Characteristics of fungal phytases from Aspergillus fumigatus and Sartorya fumigata.

Aspergillus fumigatus phytase has previously been identified as a phytase with a series of favourable properties that may be relevant in animal and human nutrition, both for maximising phytic acid degradation and for increasing mineral and amino acid availability. To study the natural variability in amino acid sequence and its impact on the catalytic properties of the enzyme, we cloned and overexpressed the phytase genes and proteins from six new purported A. fumigatus isolates.

Paracoccus zeaxanthinifaciens sp. nov., a zeaxanthin-producing bacterium.

A comprehensive taxonomic re-evaluation was performed on the marine, zeaxanthin-producing bacterium formerly classified as [Favobacterium] sp. strain R-1 512 (ATCC 21588). This strain, together with two other previously described marine isolates, [Flavobacterium] strain R-1506 and Paracoccus sp.

The consensus concept for thermostability engineering of proteins: further proof of concept.

Previously, we calculated a consensus amino acid sequence from 13 homologous fungal phytases. A synthetic gene was constructed and recombinantly expressed. Surprisingly, consensus phytase-1 was 15-26 degrees C more thermostable than all parent phytases used in its design [Lehmann et al.

Engineering of phytase for improved activity at low pH.

For industrial applications in animal feed, a phytase of interest must be optimally active in the pH range prevalent in the digestive tract. Therefore, the present investigation describes approaches to rationally engineer the pH activity profiles of Aspergillus fumigatus and consensus phytases. Decreasing the negative surface charge of the A.

Structure-based chimeric enzymes as an alternative to directed enzyme evolution: phytase as a test case.

Thermostability is a key feature for commercially attractive variants of the fungal enzyme phytase. In an initial set of experiments, we restored ionic interactions and hydrogen bonds on the surface of Aspergillus terreus phytase, which are present in the homologous but more thermostable enzyme from A. niger.

The consensus concept for thermostability engineering of proteins.

Previously, sequence comparisons between a mesophilic enzyme and a more thermostable homologue were shown to be a feasible approach to successfully predict thermostabilizing amino acid substitutions. The 'consensus approach' described in the present paper shows that even a set of amino acid sequences of homologous, mesophilic enzymes contains sufficient information to allow rapid design of a thermostabilized, fully functional variant of this family of enzymes. A sequence alignment of homologous fungal phytases was used to calculate a consensus phytase amino acid sequence.

Optimization of the catalytic properties of Aspergillus fumigatus phytase based on the three-dimensional structure.

Previously, we determined the DNA and amino acid sequences as well as biochemical and biophysical properties of a series of fungal phytases. The amino acid sequences displayed 49-68% identity between species, and the catalytic properties differed widely in terms of specific activity, substrate specificity, and pH optima. With the ultimate goal to combine the most favorable properties of all phytases in a single protein, we attempted, in the present investigation, to increase the specific activity of Aspergillus fumigatus phytase.

Active site residue 297 of Aspergillus niger phytase critically affects the catalytic properties.

The wild-type phytases from the Aspergillus niger strains NRRL 3135 and T213 display a three-fold difference in specific activity (103 versus 32 U/mg protein), despite only 12 amino acid differences that are distributed all over the sequence of the protein. Of the 12 divergent positions, three are located in or close to the substrate binding site. Site-directed mutagenesis of these residues in A.

From DNA sequence to improved functionality: using protein sequence comparisons to rapidly design a thermostable consensus phytase.

Naturally-occurring phytases having the required level of thermostability for application in animal feeding have not been found in nature thus far. We decided to de novo construct consensus phytases using primary protein sequence comparisons. A consensus enzyme based on 13 fungal phytase sequences had normal catalytic properties, but showed an unexpected 15-22 degrees C increase in unfolding temperature compared with each of its parents.

Biophysical characterization of fungal phytases (myo-inositol hexakisphosphate phosphohydrolases): molecular size, glycosylation pattern, and engineering of proteolytic resistance.

Phytases (myo-inositol hexakisphosphate phosphohydrolases) are found naturally in plants and microorganisms, particularly fungi. Interest in these enzymes has been stimulated by the fact that phytase supplements increase the availability of phosphorus in pig and poultry feed and thereby reduce environmental pollution due to excess phosphate excretion in areas where there is intensive livestock production. The wild-type phytases from six different fungi, Aspergillus niger, Aspergillus terreus, Aspergillus fumigatus, Emericella nidulans, Myceliophthora thermophila, and Talaromyces thermophilus, were overexpressed in either filamentous fungi or yeasts and purified, and their biophysical properties were compared with those of a phytase from Escherichia coli.

Comparison of the thermostability properties of three acid phosphatases from molds: Aspergillus fumigatus phytase, A. niger phytase, and A. niger PH 2.5 acid phosphatase.

Enzymes that are used as animal feed supplements should be able to withstand temperatures of 60 to 90 degrees C, which may be reached during the feed pelleting process. The thermostability properties of three histidine acid phosphatases, Aspergillus fumigatus phytase, Aspergillus niger phytase, and A. niger optimum pH 2.

Cloning of the phytases from Emericella nidulans and the thermophilic fungus Talaromyces thermophilus.

