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

Abstract

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.

Serine protease CrKP43 interacts with MAPK and regulates fungal development and mycoparasitism in Clonostachys chloroleuca

“…In contact with mycohosts or exposure to their signals, the expression of genes associated with the production of secondary metabolites, hydrolytic enzymes, and other secreted proteins is induced in C. chloroleuca ( 35 , 45 , 46 ). Proteases representing a very diverse group of hydrolases are involved in various cellular processes in organisms ( 1 , 3 , 47 ). In the current study, we found that the serine protease gene CrKP43 was markedly upregulated and expressed both in the highly efficient C. chloroleuca isolate and in mutants lacking the MAPK gene Crmapk under the induction of S. sclerotiorum sclerotia.…”

Non-Canonical Amino Acids in Analyses of Protease Structure and Function

“…Employing subtilisin from Bacillus lentus as model protein, regioisomers such as iso-His were generated via a S156C mutant, which did not significantly alter the catalytic properties [ 344 ]. By contrast, the site-specific incorporation of 3NY and 3-chlorotyrosine (3CY) instead of Phe205 near the catalytic center of the subtilisin-like protease KP-43 (S08,123) increased the activity at pH 7 to some extent [ 345 ]. Due to its unique recognition sequence ENLYFQ↓G/S, tobacco etch virus protease (TEV, C04.004) is one of most frequently used enzymes in tailoring recombinant proteins.…”