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Characterization and mutation analysis of a halotolerant serine protease from a new isolate of Bacillus subtilis
Shinji Takenaka1, Jyun Yoshinami2, Ampin Kuntiya3
1Department of Agrobioscience, Graduate School of Agricultural Science, Kobe University, 1-1 Rokkodai, Nada-ku, Kobe, 657-8501, Japan. stakenaka@people.kobe-u.ac.jp.
Objectives:
A bacterial halotolerant enzyme was characterized to understand the molecular mechanism of salt adaptation and to explore its protein engineering potential.
Results:
Halotolerant serine protease (Apr_No16) from a newly isolated Bacillus subtilis strain no. 16 was characterized. Multiple alignments with previously reported non-halotolerant proteases, including subtilisin Carlsberg, indicated that Apr_No16 has eight acidic or polar amino acid residues that are replaced by nonpolar amino acids in non-halotolerant proteases. Those residues were hypothesized to be one of the primary contributors to salt adaptation. An eightfold mutant substituted with Ala residues exhibited 1.2- and 1.8-fold greater halotolerance at 12.5% (w/v) NaCl than Apr_No16 and Carlsberg, respectively. Amino acid substitution notably shifted the theoretical pI of the eightfold mutant, from 6.33 to 9.23, compared with Apr_No16. The resulting protein better tolerated high salt conditions.
Conclusions:
Changing the pI of a bacterial serine protease may be an effective strategy to improve the enzyme's halotolerance.