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

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 degrees C, optimally at pH 9.0-9.5 and 55 degrees C. The protease is highly oxidatively stable to hydrogen peroxide and retained 58% of residual activity when incubated at 10 degrees C with 5% (v/v) H2O2 for 1 h while stimulated at 1% (v/v) H2O2. 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.

Automated summary

The study characterized a high-alkaline subtilisin and found that it retains good activity under high salt concentrations and high temperatures. It also demonstrated high oxidative stability to hydrogen peroxide, making it a promising candidate for biotechnological applications.