Cumulative stabilizing effects of glycine to alanine substitutions in Bacillus subtilis neutral protease |
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Authors: | Margarit Immaculada; Camagnoli Susanna; Frigerio Francesco; Grandi Guido; Filippis Vincenzo De; Fontana Angelo |
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Affiliation: | Laboratory of Genetic Engineering and Microbiology Enincerche SpA, San Donato Milanese, Milan
1Department of Organic Chemistry and CRIBI Biotechnology Centre, University of Padua Padua, Italy |
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Abstract: | Oligonucleotide-directed mutagenesis has been used to replaceglycine residues by alanine in neutral protease from Bacillussubtilis. One Gly to Ala substitution (G147A) was located ina helical region of the protein, while the other (G189A) wasin a loop. The effects of mutational substitutions on the functional,conformational and stability properties of the enzyme have beeninvestigated using enzymatic assays and spectroscopic measurements.Single substitutions of both G1y147 and Gly189 with Ala residuesaffect the enzyme kinetic properties using synthetic peptidesas substrates. When Gly replacements were concurrently introducedat both positions, the kinetic characteristics of the doublemutant were roughly intermediate between those of the two singlemutants, and similar to those of the wild-type protease. Bothmutants G147A and G189A were found to be more stable towardsirreversible thermal inactivation/unfolding than the wild-typespecies. Moreover, the stabilizing effect of the Gly to Alasubstitution was roughly additive in the double mutant G147A/G189A,which shows a 3.2°C increase in Tm with respect to the wild-typeprotein. These findings indicate that the Gly to Ala substitutioncan be used as a strategy to stabilize globular proteins. Thepossible mechanisms of protein stabilization are also discussed. |
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Keywords: | neutral protease/ protein engineering/ protein stability/ proteolytic enzymes/ site-directed mutagenesis |
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