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Int. J. Mol. Sci. 2017, 18(11), 2202; https://doi.org/10.3390/ijms18112202

Directed Evolution of Recombinant C-Terminal Truncated Staphylococcus epidermidis Lipase AT2 for the Enhancement of Thermostability

1
Enzyme and Microbial Technology Research Centre, Universiti Putra Malaysia, Serdang 43400, Selangor, Malaysia
2
Department of Microbiology, Faculty of Biotechnology and Biomolecular Science, Universiti Putra Malaysia, Serdang 43400, Selangor, Malaysia
3
Department of Biochemistry, Faculty of Biotechnology and Biomolecular Science, Universiti Putra Malaysia, Serdang 43400, Selangor, Malaysia
*
Author to whom correspondence should be addressed.
Received: 23 September 2017 / Revised: 9 October 2017 / Accepted: 12 October 2017 / Published: 4 November 2017
(This article belongs to the Special Issue Microbial Enzymes)
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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. View Full-Text
Keywords: directed evolution; Staphylococcal lipases; thermostability; characterization; circular dichroism directed evolution; Staphylococcal lipases; thermostability; characterization; circular dichroism
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).
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Veno, J.; Ahmad Kamarudin, N.H.; Mohamad Ali, M.S.; Masomian, M.; Raja Abd. Rahman, R.N.Z. Directed Evolution of Recombinant C-Terminal Truncated Staphylococcus epidermidis Lipase AT2 for the Enhancement of Thermostability. Int. J. Mol. Sci. 2017, 18, 2202.

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