Cloning and expression of a thermostable α-galactosidase from the thermophilic fungus Talaromyces emersonii in the methylotrophic yeast Pichia pastoris

The first gene (alpha-gal1) encoding an extracellular alpha-Dgalactosidase from the thermophilic fungus Talaromyces emersonii was cloned and characterized. The alpha-gal1 gene consisted of an open reading frame of 1,792 base pairs interrupted by six introns that encoded a mature protein of 452 amino acids, including a 24 amino acid secretory signal sequence. The translated protein had highest identity with other fungal alpha-galactosidases belonging to glycosyl hydrolase family 27. The alpha-gal1 gene was overexpressed as a secretory protein with an N-terminal histidine tag in the methylotrophic yeast Pichia pastoris. Recombinant alpha-Gal1 was secreted into the culture medium as a monomeric glycoprotein with a maximal yield of 10.75 mg/l and purified to homogeneity using Hisbinding nickel-agarose affinity chromatography. The purified enzyme was maximally active at 70 degrees C, pH 4.5, and lost no activity over 10 days at 50 degrees C. alpha-Gal1 followed Michaelis-Menten kinetics (Vmax of 240.3 micronM/min/mg, Km of 0.294 mM) and was inhibited competitively by galactose (Km obs of 0.57 mM, Ki of 2.77 mM). The recombinant T. emersonii alpha-galactosidase displayed broad substrate preference, being active on both oligo- and polymeric substrates, yet had strict specificity for the alpha-galactosidic linkage. Owing to its substrate preference and noteworthy stability, alpha-Gal1 is of particular interest for possible biotechnological applications involving the processing of plant materials.J.S. thanks her supervisor at the University of Jyvaskyla, Emily Knott, for permission to perform her Masters degree research while on exchange at NUT Galway, and for constructive comments on the manuscript. J.S. and A.G. both received scholarships under EU Erasmus/Socrates bilateral agreement. Funding for this research was provided in part to M.G.T. under the National Development Plan, through the Food Institutional Research Measure, administered by the Department of Agriculture, Fisheries and Food, Ireland

Characterization and gelling properties of a bioactive extract from Ascophyllum nodosum obtained using a chemical-free approach

peer-reviewedThe bioactivity and gelling properties of a carbohydrate-rich algal extract obtained from locally harvested Ascophyllum nodosum seaweed using a chemical-free approach were investigated for its potential interest in food applications. Physicochemical characterisation and compositional analysis of the extract, using FTIR, biochemical methods and monosaccharide analysis, confirmed the presence of alginates and fucoidans, although the main polysaccharide present in it was laminarin. Significant amounts of phenolic compounds (~9 ​mg phloroglucinol/100 ​mg sample) were also detected. As a result, the extract exhibited good antioxidant activity. It also showed promising prebiotic potential, promoting the growth of beneficial Lactobacillus sp. and Bifidobacteria sp. when compared with commercial prebiotics, but not that of pathogenic bacteria such as E. coli or P. aeruginosa. The gelling properties of the raw extract were explored to optimize hydrogel bead formation by external gelation in CaCl2 solutions. This was enhanced at neutral to alkaline pHs and high extract and CaCl2 concentrations. The mechanical strength, nano- and microstructure of the hydrogel beads prepared under optimised conditions were determined using compression tests, synchrotron small- and wide-angle X-ray scattering (SAXS/WAXS) and scanning electron microscopy (SEM). It was concluded that the raw algal extract at neutral pH had potential for use as a gelling agent, although further enrichment with alginate improved the mechanical properties of the obtained gels. The advantages and disadvantages of applying the non-purified algal extract in comparison with purified carbohydrates are discussed

Beyond the green: understanding the evolutionary puzzle of plant and algal cell walls

Niklas (2000) defined plants as “photosynthetic eukaryotes,” thereby including brown, red, and green macroalgae and microalgae. These groups share several features, including the presence of a complex, dynamic, and polysaccharide-rich cell wall. Cell walls in eukaryotes are thought to have evolved by lateral transfer from cell wall-producing organisms (Niklas, 2004). Green and red algae originate from a primary endosymbiotic event with a cyanobacterium, which is thought to have occurred over 1,500 million years ago (Palmer et al., 2004). Even though extant cyanobacteria have cell walls that are based on a peptidoglycan-polysaccharide-lipopolysaccharide matrix and thus differ markedly from the polysaccharide-rich cell walls of plants, there is preliminary evidence that they may contain some similar polysaccharides (Hoiczyk and Hansel, 2000), and genes already involved in polysaccharide synthesis or those subsequently coopted into wall biosynthesis may have been transferred during endosymbiosis. Independent secondary endosymbiotic events subsequently gave rise to the Euglenozoa (which lack cell walls) and brown algae (which have cell walls; Palmer et al., 2004). Investigations of the diversity of wall composition, structure, and biosynthesis that include algae, therefore, may lend new insights into wall evolution (Niklas, 2004)

