Cell wall structure suitable for surface display of proteins in Saccharomyces cerevisiae.

A display system for adding new protein functions to the cell surfaces of microorganisms has been developed, and applications of the system to various fields have been proposed. With the aim of constructing a cell surface environment suitable for protein display in Saccharomyces cerevisiae, the cell surface structures of cell wall mutants were investigated. Four cell wall mutant strains were selected by analyses using a GFP display system via a GPI anchor.

Synthesis and degradation of acyl peptide using enzyme from Pseudomonas aeruginosa.

The detailed properties of the enzyme from Pseudomonas aeruginosa, which catalyzes the N-acyl linkage between myristic acid and the N-terminal glycine residue of the octapeptide GNAAAARR-NH(2) (PKA) in aqueous solution without ATP and CoA, were studied. The substrate specificity for the acyl peptide in the synthetic reaction was examined, and it was found that at least eight amino acid residues are required for the reaction and that the N-terminal glycine residue is not absolutely essential for the reaction because the activity was detected using the octapeptide that has an N-terminal alanine. The activity was also strongly affected by the amino acid sequence because the activity was very weak in the reaction using GARASVLS-NH(2) (HIV-1p17(gag)).

Saccharomyces cerevisiae mutant displaying beta-glucans on cell surface.

The deletion of MCD4 leads to an increase in beta-1,6-glucan level and a decrease in glycosylphosphatidylinositol-anchored protein and mannan levels in the cell wall of Saccharomyces cerevisiae, suggesting that mcd4 deletion mutant (mcd4Delta) displays beta-glucans on the cell surface without a mannan cover. An observation of the cell surface of mcd4Delta cells and an examination of the effect of contact between mcd4Delta cells and mouse macrophages indicated that macrophages were activated by contact with mcd4Delta cells displaying beta-glucans on the cell surface. We further examined the effect of intraperitoneal ethanol-fixed mcd4Delta cells on the survival period of mice infected with Candida albicans.

New detection method for hydrogen gas for screening hydrogen-producing microorganisms using water-soluble wilkinson's catalyst derivative.

A water-soluble color indicator was developed for the effective screening of hydrogen-producing microorganisms. This indicator consists of a coloring agent and a water-soluble derivative of Wilkinson's catalyst. Wilkinson's catalyst, Tris(triphenylphosphine) rhodium chloride, had been developed as a catalyst for the hydrogenation of olefins.

Purification and characterization of enzyme responsible for N-myristoylation of octapeptide in aqueous solution without ATP and coenzyme a from Pseudomonas aeruginosa.

The enzyme that catalyzes N-acyl linkage between myristic acid and the NH(2)-terminal glycine residue of the octapeptide Gly-Asn-Ala-Ala-Ala-Ala-Arg-Arg-NH(2) in aqueous solution without ATP and coenzyme A was found in Pseudomonas aeruginosa. The enzyme was purified from cell-free crude extract using DEAE-Cellulose, Sephadex G-200, CM-Sephadex C-50, and hydroxyapatite column chromatographies, and then purified approximately 1900-fold with about 1.5% recovery of enzyme activity from the crude extract.

Relationship between cell morphology and intracellular potassium concentration in Candida albicans.

Previously we reported that valinomycin inhibited hyphal growth and induced growth as a chain of yeast cells under hyphal growth induction conditions in Candida albicans. To elucidate the hyphal growth inhibition by valinomycin, we examined the effect of various chemicals on the morphology and found that miconazole inhibited hyphal growth as well as valinomycin: both compounds promoted the leakage of potassium from cells. Analysis of intracellular potassium suggested that hyphal cells contain potassium at high concentrations in comparison with yeast cells.

Valinomycin affects the morphology of Candida albicans.

Microbial metabolites were screened for inhibitors of hyphal growth in Candida albicans. Inhibitory activity was found among metabolites of a culture of an actinomycete, which had been isolated from soil. The active substance inhibited hyphal growth and induced growth as a chain of yeast cells under hyphal growth induction conditions.

Deletion of MCD 4 involved in glycosylphosphatidylinositol (GPI) anchor synthesis leads to an increase in beta-1,6-glucan level and a decrease in GPI-anchored protein and mannan levels in the cell wall of Saccharomyces cerevisiae.

Most proteins involved in the synthesis of the GPI core structure of Saccharomyces cerevisiae are essential for growth. To explore the relationship between the GPI anchor structure and beta-1,6-glucan synthesis, we screened deletion mutants in genes involved in GPI synthesis for osmotic remedial growth. Heterozygous diploid strains were dissected on medium with osmotic support and slow growth of the mcd 4 deletion mutant was observed.

Analysis of chitin at the hyphal tip of Candida albicans using calcofluor white.

Staining with calcofluor white (CFW), which is known to bind chitin-rich areas of the cell wall, revealed a difference in the fluorescence intensity at the hyphal tip between Candida albicans hyphal cells that were grown in modified Lee (M-Lee) and SPG media. The fluorescence intensity at the tip increased with the addition of salts and sugar to SPG. The chitin levels per dry cell weight in cells grown in modified Lee and SPG with 1.

Rot1p of Saccharomyces cerevisiae is a putative membrane protein required for normal levels of the cell wall 1,6-beta-glucan.

Although ROT1 is essential for growth of Saccharomyces cerevisiae strain BY4741, the growth of a rot1Delta haploid was partially restored by the addition of 0.6 M sorbitol to the growth medium. Rot1p is predicted to contain 256 amino acids, to have a molecular mass of 29 kDa, and to possess a transmembrane domain near its C-terminus.

Characterization of a Saccharomyces cerevisiae mutant with pseudohyphae and cloning of a gene complementing the mutation.

Screening for morphological mutants of a haploid strain of Saccharomyces cerevisiae was done on the basis of their cell-shape on a solid medium containing isoamyl alcohol, which causes cell elongation, to obtain information on the morphogenesis. Mutant J19, which had pseudohyphae in liquid medium even in the absence of isoamyl alcohol, had many elongated cells. Few reports exist of haploid cells growing as pseudohyphae in liquid culture.

Characteristics of immobilized invertase.

Five kinds of immobilized invertases (IMI)-covalently of porous glass and ion-exchange resins and ionically on ion-exchange resins-have been prepared and their kinetic characteristics for sucrose hydrolysis, such as K , K, pH profile, and thermal stability were studied. Comparing the values of K and activation energy and the entropy of IMI with those of native invertase, it was concluded that the immobilization influences not binding but kinetic specificity. The effects of the immobilization method on thermal stability were also discussed.

Kinetic study on thermal stability of immobilized invertase.

The kinetic study of the thermal stability of three kinds of invertases: native, immobilized on porous glass covalently, and on ion-exchange resin ionically, has been carried out, measuring their enzymatic activity for sucrose hydrolysis. Thermal deactivations of all invertases obeyed first-order kinetics, being independent of substrate concentration, with k and ΔE , ΔS * as shown in Tables I and II, respectively. Based on these parameter values, the effects of immobilization and pH at deactivation on the stability have been considered, and it was suggested that the ionic bond gives a more loosely deformed enzyme than the covalent bond.