Biochemical characterization of a novel oxidatively stable, halotolerant, and high‐alkaline subtilisin from Alkalihalobacillus okhensisKh10‐101^T

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 Kh10‐101 T . 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) H 2 O 2 for 1 h while stimulated at 1% (v/v) H 2 O 2 . 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.

Phylogenetic survey of the subtilase family and a data-mining-based search for new subtilisins from Bacillaceae

“…Some of the high-alkaline proteases including Savinase have an extra proline at position 131, which provides extra active-site rigidity compared with other subtilisins ( Betzel et al, 1996 ). Recently, we reportet about SPAO from Alkalihalobacillus okhensis Kh10-101 T , which showed high stability against hydrogen peroxide and NaCl concentrations up to 5.0 M ( Falkenberg et al, 2022 ). SPAO can be assigned here to the holotype subtilisin sendai (S08.098) ( Figure 3 ).…”

“…KP-43 (S08.123) from Bacillus strain KSM-KP43 and the sequences within this clade form the subgroup of oxidatively stable serine proteases (OSPs) as described by Saeki et al, with a sequence identity of 92% (Saeki et al, 2000(Saeki et al, , 2002. However, it should be mentioned that subtilisins not belonging to this subgroup were reported to have higher stability against H 2 O 2 (Joshi and Satyanarayana, 2013;Falkenberg et al, 2022). The C-terminal half of KSM-KP43 downstream of the putative catalytic residue, Ser-255, is homologous to the internal segments of TagC (Saeki et al, 2002).…”

Chaperone assisted recombinant expression of a mycobacterial aminoacylase in Vibrio natriegens and Escherichia coli capable of N-lauroyl-L-amino acid synthesis

“…With the thermal shift assay, thermal denaturation of the investigated protein was accessed as previously described [ 48 ]. The fluorogenic dye Sypro orange was mixed with the protein and heated stepwise.…”

New robust subtilisins from halotolerant and halophilic Bacillaceae

“…However, extracellular subtilisins of microbial background are mainly involved in nutrient supply and are therefore directly exposed to the environmental conditions (Kalisz 1988 ). Therefore, extracellular enzymes isolated from microorganisms found in environments with extreme pH, temperature and especially salinity offer huge potential to meet the needs of industry, as shown by the growing number of newly characterised subtilisins with polyextremophilic properties (Salwan and Sharma 2019 ; Alberto Cira-Chávez et al 2019 ; Coker 2016 ; Falkenberg et al 2022b ; Mokashe et al 2018 ). Besides the labour-intensive search for microorganisms harbouring new enzymes in such extreme environments, genome sequencing and automatic annotation offer an alternative way to search for new protease genes for industrial purposes.…”

“…Within a phylogenetic tree, these sequences were categorised to the subgroups true subtilisins, PIS and high-alkaline subtilisins. We reported about SPAO from Alkalihalobacillus okhensis Kh10-101 T , which has a high stability towards H 2 O 2 and NaCl concentrations of up to 5.0 M and belongs to the subgroup of high-alkaline subtilisins (Falkenberg et al 2022b ). Here, we selected three sequences from the phylogenetic tree of true subtilisins and one sequence from the phylogenetically intermediate subtilisins obtained from halotolerant or halophilic bacteria for biochemical characterisation.…”