We 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 consisting 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.
The neutral protease II (NpII) from Aspergillus oryzae is a zinc-containing metalloprotease with some unique properties. To elucidate its structure, we isolated a full-length cDNA clone for NpII. Sequence analysis reveals that NpII has a prepro region consisting of 175 amino acids preceding the mature region, which consists of 177 amino acids. As compared with other microbial metalloproteases, NpII is found to be unique in that it shares only a limited homology with them around two zinc ligand His residues and that the positions of the other zinc ligand (Glu) and the active site (His) cannot be established by homology. When a plasmid designed to express the prepro NpII cDNA was introduced into Saccharomyces cerevisiae and the transformant was cultured in YPD medium (2% glucose, 2% polypeptone, 1% yeast extract), it secreted a proNpII. However, in a culture of the same medium containing 0.2 mM ZnCl2, it secreted a mature NpII with a specific activity and N-terminus identical to those of native NpII. This observation suggests that either an autoproteolytic activity or a yeast protease effected the processing.
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.
To understand the mechanism by which gamma-polyglutamic acid (gamma-PGA) in the sticky material of natto was synthesized, we purified the gamma-glutamyltranspeptidase (gamma-GTP) (EC 2.3.2.2) from the culture broth of Bacillus subtilis (natto) to homogeneity. gamma-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 gamma-GTP from Escherichia coli. The optimum pH and temperature of activity were 8.5 and 60 degrees C. The enzyme was inactivated by incubation for 15 min at pH 8.0 and 55 degrees C, but little loss of the activity was detected at 40 degrees C. gamma-GTP used glutamine as a gamma-glutamyl donor and acceptor for gamma-PGA synthesis. Dipeptides were better gamma-glutamyl acceptors than free amino acids.
A mixed gel composed of colloidal silica and alginate (As gel) was prepared for the immobilization of enzymes or microorganisms. The physical strength of AS gel increased with the amount of colloidal silica. The ethanol production rate of Saccharomyces cerevisiae (IFO 0224) immobilized in AS gel was higher than in alginate gel (Al gel) in the early phase of growth. At a concentration of glucose of more than 10%, the ethanol production of immobilized yeast in AS gel was higher than in Al gel. Any difference was not recognized in the diffusion coefficient of glucose between AS and Al gels. The AS gel had an ability to retain proteins such as bovine serum albumin and gamma-globulin. The alkaline protease and beta-galactosidase in AS gel continued their function for a long time, but those immobilized in Al gel did not. Immobilized beta-galactosidase in AS gel had a higher thermal stability than in Al gel or free enzymes.
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