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. 2020 Jul 28;25(15):3430.
doi: 10.3390/molecules25153430.

Ion-Pair Interaction and Hydrogen Bonds as Main Features of Protein Thermostability in Mutated T1 Recombinant Lipase Originating from Geobacillus zalihae

Siti Nor Hasmah Ishak  1   2 , Nor Hafizah Ahmad Kamarudin  1   3 , Mohd Shukuri Mohamad Ali  1   4 , Adam Thean Chor Leow  1   5   6 , Raja Noor Zaliha Raja Abd Rahman  1   2   6   7
Affiliations

Ion-Pair Interaction and Hydrogen Bonds as Main Features of Protein Thermostability in Mutated T1 Recombinant Lipase Originating from Geobacillus zalihae

Siti Nor Hasmah Ishak et al. Molecules. .

Abstract

A comparative structure analysis between space- and an Earth-grown T1 recombinant lipase from Geobacillus zalihae had shown changes in the formation of hydrogen bonds and ion-pair interactions. Using the space-grown T1 lipase validated structure having incorporated said interactions, the recombinant T1 lipase was re-engineered to determine the changes brought by these interactions to the structure and stability of lipase. To understand the effects of mutation on T1 recombinant lipase, five mutants were developed from the structure of space-grown T1 lipase and biochemically characterized. The results demonstrate an increase in melting temperature up to 77.4 °C and 76.0 °C in E226D and D43E, respectively. Moreover, the mutated lipases D43E and E226D had additional hydrogen bonds and ion-pair interactions in their structures due to the improvement of stability, as observed in a longer half-life and an increased melting temperature. The biophysical study revealed differences in β-Sheet percentage between less stable (T118N) and other mutants. As a conclusion, the comparative analysis of the tertiary structure and specific residues associated with ion-pair interactions and hydrogen bonds could be significant in revealing the thermostability of an enzyme with industrial importance.

Keywords: T1 lipase; hydrogen bonds; ion-pair interactions; site-directed mutagenesis; thermostability.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The SDS-PAGE analysis showed the purified mutated HT1 lipases after affinity chromatography. Lane 1, Marker. Lane 2, D43E. Lane 3, T118N. Lane 4, E226D. Lane 5, E250L. Lane 6, N304E. About 0.1 µg of purified mutant lipases in 20 mM sodium phosphate (pH 7.4) buffer are loaded into each well.
Figure 2
Figure 2
Effects of temperatures on activity of mutated HT1 lipase. The activity was determine using colorimetric assay and the optimum activity of each enzyme was recorded as 100%. The protein concentrations were standardized to 50 µg for each sample. The assays were conducted in triplicate.
Figure 3
Figure 3
Effects of pH on activity of mutated HT1 lipases. (a) D43E. (b) T118N. (c) E226D. (d) E250L. (e) N304E. Dashed line represent the activity of lipase in different buffers with different pH values. Straight line represents the residual activity left after treated with buffer at different pH value for 30 min (60 °C) prior to lipase assay. The enzyme concentration was standardized to 50 µg for each sample. The assay was performed in triplicate using olive oil as substrate. The higher activity of each mutant lipase was recorded as 100%.
Figure 4
Figure 4
Effect of organic solvents on the catalytic activity of HT1 lipase mutants. The relative activity of each lipase was compared to the untreated lipase without organic solvents. The value of untreated enzyme for each lipase was taken as 100% and not presented in the graph.
Figure 5
Figure 5
Circular dichroism (CD) spectra analysis of mutants.
Figure 6
Figure 6
Overall structure of HT1 lipase with mutated amino acids. The figure was generated by YASARA software [20]. Blue color helical structures represent the lids. Amino acids covered with green surface represent active sites. Mutated amino acids colored in brown. Green ball represents calcium ion, yellow ball represents chloride ion, blue ball represents sodium ion, and red ball represents zinc ion.
Figure 7
Figure 7
Hydrogen bond between residue Glu43 and Gln39 presented in homology model of D43E mutant structure. Hydrogen bond colored in magenta. The figure was generated using YASARA software [20].
Figure 8
Figure 8
Ion-pair interaction in mutant structures. (a) Ion-pair interactions in D43E mutant structure between amino acid Lys84–Glu132 (b) Ion-pair interactions in D43E mutant structure between amino acid Lys329–Asp166 (c) Ion-pair network in D43E mutant structure comprising amino acids Glu38, Arg21, and Asp36. (d) The largest ion-pair networks in E226D structure corresponding to amino acids Asp209, Arg92, and Asp205. Ion-pair interaction colored in magenta. The figure was generated using YASARA software [20].
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