Single Residue Substitution at N-Terminal Affects Temperature Stability and Activity of L2 Lipase

Abstract

Rational design is widely employed in protein engineering to tailor wild-type enzymes for industrial applications. The typical target region for mutation is a functional region like the catalytic site to improve stability and activity. However, few have explored the role of other regions which, in principle, have no evident functionality such as the N-terminal region. In this study, stability prediction software was used to identify the critical point in the non-functional N-terminal region of L2 lipase and the effects of the substitution towards temperature stability and activity were determined. The results showed 3 mutant lipases: A8V, A8P and A8E with 29% better thermostability, 4 h increase in half-life and 6.6 °C higher thermal denaturation point, respectively. A8V showed 1.6-fold enhancement in activity compared to wild-type. To conclude, the improvement in temperature stability upon substitution showed that the N-terminal region plays a role in temperature stability and activity of L2 lipase.

Keywords: homology modelling; lipase; rational design; stability prediction; thermostability.

Figure 1

Superposition of global structure of wt-L2 (blue) with A8V (orange), A8P (purple) and A8E (green). Shown in the foreground is the anchoring of the N-terminal tail towards the global structure of the lipases. The anchoring is due to the formation of molecular interactions upon substitution at position 8.

Figure 2

The hydrogen bonds (yellow dashes) and hydrophobic interactions (green line) in the catalytic triad of wt-L2 and mutant lipases. (a) wt-L2, (b) A8V, (c) A8P and (d) A8E.

Figure 3

Molecular Dynamics (MD) simulation analysis of wt-L2 and mutant lipases for 100 ns using YASARA. (a) The root mean square deviation (RMSD) values. (b) The root mean square fluctuation (RMSF).

Figure 4

The relative position of the affected local structures with the substitution site in A8V.The flexibility of one of the lid helices α7 (yellow) and the coil and turn hinge (blue) increased upon substitution with valine at the critical point. The affected structures and the α6-helix lid (green) are involved in the interfacial activation mechanism. The affected Zn²⁺ binding domain is shown in magenta. Also shown is the catalytic pocket (grey) and the substitution site (red).

Figure 5

The Zn²⁺ binding domain by residues His81 and His87 (magenta) of the α3-helix. Shown in grey are Asp61 and Asp238, the other 2 residues in Zn²⁺ binding.

Figure 6

The activity profile of wt-L2 and mutants A8V, A8P and A8E. (a) The optimum temperature profile, (b) The thermostability profile and (c) The half-life profile. The relative activity was measured against untreated lipases and assumed to be 100%.

Figure 7

Substrate specificity of wt-L2 and mutant lipases was measured with pNP carbon acyl length between 2 to 16. (a) wt-L2, (b) A8V, (c) A8P and (d) A8E.

Figure 8

Effect of pH on the activity of mutant lipases. (a–c) pH profile of A8V, A8P and A8E, respectively.

Figure 9

pH stability profile of mutant lipases. (a–c) stability profile of A8V, A8P and A8E, respectively.