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. 2019 May 24;20(10):2561.
doi: 10.3390/ijms20102561.

Changes of Thermostability, Organic Solvent, and pH Stability in Geobacillus zalihae HT1 and Its Mutant by Calcium Ion

Siti Nor Hasmah Ishak  1   2 , Malihe Masomian  3 , Nor Hafizah Ahmad Kamarudin  4   5 , Mohd Shukuri Mohamad Ali  6   7 , Thean Chor Leow  8   9   10 , Raja Noor Zaliha Raja Abd Rahman  11   12   13   14
Affiliations

Changes of Thermostability, Organic Solvent, and pH Stability in Geobacillus zalihae HT1 and Its Mutant by Calcium Ion

Siti Nor Hasmah Ishak et al. Int J Mol Sci. .

Abstract

Thermostable T1 lipase from Geobacillus zalihae has been crystallized using counter-diffusion method under space and Earth conditions. The comparison of the three-dimensional structures from both crystallized proteins show differences in the formation of hydrogen bond and ion interactions. Hydrogen bond and ion interaction are important in the stabilization of protein structure towards extreme temperature and organic solvents. In this study, the differences of hydrogen bond interactions at position Asp43, Thr118, Glu250, and Asn304 and ion interaction at position Glu226 was chosen to imitate space-grown crystal structure, and the impact of these combined interactions in T1 lipase-mutated structure was studied. Using space-grown T1 lipase structure as a reference, subsequent simultaneous mutation D43E, T118N, E226D, E250L, and N304E was performed on recombinant wild-type T1 lipase (wt-HT1) to generate a quintuple mutant term as 5M mutant lipase. This mutant lipase shared similar characteristics to its wild-type in terms of optimal pH and temperature. The stability of mutant 5M lipase improved significantly in acidic and alkaline pH as compared to wt-HT1. 5M lipase was highly stable in organic solvents such as dimethyl sulfoxide (DMSO), methanol, and n-hexane compared to wt-HT1. Both wild-type and mutant lipases were found highly activated in calcium as compared to other metal ions due to the presence of calcium-binding site for thermostability. The presence of calcium prolonged the half-life of mutant 5M and wt-HT1, and at the same time increased their melting temperature (Tm). The melting temperature of 5M and wt-HT1 lipases increased at 8.4 and 12.1 °C, respectively, in the presence of calcium as compared to those without. Calcium enhanced the stability of mutant 5M in 25% (v/v) DMSO, n-hexane, and n-heptane. The lipase activity of wt-HT1 also increased in 25% (v/v) ethanol, methanol, acetonitrile, n-hexane, and n-heptane in the presence of calcium. The current study showed that the accumulation of amino acid substitutions D43E, T118N, E226D, E250L, and N304E produced highly stable T1 mutant when hydrolyzing oil in selected organic solvents such as DMSO, n-hexane, and n-heptane. It is also believed that calcium ion plays important role in regulating lipase thermostability.

