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Substitution of three aromatic residues with threonine (W385T), tyrosine (F417Y), and alanine (W525A), present at the corresponding positions in EcGGT, resulted in enhanced transpeptidase activity for all three mutants, while a 514% decrease in hydrolytic activity was observed

Cooperativity and flexibility in enzyme evolution

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This was followed by the addition of 300 l of water and the resulting suspension was centrifuged at 18,500 g for 10 minutes at 4 C