The Protein Engineering of Zearalenone Hydrolase Results in a Shift in the pH Optimum of the Relative Activity of the Enzyme

Abstract

An acidic shift in the pH profile of Clonostachys rosea zearalenone hydrolase (ZHD), the most effective and well-studied zearalenone-specific lactone hydrolase, is required to extend the range of applications for the enzyme as a decontamination agent in the feed and food production industries. Amino acid substitutions were engineered in the active center of the enzyme to decrease the pKa values of the catalytic residues E126 and H242. The T216K substitution provided a shift in the pH optimum by one unit to the acidic region, accompanied by a notable expansion in the pH profile under acidic conditions. The engineered enzyme demonstrated enhanced activity within the pH range of 3-5 and improved the activity within the pH ranging from 6 to 10. The D31N and D31A substitutions also resulted in a two-unit shift in the pH optimum towards acidic conditions, although this was accompanied by a significant reduction in the enzyme activity. The D31S substitution resulted in a shift in the pH profile towards the alkaline region. The alterations in the enzyme properties observed following the T216K substitution were consistent with the conditions required for the ZHD application as decontamination enzymes at acidic pH values (from 3.0 to 6.0).

Keywords: enzymatic degradation; lactonohydrolase; pH profile; protein engineering; recombinant proteins; zearalenone.

Figure 1

The tertiary structure of C. rosea ZHD. The S102-H242-E126 catalytic triad in the active center of the enzyme; the residues S102, H242, and E126 are shown in green (a); (b) the pocket of the active center. The molecule of the substrate (zearalenone) is shown in gray; atoms of nitrogen, oxygen, and sulfur in the structure of the enzyme are shown in blue, red, and yellow, respectively.

Figure 2

Location of the residues G213, T216, and F221 near the entrance to the pocket and the residue D31 at the bottom of the active center pocket of C. rosea ZHD. The substrate molecule (zearalenone) is shown in gray; the surface of the pocket of the active center is shown in transparent gray; the residues D31, G213, T216, and F221 are shown in green spheres, with nitrogen and oxygen atoms shown in blue and red, respectively.

Figure 3

The tertiary structure of C. rosea ZHD with the T216K and T216R amino acid substitutions. The S102-H242-E126 catalytic triad in the active center of the enzyme is shown in green, the substrate molecule (zearalenone) is shown in gray, and the T216K and T216R substitutions are shown in dark gray. Nitrogen and oxygen atoms are shown in blue and red, respectively.

Figure 4

The tertiary structure of C. rosea ZHD with the T216K amino acid substitution (a) and the wild-type enzyme (b). The surface of the active center pocket is shown in transparent gray; the molecule of the substrate zearalenone is shown in gray and magenta for the modes at the bottom of the pocket and closer to the entrance to the pocket, respectively; the residues K216 and T216 are shown in green spheres, with nitrogen and oxygen atoms shown in blue and red, respectively.

Figure 5

The tertiary structure of C. rosea ZHD with the residue D31 at the bottom of the active center pocket. The S102-H242-E126 catalytic triad in the active center of the enzyme is shown in green, and the molecule of the substrate zearalenone is shown in gray. Nitrogen and oxygen atoms are shown in blue and red, respectively.

Figure 6

SDS-PAGE electropherogram of the crude lysates of E. coli cells (C—non-induced control; 1—native ZHD; 2—ZHD(T216K); 3—ZHD(D31A); 4—ZHD(D31S); 5—ZHD(D31N) and M—molecular weight marker).

Figure 7

The influence of pH on the zearalenone conversion by mutant and wild-type forms of C. rosea ZHD. Y-bars on curves show standard deviation (p < 0.05).