Enhanced salt-tolerance of Bacillus subtilis glutaminase by fusing self-assembling amphipathic peptides at its N-terminus

Abstract

Glutaminase (EC 3.5.1.2) can catalyze the deamidation of glutamine, which has been used to improve umami taste in oriental fermented foods. However, a high salt concentration is still a fundamental challenge for glutaminase application, especially in soy sauce production. To improve the salt tolerance of glutaminase, the self-assembling amphiphilic peptides EAK16 and ELK16 were fused to the N-terminus of a mutant (E3C/E55F/D213T) derived from Bacillus subtilis glutaminase, yielding the fusion enzymes EAK16-E3C/E55F/D213T and ELK16-E3C/E55F/D213T, respectively. As ELK16-E3C/E55F/D213T was expressed as insoluble active inclusion bodies, only the purified EAK16-E3C/E55F/D213T was subjected to further analyses. After the incubation with 18% (w/v) NaCl for 200 min, the residual activities of EAK16-E3C/E55F/D213T in a NaCl-free solution reached 43.6%, while E3C/E55F/D213T was completely inactivated. When the enzyme reaction was conducted in the presence of 20% NaCl, the relative activity of EAK16-E3C/E55F/D213T was 0.47-fold higher than that of E3C/E55F/D213T. As protein surface hydrophobicity and protein particle size analysis suggested, oligomerization may play an important role in the salt-tolerance enhancement of the fusions. Furthermore, EAK16-E3C/E55F/D213T achieved a 0.88-fold increase in the titer of glutamic acid in a model system of soy sauce fermentation compared to E3C/E55F/D213T. Therefore, the fusion with self-assembling amphiphilic peptides is an efficient strategy to improve the salt-tolerance of glutaminase.

Sequence-Structure Based Comparison of Structurally Homologous Thermophilic and Mesophilic Polyethylene Terephthalate (PET) Hydrolases

“…Amino acid preference, the ratio of charged versus uncharged amino acids, ionic interactions, codon use, hydrophobicity, and protein surface area are examples of these (Zhou et al 2008). Even though the enzymes used as examples originate from hyperthermophilic sources (Vieille and Zeikus 2001), to date, the publication written two decades ago has become guidance for scientists conducting protein engineering Van Wyk et al 2022;Liu et al 2022;Srivastava et al 2023). Since there, numerous engineering methods to obtain thermostable enzymes or other purposes have been documented (Wang et al 2006;Shirke et al 2018;Khersonsky et al 2018;Noda-Garcia et al 2018;Deng et al 2023).…”