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The Escherichia coli enzyme EcAIII catalyzes the hydrolysis of L‐Asn to L‐Asp and ammonia. Using a nature‐inspired mutagenesis approach, we designed and produced five new EcAIII variants (M200I, M200L, M200K, M200T, M200W). The modified proteins were characterized by spectroscopic and crystallographic methods. All new variants were enzymatically active, confirming that the applied mutagenesis procedure...