzymes relevant as wheat dough improvers. The presence of known and de novo enzyme sequences were confirmed through NanoLC-ESI-MS/MS and resultant peptide sequences were identified using SWISS PROT databases. The de novo protein sequences were assigned identity based on homology to known fungal proteins. Other proteins were assigned function based on the limited T. emersonii genome coverage. This approach allowed the identification of enzymes with relevance as wheat dough improvers. Rheological examination of wheat dough and wheat flour components treated with the thermostable fungal enzyme cocktail revealed structural alterations that can be extrapolated to the baking process.Thermoactive amylolytic, xylanolytic, glucanolytic, proteolytic and lipolytic enzyme activities were observed. Previously characterized T. emersonii enzymes present included; β-glucosidase, xylan-1,4-β-xyloxidase, acetylxylan esterase, acid trehalase, avenacinase, cellobiohydrolase and endo-glucanase. De novo sequence analysis confirmed peptides as being; α-glucosidase, endo-1,4-β-xylanase, endo-arabinase, endo-glucanase, exo-β-1,3-glucanase, glucanase/cellulase, endopeptidase and lipase/acylhydrolase. Rheology tests using wheat dough and fractioned wheat flour components in conjunction with T. emersonii enzymes show the role of these novel biocatalysts in altering properties of wheat substrates. Enzyme treated wheat flour fractions showed the effects of particular enzymes on appropriate substrates. This proteomic approach combined with rheological characterization is the first such report to the authors’ knowledge.     

Effect of pH on the stability of Pleurotus eryngii versatile peroxidase during heterologous production in Emericella nidulans

Complementary DNA (cDNA) encoding the new versatile peroxidase from the ligninolytic basidiomycete Pleurotus eryngii has been expressed in the ascomycete Emericella nidulans. In recombinant E. nidulans cultures, the pH reached values as high as 8.3, correlating with a sharp decrease in peroxidase activity. Peroxidase was rapidly inactivated at alkaline pH, but was comparatively stable at acidic pH. The peroxidase inactivation in alkaline buffer could be reversed by adding Ca²⁺ and lowering the pH. However, reactivation did not result after incubating the enzyme in non-buffered E. nidulans cultures that reached pH 7.5. To optimize recombinant peroxidase production, the effect of controlling the pH in E. nidulans bioreactor cultures was studied. An extended growth period, and a significant increase in the recombinant peroxidase level (5.3-fold higher activity than in the bioreactor without pH control) was obtained when the pH was maintained at 6.8, showing that culture pH is an important parameter for recombinant peroxidase production.     

Production of xylanase by Emericella nidulans NK-62 on low-value lignocellulosic substrates

Thermotolerant Emericella nidulans NK-62 was isolated from bird nesting material and was tested for its ability to produce xylanase. The fungus when grown on a medium containing wheat bran (2% w/v) supplemented with Czapek's mineral salt solution at 45 °C for 7 days produced 362 IU/ml of xylanase (EC 3.2.1.8). The specific activity of E. nidulans NK-62 xylanase was found to be 275 IU/mg of total protein. The enzyme was found to be active over a broad temperature and pH range with 60 °C as optimum temperature for enzyme activity. The enzyme was stable at 50 °C and its half-life at 55 °C was 45 min. -xylosidase (EC 3.2.1.37) and carboxymethylcellulase (EC 3.2.1.4) activities, 0.018 and 0.21 IU/ml respectively, were also noticed. The fungus was screened for its ability to produce xylanase on four different lignocellulosic substrates. It produced 318.9 IU/ml of cellulase-free xylanase on corn cobs. The fungus could also utilize lentil bran (seed husk of Lens esculentus) and meal of groundnut shells to produce 84.8 and 17.3 IU/ml xylanase respectively.     

