Abstract
BackgroundLipases 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. ResultsLipEMP48-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%. ConclusionsThis 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.
Highlights
Lipases are promising biocatalysts for industrial applications and attract attention to be explored
Optimization of LipEMP48-D lipase gene The entire Open reading frame (ORF) of the LipEMP48-D lipase gene consisted of 1356 bp encoding 451 amino acid residues, including 31 amino acids of native signal sequence of M. luteus
The native signal sequence was eliminated by EcoRI at the sites between 93 and 94 bases, it resulting in 1263 bp full ORF without signal sequence (Fig. 1)
Summary
Lipases are promising biocatalysts for industrial applications and attract attention to be explored. Lipases (triacylglycerol acyl-hydrolase, EC 3.1.1.3) are enzymes that have the responsibility to catalyze the hydrolysis of triglyceride ester bonds into diglycerides, monoglycerides, fatty acid, and glycerol, and work for the synthesis of long fatty acid chains acylglycerol at the interface between oil and water [1,2,3] Owing to their enzymatic properties, lipases have. The research about acidic lipase that has been reported is LipEMP48-D lipase, isolated from the lipolytic bacteria Microccoccus luteus EMP48-D screened from tempeh [28]. This lipase is relatively new because there has been no literature available for M. luteus lipase. It makes the P. pastoris become the most frequently used host for heterologous production of recombinant proteins [33]
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