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

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.

Structure elucidation and docking analysis of 5M mutant of T1 lipase Geobacillus zalihae

“…The 5M mutant lipase derived from genetically modified of T1 lipase Geobacillus zalihae consists of 388 amino acids with a molecular weight of 43 kDa. This lipase has five mutations (D43E, T118N, E226D, E250L, and N304E) which have been genetically modified based on the comparison of earth-and space-grown T1 lipase structures [19,20]. This mutant lipase showed similar characteristics in temperature and pH optimum as wild-type T1 lipase, which optimally active at temperature 70˚C and pH 9.…”

“…However, the comparison of thermostability with its parent lipase indicates that 5M lipase has a low tolerance against high temperature based on the half-life study, and melting point analysis. Analysis of the model structure of 5M lipase suggests that the number of molecular interactions such as hydrogen bonds and ion interactions have decreased due to the mutations [20]. Thus, subsequent study has been conducted to solve the three-dimensional structure of 5M lipase and revealed the factor contributes to the decrease in lipase stability using the crystal structure of 5M lipase.…”

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

“…Previously, biochemical characterization of wildtype HT1 lipase has been conducted across a wide range of parameters, including temperature, pH, metal ions, organic solvents and thermostability [ 16 ]. In terms of targeted mutations, there was a shift in optimum temperature for E226D, E250L and N304E in comparison to wildtype HT1 [ 16 ]. The change in optimum temperature was also observed in Y224C and Y224P from thermophilic lipase from Geobacillus sp.…”

“…Comparison of mutated HT1 lipases with previously characterized wildtype HT1 lipase has revealed that mutants in the form of D43E, E226D, E250L and N304E have a longer half-life and higher melting temperature ( Table 5 ). The wildtype HT1 had a half-life time of 540, 85 and 16 min at 60, 70, and 80 °C, respectively, with a melting temperature at 70.9 °C [ 16 ]. A previous study by Syal et al [ 24 ] demonstrated that YLIPL1 and YLIP9L1Bp3 lipases exhibited better thermostability compared to their wildtype, YLIP9 lipase.…”

“…The interactions between residues Gln59-Thr118, Asp43–Gln39, Glu226–Gln254, Glu250–Arg330, and Asn304–Thr306 were absent in the Earth-grown structure [ 15 ]. In another work, a mutant of T1 lipase with five mutation sites was constructed in an attempt to mimic the space-grown structure [ 16 ]. Nonetheless, the multiple mutations failed in creating non-covalent interactions between the targeted residues such as those observed in space-grown structure and which compromised the stability.…”