Molecular cloning and nucleotide sequence of the 90k serine protease gene,hspK, from Bacillus subtilis (natto) No. 16

We previously reported purification and characterization of a 90k serine protease with pI 3.9 from Bacillus subtilis (natto) No. 16 [Kato et al. 1992 Biosci Biotechnol Biochem 56:1166]. The enzyme showed different and unique substrate specificity towards the oxidized B-chain of insulin from those of well-known bacterial serine proteases from Bacillus subtilisins. The structural gene, hspK, for the 90k serine protease was cloned and sequenced. The cloned DNA fragment contained a single open reading frame of 4302 bp coding a protein of 1433 amino acid residues. The deduced amino acid sequence of the 90k-protease indicated the presence of a typical signal sequence of the first 30 amino acids region and that there was a pro-sequence of 164 amino acid residues after the signal sequence. The mature region of the 90k-protease started from position 195 of amino acid residue, and the following peptide consisted of 1239 amino acid residues with a molecular weight of 133k. It might be a precursor protein of the 90k-protease, and the C-terminal region of 43k might be degraded to a mature protein from the precursor protein. The catalytic triad was thought to consist of Asp33, His81, and Ser259 from comparison of the amino acid sequence of the 90k-protease with those of the other bacterial serine proteases. The high-molecular-weight serine protease, the 90k-protease, may be an ancient form of bacterial serine proteases.     

Mode of Action of a New Aspergillus oryzae Carboxypeptidase O-1

DNA breakage by hydrolyzable tannins of known structures was investigated by agarose or polyacrylamide gel electrophoretic analysis. Hydrolyzable tannins could cause both double-strand and single-strand breakages in λDNA in the presence of Cu²⁺. The breaking reaction was strongly suppressed at concentrations higher than 100 μm of hydrolyzable tannins. DNA breakage by various tannins was dependent upon their sorts, numbers, and binding sites of the constituent acids and polyalcohols. Metallic ions other than Cu²⁺ had little effect on the breaking reaction.     

Degradation of α-amylase fromAspergillus oryzae with firmly bound proteinases at pH 3.0

Incubation of highly purified -amylase fromAspergillus oryzae (EC 3.2.1.1) with 0.01M acetate buffer, pH 3.0, resulted in degradation of the -amylase. The molecular weight values of degradation products were 42 K, 37 K, and 28 K. Incubation of the purified -amylase in 0.02m phosphate buffer, pH 7.5, at 30°C for 17 h, however, resulted in no degradation of the -amylase molecule.Incubation of the purified -amylase with proangiotensin at pH 3.0 for 24 h resulted in cleavage of Tyr⁴-Ile⁵, His⁶-Pro⁷, Pro⁷-Phe⁸, Phe⁸-His⁹, and His⁹-Leu¹⁰. Thus, it appears that proteolytic activities firmly bound to -amylase are identical withAspergillus aspartic proteinase (EC 3.4.23.6) andAspergillus acid carboxypeptidase (EC 3.4.16.1).     

The molecular surface of proteolytic enzymes has an important role in stability of the enzymatic activity in extraordinary environments.

It is scientifically and industrially important to clarify the stabilizing mechanism of proteases in extraordinary environments. We used subtilisins ALP I and Sendai as models to study the mechanism. Subtilisin ALP I is extremely sensitive to highly alkaline conditions, even though the enzyme is produced by alkalophilic Bacillus, whereas subtilisin Sendai from alkalophilic Bacillus is stable under conditions of high alkalinity. We constructed mutant subtilisin ALP I enzymes by mutating the amino acid residues specific for subtilisin ALP I to the residues at the corresponding positions of amino acid sequence alignment of alkaline subtilisin Sendai. We observed that the two mutations in the C-terminal region were most effective for improving stability against surfactants and heat as well as high alkalinity. We predicted that the mutated residues are located on the surface of the enzyme structures and, on thebasis of three-dimensional modelling, that they are involved in stabilizing the conformation of the C-terminal region. As proteolytic enzymes frequently become inactive due to autocatalysis, stability of these enzymes in an extraordinary environment would depend on the conformational stability of the molecular surface concealing scissile peptide bonds. It appeared that the stabilization of the molecular surface structure was effective to improve the stability of the proteolytic enzymes.     

