Subtilisin-like serine protease from hyperthermophilic archaeon Thermococcus kodakaraensis with N- and C-terminal propeptides

Abstract

The genome of the hyperthermophilic archaeon Thermococcus kodakaraensis contains three genes encoding subtilisin-like serine proteases, Tk-1689, Tk-0076 and Tk-subtilisin. Of them, the structure and function of Tk-subtilisin have been extensively studied. To examine whether Tk-1689 is matured to an active form and functions as a hyperthermostable protease as is Tk-subtilisin, the gene encoding the Tk-1689 derivative without a putative N-terminal signal sequence, termed Pro-Tk-SP, was overexpressed in Escherichia coli. Pro-Tk-SP is composed of 640 amino acid residues and its molecular mass is 68.6 kDa. The recombinant protein was purified, however, as an active 44 kDa protease, termed Tk-SP, which lacks the N-terminal 113 and C-terminal 101 amino acid residues. This result suggests that Pro-Tk-SP consists of an N-terminal propeptide (Ala1-Ala113), a mature domain (Tk-SP, Val114-Val539) and a C-terminal propeptide (Asp540-Gly640). Like Tk-subtilisin, Tk-SP showed a broad substrate specificity and was highly thermostable. Its optimum temperature for activity was approximately 100 degrees C and its half-life at 100 degrees C was 100 min. It was fully resistant to treatment with 5% SDS, 8 M urea or 10% Triton X-100. However, unlike Tk-subtilisin and bacterial subtilisins, Tk-SP requires neither Ca2+ nor propeptide for folding. As a result, Tk-SP was fully active even in the presence of 10 mM EDTA. Thus, Tk-SP has a great advantage over other proteases in high resistance to heat, denaturants, detergents and chelating agents and therefore has great potential for application in biotechnology fields.

Proteolysin, a Novel Highly Thermostable and Cosolvent-Compatible Protease from the Thermophilic Bacterium Coprothermobacter proteolyticus

“…Proteolysin can hydrolyze the large globular protein azocasein at temperatures as high as 90°C. The activity toward the synthetic substrate N-suc-AAPF-pNA is comparable to that of other thermophilic subtilases (20,25,26). At elevated temperature (70 to 80°C), the enzyme is stable and displays a higher catalytic activity than at 40°C.…”

“…Samples were added to wells in the agar, and proteolytic activity was scored after overnight incubation at 37°C by inspection for clearing zones. For further analysis, an azocasein assay (20) and an amidolytic assay (37) were employed. For the azocasein assay, an azocasein solution (1.25% [wt/vol] in HEPES · NaOH buffer [100 mM, pH 7.5] containing CaCl 2 [1 mM]) was used as the substrate.…”

“…They are extensively used in food, textile, detergent, pharmaceutical, and leather industries (10,11,12,13,14,15). A few thermostable subtilisin homologs (subtilases) have been isolated and characterized from, for example, Archaea (e.g., pyrolysin [16], stetterlysin [17], pernisine [18], Tk-SP [19], Tk-1689, and Tksubtilisin [20,21]) and from thermophilic bacteria (fervidolysin [22], Ak.1 protease [23], Rt41A proteinase [24,25], aqualysin I [26], islandisin [27], and thermitase [28]). Since cultivation of extremophiles is associated with potential difficulties, cloning and expression of protease genes into a mesophilic host that is easy to grow (Escherichia coli, Bacillus subtilis, and yeast) is of importance for biochemical investigation, protein engineering studies, and practical enzyme production.…”

A Review: Biodegradation and Applications of Keratin Degrading Microorganisms and Keratinolytic Enzymes, Focusing on Thermophiles and Thermostable Serine Proteases

“…Thermococcus kodakaraensis KOD1 was found containing three subtilases: TK-subtilisin, alkaline serine protease (TK-SP) and subtilisin-like Tk-0076 (Tanaka et al, 2007;Foophow et al, 2010). The Tk-subtilisin and alkaline serine protease exhibited optimum enzymatic activity at 90°C and 100°C and halflives at 100°C of 50 min and 100 min, respectively.…”

Autocatalytic Maturation of the Tat-Dependent Halophilic Subtilase Nep Produced by the Archaeon Natrialba magadii

“…Comparatively less is known regarding how extracellular proteases are transported through the archaeal membrane and processed into fully active enzymes. In this context, the maturation mechanism of the Sec-dependent subtilisin-like proteases secreted by the hyperthermophilic archaeon Thermococcus kodaraensis have been extensively characterized (8,33). Many halophilic bacteria and archaea produce extracellular proteases, however, there is limited information on the pathways used by halophiles to deliver secretory proteases (27,28), and the mechanism that leads to enzyme activation has not been examined.…”