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.2022 Oct;12(10):1729-1746.
doi: 10.1002/2211-5463.13457. Epub 2022 Jul 6.

Biochemical characterization of a novel oxidatively stable, halotolerant, and high-alkaline subtilisin from Alkalihalobacillus okhensis Kh10-101T

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Biochemical characterization of a novel oxidatively stable, halotolerant, and high-alkaline subtilisin from Alkalihalobacillus okhensis Kh10-101T

Fabian Falkenberg et al. FEBS Open Bio.2022 Oct.

Abstract

Halophilic and halotolerant microorganisms represent a promising source of salt-tolerant enzymes suitable for various biotechnological applications where high salt concentrations would otherwise limit enzymatic activity. Considering the current growing enzyme market and the need for more efficient and new biocatalysts, the present study aimed at the characterization of a high-alkaline subtilisin from Alkalihalobacillus okhensis Kh10-101T . The protease gene was cloned and expressed in Bacillus subtilis DB104. The recombinant protease SPAO with 269 amino acids belongs to the subfamily of high-alkaline subtilisins. The biochemical characteristics of purified SPAO were analyzed in comparison with subtilisin Carlsberg, Savinase, and BPN'. SPAO, a monomer with a molecular mass of 27.1 kDa, was active over a wide range of pH 6.0-12.0 and temperature 20-80 °C, optimally at pH 9.0-9.5 and 55 °C. The protease is highly oxidatively stable to hydrogen peroxide and retained 58% of residual activity when incubated at 10 °C with 5% (v/v) H2 O2 for 1 h while stimulated at 1% (v/v) H2 O2 . Furthermore, SPAO was very stable and active at NaCl concentrations up to 5.0 m. This study demonstrates the potential of SPAO for biotechnological applications in the future.

Keywords: Alkalihalobacillus okhensis; detergent protease; halotolerant protease; high-alkaline subtilisin; oxidative stable protease.

