Expression of novel acidic lipase from Micrococcus luteus in Pichia pastoris and its application in transesterification

Abstract

Background Lipases are promising biocatalysts for industrial applications and attract attention to be explored. A novel acidic lipase has been isolated from the lipolytic bacteria Micrococcus luteus EMP48-D (LipEMP48-D) screened from tempeh. The lipase gene had previously been overexpressed in Escherichia coli BL21, but the expression level obtained was relatively low. Here, to improve the expression level, the lipase gene was cloned to Pichia pastoris. We eliminated the native signal sequence of M. luteus and replaced it with α-mating factor (α-MF) signal sequence. We also optimized and synthesized the lipase gene based on codon preference in P. pastoris. Results LipEMP48-D lipase was expressed as an extracellular protein. Codon optimization has been conducted for 20 codons, with the codon adaption index reaching 0.995. The highest extracellular lipase activity obtained reached 145.4 ± 4.8 U/mg under AOX1 promoter in P. pastoris KM71 strain, which was 9.7-fold higher than the previous activity in E. coli. LipEMP48-D showed the highest specific activity at pH 5.0 and stable within the pH range 3.0–5.0 at 40 °C. LipEMP48-D also has the capability of hydrolyzing various long-chain triglycerides, particularly olive oil (100%) followed by sunflower oil (88.5%). LipEMP48-D exhibited high tolerance for various polar organic solvents with low log P, such as isopropanol (115.7%) and butanol (114.6%). The metal ions (Na+, K+, Ca2+, Mg2+, Mn+) decreased enzyme activity up to 43.1%, while Fe2+ increased relative activity of enzymes up to 200%. The conversion of free fatty acid (FFA) into fatty acid methyl ester (FAME) was low around 2.95%. Conclusions This study was the first to report overexpression of Micrococcus lipase in yeast. The extracellular expression of this acidic lipase could be potential for biocatalyst in industrial fields, especially organic synthesis, food industry, and production of biodiesel.

A Valuable Product of Microbial Cell Factories: Microbial Lipase

“…The optimum pH of a solvent-resistant lipase from Micrococcus luteus EMP48-D was 5.0; enzyme activity significantly decreased up to pH 6.0 and reducing continuously from pH 6.0 to pH 10.0, although was more than 85% of the initial activity of LipEMP48-D after incubation for 120 min at 40 °C ( Adina et al, 2021 ). The heat and organic solvent-resistant lipase NS-40116 from T. lanuginosus displays highest activity at pH = 7.0 and maintains stability between pH 5.0–9.0 ( Facin et al, 2018 ).…”

Cloning and Co-Expression of Lipase and its Specific Foldase from Ralstonia pickettii BK6

“…Organic solvents can increase the hydrophobicity of the α-helix structure of the lipase which has implications for supporting the interaction between the substrate and the active site of the enzyme. LipRM stability to ethanol and methanol plays an important role in biodiesel production through a transesterification reaction (Adina et al 2021;Jayaraman et al 2020).…”

Aplicação de microrganismos lipolíticos em alimentos e na biodegradação de poliuretanos

“…Finally, the enzyme was immobilized on different hydrophobic supports for the transesterification reaction. An acidic lipase gene from the lipolytic bacteria Micrococcus luteus was successfully cloned to the yeast Pichia pastoris and utilizing combined codon optimization and optimization of signal sequence for extracellular expression resulted in increased gene expression compared to the previous expression in E. coli (Adina et al, 2021).…”