Purification and characterization of a thermostable, haloalkaliphilic extracellular serine protease from the extreme halophilic archaeon Halogeometricum borinquense strain TSS101

Abstract

A novel haloalkaliphilic, thermostable serine protease was purified from the extreme halophilic archaeon, Halogeometricum borinquense strain TSS101. The protease was isolated from a stationary phase culture, purified 116-fold with 18% yield and characterized biochemically. The molecular mass of the purified enzyme was estimated to be 86 kDa. The enzyme showed the highest activity at 60 degrees C and pH 10.0 in 20% NaCl. The enzyme had high activity over the pH range from 6.0 to 10.0. Enzymatic activity was strongly inhibited by 1 mM phenyl methylsulfonyl fluoride, but activity was increased 59% by 0.1% cetyltrimethylammonium bromide. The enzyme exhibited relatively high thermal stability, retaining 80% of its activity after 1 h at 90 degrees C. Thermostability increased in the presence of Ca2+. The stability of the enzyme was maintained in 10% sucrose and in the absence of NaCl.

Figure 1.

The sodium dodecyl sulfate polyacrylamide gel electrophoresis of the purified protease from Halogeometricum borinquense strain TSS101. Lane A shows relative molecular mass standards: phosphorylase b (97.4 kDa); bovine serum albumin-(68 kDa); ovalbumin (43 kDa); carbonic anhydrase (29 kDa); soyabean trypsin inhibitor (20 kDa); and lysozyme (14.3 kDa). Lane B shows the purified protease (86 kDa, indicated by arrow).

Figure 2.

Activity of purified protease from Halogeometricum borinquense strain TSS101 at different pH values. Azocaseinolytic activity was measured at 60 °C in the presence of 20% NaCl in the indicated buffers at a concentration of 0.1 M. Each value represents the mean ± SE of three independent experiments.

Figure 3.

Effect of temperature on purified protease activity. Azocaseinolytic activity was determined in 0.1 M glycine-NaOH buffer (pH 10.0) with 20% NaCl at each temperature after incubation for 15 min with 100 mM Ca²⁺ (open squares) and without 100 mM Ca²⁺ (filled triangles). Each value represents the mean ± SE of three independent experiments.

Figure 4.

Effect of temperature on the stability of purified protease. The enzyme in glycine-NaOH buffer (pH 10.0) with 100 mM Ca²⁺ (open squares) and without 100 mM Ca²⁺ (open triangles) was preincubated for 1 h at the specified temperatures. The residual enzymatic activity was measured under the standard conditions. Each value represents the mean ± SE of three independent experiments.

Figure 5.

Effect of NaCl concentration on purified protease activity. Azocaseinolytic activity of protease was determined in 0.1 M glycine-NaOH buffer (pH 10.0) at 60 °C in the presence of the indicated concentrations of NaCl, after incubation for 15 min. Each point represents the mean ± SE of three independent experiments.

Figure 6.

Effect of Ca²⁺ on purified protease activity. The enzyme was incubated with different concentrations of Ca²⁺ at 60 °C, 20% NaCl and pH 10.0, and the relative activity was measured. Each value represents the mean ± SE of three independent experiments.

Figure 7.

Effects of osmolytes on the purified protease activity in the absence of NaCl. The enzyme was incubated in the various concentrations of osmolytes: sucrose (open diamonds); mannitol (fillled triangles); glycerol (open triangles); and betaine (filled squares) without NaCl in standard assay conditions. Each value represents the mean ± SE of three independent experiments.