Rapid and cost-efficient microplate assay for the accurate quantification of total phenolics in seaweeds

Brown seaweeds (Phaeophyceae) are a rich source of polyphenols (up to 20% dry weight) with a structure based on phloroglucinol (1,3,5-trihydroxybenzene). To-date the determination of total phenolics content (TPC) involves a redox reaction with the Folin-Ciocalteu (FC) reagent. However, side reactions with other reducing substances preclude accurate, direct measurement of TPC. This research reports a novel microplate assay involving a coupling reaction between phloroglucinol with Fast Blue BB (FBBB) diazonium salt, at basic pH, to form a stable tri-azo complex with maximum absorbance at 450 nm. Linear regression correlation values (R2) were ≥0.99 with phloroglucinol as standard. Direct quantification of TPCs (phloroglucinol equivalents, PGEs) in crude aqueous and ethanolic extracts from A. nodosum demonstrated that the new FBBB assay is not subject to side-redox interference and provides a more accurate estimate of TPC (1.2–3.9-fold lower than with the FC assay) in a relatively rapid (30 min), cost-effective (0.24€/test) microplate format

ENZYME SYSTEMS FROM THE THERMOPHILIC FUNGUS TALAROMYCES EMERSONII FOR SUGAR BEET BIOCONVERSION

The thermostable enzyme systems produced by the thermophilic ascomycete fungus Talaromyces emersonii cultivated on various carbon sources were investigated for the production of high value products from sugar beet. A broad range of enzymatic activities relevant to cellulose, hemicellulose, and pectin hydrolysis were identified in T. emersonii culture filtrates. In hydrolysis experiments conducted at 71ºC, the enzyme cocktails generated sugar-rich syrups from untreated sugar beet plants. Maximal levels of sugar beet hydrolysis were obtained with T. emersonii enzyme cocktails induced with sorghum/ beet pulp (68%) and sugar beet plant (56%). The principle monosaccharides released were glucose, xylose, and arabinose with minor amounts of galactose and galacturonic acid. Northern analysis of RNA isolated from T. emersonii when sugar beet plants were used as the sole carbon inducing source showed that genes required for polysaccharide hydrolysis and five carbon monosaccharide metabolism were co-ordinately expressed

Biofuel TechnologiesRecent Developments /

XVII, 534 p. 93 illus., 44 illus. in color.onlin

Mitochondrial malate dehydrogenase from the thermophilic, filamentous fungus talaromyces emersonii

Mitochondrial malate dehydrogenase (m-MDH; EC 1.1.1.37), from mycelial extracts of the thermophilic, aerobic fungus Talaromyces emersonii, was purified to homogeneity by sequential hydrophobic interaction and biospecific affinity chromatography steps. Native m-MDH was a dimer with an apparent monomer mass of 35 kDa and was most active at pH 7.5 and 52 degreesC in the oxaloacetate reductase direction. Substrate specificity and kinetic studies demonstrated the strict specificity of this enzyme, and its closer similarity to vertebrate m-MDHs than homologs from invertebrate or mesophilic fungal sources. The full-length m-MDH gene and its corresponding cDNA were cloned using degenerate primers derived from the N-terminal amino acid sequence of the native protein and multiple sequence alignments from conserved regions of other m-MDH genes. The m-MDH gene is the first oxidoreductase gene cloned from T. emersonii and is the first full-length m-MDH gene isolated from a filamentous fungal species and a thermophilic eukaryote. Recombinant m-MDH was expressed in Escherichia coli, as a His-tagged protein and was purified to apparent homogeneity by metal chelate chromatography on an Ni(2+)-nitrilotriacetic acid matrix, at a yield of 250 mg pure protein per liter of culture. The recombinant enzyme behaved as a dimer under nondenaturing conditions. Expression of the recombinant protein was confirmed by Western blot analysis using an antibody against the His-tag. Thermal stability studies were performed with the recombinant protein to investigate if results were consistent with those obtained for the native enzyme