A serine proteinase of an archaebacterium, Halobacterium mediterranei. A homologue of eubacterial subtilisins

Abstract

A homogeneous serine proteinase secreted by the extreme halophilic bacterium Halobacterium mediterranei 1538 was isolated by affinity chromatography on bacitracin-Sepharose with a yield of 48% (260-fold purification). The enzyme reveals an optimum for pyroglutamyl-Ala-Ala-Leu p-nitroanilide hydrolysis at pH 8.0-8.5 (Km 0.14 mM; k(cat). 36.9 s-1). Its activity increases linearly with NaCl concentration over the range 2-5 M. The substrate specificity of the enzyme is comparable with that of secretory subtilisins, the extent of protein degradation approaching that attained with proteinase K. The enzyme has a molecular mass of 41 kDa and a pI of 7.5. The N-terminal sequence of H. mediterranei serine proteinase reveals a 50% identity with that of Thermoactinomyces vulgaris serine proteinases, indicating that the enzyme belongs to the subtilisin family. Hence the serine proteinase secreted by the halophilic bacterium should be considered as a functional analogue, and a structural homologue, of eubacterial serine proteinases (subtilisins).

Purification and biochemical characterization of the haloalkaliphilic archaeonNatronococcus occultus extracellular serine protease

“…Several extracellular proteases have been purified and characterized from haloneutrophilic halobacteria (IZOTOVA et al 1989, KAMEKURA and SENO 1990, KAMEKURA et al 1992, SCHMITT et al 1990, STEPANOV et al 1992, KAMEKURA et al 1996. However, at present, only two secretory proteases have been studied from the haloakaliphilic group: a serine protease from strain A2 (unidentified but presumably from the genus Natronobacterium) partiallly purified and characterized by YU et al (1990) and a serine protease from Natrialba magadii purified and characterized in our laboratory (GIMENEZ et al 2000).…”

“…A preliminary characterization of the proteolytic activity present in the ethanol-precipitated culture medium was performed in our laboratory N. occultus EP was purified 328-fold with a total yield of 19% by ethanol precipitation and bacitracin-Sepharose affinity chromatography (Table 1). Like other extracellular proteolytic enzymes so far purified from halobacteria (IZOTOVA et al 1983, KAMEKURA and SENO 1990, SCHMITT et al 1990, YU 1991, KAMEKURA et al 1992, STEPANOV et al 1992 N. occultus EP is a serine protease (inhibited by PMSF and chymostatin, Table 2), is dependent on high salt concentrations for activity and stability (Fig. 1) and is rather thermophilic, showing maximal activity at 60 °C in 2 M Na (Fig.…”

Antagonistic Bacillus cereus TC-1 Isolated from Solar Salt Work in Southern India

“…Similar results have also been reflected by the haloalkalophilic archaeon, Natronococcus occultus in which protease secretion was optimum at 1-2 M NaCl 21 . However, in the case of the archaebacterium Halobacterium mediterranei, a much higher salt requirement (25%, w/v) for serine protease secretion was reported 22 .…”

Molecular Cloning and Sequence Analysis of Two Distinct Halotolerant Extracellular Proteases fromBacillus subtilisFP-133

“…It has been documented that a remarkably high number of Asp and Glu and/or Asn and Gln residues are present on the surfaces of halophilic bacterial proteins, including proteases, and the negative charge is thought to enable the stabilization of specific-water and/or ion-binding required to establish the proper tertiary or quaternary structure. 18,19) The negative charge also prevents protein aggregation and leads to higher efficiency in protein refolding. 19) Negatively charged amino acid residues are found predominately, for example, in the mature zincmetalloprotease of Salinivibrio sp.…”