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.
Highlights
Lipase is a class of enzyme known as serine hydrolases which function as biocatalyst for many applications such as hydrolysis, esterification, interesterification, Molecules 2020, 25, 3430; doi:10.3390/molecules25153430 www.mdpi.com/journal/moleculesMolecules 2020, 25, 3430 and alcoholysis [1,2,3,4]
All mutated lipases were subjected to purification by affinity chromatography (Ni-Sepharose High Performance, GE Healthcare, Chicago, IL, USA), in which the recovery rates of (Ni-Sepharose High Performance, GE Healthcare, Chicago, IL, USA), in which the recovery rates of purification for mutant D43E, T118N, E226D, E250L, and N304E were 58.7%, 58.2%, 53.3%, 58.0%, purification for mutant D43E, T118N, E226D, E250L, and N304E were 58.7%, 58.2%, 53.3%, 58.0%, and 59.9%, for respective protein yields of 456, 426, 438, 798, and 606 mg/L
The results showed that D43E and T118N had their relative lipase activity improved correspond to dimethyl sulfoxide (DMSO) at 129.4% and 120.6%, respectively
Summary
Lipase (triacylglycerol acylhydrolase E.C.3.1.1.3) is a class of enzyme known as serine hydrolases which function as biocatalyst for many applications such as hydrolysis, esterification, interesterification, Molecules 2020, 25, 3430; doi:10.3390/molecules25153430 www.mdpi.com/journal/moleculesMolecules 2020, 25, 3430 and alcoholysis [1,2,3,4]. Lipase (triacylglycerol acylhydrolase E.C.3.1.1.3) is a class of enzyme known as serine hydrolases which function as biocatalyst for many applications such as hydrolysis, esterification, interesterification, Molecules 2020, 25, 3430; doi:10.3390/molecules25153430 www.mdpi.com/journal/molecules. As a leading industrial biocatalyst, lipases can tolerate heat and to remain stable at high temperatures. The ability to tolerate heat is generally obtained from a source where catalytic reaction is naturally performed at an elevated temperature. The use of a thermostable lipase is crucial for industrial processes, because of their ability to slow the denaturation at high temperatures, which contributes to enzyme deactivation [5]. Thermostable enzymes are active at temperatures between 60 ◦ C and 125 ◦ C [6]. Thermostable lipases are known to be resistant to the presence of chemical denaturants, detergents, organic solvents, and protein inhibitors [7]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
