Engineering subtilisin E for enhanced stability and activity in polar organic solvents.
We examined the effect of a novel disulfide bond engineered in subtilisin E from Bacillus subtilis based on the structure of a thermophilic subtilisin-type serine protease aqualysin I. Four sites (Ser163/Ser194, Lys170/Ser194, Lys170/Glu195, and Pro172/Glu195) in subtilisin E were chosen as candidates for Cys substitutions by site-directed mutagenesis. The Cys170/Cys195 mutant subtilisin formed a disulfide bond in B. subtilis, and showed a 5-10-fold increase in specific activity for an authentic peptide substrate for subtilisin, N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide, compared with the single-Cys mutants. However, the disulfide mutant had a 50% decrease in catalytic efficiency due to a smaller k(cat) and was thermolabile relative to the wild-type enzyme, whereas it was greatly stabilized relative to its reduced form. These results suggest that an electrostatic interaction between Lys170 and Glu195 is important for catalysis and stability in subtilisin E. Interestingly, the disulfide mutant was found to be more stable in polar organic solvents, such as dimethylformamide and ethanol, than the wild-type enzyme, even under reducing conditions; this is probably due to the substitution of uncharged Cys by charged surface residues (Lys170 and Glu195). Further, the amino-terminal engineered disulfide bond (Gly61Cys/Ser98Cys) and the mutation Ile31Leu were introduced to enhance the stability and catalytic activity. A prominent 3-4-fold increase in the catalytic efficiency occurred in the quintet mutant enzyme over the range of dimethylformamide concentration (up to 40%).
Takagi
H
H
Department of Bioscience, Fukui Prefectural University, Kenjojima, Matsuoka-cho, Fukui 910-1195, Japan. hiro@fpu.ac.jp
Hirai
K
K
Maeda
Y
Y
Matsuzawa
H
H
Nakamori
S
S
D001412:Bacillus subtilis / Q000201:enzymology*; D004126:Dimethylformamide; D004220:Disulfides / Q000737:chemistry; D004795:Enzyme Stability / Q000235:genetics; D000431:Ethanol; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000378:metabolism; D015202:Protein Engineering; D012997:Solvents; D055672:Static Electricity; D013381:Subtilisins / Q000235:genetics* / Q000378:metabolism
D004220:Disulfides; D010455:Peptides; D012997:Solvents; D000431:Ethanol; D004126:Dimethylformamide; D013381:Subtilisins