A Novel Thermostable and Alkaline Protease Produced from Bacillus stearothermophilus Isolated from Olive Oil Mill Sols Suitable to Industrial Biotechnology

Abstract

This study was conducted to identify a new alkaline and thermophilic protease (Ba.St.Pr) produced from Bacillus stearothermophilus isolated from olive oil mill sols and to evaluate its culture conditions, including temperature, pH, carbon and nitrogen sources, and incubation time. The optimum culture conditions for cell growth (10 g/L) and protease production (5050 U/mL) were as follows: temperature 55 °C, pH 10, inoculation density 8 × 10⁸ CFU/mL, and incubation time 24 h. The use of 3% yeast extract as the nitrogen sources and galactose (7.5 g/L) as the carbon sources enhanced both cell growth and protease production. Using reversed-phase analytical HPLC on C-8 column, the new protease was purified with a molecular mass of approximately 28 kDa. The N-terminal sequence of Ba.St.Pr exhibited a high level of identity of approximately 95% with those of Bacillus strains. Characterization under extreme conditions revealed a novel thermostable and alkaline protease with a half-life time of 187 min when incubated with combined Ca²⁺/mannitol. Ba.St.Pr demonstrated a higher stability in the presence of surfactant, solvent, and Ca²⁺ ions. Consequently, all the evaluated activity parameters highlighted the promising properties of this bacterium for industrial and biotechnological applications.

Keywords: Bacillus stearothermophilus; characterization; protease; thermostable.

Figure 1

Effects of (A) temperature and (B) pH on growth (triangles) and protease production (bars) from Bacillus stearothermophilus. Data are expressed as mean (n = 3) ± SD.

Figure 2

Effects of carbon source (A) and galactose concentration (B) on growth (triangles) and protease production (bars) from Bacillus stearothermophilus. Data are expressed as mean (n = 3) ± SD.

Figure 3

Effects of nitrogen source (A) and selected yeast extract concentration (B) on growth (triangles) and protease production (bars) from Bacillus stearothermophilus. Data are expressed as mean (n = 3) ± SD.

Figure 4

Effect of incubation time (A) on growth (white squares) and protease activity (white circles) and effect of inoculum size (B) on protease activity; (green line; 10⁸ CFU/mL) (black line; 2 × 10⁸ CFU/mL) (blue line; 4 × 10⁸ CFU/mL) (purple line; 8 × 10⁸ CFU/mL).

Figure 5

(A) Reversed-phase analytical HPLC on C8 matrix. Reversed-phase (RP)-HPLC eurospher 100, C-8 column (250 × 4.6 mm), was equilibrated in water. Protein elution was performed with an acetonitrile linear gradient (0–80%) at a flow rate of 1 mL/min over 60 min. 20 µL of protease (2 mg/mL). (B) SDS-PAGE of pure Ba.St.Pr (10 µg). (C) Local alignment of the Ba.St.Pr N-terminal sequence (present work) with the highest identity protease sequences from Bacillus strains. Identical amino acids in bold and different amino acids are in red.

Figure 6

Evaluation of pH effect on the activity (white squares) and stability (black squares) of Ba.St.Pr. (A). Effect of alkali pH on Ba.St.Pr stability at various incubation times from 0 to 36 h (B). Evaluation of the effect of temperature on protease activity without Ca²⁺ ions (white squares) and with Ca²⁺ ions (black squares) of Ba.St.Pr. (C). Effect of extreme temperatures on Ba.St.Pr stability at various incubation times from 0 to 10 h (D).

Figure 7

Effects of surfactants (A), oxidizing agents (B), organic solvents (C), and divalent ions (D) on Ba.St.Pr stability. The enzyme was incubated with the appropriate agent for 1 h, and the residual activity was measured at the optimal conditions. A commercial Alcalase 2.5 L, type DX, was used as a positive control.

Figure 8

Stability and compatibility of Ba.St.Pr with solid (A) and liquid (B) commercial laundry detergents compared with commercial Alcalase 2.5 L measured under the same conditions.