Talaromyces euchlorocarpius, a new species from soil

A new species ofTalaromyces, characterized by development of unusual deep green ascomata on common media, is described and given the nameTalaromyces euchlorocarpius. This species, isolated from soil, also produces ellipsoidal, spinose ascospores, typically biverticillate penicilli, large ellipsoidal, smooth-walled conidia, and is assigned to the seriesLutei of the sectionTalaromyces.     

Emericella appendiculata, a new species from Chinese soil

Emericella appendiculata, a new species isolated from soil of the Pamire Plateau, is described and illustrated. It is characterized by grayish green non-ostiolate ascomata surrounded by a thick layer of hülle cells, membranaceous peridium, prototunicate asci, violet-brown, lenticular ascospores which are ornamented by two stellate equatorial crests, capitate convex surfaces, and long filiform appendages, and anAspergillus anamorph with biseriate conidiogenous cells.     

Emericella omanensis,a new species from Oman soil

A new species ofEmericella isolated from forest soil in the Oman,E. omanensis, is described and illustrated. It differs from the other known species of the genus in having bivalvate ascospores with a tuberculate or verruculose convex wall. The new species is compared with the closely related speciesE. desertorum andE. echinulata.     

Emericella qinqixianii, a new species from desert soil in China

Emericella qinqixianii, a new species isolated from desert soil from Sanchakou, Aksu, Qiemo, Yuli, Yutian, and the Taklimakan desert 100 km inland from Minfeng, Xinjiang Province, China, is described and illustrated. It is characterized by grayish yellow to olive brown, non-ostiolate ascomata surrounded by hyaline to pale yellowish brown hülle cells, membranaceous peridium, prototunicate asci, and violet-brown, lenticular ascospores with two equatorial crests, smooth convex surfaces, and long filiform appendages. It hasAspergillus anamorph with biseriate aspergilla.     

Cloning, characterisation and expression analysis of α-glucuronidase from the thermophilic fungus Talaromyces emersonii

The aguA gene encoding α-glucuronidase was isolated from the thermophilic fungus Talaromyces emersonii by degenerate PCR. AguA has no introns and consists of an open reading frame of 2511 bp, encoding a putative protein of 837 amino acids. The N-terminus of the protein contains a putative signal peptide of 17 amino acids yielding a mature protein of 820 amino acids with a predicted molecular mass of 91.6 kDa. Twenty putative N-glycosylation sites and four O-glycosylation were identified. The T. emersonii α-glucuronidase falls into glycosyl hydrolase family 67, showing approximately 63% identity to similar enzymes from other fungi. Analysis of the aguA promoter revealed several possible regulatory motifs including two XlnR and a CreA binding site. Enzyme activity was optimal at 50 °C and pH 5. Enzyme production was investigated on a range of carbon sources and showed induction on beechwood, oat spelt and birchwood xylan, and repression by glucose or glucuronic acid.     

Large-subunit rRNA sequence of the chytridiomyceteBlastocladiella emersonii,and implications for the evolution of zoosporic fungi

The 5.8S and 28S ribosomal RNA sequences of the chytridiomyceteBlastocladiella emersonii were determined. These data were combined with 18S rRNA sequences in order to carry out a phylogenetic analysis based on distance matrix, parsimony, and maximum likelihood methods. The new data confirmed that chytridiomycetes are true fungi and not protists, as was already suggested on the basis of biochemical, ultrastructural, and 18S rRNA data. Within the fungal clade,B. emersonii formed the first line of divergence. The position of the fungi within the eukaryotic “crown” taxa was also reassessed, and the alveolate-stramenopile cluster appeared as their sister group. The stramenopiles also comprise a number of zoosporic fungi, which resemble chytridiomycetes in so many respects, e.g., production of motile spores, thallus morphology, and absorptive nutrition, that they have been classified together with them in the past. This suggests that the possible common ancestor of the fungi, stramenopiles, and alveolates may have been a zoosporic fungus, which would mean that zoosporic fungi are paraphyletic instead of polyphyletic as previously suggested. Correspondence to: R. De Wachter     