Identification of N-terminal autodigestion target site in subtilisin ALP I.

Autodigestion of subtilisin ALP I (ALP I), secreted from the alkalophilic Bacillus sp. NKS-21 and its predicted amino acid sequence having about 60% identity with other alkaline subtilisins, was examined under alkaline conditions. At the alkaline pH of 12, ALP I was rapidly degraded, and almost no breakdown products were detectable. However, by incubating ALP I at 5 degrees C for an extended time, a couple of specific peptides (26.7 kDa and 25.6 kDa) were accumulated. Each of them was purified and amino acid sequences of these fragments were found. Both peptides appeared to start at Gly-19 of the mature sequence of ALP I.     

Functional Changes of Polyethylene Glycol-modified Serine Proteinase from Aspergillus sojae and Interaction with α2-Macroglobulin

The covalent attachment of activated polyethylene glycol₂ (PEG₂) of 10,000 daltons to non- essential groups on a serine proteinase II (SepII) from Aspergillus sojae produced two modified preparations (PEG₂-SepII-S and PEG₂-SepII-L). The molecular weights of PEG₂-SepII-S and PEG₂-SepII-L were about 170,000 and 280,000, respectively. The PEG₂-SepII-S lost about 80 % of its antigenicity, while the PEG₂-SepU-L completely lost its antigenicity. In comparison of kinetic parameters with SepII there was less than 40 % variation in Km, but the values of k_cat towards succinyl-l-leucy 1-l-leucy 1-l-valy 1-l-tyrosine 4-methylcoumaryl-7-amide (Suc-LLVY-MCA) or succinyl-l- alanyl-l-alanyl-l-valyl-l-alanine β-nitroanilide (Suc-AAVA-/>NA) decreased to about 70% less than that of SepII. The modified preparations have about 20 % activity towards fibrin hydrolysis and a low affinity for a protein proteinase inhibitor, Streptomyces subtilisin inhibitor (SSI), with a molecular weight of 23,000, while the preparations have high affinity for a low molecular weight microbial inhibitor, chymostatin. The stoichiometry of the reaction of a₂-macroglobulin (α₂M) with PEG₂-SepII-S showed that PEG₂-SepII-L bound to α₂M in a molar ratio of 1:1. No appreciable differences were observed in the pH stabilities of the modified enzymes and the native one at pH 3.6, while the modified enzymes were more stable than that of the native one at pH 11.5. The two modified preparations were labile at 50°C, but the native enzyme was completely stable at 50°C.     

Initial Cleavage Site of Bacillus Ribosomal Proteinase

Purified N-acetylmuramidase (EC 3.2.1.17) produced by Streptomyces rutgersensis H-46 was used as a preservative for bean paste made of adzuki beans (Vigna angularis) without addition of sugar, called raw an. The enzyme was stable during the storage of the sample. The viable cell count in the enzyme-treated sample first decreased as susceptible microorganisms were killed as a result of cell wall degradation by the enzyme and then increased as resistant microorganisms grew. The shelf life of raw an stored at 10°C can be retarded from 48 hr to 109 hr by the addition of enzyme at a concentration of ■ 0.109%. Differences in microflora between control and the enzyme-treated sample was described. Synergistic effects of glycine, EDTA, acetic acid, and lactic acid with the enzyme were also investigated.     

Intracellular acid carboxypeptidase ofPenicillium roqueforti isolated from blue cheese

We found thatPenicillium roqueforti isolated from a commercial blue cheese produced an acid carboxypeptidase. The acid carboxypeptidase was present in mycelia and little was detected in the liquid medium. The optimum pH for benzyloxycarbonyl-Glu-Tyr was 3.6. The enzyme had the ability to liberate the carboxyterminal amino acid (leucine) of angiotensin I at pH 3.6. Furthermore, the enzyme liberated the carboxyterminal proline from benzyloxycarbonyl-Gly-Pro-Leu-Gly-Pro. The molecular weight of the enzyme determined by gel filtration on Sephadex G-200 was 155,000. The acid carboxypeptidase was inhibited by phenylmethanesulfonyl fluoride and hydrocinnamic acid.