Keywords: Calcium ion; Geobacillus zalihae; T1 lipase; metal-binding site; organic solvent tolerance; thermostability.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Alignment of 5M mutant with T1 lipase for verification of mutation at amino acid position 43, 118, 226, 250, and 304. The mutated residues are marked in red box.
Figure 2
Figure 2
SDS-PAGE analysis of the wt-HT1 and 5M mutant lipases. The concentration of each samples (crude and purified enzymes) were standardized to 100 µg. Lane M; Molecular weight marker. Lane C1; crude enzyme of wt-HT1 lipase. Lane C2; crude enzyme of 5M mutant lipase. Lane 1; purified wt-HT1 lipase. Lane 2; purified 5M mutant lipase.
Figure 3
Figure 3
Effects of temperatures on activity and stability of lipases. (a) Optimum temperature of wt-HT1 and mutant 5M lipases. (b) Temperature stability of lipases from 30 to 90 °C.
Figure 4
Figure 4
Optimum pH and stability of lipases in different pH values in buffer systems as follow; Sodium acetate buffer (pH 4–6), Sodium phosphate buffer (pH 6–7), Tris-HCl (pH 7–9) and Glycine-NaOH (pH 9–11). Sodium acetate buffer (pH 4–6), Sodium phosphate buffer (pH 6–7), Tris-HCl (pH 7–9) and Glycine-NaOH (pH 9–11). (a) Optimum pH of wt-HT1 lipase. (b) Optimum pH of 5M mutant lipase. (c) pH stability of wt-HT1 lipase. (d) pH stability of 5M mutant lipase.
Figure 4
Figure 4
Optimum pH and stability of lipases in different pH values in buffer systems as follow; Sodium acetate buffer (pH 4–6), Sodium phosphate buffer (pH 6–7), Tris-HCl (pH 7–9) and Glycine-NaOH (pH 9–11). Sodium acetate buffer (pH 4–6), Sodium phosphate buffer (pH 6–7), Tris-HCl (pH 7–9) and Glycine-NaOH (pH 9–11). (a) Optimum pH of wt-HT1 lipase. (b) Optimum pH of 5M mutant lipase. (c) pH stability of wt-HT1 lipase. (d) pH stability of 5M mutant lipase.
Figure 5
Figure 5
Thermostability of wt-HT1 and 5M mutant lipases. (a) Temperature profile at 60 °C. (b) Temperature profile at 70 °C. (c) Temperature profile at 80 °C.
Figure 6
Figure 6
Effect of organic solvent on lipases activities with and without the presence of 1 mM Calcium ion.
Figure 7
Figure 7
Conformational changes in the secondary structure of wt-HT1 and 5M mutant lipases at 20 °C.
Figure 8
Figure 8
Superimposed of 5M mutant modeled structure (color in gold) with T1 lipase crystal structure (color in green). The calcium ion, zinc ion, sodium ion, and chloride ion shown as sphere color by yellow, magenta, red, and green, respectively.
Figure 9
Figure 9
Zinc binding site and calcium-binding site in modeled structure of 5M mutant and Earth-grown T1 lipase crystal structure. (a) Zinc binding site in Earth-grown T1 lipase structure. The Zinc ion (Magenta sphere) are coordinated in tetrahedral form by Asp61, His81, His87, and Asp238. (b) Zinc binding site in 5M mutant coordinated by Asp61, Tyr77, His81, His87, and Asp238. (c) Calcium-binding site in Earth-grown T1 lipase structure coordinated by residues Asp365, Glu360, Gly286, and Pro366. (d) Calcium-binding site in 5M mutant coordinated by residues Asp365, Glu360, Gly286, and Pro366.
Figure 10
Figure 10
Structure of predicted structure of 5M mutant lipase with mutation location sites. The catalytic triad residues (Ser113, Asp317 and His358) are colored as green surface. Mutated residues (Glu43, Asn118, Asp226, Glu250 and Glu304) colored by element.
Figure 11
Figure 11
Structure model of 5M mutant and three-dimensional structure of Earth-grown T1 lipase crystal structure with hydrogen bonds and ion interaction. (a) Hydrogen bond between residues Gln39 and Glu43 in 5M. (b) Hydrogen bond between residues Gln39 and Glu43 in T1. (c) Hydrogen bond between residues Asn59 and Asn118 in 5M. (d) Hydrogen bond between residues Asn59 and Thr118 in T1. (e) Hydrogen bond between residues Asp226 and Arg230 in 5M. (f) Hydrogen bond between residues Glu226 and Arg230 in T1. (g) Hydrogen bond between residues Leu250, Arg330, and Gln254 in 5M. (h) Hydrogen bond and ion interaction between residues Glu250, Arg330, and Gln254 in T1. (i) Hydrogen bond between residues Glu304 and Thr306 in 5M. (j) Hydrogen bond between residues Glu304 and Thr306 in T1.
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