Improvement of thermal stability via outer-loop ion pair interaction of mutated T1 lipase from Geobacillus zalihae strain T1

Abstract

Mutant D311E and K344R were constructed using site-directed mutagenesis to introduce an additional ion pair at the inter-loop and the intra-loop, respectively, to determine the effect of ion pairs on the stability of T1 lipase isolated from Geobacillus zalihae. A series of purification steps was applied, and the pure lipases of T1, D311E and K344R were obtained. The wild-type and mutant lipases were analyzed using circular dichroism. The T(m) for T1 lipase, D311E lipase and K344R lipase were approximately 68.52 °C, 70.59 °C and 68.54 °C, respectively. Mutation at D311 increases the stability of T1 lipase and exhibited higher T(m) as compared to the wild-type and K344R. Based on the above, D311E lipase was chosen for further study. D311E lipase was successfully crystallized using the sitting drop vapor diffusion method. The crystal was diffracted at 2.1 Å using an in-house X-ray beam and belonged to the monoclinic space group C2 with the unit cell parameters a = 117.32 Å, b = 81.16 Å and c = 100.14 Å. Structural analysis showed the existence of an additional ion pair around E311 in the structure of D311E. The additional ion pair in D311E may regulate the stability of this mutant lipase at high temperatures as predicted in silico and spectroscopically.

Keywords: CD spectral analysis; X-ray diffraction; ion pair interaction; lipase crystal; thermal stability; thermostable lipase.

Figure 1

Protein tailoring of D311E lipase forming inter-loops networking, whereby K344R forming intra-loop networking. The green dash line indicated ion pair and hydrogen bond interactions.

Figure 2

Denatured protein analysis by circular dichroism. T_m of T1 lipase (blue) was at ~68.52 °C, K344R lipase (green) at ~68.54 °C and D311E lipase (red) at ~70.59 °C.

Figure 3

CD spectra of T1 lipase (green) and mutant D311E (blue) and K344R (red).

Figure 4

Effect of temperature on D311E and T1 lipase activity and stability. (A) The optimum temperature of D311E and T1 lipase; (B) Effect of temperature on lipase stability, pre-incubated at 70 °C; (C) Effect of temperature on lipase stability, pre-incubated at 60 °C.

Figure 5

(A) SDS-PAGE (12%) of crude mutant lipase D311E. M: standard protein markers (kDa); (B) SDS-PAGE (12%) of crude native T1 lipase. M: standard protein markers.

Figure 6

(A) SDS-PAGE (12%) of mutant lipase D311E. M: standard protein markers (kDa); purified lipase (lane 1 and 2); (B) Single band of D311E lipase on Native PAGE analysis after ion exchange chromatography.

Figure 7

D311E lipase crystal grown using sitting drop method with 0.1 M MES pH 5.5, 0.1 M Sodium phosphate, 0.1 M Potassium phosphate, and 1.5 M NaCl as precipitant reagent.

Figure 8

Diffraction patterns of thermostable mutant lipase D311E. The resolution is ~2.1 Å at the edge with starting position; distance: 50.00 mm, 2 theta: 22.00°, Omega: 31.64°, Phi: 271.48°, Chi: −30.01° and ending position; distance: 50.00 mm, 2 theta: 22.00°, Omega: 31.64°, Phi: 271.98°, Chi: −30.01°.

Figure 9

Snapshoot of ion pair interaction formation of D311E lipase crystal structure at residue between Glu311 and four residues (Arg 274, Thr 278, Gly 279 and Arg 303). * The generated 2Fo-Fc electron-density maps with 1.0 sigma level.

Figure 10

Schematic diagram of the parental plasmid pGEX/T1S. Expression of the T1, D311E and K344R lipase gene were done as the fusion protein.