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Characterization of Organic Solvent-Tolerant Lipolytic Enzyme from Marinobacter lipolyticus Isolated from the Antarctic Ocean

Park, Se Hyeon, Kim, Soo-jin, Park, Seongsoon, Kim, Hyung Kwoun
Applied biochemistry and biotechnology 2019 v.187 no.3 pp. 1046-1060
DNA, Escherichia coli, Marinobacter, amino acids, anion exchange, catalytic activity, esterases, gel chromatography, genes, marine environment, models, molecular weight, open reading frames, pH, sequence analysis, site-directed mutagenesis, solvents, substrate specificity, temperature, translation (genetics), triacylglycerols, Antarctic region
The Antarctic marine environment provides a good source of novel lipolytic enzymes that possess beneficial properties, i.e., resistance to extreme physical and chemical conditions. We found a lipolytic Escherichia coli colony that was transformed using genomic DNA from Marinobacter lipolyticus 27-A9 isolated from the Antarctic Ross Sea. DNA sequence analysis revealed an open reading frame of lipolytic enzyme gene. The gene translates a protein (LipA9) of 404 amino acids with molecular mass of 45,247 Da. Recombinant LipA9 was expressed in E. coli BL21 (DE3) cells and purified by anion exchange and gel filtration chromatography. The kcₐₜ/Kₘ of LipA9 was 175 s⁻¹ μM⁻¹, and the optimum temperature and pH were 70 °C and pH 8.0, respectively. LipA9 had quite high organic solvent stability; it was stable toward several common organic solvents up to 50% concentration. Substrate specificity studies showed that LipA9 preferred a short acyl chain length of p-nitrophenyl ester and triglyceride. Sequence analysis showed that LipA9 contained catalytic Ser⁷² and Lys⁷⁵ in S-x-x-K motif, like family VIII esterases. Homology modeling and site-directed mutagenesis studies revealed that Tyr¹⁴¹ and Tyr¹⁸⁸ residues were located near the conserved motif and played an important role in catalytic activity.