Citation
Ishak, Siti Nor Hasmah
(2019)
Redesigning of Geobacillus zalihae T1 lipase based on spacegrown crystal structure.
Doctoral thesis, Universiti Putra Malaysia.
Abstract
A microgravity environment is a favorable condition meant for growing a
protein crystal, due to less sedimentation and convection. These factors would
have benefited the protein crystal in terms of morphologies, crystal quality and
appearance, which are important in producing a high quality electron density
map. Nevertheless, the differences of structural architecture and features in
protein related to the formation of hydrogen bonds and ion interactions remains
unclear. In order to understand the relative contributions of a space atomic
model in protein structure stability, it was necessary to compare the structure
with the one grown on earth condition. There are existing limitations about
manipulation of structural information for production of new enzyme due to
insufficient analysis of both structures. Therefore, an earth and space condition
crystal structures from a thermostable T1 lipase of Geobacillus zalihae were
analyzed and compared. It was anticipated that the differences in hydrogen
bonds and ion interactions are the main contributing factors towards protein
stabilization. A molecular dynamics simulations approach was used to study
differences of atomic fluctuations and conformational changes of both T1 lipase
structures. From here, the structures stability was determined by a set of
parameters comprising root mean square deviation (RMSD), radius of gyration,
and root mean square fluctuation (RMSF) in which the results showed a more
stable space-grown structure compared to the earth-grown structure due to the
presence of more hydrogen bonds. According to the in silico data, hydrogen bond
interactions at position Asp43, Thr118, Glu250 and Asn304 and ion interaction at position Glu226 were chosen to imitate the space-grown crystal structure.
Following that, the impact of combined interactions in mutated structure of T1
lipase was studied. The molecular interactions of five single mutants and the one
that combined all five mutations, 5M were predicted based on structural changes
and energy landscape by GROMACS simulation package. Site directed
mutagenesis was applied on wild-type HT1 (wt-HT1) lipase to generate five
single mutants (D43E, T118N, E226D, E250L and N304E), in which these sites
were further combined by a gene synthesis to generate a new mutant showing
five mutation points (D43E/T118N/E226D/E250L/N304E). The native lipase wt-
HT1, single mutants and 5M mutant lipases were purified by affinity
chromatography showing a recovery between 49.6 to 59.9% and a purification
fold of 2.5 to 3.3. All lipases exhibited high activity at 60 to 80 °C. Mutants E250L
and N304E shifted in optimum temperature to 80 °C as compared to wt-HT1
lipase. All lipases showed high activity at alkaline conditions of pH 6.0 to 9.0.
The thermostability study indicates the mutant E226D as the most stable lipase
having prolonged half-life (T1/2) values and melting temperature. A T1/2 value of
E226D was found at 28 hours, 165 minutes and 47 minutes at 60 °C, 70 °C and 80
°C, respectively where the mutant reportedly showing a melting temperature
(Tm) of 77.4 ± 2.6 °C. In contrast, mutation of all five positions in the 5M mutant
failed to increase the stability of lipase as the half-life at 60 °C exhibited a decline
from 9 hours to 6 hours. At 70 °C and 80 °C, the half-life was found to be 23
minutes and 8 minutes, respectively. The melting temperature decreased 3.3 °C
to 67.6 ± 0.8 °C. The presence of metal ions, especially calcium ion, had a positive
effect on the stability of D43E, T118N, E250L and 5M lipases, which increased as
more calcium was added. Meanwhile, Zn2+, Cu2+, Mg2+ and Fe3+ ions inhibited the
activity of lipases. In addition, the activities of D43E, T118N and 5M lipases
increased in the presence of DMSO. All lipases showed a good hydrolysis rate
in natural oil, except for coconut oil. All lipases shown to have loss in activities
in the presence of surfactants and sodium dodecyl sulfate (SDS). In the presence
of calcium ion, the stability of 5M mutant and wt-HT1 lipases were increased
towards high temperatures and organic solvents. The presence of calcium
prolonged the half-life of 5M and wt-HT1, and increased the Tm at 8.4 and 12.1
°C, respectively. The combination of substituted amino acid had produced a
highly stable mutant hydrolyzing oil in selected organic solvents such as DMSO,
n-hexane and n-heptane. To correlate mutations in 5M mutant with its structural
transition, 5M mutant lipase was subjected to crystallization in 0.5 M sodium
cacodylate trihydrate, 0.4 M sodium citrate tribasic pH 6.5 supplemented with
0.2 M sodium chloride (NaCl). The protein structure was elucidated at resolution
2.64 Å with 90.9% completeness. The crystal structure of 5M mutant consists of
two asymmetric units that are similar to each other, with RMSD value of 0.7789
Å after superimpositions of chains A and B. The structure analysis revealed that
5M failed to introduce hydrogen bonds and ionic interaction at the intended
positions. The cumulative mutations also resulted in decreasing in molecular interactions such as hydrogen bonds and interactions. The impacts of the
mutations resulted in decreasing in stability and half-life of lipase against high
temperature. As a conclusion, it is difficult to emulate the cumulative
interactions happened in the space-grown T1 lipase as shown by mutant 5M.
Nonetheless, lipases containing a single mutant of D43E and E226D were found
to be successful in introducing and increasing the mutant stability, where the
stability of protein structure was highly dependent on the role of hydrogen
bonds and ion interactions.
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