Variants of the industrially relevant protease KP-43 with suppressed activity under alkaline conditions developed using expanded genetic codes.

In the present study, we attempted to control the pH profile of the catalytic activity of the industrially relevant alkaline protease KP-43, by incorporating 3-nitro-l-tyrosine and 3-chloro-l-tyrosine at and near the catalytic site. Thirty KP-43 variants containing these non-natural amino acids at the specific positions were synthesized in Escherichia coli host cells with expanded genetic codes. The variant with 3-nitrotyrosine at position 205, near the substrate binding site, retained its catalytic activity at the neutral pH and showed a 60% activity reduction at pH 10.5. This reduction in the alkaline domain is desirable for enhancing the stability of the enzyme in the liquid laundary detergent, whereas the wild-type molecule showed a 20% increase in response to the same pH shift. The engineered pH dependency of the activity of the variant was ascribed partly to a lowered substrate affinity under the alkaline conditions, in which the incorporated 3-nitrotyrosine was probably charged negatively due to the phenolic pKa lower than that of tyrosine.