Improvement of the halotolerance of a Bacillus serine protease by protein surface engineering

Abstract

A moderately halotolerant serine protease was previously isolated from Bacillus subtilis from salted, fermented food. Eight mutation sites on the protein surface were selected for protein engineering based on sequence and structural comparisons with moderately halotolerant proteases and homologous non-halotolerant proteases. The newly constructed multiple mutants with substituted Asp and Arg residues were compared with the recombinant wild type (rApr) and the previously constructed mAla-8 substituted with Ala to analyze the contribution of protein surface charge to the salt adaptation of the protease. The three mutants showed >1.2-fold greater halotolerance than rApr. In addition, the mutants showed a broader range of pH stability than rApr, retaining >80% of their maximum activity in the pH range 5.0-11. The mutants also retained >75% of their activity after incubation for 1 h at pH 8.0 and 55°C or at pH 11.5 and 25°C. The Asp and Arg residues exchanged by multiple substitution probably played a role in increasing protein surface hydration and solubility in high salt conditions. This study illustrated that increasing a high proportion of the negative or positive charge on the surface of the Bacillus serine protease stably improved the protein's salt adaptation.

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

“…BPN' has five of the seven amino acid positions identified by Takenaka et al. [ 86 ] as favorable for salt adaptation and therefore, unlike subtilisin Carlsberg, shows good adaptation to high salt concentrations.…”

“…4), and in general, a high proportion of negative or positive charges on the surface of the enzyme improves salt adaptation [86]. BPN' has five of the seven amino acid positions identified by Takenaka et al [86] as favorable for salt adaptation and therefore, unlike subtilisin Carlsberg, shows good adaptation to high salt concentrations.…”

“…However, the high Arg content of SPAO and Savinase, which favors adaptation to highly alkaline conditions, results in a predominantly positive charge on the protein surface in SPAO (Fig. 4), and in general, a high proportion of negative or positive charges on the surface of the enzyme improves salt adaptation [86]. BPN' has five of the seven amino acid positions identified by Takenaka et al [86] as favorable for salt adaptation and therefore, unlike subtilisin Carlsberg, shows good adaptation to high salt concentrations.…”

New robust subtilisins from halotolerant and halophilic Bacillaceae

“…In contrast to the true subtilisins studied here, BPN′, which belongs to the same subgroup, showed higher activity with increasing NaCl levels (Falkenberg et al 2022b ). This is probably because BPN′ has five of the seven amino acid positions identified that are beneficial for salt adaptation (Takenaka et al 2022 ). The finding that SPAH lost more activity at higher NaCl concentrations could be related to the constant activity at high salt concentrations and thus a higher autoproteolytic activity, which was also observed for subtilisin Carlsberg (Falkenberg et al 2022b ).…”

“…3 ). As mentioned above, salt adaptation is increased by a high number of negative or positive charges on the surface of the enzyme (Takenaka et al 2022 ). The differences in the AB ratio of all residues seems to have no influence on the salt tolerance, as the AB ratio of SPPM (1.9) and SPAH (1.7) is quite high, but much lower for SPMI (1.0) and SPLA (1.1) (Fig.…”