Purification and characterisation of an F16L mutant of a thermostable lipase.

The Protein Journal
Mohd Shukuri Mohamad AliAbu Bakar Salleh

Abstract

A mutant of the lipase from Geobacillus sp. strain T1 with a phenylalanine to leucine substitution at position 16 was overexpressed in Escherichia coli strain BL21(De3)pLysS. The crude enzyme was purified by two-step affinity chromatography with a final recovery and specific activity of 47.4 and 6,315.8 U/mg, respectively. The molecular weight of the purified F16L lipase was approximately 43 kDa by 12% SDS-PAGE analysis. The F16L lipase was demonstrated to be a thermophilic enzyme due its optimum temperature at 70 °C and showed stability over a temperature range of 40-60 °C. The enzyme exhibited an optimum pH 7 in phosphate buffer and was relatively stable at an alkaline pH 8-9. Metal ions such as Ca(2+), Mn(2+), Na(+), and K(+) enhanced the lipase activity, but Mg(2+), Zn(2+), and Fe(2+) inhibited the lipase. All surfactants tested, including Tween 20, 40, 60, 80, Triton X-100, and SDS, significantly inhibited the lipolytic action of the lipase. A high hydrolytic rate was observed on long-chain natural oils and triglycerides, with a notable preference for olive oil (C18:1; natural oil) and triolein (C18:1; triglyceride). The F16L lipase was deduced to be a metalloenzyme because it was strongly inhibited by 5 mM EDTA. Moderate ...Continue Reading

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Citations

Sep 8, 2012·Applied Biochemistry and Biotechnology·Saisubramanian Nagarajan
Dec 23, 2014·BioMed Research International·Lívia Tereza Andrade SouzaRodrigo R Resende
May 20, 2017·Antonie van Leeuwenhoek·Daniel Borda-MolinaSandra Baena
Aug 1, 2020·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Siti Nor Hasmah IshakRaja Noor Zaliha Raja Abd Rahman

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