© 2022 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1
Fig. 1
Evolutionary phylogenetic tree of various subtilisins from different species of the familyBacillaceae. Maximum‐likelihood phylogenetic analysis of the mature protease domains was performed using thePhylogeny.fr server. Numbers at nodes indicate support for the internal branches within the tree obtained by approximate likelihood ratio test (SH‐like aLRT).
Fig. 2
Fig. 2
Multiple sequence alignment (MSA) of SPAO with Savinase (WP_094423791.1), subtilisin Carlsberg (WP_020450819.1), and BPN' (WP_013351733.1). The alignment was calculated byclustal omega and drawn using ESPript 3.0 and Savinase (PDB:1C9J) as a template. Solid green and blue bars indicate the signal peptide sequence and propeptide of SPAO. Secondary structure elements are presented on top (helices with squiggles, β‐strands with arrows, and turns with TT letters). Orange boxes show residues comprising the catalytic triad (Asp145, His175, Ser328; SPAO numbering).
Fig. 3
Fig. 3
Homology model of mature SPAO usingi‐tasser software.In silico metal‐binding analysis predicted the existence of two Ca2+ binding sites (yellow balls). The catalytic residues Asp32, His62, and Ser215 are shown in red.
Fig. 4
Fig. 4
Protein surface electrostatic potential calculations for the structural model of SPAO. (Left) front view of the active site; (right) back of the active site. Electrostatic potential at pH 7.0 is shown as red (negative) and blue (positive) and was calculated using the Swiss‐PdbViewer.
Fig. 5
Fig. 5
SDS/PAGE and IEF‐PAGE analysis of recombinant SPAO. Samples were electrophoresed using an 8–20% SDS/polyacrylamide gel. As protein standards, the LM mixture Bio‐Rad Precision Plus Dual Color was used. Culture supernatant ofB. subtilis DB104 carrying pFF‐RED (lane 1); culture supernatant ofB. subtilis DB104 carrying pFF003 producing SPAO (lane 2); and SPAO after purification (lane 3). Purified and desalted SPAO on an IEF gel SERVALYT™ PRECOTES™ wide range pH 3–10 (lane 4) and marker (M; SERVA IEF marker 3–10).
Fig. 6
Fig. 6
Melting curves of purified SPAO in comparison with BPN' and subtilisin Carlsberg. The effect of temperature on the stability of the enzyme using SYPRO® Orange as a fluorescence probe, based on the changes in fluorescence emission intensity (Ex/Em = 470/550 nm; 5 × SYPRO® Orange, 10 mm HEPES/NaOH, pH 8.0, 3 mm PMSF), is shown as normalized denaturation curves of the thermal shift assay for the proteases SPAO (closed circles), BPN' (squares), and subtilisin Carlsberg (open circles). The inflection point corresponds to the melting temperature (Tm), at which 50% of the protein is unfolded (−). The experiment was performed in triplicates, and data are plotted as mean values ± SD.
Fig. 7
Fig. 7
Effect of temperature on the activity of purified SPAO, BPN', Savinase, and subtilisin Carlsberg. The activities of the proteases were determined by assaying protease activity at temperatures between 20 and 90 °C with suc‐AAPF‐pNA assay. The maximum activity for each protease was considered as 100% activity; SPAO (closed circles; 1076 U·mg−1), BPN' (squares; 1007 U·mg−1), Savinase (triangles; 2264 U·mg−1), and subtilisin Carlsberg (open circles; 2767 U·mg−1). *The enzyme was not stable for the intended 5 min. The experiment was performed in triplicates, and data are plotted as mean values ± SD.
Fig. 8
Fig. 8
Temperature stability of purified SPAO, BPN', Savinase, and subtilisin Carlsberg. Stability was investigated at 20 °C in 10 mm HEPES/NaOH buffer, pH 8.0. The activity was measured with the suc‐AAPF‐pNA assay in reaction buffer at 30 °C. The activity at 0 min was considered as 100% activity; SPAO (closed circles; 325 U·mg−1), BPN' (squares; 343 U·mg−1), Savinase (triangles; 367 U·mg−1), and subtilisin Carlsberg (open circles; 635 U·mg−1). The experiment was performed in triplicates, and data are plotted as mean values ± SD.
Fig. 9
Fig. 9
Effect of pH on the activity of purified SPAO, BPN', Savinase, and subtilisin Carlsberg. The activity was measured with the suc‐AAPF‐pNA assay at 30 °C in the pH range of 5.0–12.0. The maximum activity for each protease was considered as 100% activity; SPAO (closed circles; 358 U·mg−1), BPN' (squares; 604 U·mg−1), Savinase (triangles; 705 U·mg−1), and subtilisin Carlsberg (open circles; 1193 U·mg−1). The experiments were performed in triplicates, and data are plotted as mean values ± SD.
Fig. 10
Fig. 10
Effect of NaCl on the activity of the purified SPAO, BPN', Savinase, and subtilisin Carlsberg. The activity was measured in standard buffer (pH 8.6) for suc‐AAPF‐pNA assay at 30 °C with different NaCl concentrations of 0–5 m. The maximum activity for each protease was considered as 100% activity; SPAO (closed circles; 1205 U·mg−1), BPN' (squares; 560 U·mg−1), Savinase (triangles; 757 U·mg−1), and subtilisin Carlsberg (open circles; 846 U·mg−1). The experiment was performed in triplicates, and data are given as mean values ± SD.
Fig. 11
Fig. 11
Stability against NaCl of purified SPAO, BPN', Savinase, and subtilisin Carlsberg. Stability was tested in 10 mm HEPES/NaOH buffer, pH 8.0, with different NaCl concentrations (0–5 m) after 2 h at 20 °C. The activity was measured with the suc‐AAPF‐pNA assay in standard buffer at pH 8.6. Activity at 0 h for each NaCl concentration was considered as 100% activity; SPAO (circles), BPN' (squares), Savinase (triangles), and subtilisin Carlsberg (open circles). The experiment was performed in triplicates, and data are given as mean values ± SD.
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