Aqualysin I is an alkaline serine protease which is secreted into the culture medium by Thermus aquaticus YT-1. Aqualysin I was purified, and its apparent relative molecular mass was determined to be 28 500. The enzyme contained four Cys residues (probably as two cystines), and its amino acids composition was similar to those of cysteine-containing serine proteases (proteinase K, etc.) as well as those of subtilisins. The NH,-terminal sequence of aqualysin I showed homology with those of the microbial serine proteases. The optimum pH for the proteolytic activity of aqualysin 1 was around 10.0. Ca2+ stabilized the enzyme to heat treatment, and the maximum proteolytic activity was observed at 80°C. Aqualysin I was stable to denaturing reagents (7 M urea, 6 M guanidine . HCl and 1 YO SDS) at 23 "C for 24 h. The enzyme hydrolyzed the ester bond of an alanine ester and succinyl-Ala-Ala-Ala p-nitroanilide, a synthetic substrate for mammalian elastase. The cleavage sites for aqualysin I in oxidized insulin B chain were not specific when it was digested completely.The enzymes of thermophilic bacteria are generally heatstable [I], and the stability of these proteins is interesting for protein chemistry. Besides their scientific interest, these heatstable enzymes are valuable because of their bioengineering and biotechnological applications. We have studied the enzymes of extremely thermophilic bacteria belonging to the genus Thermus, mainly L-lactate dehydrogenases [2 -51 and proteases [6,7]. Heat-stable proteases are expected to be useful for application [8].At present three Thermus species strains are known which produce heat-stable extracellular proteases. A metal protease (called caldolysin) produced by Thermus T-351 has been purified and characterized [9]. We reported that Thermus caldophilus GK24 produces a neutral serine protease which forms a complex with several kinds of proteins and which appears homogeneous on chromatographic analysis [6]. These two proteases are extremely heat-stable and resistant to denaturing treatments [6, 91. We previously reported the production of two kinds of extracellular proteases, aqualysins I and I1 [7], by Thermus aquaticus YT-1 [lo]. Aqualysin I is an alkaline serine protease, and aqualysin I1 an chelator-sensitive, neutral serine protease, the optimum temperatures for the proteolytic activities of the two enzymes being around 75°C and 95°C respectively [7].
Correspoiidence to H. Matsuzawa, Department of AgriculturalChemistry, The University of Tokyo, Bunkyo-ku, Tokyo, Japan 113Abbreviations. iPr,P-F, diisopropylfluorophosphate; Z-AlaGly-PheCH2C1, benzyloxycarbonyl-Ala-Gly-Phe-chloromethane; CIHgBzOH, p-chloromercuribenzoate; TosPheCH2C1, N-tosylphenylalanylchloromethane; TosLysCH2Cl, Nu-tosyl-L-lysylchloromethane; Suc-Ala-Ala-Ala-NH-Np, succinyl-Ala-Ala-Ala p-nitroanilide; Cya, cysteic acid. Except where otherwise specified, the constituent amino acids were all of the L configuration.Enzymes. Serine proteinase (EC 3.4.21 .-); microbial serine proteinase (EC 3.4.21.14); subtili...