Purification and partial characterization of two chitinases from the mycoparasitic fungus <Emphasis Type="Italic">Talaromyces flavus</Emphasis>

Chitinases were produced by Talaromyces flavus CGMCC 3.4301 when it was grown in the presence of chitin. Two chitinases from the culture filtrate of T. flavus were purified to homogeneity by fractional ammonium sulphate precipitation, ion-exchange chromatography on DEAE–Sepharose and Phenyl–Sepharose hydrophobic interaction chromatography. By SDS–PAGE, the molecular weight of the two enzymes was estimated to be 41 and 32 kDa, respectively. The 41 kDa chitinase (CHIT41) had a 4.0 pH optimum; the 32 kDa chitinase (CHIT32) optimum activity was at pH 5.0. The optimum temperature for the two chitinase activities was 40 °C. The two chitinases had activity against cell wall of Verticillium dahliae, Sclerotinia sclerotiorum and Rhizoctonia solani, and inhibited spore germination and germ tube elongation of Alternaria alternata, Fusarium moniliforme, and Magnaporthe grisea.     

Talaromyces lagunensis,a new species from Philippine soil

A new species ofTalaromyces (Ascomycetes; Trichocomaceae) with aPenicillium anamorph,T. lagunensis, is described and illustrated. This fungus is characterized by its extremely restricted growh on Czapek-yeast extract agar, light yellow to light orange ascomata with a telaperidium, catenate, pyriform or ellipsoidal asci, ellipsoidal or subglobose ascospores with a microtuberculate wall, short conidiophores with an irregular, mostly monoverticillate to biverticillate penicillus, and subglobose to ovoid conidia. The holotype was isolated from forest soil in the Philippines.     

Quantification of intra- and extra-cellular thermophilic lipase/esterase production by Thermus sp.

Four Thermus strains produced lipolytic activity when grown in liquid medium for 30 h at 70 degrees C. The highest total lipase/esterase activity (57 U l(-1)) was in Thermus aquaticus YT-1, followed by Thermus thermophilus HB27 and HB8 (33 and 25 U l(-1), respectively), and finally by Thermus sp. (16 U l(-1)). Extra-cellular activity was detected in T. aquaticus YT-1 and T. thermophilus HB27 (33 and 17 U l(-1)). All enzymes were stable at 80 degrees C over 30 min, and their activity towards fatty acid esters increased as substrate chain-length diminished (i.e. hydrolysis rate was up to 6-fold higher on p-nitrophenyl caproate than on laurate).     

Isolation and properties of an isocitrate dehydrogenase from Anacystis nidulans

A NADP⁺-specific isocitrate dehydrogenase (EC 1.1.1.42) was isolated and purified over 400-fold from Anacystis nidulans. The enzyme activity responded slowly to rapid changes in ligand (NADP⁺, isocitrate, Mg²⁺-ions) or enzyme concentration as well as to rapid changes in temperature. These are properties characteristic of the hysteretic enzymes. In addition, the enzyme activity was subject to product (-ketoglutarate) inhibition. ATP, ADP and CDP also inhibited the enzyme. Unlike several other cyanobacterial enzymes, the isocitrate dehydrogenase of Anacystis is not under redox control.     

Allocation of random amplified polymorphic DNA markers and enzyme activities toAspergillus nidulans andAspergillus tetrazonus chromosomes

Chromosome-substituted haploid segregants of anA. nidulans × A. tetrazonus somatic hybrid were used to allocate several random amplified polymorphic DNA and isoenzyme markers to parental chromosomes. Twenty-six amplified DNA fragments, and nine isoenzyme activities, including lactate dehydrogenase, superoxide dismutase, and arylesterase isoenzymes were assigned to chromosomes. Chromosome-specific markers were found for eachA. nidulans andA. tetrazonus chromosome. These markers could be used to saturate the genetic map ofA. nidulans. The formation of two secondary metabolites was also assigned to chromosomes III and VIII. Attempts were made to allocate extracellular enzyme activities to parental chromosomes, mostly without success, possibly because multiple enzyme forms located on different chromosomes could be responsible for the production of an enzyme activity.     

