Development of host strains and vector system for an efficient genetic transformation of filamentous fungi.

An ability to synthesize extracellular enzymes degrading a wide spectrum of plant and algae polymeric substrates makes many fungi relevant for biotechnology. The terrestrial thermophilic and marine fungal isolates capable of plant and algae degradation have been tested for antibiotic resistance for their possible use in a new genetic transformation system. Plasmids encoding the hygromycin B phosphotransferase (hph) under the control of the cauliflower mosaic virus 35S promoter, the trpC gene promoter of Aspergillus nidulans, and the Aureobasidium pullulans TEF gene promoter were delivered into the fungal cells by electroporation. The effectiveness of different promoters was compared by transformation and growth of Thermothelomyces thermophila (formerly Myceliophthora thermophila) on the selective medium and by real-time PCR analysis. A highly efficient transformation was observed at an electric-pulse of 8.5 kV/cm by using 10 µg of DNA per 1 × 105 conidia. Although all promoters were capable of hph expression in the Th. thermophila cells, the trpC promoter provided the highest level of hygromycin resistance. We further successfully applied plant binary vector pPZP for co-transformation of hph gene and enhanced green fluorescent protein gene that confirmed this transformation system could be used as an appropriate tool for gene function studies and the expression of heterologous proteins in micromycetes.

Hooked on α-d-galactosidases: from biomedicine to enzymatic synthesis.

α-d-Galactosidases (EC 3.2.1.22) are enzymes employed in a number of useful bio-based applications. We have depicted a comprehensive general survey of α-d-galactosidases from different origin with special emphasis on marine example(s). The structures of natural α-galactosyl containing compounds are described. In addition to 3D structures and mechanisms of action of α-d-galactosidases, different sources, natural function and genetic regulation are also covered. Finally, hydrolytic and synthetic exploitations as free or immobilized biocatalysts are reviewed. Interest in the synthetic aspects during the next years is anticipated for access to important small molecules by green technology with an emphasis on alternative selectivity of this class of enzymes from different sources.

Molecular characterization and therapeutic potential of a marine bacterium Pseudoalteromonas sp. KMM 701 alpha-galactosidase.

An alpha-galactosidase capable of converting B red blood cells into the universal blood type cells at the neutral pH was produced by a novel obligate marine bacterium strain KMM 701 (VKM B-2135 D). The organism is heterotrophic, aerobic, and halophilic and requires Na+ ions and temperature up to 34 degrees C for its growth. The strain has a unique combination of polysaccharide-degrading enzymes. Its single intracellular alpha-galactosidase exceeded other glycoside hydrolases in the level of expression up to 20-fold. The alpha-galactosidase was purified to determine the N-terminal amino acid sequences and new activities. It was found to inhibit Corynebacterium diphtheria adhesion to host buccal epithelium cell surfaces with high effectiveness. The nucleotide sequence of the homodimeric alpha-galactosidase indicates that its subunit is composed of 710 amino acid residues with a calculated Mr of 80,055. This alpha-galactosidase shares structural property with 36 family glycoside hydrolases. The properties of the enzyme are likely to be highly beneficial for medicinal purposes.

An endo-(1-->3)-beta-D-glucanase from the scallop Chlamys albidus: catalytic properties, cDNA cloning and secondary-structure characterization.

An endo-(1-->3)-beta-d-glucanase (L(0)) with molecular mass of 37 kDa was purified to homogeneity from the crystalline style of the scallop Chlamys albidus. The endo-(1-->3)-beta-d-glucanase was extremely thermolabile with a half-life of 10 min at 37 degrees C. L(0) hydrolyzed laminaran with K(m) approximately 0.75 mg/mL, and catalyzed effectively transglycosylation reactions with laminaran as donor and p-nitrophenyl betad-glucoside as acceptor (K(m) approximately 2mg/mL for laminaran) and laminaran as donor and as acceptor (K(m) approximately 5mg/mL) yielding p-nitrophenyl betad-glucooligosaccharides (n=2-6) and high-molecular branching (1-->3),(1-->6)-beta-d-glucans, respectively. Efficiency of hydrolysis and transglycosylation processes depended on the substrate structure and decreased appreciably with the increase of the percentage of beta-(1-->6)-glycosidic bonds, and laminaran with 10% of beta-(1-->6)-glycosidic bonds was the optimal substrate for both reactions. The CD spectrum of L(0) was characteristic for a protein with prevailing beta secondary-structural elements. Binding L(0) with d-glucose as the best acceptor for transglycosylation was investigated by the methods of intrinsic tryptophan fluorescence and CD. Glucose in concentration sufficient to saturate the enzyme binding sites resulted in a red shift in the maximum of fluorescence emission of 1-1.5 nm and quenching the Trp fluorescence up to 50%. An apparent association constant of L(0) with glucose (K(a)=7.4 x 10(5)+/-1.1 x 10(5)M(-1)) and stoichiometry (n=13.3+/-0.7) was calculated. The cDNA encoding L(0) was sequenced, and the enzyme was classified in glycoside hydrolases family 16 on the basis of the amino acid sequence similarity.

A new precursor for the immobilization of enzymes inside sol-gel-derived hybrid silica nanocomposites containing polysaccharides.

Tetrakis(2-hydroxyethyl) orthosilicate (THEOS) introduced by Hoffmann et al. (J. Phys. Chem. B., 106 (2002) 1528) was first used to prepare hybrid nanocomposites containing various polysaccharides and immobilize enzymes in these materials. Two different types of O-glycoside hydrolyses (EC3.2.1), 1-->3-beta-D-glucanase LIV from marine mollusk Spisula sacchalinensis and alpha-D-galactosidase from marine bacterium Pseudoalteromonas sp. KMM 701, were taken for the immobilization. To reveal whether the polysaccharide inside the hybrid material influences the enzyme entrapment and functioning, negatively charged xanthan, cationic derivative of hydroxyethylcellulose and uncharged locust bean gum were examined. The mechanical properties of these nanocomposites were characterized by a dynamic rheology and their structure by a scanning electron microscopy. It was found that 1-->3-beta-D-glucanase was usually immobilized without the loss of its activity, while the alpha-D-galactosidase activity in the immobilized state depended on the polysaccharide type of material. An important point is that the amount of immobilized enzymes was small, comparable to their content in the living cells. It was shown by the scanning electron microscopy that the hybrid nanocomposites are sufficiently porous that allows the enzymatic substrates and products to diffuse from an external aqueous solution to the enzymes, whereas protein molecules were immobilized firmly and not easily washed out of the silica matrix. A sharp increase of the enzyme lifetime (more than a hundred times) was observed after the immobilization. As established, the efficient entrapment of enzymes is caused by few advantages of new precursor over the currently used TEOS and TMOS: (i) organic solvents and catalysts are not needed owing to the complete solubility of THEOS in water and the catalytic effect of polysaccharides on the sol-gel processes; (ii) the entrapment of enzymes can be performed at any pH which is suitable for their structural integrity and functionality; (iii) a gel can be prepared at reduced concentrations of THEOS (1-2%) in the initial solution that excludes a notable heat release in the course of its hydrolysis.