title: Isolation and characterization of EstC, a new cold-active esterase from Streptomyces coelicolor A3(2).
abstract: The genome sequence of Streptomyces coelicolor A3(2) contains more than 50 genes coding for putative lipolytic enzymes. Many studies have shown the capacity of this actinomycete to store important reserves of intracellular triacylglycerols in nutrient depletion situations. In the present study, we used genome mining of S. coelicolor to identify genes coding for putative, non-secreted esterases/lipases. Two genes were cloned and successfully overexpressed in E. coli as His-tagged fusion proteins. One of the recombinant enzymes, EstC, showed interesting cold-active esterase activity with a strong potential for the production of valuable esters. The purified enzyme displayed optimal activity at 35°C and was cold-active with retention of 25% relative activity at 10°C. Its optimal pH was 8.5-9 but the enzyme kept more than 75% of its maximal activity between pH 7.5 and 10. EstC also showed remarkable tolerance over a wide range of pH values, retaining almost full residual activity between pH 6-11. The enzyme was active toward short-chain p-nitrophenyl esters (C2-C12), displaying optimal activity with the valerate (C5) ester (k(cat)/K(m) = 737±77 s(-1) mM(-1)). The enzyme was also very active toward short chain triglycerides such as triacetin (C2:0) and tributyrin (C4:0), in addition to showing good primary alcohol and organic solvent tolerance, suggesting it could function as an interesting candidate for organic synthesis of short-chain esters such as flavors.
authors.affiliation: INRS-Institut Armand-Frappier, Université du Québec, Laval, Québec, Canada.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Brault
authors.forename: François
authors.initials: F
authors.lastname: Shareck
authors.forename: Yves
authors.initials: Y
authors.lastname: Hurtubise
authors.forename: François
authors.initials: F
authors.lastname: Lépine
authors.forename: Nicolas
authors.initials: N
authors.lastname: Doucet
mesh_terms: D000595:Amino Acid Sequence; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000235:genetics; D003080:Cold Temperature; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D004950:Esterases / Q000737:chemistry*; D004952:Esters; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D048372:Streptomyces coelicolor / Q000378:metabolism*; D013696:Temperature
chemical_list: D004952:Esters; D004950:Esterases; D002265:Carboxylic Ester Hydrolases; D008049:Lipase