Abstract
In the industrial processes, lipases are expected to operate at temperatures above 45 °C and could retain activity in organic solvents. Hence, a C-terminal truncated lipase from Staphylococcus epidermis AT2 (rT-M386) was engineered by directed evolution. A mutant with glycine-to-cysteine substitution (G210C) demonstrated a remarkable improvement of thermostability, whereby the mutation enhanced the activity five-fold when compared to the rT-M386 at 50 °C. The rT-M386 and G210C lipases were purified concurrently using GST-affinity chromatography. The biochemical and biophysical properties of both enzymes were investigated. The G210C lipase showed a higher optimum temperature (45 °C) and displayed a more prolonged half-life in the range of 40–60 °C as compared to rT-M386. Both lipases exhibited optimal activity and stability at pH 8. The G210C showed the highest stability in the presence of polar organic solvents at 50 °C compared to the rT-M386. Denatured protein analysis presented a significant change in the molecular ellipticity value above 60 °C, which verified the experimental result on the temperature and thermostability profile of G210C.
Highlights
Lipases are versatile due to their capability to react in both aqueous and non-aqueous environments
An attempt to randomly modify the structure of rT-M386 lipase was taken to achieve greater thermostability
The greatest improvement in activity was exhibited by mutant no. 7, which had approximately five-fold higher activity compared to the rT-M386 lipase
Summary
Lipases are versatile due to their capability to react in both aqueous and non-aqueous environments. Since many industrial applications operate at temperatures above 45 ◦C, lipases should preferably have enzymatic activity and thermal stability approximately at 50 ◦C [1]. The thermostability of an enzyme is closely associated with its expression, folding, activities, and functions, thereby thermal stability is a fundamental property of an enzyme [2]. Lipases with increased thermal properties are needed for industrial lipase-catalyzed reactions as they can increase the conversion rate of lipid substrates, especially with a high melting point, resist chemical modifications at elevated temperature and prevent contamination by microorganism [6,7]. Organic tolerant lipases with increased thermal properties are needed for the industrial lipase-catalyzed reactions as they can resist chemical modifications, prevent contamination by microorganism and increase the conversion rate of lipid substrates, especially with a high melting point [6,7]. Many researchers have characterized thermostable microbial lipases, only a few lipases have been reported to possess both thermostable and organic solvent-tolerant properties
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