Yoshihiro Ogawa

A full length cDNA clone for the alkaline protease from Aspergillus oryzae: Structural analysis and expression in Saccharomyces cerevisiae

SummaryWe have cloned and determined the nucleotide sequence of a cDNA fragment for the entire coding region of the alkaline protease (Alp) from a filamentous ascomycete Aspergillus oryzae. According to the deduced amino acid sequence, Alp has a putative prepro region of 121 amino acids preceding the mature region, which consists of 282 amino acids. A consensus sequence of a signal peptide consiting of 21 amino acids is found at the N-terminus of the prepro region. The primary structure of the mature region shares extensive homology (29%–44%) with those of subtilisin families, and the three residues (Asp 32, His 64 and Ser 221 in subtilisin BPN′) composing the active site are preserved. The entire cDNA, coding for prepro Alp, when introduced into the yeast Saccharomyces cerevisiae, directed the secretion of enzymatically active Alp into the culture medium, with its N-terminus and specific activity identical to native Aspergillus Alp.

Efficient Production of γ-Polyglutamic Acid by Bacillus subtilis (natto) in Jar Fermenters

The large scale fermentation of γ-polyglutamic acid (γ-PGA) by Bacillus subtilis (natto) was done using a 30-liter jar fermenter. A stable cultivation without foaming could be done with addition of 3% NaCl to the medium. The γ-PGA productivity became higher with increasing speed of agitation and amounts of glutamic acid added to the broth. Finally, we were able to obtain about 35 mg/ml of γ-PGA under the optimum conditions. The glutamic acid added to the medium was efficiently converted into γ-PGA in the stationary phase. To discover the role of l-glutamic acid added to the medium for γ-PGA biosynthesis by Bacillus subtilis (natto), the radioactivity incorporated into γ-PGA from (14)C-l-glutamic acid was measured. As a result, radioactive γ-PGA was detected in the medium. Then, the glutamic acid in the medium was transported into the cells and actually polymerized as the glutamic acid unit of γ-PGA.

Detection of γ-Polyglutamic Acid (γ-PGA) by SDS-Page

γ-Polyglutamic acid (γ-PGA) produced by Bacillus suhtilis (natto) was detected by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and basic dye staining. Using this method, the molecular weight of γ-PGA was estimated at 275,000. This value was almost the same in all bacterial strains tested. As other applications of this SDS-PAGE method, degradation of γ-PGA by acid and heat treatment and a cross-linking reaction with carbodiimide and ethylenediamine were made visible in acrylamide gel. In the growth curve of the bacteria, γ-PGA production was detected in early stationary phase by SDS-PAGE.

Purification and Properties of γ-Glutamyltranspeptidase from Bacillus subtilis (natta)

To understand the mechanism by which γ-poly glutamic acid (γ-PGA) in the sticky material of natto was synthesized, we purified the γ-glutamyltranspeptidase (γ-GTP) (EC 2.3.2.2) from the culture broth of Bacillus subtilis (natto) to homogeneity. γ-GTP was composed of two subunits with molecular weight of 45,000 and 22,000. The N-terminal amino acid sequence of light subunit was homologous with that of γ-GTP from Escherichia coli. The optimum pH and temperature of activity were 8.5 and 60°C. The enzyme was inactivated by incubation for 15 min at pH 8.0 and 55°C, but little loss of the activity was detected at 40°C. γ-GTP used glutamine as a γ-glutamyl donor and acceptor for γ-PGA synthesis. Dipeptides were better γ-glutamyl acceptors than free amino acids.