Abstract
5M mutant lipase was derived through cumulative mutagenesis of amino acid residues (D43E/T118N/E226D/E250L/N304E) of T1 lipase from Geobacillus zalihae. A previous study revealed that cumulative mutations in 5M mutant lipase resulted in decreased thermostability compared to wild-type T1 lipase. Multiple amino acids substitution might cause structural destabilization due to negative cooperation. Hence, the three-dimensional structure of 5M mutant lipase was elucidated to determine the evolution in structural elements caused by amino acids substitution. A suitable crystal for X-ray diffraction was obtained from an optimized formulation containing 0.5 M sodium cacodylate trihydrate, 0.4 M sodium citrate tribasic pH 6.4 and 0.2 M sodium chloride with 2.5 mg/mL protein concentration. The three-dimensional structure of 5M mutant lipase was solved at 2.64 Å with two molecules per asymmetric unit. The detailed analysis of the structure revealed that there was a decrease in the number of molecular interactions, including hydrogen bonds and ion interactions, which are important in maintaining the stability of lipase. This study facilitates understanding of and highlights the importance of hydrogen bonds and ion interactions towards protein stability. Substrate specificity and docking analysis on the open structure of 5M mutant lipase revealed changes in substrate preference. The molecular dynamics simulation of 5M-substrates complexes validated the substrate preference of 5M lipase towards long-chain p-nitrophenyl–esters.
Highlights
Lipase is a class of enzymes belonging to the serine hydrolases and is widely known as important biocatalysts
The 5M mutant is a hydrolase enzyme derived from genetically modified T1 lipase of Geobacillus zalihae in the aim of introducing extra hydrogen bonds and ion interactions in its structure
Biochemical characterization was conducted across parameters, such as temperature, pH, metal ions, and organic solvents
Summary
Lipase (triacylglycerol acylhydrolase E.C.3.1.1.3) is a class of enzymes belonging to the serine hydrolases and is widely known as important biocatalysts. A lipase needs to offer stability and flexibility while effectively catalyzing broad reactions in organic solvents, high temperatures, and salinity. Organic solvent and temperature tolerant lipase are important in the application of meat degradation, fatty acid ester synthesis, biodiesel processes as well as food and detergent industries [11,12,13,14]. These enzymes allow catalytic reactions to be conducted at higher temperatures and withstand denaturation, which is the main cause of enzyme deactivation [15]
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