Isolation of fungi and optimization of process parameters for decolorization of distillery mill effluent

Five different fungi isolated from distillery mill site in which two isolates (DF3 and DF4) had higher capabilities to remove color were identified as Emericella nidulans var. lata and Neurospora intermedia, respectively. Optimization of process parameter for decolorization was initially performed to select growth factors which were further substantiated by Taguchi approach in which seven factors, %carbon, %nitrogen, duration, pH, temperature, stirring and inoculum size, at two levels applying L-8 orthogonal array were taken for both fungi. Maximum color was removed at pH 3, temperature 30°C, stirring 125 rpm, dextrose (0.05%) and sodium nitrate (0.025%) by both fungi. After optimization, there was two-fold increase in color removal from 38 to 62% (DF3) and 31 to 64% (DF4) indicating significance of Taguchi approach in decolorization of distillery mill effluent. The mechanism for decolorization was determined by enzyme analysis, laccase and glucose oxidase, and indicated significant activity in DF3 as compared to DF4.     

Experimental acute poisoning in mice induced by emestrin,a new mycotoxin isolated from Emericella species

The effects of emestrin (EMS), a secondary metabolite of the Emericella species, on male ICR mice were examined. The intraperitoneal LD₅₀ values of EMS were 17.7 and 13.0 mg/kg at 24 and 48 hr, respectively. The target organs of EMS were the heart, liver and thymus. In doses over 30 mg/kg the experimental animals died from cardiac failure shortly after the injections. Several survivors that were given EMS in doses under 20 mg/kg showed severe centrilobular necrosis in the liver at 24 hr. Marked degeneration of mitochondria was seen in electron micrographs of both cardiac muscle cells and hepatocytes. In the degenerated hepatocytes, prominent proliferation of RER, membrane-limited inclusions containing both ribosome-like granules and RER, and fenestrated lamella-like structures were observed. Massive necrosis of lymphocytes was always observed in the cortical layer of the thymus of the survivors within 24 hr, while bilateral adrenalectomized mice showed no discernible pathomorphological changes in the lymphoid tissues. Pretreatment of mice with diethyl maleate increased the incidence and severity of hepatic necrosis, whereas that with either cysteine or CoCl₂ reduced the severity of centrilobular necrosis of the liver. Pretreatment with phenobarbital had no significant effect on EMS-induced hepatic lesions.     

Chitosome-like vesicles from gamma particles of Blastocladiella emersonii synthesize chitin

When gamma particles isolated from the aquatic fungus, Blastocladiella emersonii, were incubated in a supernatant derived from a homogenate of zoospores previously triggered to encyst with 50 mM KCl, they exhibited a three-fold increase in chitin synthetase activity and produced chitosome-like vesicles. Passage of such vesicles through a linear sucrose gradient resulted in a symmetrical distribution of chitin synthetase activity over a broad portion of the gradient, and the specific activity of the peak fraction was seven times greater than that of the gamma particles. After isopycnic sucrose gradient centrifugation, chitin synthetase activity occurred in a band of particles with a peak buoyant density of 1.18 g/cm³. Ultrastructural examination of negatively stained samples from the peak fraction revealed spheroidal, chitosome-like particles 70–120 nm in diameter. Suspension of these particles produced chitin microfibrils when incubated with uridine diphosphate N-acetylglucosamine, the substrate for chitin synthetase.Non Standard Abbreviations Used GlcNAc N-acetylglucosamine - UDP-GlcNAc uridine diphosphate N-acetylglucosamine - PYG agar 1.25 g of peptone, 1.25 g of yeast extract, 3 g of glucose, and 20 of agar per 1000 ml of water, the pH being adjusted to 6.8 with KOH after autoclaving - EGTA ethyleneglycol-bis (-aminoethylether)-N,N-tetraacetic acid