Naofumi Shiomi

Nucleotide sequence and characterization of a Gene conferring resistance to ethionine in yeast Saccharomyces cerevisiae

Abstract The nucleotide sequence of a DNA fragment, when present on a multi-copy plasmid, conferring ethionine resistance to Saccharomyces cerevisiae cells was determined. The fragment contained one long open reading frame and the frame was confirmed to be an ethionine resistant gene caused by frame-shift mutation. Other than ethionine resistance, an increased dosage of the gene directed overaccumulation in cells of S- adenosyl- l -methionine . The protein deduced from the open reading frame consisted of 617 amino acid residues with a calculated molecular weight of 67,977.

Production of Glucoamylase by Passively Immobilized Cells of a Flocculent Yeast, Saccharomyces diastaticus

Abstract Production of glucoamylase by cells of a flocculent yeast, Saccharomyces diastaticus, immobilized within porous biomass support particles (BSPs) was studied in repeated batch fermentation. Since many of the immobilized cells could be utilized as seeds for each subsequent batch cycle, the glucoamylase productivity in repeated batch fermentation was 70% higher than that obtained in batch fermentation without BSPs.

Characteristics of Neutralization of Acids by Newly Isolated Fungal Cells

Soil microorganisms play an important role in maintaining soil pH at levels suitable for other soil organisms. To clarify the biological neutralization mechanism in soil, we isolated soil microorganisms showing a high ability to neutralize acids and studied their characteristics. From our taxonomic study, three isolated strains were identified as filamentous fungi, namely Mucor sp., Aspergillus fumigatus, and Aureobasidium pullulans. These strains could secrete basic materials, such as ammonia, for neutralization, grow in the medium at pH 4.0 and increase the pH of the medium to approximately 8.0. These microbial cells could neutralize not only nitric acid but also sulfuric and hydrochloric acids. The strains could also grow by utilizing nitric acid as a sole nitrogen source. In the soil containing these organisms, the pH was maintained in the neutral range by the buffering action of basic materials that they secrete. These results suggest that these fungal cells are useful for protecting the soil from acidification by acid rain.

Cloning of a gene for S-adenosylmethionine synthesis in Saccharomyces cerevisiae

SummaryThe gene for ethionine resistance was isolated, and its phenotypic characteristics were investigated. The cells transformed with this gene showed strong resistance to DL-ethionine, and S-adenosylmethionine (SAM) was remarkably accumulated within the cells. Judging from the restriction map of this gene, it suggests that the gene is not the gene SAM1 but SAM2.

Utilization of antipeptide antibodies as affinity ligands in immunoaffinity purification

Anti-peptide antibodies against the C-terminal regions of chimeric alpha-amylase, recombinant CD2 and insulin B-chain were obtained by using peptides corresponding to the C-terminal regions as immunogens. These anti-peptide antibodies adsorbed the native proteins, as well as the antigen peptides. The proteins were purified to high purity using the anti-peptide antibodies as affinity ligands. These ligands could discriminate the target proteins having different C-terminal regions. The adsorbed proteins were specifically eluted by the eluents containing the antigen peptides.

Cloning of genes for spermine resistance in Saccharomyces cerevisiae and their effects on S-adenosyl-l-methionine accumulation

Abstract Among the polyamines tested, spermine strongly inhibited the growth of Saccharomyces cerevisiae DKD-5D-H. Two kinds of DNA fragments that confer strong and weak resistances to spermine were cloned onto a vector plasmid, YEp13. The restriction map of the DNA fragment conferring strong resistance was the same as that of a DNA fragment responsible for ethionine resistance in the same yeast cells. (Shiomi et al., Appl. Microbiol. Biotechnol., 29, 302–304, 1988) The yeast cells with the DNA fragment conferring strong resistance to spermine were resistant to ethionine and accumulated S- adenosyl- l -methionine intracellularly.

Production and purification of soluble human CD2 secreted from recombinant Pichia pastoris

Abstract The solubilized form of human CD2 (sCD2), which consists of D1 and D2 domains and can bind specifically to LFA-3, was produced by secretion from Pichia pastoris by use of the AOX1 promoter and the α-factor signal sequence. An anti-peptide antibody against the C-terminal region of sCD2 (anti-PC-CD2-12 antibody) was used for ELISA, purification and characterization of sCD2. Secreted sCD2 was purified from fermentation broth with an affinity column coupled with anti-PC-CD2-12 antibody. The concentrations of sCD2 in fermentation broth and purified fractions were measured with the anti-peptide antibody and a monoclonal antibody recognizing the D1 domain. These results suggest that a large portion of secreted sCD2 lacked several amino acids in the C-terminal region probably because of digestion by proteases. Therefore, purification by the anti-peptide antibody recognizing the C-terminal region is a promising method for the purification of sCD2 having the complete C-terminal region.