Biochemical characterization and application of a new lipase and its cognate foldase obtained from a metagenomic library derived from fat-contaminated soil

• Published in: International journal of biological macromolecules 2019-09, Vol.137, p.442-454
• Main Authors: Almeida, Janaina Marques, Martini, Viviane Paula, Iulek, Jorge, Alnoch, Robson Carlos, Moure, Vivian Rotuno, Müller-Santos, Marcelo, Souza, Emanuel Maltempi, Mitchell, David Alexander, Krieger, Nadia
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Chromobacterium - enzymology; Co-expression; Enzyme Stability; Enzymes, Immobilized - chemistry; Enzymes, Immobilized - metabolism; Fatty Acids - analysis; Foldase; Gene Library; Hydrogen-Ion Concentration; Hydrolysis; Immobilization; Kinetics; Lipase; Lipase - chemistry; Lipase - metabolism; Metagenome; Metagenomics; Models, Molecular; Protein Conformation; Soil - chemistry; Soil Microbiology; Solvents - pharmacology; Temperature; Triglycerides - metabolism
• ISSN: 0141-8130; 1879-0003
• DOI: 10.1016/j.ijbiomac.2019.06.203

LipMF3 is a new lipase isolated from a metagenomic library derived from a fat-contaminated soil. It belongs to the lipase subfamily I.1 and has identities of 68% and 67% with lipases of Chromobacterium violaceum and C. amazonense, respectively. Genes encoding LipMF3 and its cognate foldase, LifMF3, were cloned and co-expressed in Escherichia coli. The highest hydrolytic activity of purified Lip-LifMF3 was at 40 °C and pH 6.5. Under these conditions, the highest activity was against tributyrin (1650 U mg−1), but it also had high activity against olive oil (862 U mg−1). It was stable in hydrophilic organic solvents (25%, v/v in water) with residual activity around 100% after 24 h. It also showed stability over a wide pH range (5.5 to 11) with residual activity above 80% after 24 h. Lip-LifMF3 was immobilized by covalent bonding onto Immobead 150P and by adsorption onto Sepabeads FP-BU. The latter preparation gave the best results, producing 94% conversion after 5 h for the synthesis of ethyl oleate and a 90% enantiomeric excess of the product (R)‑1‑phenylethyl acetate for the kinetic resolution of (R,S)‑1‑phenyl‑1‑ethanol. The results obtained in this work provide a basis for the development of applications of Lip-LifMF3 in biocatalysis.