Katsumi Takada

A Halophilic Serine Proteinase from Halobacillus sp. SR5-3 Isolated from Fish Sauce: Purification and Characterization

A halophilic bacterium was isolated from fish sauce, classified, and named Halobacillus sp. SR5-3. A purified 43-kDa proteinase produced by this bacterium showed optimal activity at 50 °C and pH 9–10 in 20% NaCl. The activity of the enzyme was enhanced about 2.5-fold by the addition of 20–35% NaCl, and the enzyme was highly stabilized by NaCl. It was found to be a serine proteinase related to either chymotrypsin or subtilisin. It absolutely preferred Ile at the P2 position of substrates. Thus, the enzyme was found to be a halophilic serine proteinase with unique substrate specificity.

Purification and Characterization of Serine Proteinase from a Halophilic Bacterium, Filobacillus sp. RF2-5

In order to find a unique proteinase, proteinase-producing bacteria were screened from fish sauce in Thailand. An isolated moderately halophilic bacterium was classified and named Filobacillus sp. RF2-5. The molecular weight of the purified enzyme was estimated to be 49 kDa. The enzyme showed the highest activity at 60 °C and pH 10–11 under 10% NaCl, and was highly stable in the presence of about 25% NaCl. The activity was strongly inhibited by phenylmethane sulfonyl fluoride (PMSF), chymostatin, and α-microbial alkaline proteinase inhibitor (MAPI). Proteinase activity was activated about 2-fold and 2.5-fold by the addition of 5% and 15–25% NaCl respectively using Suc–Ala–Ala–Phe–pNA as a substrate. The N-terminal 15 amino acid sequence of the purified enzyme showed about 67% identity to that of serine proteinase from Bacillus subtilis 168 and Bacillus subtilis (natto). The proteinase was found to prefer Phe, Met, and Thr at the P1 position, and Ile at the P2 position of peptide substrates, respectively. This is the first serine proteinase with a moderately thermophilic, NaCl-stable, and NaCl-activatable, and that has a unique substrate specificity at the P2 position of substrates from moderately halophilic bacteria, Filobacillus sp.

A membrane-bound, calcium-dependent protease in yeast α-cell cleaving on the carboxyl side of paired basic residues

Paired basic residues are known as a typical site for proteolytic processing of precursors of bioactive peptides. By using a fluorogenic substrate Boc-Gln-Arg-Arg-MCA, a unique endoprotease exhibiting hydrolytic specificity toward the carboxyl side of paired basic residues was partially purified (about 4600-fold) from the membrane fraction of yeast Saccharomyces cerevisiae alpha-cells. The enzyme is a calcium-dependent thiol protease, with optimal pH at 7.0. It is a glycoprotein, with an apparent molecular weight of about 100,000-120,000. It cleaves fluorogenic substrates and a synthetic model peptide at the carboxyl side of paired basic residues. From its unique substrate specificity, this enzyme may be involved in precursor processing in vivo.

Catalytic residues and substrate specificity of scytalidoglutamic peptidase, the first member of the eqolisin in family (G1) of peptidases

Scytalidoglutamic peptidase (SGP) is the first‐discovered member of the eqolisin family of peptidases with a unique structure and a presumed novel catalytic dyad (E136 and Q53) [Fujinaga et al., PNAS 101 (2004) 3364–3369]. Mutants of SGP, E136A, Q53A, and Q53E lost both the autoprocessing and enzymatic activities of the wild‐type enzyme. Coupled with the results from the structural analysis of SGP, Glu136 and Gln53 were identified as the catalytic residues. The substrate specificity of SGP is unique, particularly, in the preference at the P3 (basic amino acid), P 1 ′ (small a.a.), and P 3 ′ (basic a.a.) positions. Superior substrates and inhibitors have been synthesized for kinetic studies based on the results reported here. k cat, K m, and k cat/K m of SGP for d‐Dap(MeNHBz)‐GFKFF*ALRK(Dnp)‐d‐R‐d‐R were 34.8 s−1, 0.065 μM, and 535 μM−1 s−1, respectively. K i of Ac‐FKF‐(3S,4S)‐phenylstatinyl‐LR‐NH2 for SGP was 1.2 × 10−10 M. Taken together, we can conclude that SGP has not only structural and catalytic novelties but also a unique subsite structure.

Exploring the subsite‐structure of vimelysin and thermolysin using FRETS‐libraries

Vimelysin is a metalloproteinase with high activity at low temperature and an unusual resistance to organic solvents. Substrate specificities of vimelysin and thermolysin were examined using FRETS‐libraries, revealing a significant difference at the P3′ position: vimelysin preferred acidic amino acid residues, whereas thermolysin preferred basic residues. Homology modeling of vimelysin suggests that oppositely charged residues in the S3′ subsites (R215 in vimelysin and D213 in thermolysin) may be responsible for this specificity difference. This hypothesis was confirmed by examining the R215D mutant of vimelysin, which showed a substrate specificity profile intermediate between thermolysin and vimelysin.

Novel peptides with orally active and long-lasting antihypertensive activity

A series of N-(P-substituted phosphinoyl)peptides were synthesized and their antihypertensive activities were tested in spontaneously hypertensive rats (SHR). Among them, N-(dibenzyloxyphosphinoyl)-L-Ala-L-Pro-L-Pro-OH showed the most potent and long-lasting antihypertensive activity in SHR when administered orally. Although the inhibitory activity of this peptide against the angiotensin-converting enzyme was about one-hundredth of that of Captopril, the antihypertensive activity in SHR was significantly higher and longer-lasting than that of Enalapril which has been reported to be the most potent agent among similar converting enzyme inhibitors.

An Alkaline Serine-Proteinase from a Bacterium Isolated from Bat Feces: Purification and Characterization

An alkaline serine-proteinase from Bacillus sp. PN51 isolated from bat feces collected in Phang Nga, Thailand, was purified and characterized. The molecular mass was estimated to be 35.0 kDa. The N-terminal 25 amino acid sequence was about 70% identical with that of Natrialba magadii halolysin-like extracellular serine protease. The enzyme showed the highest proteinase activity at 60 °C at pH 10.0. The activity was strongly inhibited by PMSF and chymostatin. The proteinase activity was not affected by the presence of 2% urea, 2% H2O2, 12% SDS, 15% triton X-100, or 15% tween 80. The proteinase preferred Met, Leu, Phe, and Tyr residues at the P1 position, in descending order. The k cat, K m and k cat/K m values for Z-Val-Lys-Met-MCA were 16.8±0.14 min−1, 5.1±0.28 μM, and 3.3±0.28 μM−1 min−1 respectively. This is the first report of an alkaline serine-proteinase with extremely high stability against detergents such as SDS.

The model of media conference

Abstract The routine conferences are classified into four types: i.e. 1-message transfer, 2-transaction, 3- coordination, 4- tactic decision type. General conference models of the transaction and coordination type are formulated and experimental analysis of the model are described in this paper

The Roles of the Multimedia Communications on the Plant

Abstract Modality choice of participants in media conferencing is analyzed in this paper. Firstly, the role of multimedia in plant operations is mentioned. Secondly, the problem of communication by telephone among the plant operators is described. Thirdly, model conferencing is done in nine combinations of media. which include text, sound and image. The following conclusions were obtained from the results. 1) the task efficiency depends on the combination of media. 2) sound should be used in simple task, and 3) image should be used when solving a complicated problem.