Phytases (EC 3.1.3.

Gene cloning, purification, and characterization of a heat-stable phytase from the fungus Aspergillus fumigatus.

The finding of heat-stable enzymes or the engineering of moderately thermostable enzymes into more stable ones by random or site-directed mutagenesis has become a main priority of modern biotechnology. We report here for the first time a heat-stable phytase able to withstand temperatures up to 100 degrees C over a period of 20 min, with a loss of only 10% of the initial enzymatic activity. The gene (phyA) encoding this heat-stable enzyme has been cloned from Aspergillus fumigatus and overexpressed in Aspergillus niger.

Isolation and characterization of the carotenoid biosynthesis genes of Flavobacterium sp. strain R1534.

The Gram-negative bacterium Flavobacterium sp. strain R1534 is a natural producer of zeaxanthin. A 14 kb genomic DNA fragment of this organism has been cloned and a 5.

The phytase subfamily of histidine acid phosphatases: isolation of genes for two novel phytases from the fungi Aspergillus terreus and Myceliophthora thermophila.

Phytases catalyse the hydrolysis of phytate (myo-inositol hexakisphosphate) to myo-inositol and inorganic phosphate. In this study genes encoding novel phytases from two different filamentous fungi, Aspergillus terreus strain 9A-1 and Myceliophthora thermophila were isolated. The encoded PhyA phytase proteins show 60% (A.

Reversible dissociation of the poliovirus replication complex: functions and interactions of its components in viral RNA synthesis.

Membrane-bound replication complexes containing transcriptionally active replicative intermediates (RI) can be isolated from poliovirus-infected HEp-2 cells and consist of rosette-like structures of virus-induced vesicles surrounding the replicating viral RNA. At low ionic strength and low temperature, the rosettes reversibly dissociate into individual tubulated vesicles. As determined by immunoelectron microscopy and immunoprecipitation, the vesicles carry a set of viral structural and nonstructural proteins as well as RI RNA.

Reverse transcription multiplex PCR for differentiation between polio- and enteroviruses from clinical and environmental samples.

For the rapid detection of polioviruses and their differentiation from nonpoliovirus enteroviruses, we developed a protocol in which clinical or environmental specimens are first inoculated onto cell cultures in tubes. After overnight incubation, the cultures are subjected to reverse transcription multiplex PCR with a primer pair which detects all enteroviruses (T. Hyypiä, P.

Cloning and characterization of a surface antigen of Eimeria tenella merozoites and expression using a recombinant vaccinia virus.

A rabbit serum raised against Eimeria tenella merozoites was used to screen a lambda gt11 cDNA library made from merozoite mRNA of E. tenella. The insert of the phage clone lambda Mz 5-7 revealed an open reading frame consisting of 945 nucleotides, encoding a 33-kDa protein.

Immunocytochemical localization of capsid-related particles in subcellular fractions of poliovirus-infected cells.

The structural proteins of poliovirus can assemble into a series of different configurations (capsid-related particles, CRP). Only some seem to be true capsid precursors and the role of most CRP in morphogenesis is unclear. We used electron microscopic immunocytochemistry with monoclonal antibodies recognizing different CRP [protomers, pentamers, 65S empty capsids (EC), 74S-EC, and virions] to locate CRP in subcellular fractions containing virus-induced vesicles associated with the viral replication complex.

Direct identification of recombinant vaccinia virus plaques by PCR.

A fast method for the identification of recombinant vaccinia viruses directly from individual plaques is described. Plaques are picked, resuspended in PBS-A and processed for PCR using two 'universal' primers. The amplified sequences are analyzed by agarose gel electrophoresis.

In situ hybridization for light and electron microscopy: a comparison of methods for the localization of viral RNA using biotinylated DNA and RNA probes.

We report procedures for in situ hybridization at the light and electron microscopic level for localization of viral RNA in poliovirus-infected, Lowicryl-embedded cells. We compare specificity and signal intensity of biotinylated, double-stranded DNA and single-stranded, strand-specific RNA probes, both corresponding to the same region of the poliovirus genome. The hybrids were detected with antibiotin antibodies or streptavidin with colloidal gold as a marker.

Structural organization of poliovirus RNA replication is mediated by viral proteins of the P2 genomic region.

Transcriptionally active replication complexes bound to smooth membrane vesicles were isolated from poliovirus-infected cells. In electron microscopic, negatively stained preparations, the replication complex appeared as an irregularly shaped, oblong structure attached to several virus-induced vesicles of a rosettelike arrangement. Electron microscopic immunocytochemistry of such preparations demonstrated that the poliovirus replication complex contains the proteins coded by the P2 genomic region (P2 proteins) in a membrane-associated form.

Association of polioviral proteins of the P2 genomic region with the viral replication complex and virus-induced membrane synthesis as visualized by electron microscopic immunocytochemistry and autoradiography.

Using high resolution electron microscopic autoradiography and immunocytochemistry with monoclonal antibodies against poliovirus proteins of the P2 genomic region, the location of these proteins in respect to the virus-induced vesicle formation and the viral RNA synthesis was followed during the viral replication cycle. It was found that P2 proteins become rER associated soon after their synthesis. At the site of protein and rER interaction, electron-dense patches appear.