Abstract
KP-43, a 43-kDa alkaline serine protease, is resistant to chemical oxidants and surfactants, making it suitable for use in laundry detergents. An amino acid residue at position 195, in a unique flexible loop that binds a Ca2+ ion, dramatically affects the proteolytic activity and thermal stability of KP-43. In the present study, we obtained 20 variants with substitutions at position 195 and investigated how these residues affect hydrolytic activity toward a macromolecular substrate (casein) and a synthetic tetra-peptide (AAPL). At pH 10, the variant with the highest caseinolytic activity, Tyr195Gln, exhibited 4.4-fold higher activity than the variant with the lowest caseinolytic activity, Tyr195Trp. A significant negative correlation was observed between the hydrophobicity of the residue at position 195 and caseinolytic activity at pH 8–10. At pH 7, the correlation became weak; at pH 6, the correlation reversed to positive. Unlike casein, in the case of hydrolysis of AAPL, no correlation was observed at pH 10 or pH 6. Because the amino acid residue at position 195 is located on the protein surface and considered sufficiently far from the active cleft, the variation in caseinolytic activity between the 20 variants was attributed to changes in interaction efficiency with different states of casein at different pH values. To improve the enzymatic activity, we propose substituting amino acid residues on the protein surface to change the efficiency of interaction with the macromolecular substrates.Key points• A single amino acid residue on the protein surface markedly changed enzyme activity.• The hydrophobicity of the amino acid residue and enzyme activity had a correlation.• The key amino acid residue for substrate recognition exists on the protein surface.
Highlights
Proteases are enzymes widely used in a variety of industrial applications, such as detergents, food and feed production, Electronic supplementary material The online version of this article contains supplementary material, which is available to authorized users.Analyses of three-dimensional structures of subtilisins and subtilisin/inhibitor complexes revealed the residues involved in substrate binding in the active cleft (Bode et al 1986; Hirono et al 1984; Mcphalen et al 1985)
The crystal structure indicates that KP43 consists of two domains, a subtilisin-like α/β domain (Ndomain) and a C-terminal jelly roll β-barrel domain (C-domain)
A previous study revealed that substitution of Tyr with Cys at position 195, located in the surface loop of KP-43, simultaneously increases the enzyme’s proteolytic activity and thermal/surfactant stability (Okuda et al 2013)
Summary
Analyses of three-dimensional structures of subtilisins and subtilisin/inhibitor complexes revealed the residues involved in substrate binding in the active cleft (Bode et al 1986; Hirono et al 1984; Mcphalen et al 1985). These residues were the first targets for protein engineering, for improving the catalytic activity of the. The P1 substrate preference of the triple mutant changed to that of subtilisin Carlsberg (Wells et al 1987a, 1987b) These mutations in the catalytic cleft tend to result in biased substrate preference. A mutant that prefers acidic P1 residues does not prefer basic P1 residues, and a mutant that prefers bulky P1 residues does not prefer small P1 residues (Estell et al 1986; Wells et al 1987a, 1987b)
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