Biochemical characterisation of a novel broad pH spectrum subtilisin from Fictibacillus arsenicus DSM 15822T

Abstract

Subtilisins from microbial sources, especially from the Bacillaceae family, are of particular interest for biotechnological applications and serve the currently growing enzyme market as efficient and novel biocatalysts. Biotechnological applications include use in detergents, cosmetics, leather processing, wastewater treatment and pharmaceuticals. To identify a possible candidate for the enzyme market, here we cloned the gene of the subtilisin SPFA from Fictibacillus arsenicus DSM 15822^T (obtained through a data mining-based search) and expressed it in Bacillus subtilis DB104. After production and purification, the protease showed a molecular mass of 27.57 kDa and a pI of 5.8. SPFA displayed hydrolytic activity at a temperature optimum of 80 °C and a very broad pH optimum between 8.5 and 11.5, with high activity up to pH 12.5. SPFA displayed no NaCl dependence but a high NaCl tolerance, with decreasing activity up to concentrations of 5 m NaCl. The stability enhanced with increasing NaCl concentration. Based on its substrate preference for 10 synthetic peptide 4-nitroanilide substrates with three or four amino acids and its phylogenetic classification, SPFA can be assigned to the subgroup of true subtilisins. Moreover, SPFA exhibited high tolerance to 5% (w/v) SDS and 5% H₂ O₂ (v/v). The biochemical properties of SPFA, especially its tolerance of remarkably high pH, SDS and H₂ O₂ , suggest it has potential for biotechnological applications.

Keywords: Bacillaceae; biotechnological application; broad pH spectrum; subtilases; subtilisin.

Fig. 1

Multiple sequence alignment (MSA) of SPFA with Savinase (WP_094423791.1), subtilisin Carlsberg (WP_020450819.1) and BPN’ (WP_013351733.1). clustal omega was used for the alignment [59]. ESPript 3.0 with Savinase (PDB: 1C9J) as a template was used to analyse the MSA [60]. Signal peptide sequence (green bars); propeptide (blue bars) of SPFA. Red bars indicate individual signal peptide cleavage sites. Secondary structure elements: helices with squiggles, β‐strands with arrows and turns with TT letters. The catalytic triad (Asp¹⁴³, His¹⁷³, Ser³²⁶; Savinase numbering) is marked with orange boxes.

Fig. 2

Structural model of SPFA with its calculated surface electrostatic potential. (Left) top view of the active site; (right) rear view to the active site. Swiss‐PdbViewer was used to calculate the electrostatic potential at pH 7.0 and negative charges (red), positive (blue) and neutral (white) are shown.

Fig. 3

SDS/PAGE analysis of recombinant SPFA. Electrophoresis was performed using an 8–20% SDS polyacrylamide gel. Bio‐Rad Precision Plus Dual Color length marker (LM); culture supernatant of B. subtilis DB104 carrying pFF‐RED (1); culture supernatant of B. subtilis DB104 carrying pFF producing SPFA (2), after purification (3).

Fig. 4

Influence of pH on the activity and stability of purified SPFA. Enzyme activity was determined using the suc‐AAPF‐pNA assay at 30 °C in a pH range of 5.0–12.5 (closed circles). The average maximum activity was considered as 100%: 64 U·mg⁻¹. The effect of pH on the stability of purified SPFA (squares). The residual activity was measured with the standard suc‐AAPF‐pNA assay after incubation for 24 h at 4 °C in Tris‐maleate buffer (pH 5–7), in Tris–HCl (pH 7–9) and in glycine‐NaOH (pH 9–12). The activity at 0 h was considered as 100%; highest residual activity: 106 U·mg⁻¹. Experiments were performed in triplicate, and data are presented as means ± SD.

Fig. 5

Effect of temperature on the activity (A) and stability (B) of purified SPFA. Enzyme activity was analysed at temperatures between 20 and 90 °C using the suc‐AAPF‐assay. The maximum activity was defined as 100%: 272 U·mg⁻¹. * Enzyme stability did not last the intended 5 min. Stability was examined at 20 and 50 °C in 10 mm HEPES‐NaOH buffer, pH 8.0. Residual activity was measured using the suc‐AAPF pNA assay at 30 °C. The activity at 0 min was set at 100%: 85 U·mg⁻¹. Experiments were performed in triplicate, and data are plotted as mean values ± SD.

Fig. 6

Activity and stability of purified SPFA at different NaCl concentrations. Activity was measured with the suc‐AAPF‐pNA assay in standard buffer (pH 8.6) at 30 °C with different NaCl concentrations (0–5 m). Maximum activity was defined as 100%: 104 U·mg⁻¹. Stability was tested in 10 mm HEPES‐NaOH buffer, pH 8.0, with NaCl (0–5 m). The residual activity was measured with the suc‐AAPF‐pNA assay in standard buffer at pH 8.6 after incubation for 2 h at 20 °C. The activity before incubation for each NaCl concentration was defined as 100%. The experiments were performed in triplicate, and data are displayed as means ± SD.